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Sample records for brefeldin a-inhibited guanine

  1. BIG1, a brefeldin A-inhibited guanine nucleotide-exchange protein regulates neurite development via PI3K-AKT and ERK signaling pathways.

    Science.gov (United States)

    Zhou, C; Li, C; Li, D; Wang, Y; Shao, W; You, Y; Peng, J; Zhang, X; Lu, L; Shen, X

    2013-12-19

    The elongation of neuron is highly dependent on membrane trafficking. Brefeldin A (BFA)-inhibited guanine nucleotide-exchange protein 1 (BIG1) functions in the membrane trafficking between the Golgi apparatus and the plasma membrane. BFA, an uncompetitive inhibitor of BIG1 can inhibit neurite outgrowth and polarity development. In this study, we aimed to define the possible role of BIG1 in neurite development and to further investigate the potential mechanism. By immunostaining, we found that BIG1 was extensively colocalized with synaptophysin, a marker for synaptic vesicles in soma and partly in neurites. The amount of both protein and mRNA of BIG1 were up-regulated during rat brain development. BIG1 depletion significantly decreased the neurite length and inhibited the phosphorylation of phosphatidylinositide 3-kinase (PI3K) and protein kinase B (AKT). Inhibition of BIG1 guanine nucleotide-exchange factor (GEF) activity by BFA or overexpression of the dominant-negative BIG1 reduced PI3K and AKT phosphorylation, indicating regulatory effects of BIG1 on PI3K-AKT signaling pathway is dependent on its GEF activity. BIG1 siRNA or BFA treatment also significantly reduced extracellular signal-regulated kinase (ERK) phosphorylation. Overexpression of wild-type BIG1 significantly increased ERK phosphorylation, but the dominant-negative BIG1 had no effect on ERK phosphorylation, indicating the involvement of BIG1 in ERK signaling regulation may not be dependent on its GEF activity. Our result identified a novel function of BIG1 in neurite development. The newly recognized function integrates the function of BIG1 in membrane trafficking with the activation of PI3K-AKT and ERK signaling pathways which are critical in neurite development. Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

  2. Brefeldin A inhibits pestivirus release from infected cells, without affecting its assembly and infectivity

    International Nuclear Information System (INIS)

    Macovei, Alina; Zitzmann, Nicole; Lazar, Catalin; Dwek, Raymond A.; Branza-Nichita, Norica

    2006-01-01

    The enveloped bovine viral diarrhea virus (BVDV) is a member of the Pestivirus genus within the Flaviviridae family. While considerable information has been gathered on virus entry into the host cell, genome structure and protein function, little is known about pestivirus morphogenesis and release from cells. Here, we analyzed the intracellular localization, N-glycan processing and secretion of BVDV using brefeldin A (BFA), which blocks protein export from the endoplasmic reticulum (ER) and causes disruption of the Golgi complex with subsequent fusion of its cis and medial cisternae with the ER. BFA treatment of infected cells resulted in complete inhibition of BVDV secretion and increased co-localization of the envelope glycoproteins with the cis-Golgi marker GM 130. Processing of the N-linked glycans was affected by BFA, however, virus assembly was not perturbed and intracellular virions were fully infectious, suggesting that trafficking beyond the cis-Golgi is not a prerequisite for pestivirus infectivity

  3. Brefeldin A inhibits pestivirus release from infected cells, without affecting its assembly and infectivity.

    Science.gov (United States)

    Macovei, Alina; Zitzmann, Nicole; Lazar, Catalin; Dwek, Raymond A; Branza-Nichita, Norica

    2006-08-04

    The enveloped bovine viral diarrhea virus (BVDV) is a member of the Pestivirus genus within the Flaviviridae family. While considerable information has been gathered on virus entry into the host cell, genome structure and protein function, little is known about pestivirus morphogenesis and release from cells. Here, we analyzed the intracellular localization, N-glycan processing and secretion of BVDV using brefeldin A (BFA), which blocks protein export from the endoplasmic reticulum (ER) and causes disruption of the Golgi complex with subsequent fusion of its cis and medial cisternae with the ER. BFA treatment of infected cells resulted in complete inhibition of BVDV secretion and increased co-localization of the envelope glycoproteins with the cis-Golgi marker GM 130. Processing of the N-linked glycans was affected by BFA, however, virus assembly was not perturbed and intracellular virions were fully infectious, suggesting that trafficking beyond the cis-Golgi is not a prerequisite for pestivirus infectivity.

  4. Enantioselective total synthesis of (+)-brefeldin A and 7-epi-brefeldin A.

    Science.gov (United States)

    Wu, Yikang; Shen, Xin; Yang, Yong-Qing; Hu, Qi; Huang, Jia-Hui

    2004-05-28

    A convergent enantioselective route to brefeldin A (BFA) and 7-epi-BFA was developed. The key C-4/C-5 chiral centers were established by using chiral auxiliary induced intermolecular asymmetric aldolization in the presence of TiCl(4) and TMEDA. The results with the thiazolidinethione/TiCl(4) mediated intermolecular asymmetric aldolization added some new information about the scope and limitations to the existing knowledge of that type of reactions (which so far was essentially limited to the reactions with N-propionyl thiazolidinethiones). During the course a method for protecting the liable aldol hydroxyl groups by using inexpensive TBSCl in DMF with 2,6-lutidine as the base was developed to replace the otherwise unavoidable TBSOTf procedure. Due to the excessive steric hindrance, removal of the auxiliary was much more difficult than most literature cases. Cleavage of the oxazolidinone by reduction was almost impossible. The thiazolidinethione auxiliary was relatively easier to remove. However, several reactions reported for facile removal of thiazolidinethione auxiliaries in the literature still failed. Reductive removal of the thiazolidinethione auxiliary was most effectively realized with LiBH(4) in diethyl ether in the presence of 1 equiv of MeOH (a modification of a literature procedure for removal of oxazolidinone auxiliaries in less hindered substrates). Apart from the auxiliary removal, oxidation of the alcohol into aldehyde and the deprotection of the dithiolane protecting group were also rather difficult in the present context. A range of methods were screened before final solutions were found. The five-membered ring was constructed by employing an intramolecular Mukaiyama reaction after many attempts with the intramolecular aldolization under a variety of conditions failed. The rate of elimination of the alkoxyl to form the alpha,beta-double bond of the key intermediate cyclopentenone 49 with DBU was highly solvent dependent (very sluggish in CH(2)Cl(2

  5. 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...

  6. New analogues of brefeldin A from sediment-derived fungus Penicillium sp. DT-F29.

    Science.gov (United States)

    Hu, Zhi-Fei; Qin, Le-Le; Ding, Wan-Jing; Liu, Yu; Ma, Zhong-Jun

    2016-10-01

    Four new analogues of brefeldin A named 7, 7-dimethoxybrefeldin C (3), 6β-hydroxybrefeldin C (4), 4-epi-15-epi-brefeldin A (5), 4-epi-8α-hydroxy-15-epi-brefeldin C (6), together with four known analogues (1, 7-9) were isolated from a fermentation of the sediment-derived fungus Penicillium sp. DT-F29. The structures of these compounds were elucidated on the basis of extensive spectroscopic and chemical methods. In the bioactivity assays, only compounds 1 and 8 showed significant inhibitory activities against human lung adenocarcinoma cell. In addition, compound 1 was first reported for the potent ability to reactivate latent HIV with EC50 value of 0.03 μM.

  7. 21 CFR 73.1329 - Guanine.

    Science.gov (United States)

    2010-04-01

    ... in this subpart as safe and suitable for use in color additive mixtures for coloring externally... ADDITIVES EXEMPT FROM CERTIFICATION Drugs § 73.1329 Guanine. (a) Identity. (1) The color additive guanine is the crystalline material obtained from fish scales and consists principally of the two purines...

  8. Synthesis of Lipophilic Guanine N-9 Derivatives

    DEFF Research Database (Denmark)

    Wamberg, Michael C; Pedersen, Pernille L; Löffler, Philipp M G

    2017-01-01

    the synthesis of five new guanine-N9 derivatives bearing alkyl chains with different attachment chemistries, exploiting a synthesis pathway that allows a flexible choice of hydrophobic anchor moiety. In this study, these guanine derivatives were functionalized with C10 chains for insertion into decanoic acid...... bilayer structures, in which both alkyl chain length and attachment chemistry determined their interaction with the membrane. Incubation of these guanine conjugates, as solids, with a decanoic acid vesicle suspension, showed that ether- and triazole-linked C10 anchors yielded an increased partitioning...... of the guanine derivative into the membranous phase compared to directly N-9-linked saturated alkyl anchors. Decanoic acid vesicle membranes could be loaded with up to 5.5 mol % guanine derivative, a 6-fold increase over previous limits. Thus, anchor chemistries exhibiting favorable interactions with a bilayer...

  9. Effects of brefeldin A on sphingomyelin transport and lipid synthesis in BHK21 cells.

    OpenAIRE

    Kallen, K J; Quinn, P; Allan, D

    1993-01-01

    1. Addition of brefeldin A (BFA) to BHK cells incubated for 4 h with [3H]acetate led to a 3-4-fold increase in incorporation of label into sphingomyelin, monoglucosylceramide and cholesterol ester compared with untreated controls. There was a similar increase in incorporation of [3H]choline into sphingomyelin. The level of cholesterol ester increased 3-fold when BFA was added to cells labelled to equilibrium with [3H]acetate, but no statistically significant changes in the levels of other lip...

  10. Exposure to Brefeldin A promotes initiation of meiosis in murine female germ cells.

    Science.gov (United States)

    Zhang, Lian-Jun; Chen, Bo; Feng, Xin-Lei; Ma, Hua-Gang; Sun, Li-Lan; Feng, Yan-Min; Liang, Gui-Jin; Cheng, Shun-Feng; Li, Lan; Shen, Wei

    2015-01-01

    In mammals, ontogenesis starts from a fusion of spermatozoon and oocyte, which are produced by reductive nuclear division of a diploid germ cell in a specialised but complex biological process known as meiosis. However, little is known about the mechanism of meiotic initiation in germ cells, although many factors may be responsible for meiosis both in male and female gonads. In this study, 11.5 days post coitum (dpc) female fetal mouse genital ridges were cultured in vitro with exposure to Brefeldin A (BFA) for 6h, and the changes in meiosis were detected. Synaptonemal-complex analysis implied that BFA played a positive role in meiosis initiation and this hypothesis was confirmed by quantitative PCR of meiosis-specific genes: stimulated by retinoic acid gene 8 (Stra8) and deleted in a zoospermia-like (DAZL). At the same time, mRNA expression of retinoic acid synthetase (Raldh2) and retinoic acid (RA) receptors increased in female gonads with in vitro exposure to BFA. Transplanting genital ridges treated with BFA into the kidney capsule of immunodeficient mice demonstrated that the development capacity of female germ cells was normal, while formation of primordial follicles was seen to be a result of accelerated meiosis after exposure to BFA. In conclusion, the study indicated that BFA stimulated meiosis initiation partly by RA signalling and then promoted the development of follicles.

  11. Rapid induction of lipid droplets in Chlamydomonas reinhardtii and Chlorella vulgaris by Brefeldin A.

    Directory of Open Access Journals (Sweden)

    Sangwoo Kim

    Full Text Available Algal lipids are the focus of intensive research because they are potential sources of biodiesel. However, most algae produce neutral lipids only under stress conditions. Here, we report that treatment with Brefeldin A (BFA, a chemical inducer of ER stress, rapidly triggers lipid droplet (LD formation in two different microalgal species, Chlamydomonas reinhardtii and Chlorella vulgaris. LD staining using Nile red revealed that BFA-treated algal cells exhibited many more fluorescent bodies than control cells. Lipid analyses based on thin layer chromatography and gas chromatography revealed that the additional lipids formed upon BFA treatment were mainly triacylglycerols (TAGs. The increase in TAG accumulation was accompanied by a decrease in the betaine lipid diacylglyceryl N,N,N-trimethylhomoserine (DGTS, a major component of the extraplastidic membrane lipids in Chlamydomonas, suggesting that at least some of the TAGs were assembled from the degradation products of membrane lipids. Interestingly, BFA induced TAG accumulation in the Chlamydomonas cells regardless of the presence or absence of an acetate or nitrogen source in the medium. This effect of BFA in Chlamydomonas cells seems to be due to BFA-induced ER stress, as supported by the induction of three homologs of ER stress marker genes by the drug. Together, these results suggest that ER stress rapidly triggers TAG accumulation in two green microalgae, C. reinhardtii and C. vulgaris. A further investigation of the link between ER stress and TAG synthesis may yield an efficient means of producing biofuel from algae.

  12. Haploinsufficiency of the Sec7 guanine nucleotide exchange factor gea1 impairs septation in fission yeast.

    Directory of Open Access Journals (Sweden)

    Alan M Eckler

    Full Text Available Membrane trafficking is essential to eukaryotic life and is controlled by a complex network of proteins that regulate movement of proteins and lipids between organelles. The GBF1/GEA family of Guanine nucleotide Exchange Factors (GEFs regulates trafficking between the endoplasmic reticulum and Golgi by catalyzing the exchange of GDP for GTP on ADP Ribosylation Factors (Arfs. Activated Arfs recruit coat protein complex 1 (COP-I to form vesicles that ferry cargo between these organelles. To further explore the function of the GBF1/GEA family, we have characterized a fission yeast mutant lacking one copy of the essential gene gea1 (gea1+/-, the Schizosaccharomyces pombe ortholog of GBF1. The haploinsufficient gea1+/- strain was shown to be sensitive to the GBF1 inhibitor brefeldin A (BFA and was rescued from BFA sensitivity by gea1p overexpression. No overt defects in localization of arf1p or arf6p were observed in gea1+/- cells, but the fission yeast homolog of the COP-I cargo sac1 was mislocalized, consistent with impaired COP-I trafficking. Although Golgi morphology appeared normal, a slight increase in vacuolar size was observed in the gea1+/- mutant strain. Importantly, gea1+/- cells exhibited dramatic cytokinesis-related defects, including disorganized contractile rings, an increased septation index, and alterations in septum morphology. Septation defects appear to result from altered secretion of enzymes required for septum dynamics, as decreased secretion of eng1p, a β-glucanase required for septum breakdown, was observed in gea1+/- cells, and overexpression of eng1p suppressed the increased septation phenotype. These observations implicate gea1 in regulation of septum breakdown and establish S. pombe as a model system to explore GBF1/GEA function in cytokinesis.

  13. Hepatitis C virus replication and Golgi function in brefeldin a-resistant hepatoma-derived cells.

    Directory of Open Access Journals (Sweden)

    Rayan Farhat

    Full Text Available Recent reports indicate that the replication of hepatitis C virus (HCV depends on the GBF1-Arf1-COP-I pathway. We generated Huh-7-derived cell lines resistant to brefeldin A (BFA, which is an inhibitor of this pathway. The resistant cell lines could be sorted into two phenotypes regarding BFA-induced toxicity, inhibition of albumin secretion, and inhibition of HCV infection. Two cell lines were more than 100 times more resistant to BFA than the parental Huh-7 cells in these 3 assays. This resistant phenotype was correlated with the presence of a point mutation in the Sec7 domain of GBF1, which is known to impair the binding of BFA. Surprisingly, the morphology of the cis-Golgi of these cells remained sensitive to BFA at concentrations of the drug that allowed albumin secretion, indicating a dichotomy between the phenotypes of secretion and Golgi morphology. Cells of the second group were about 10 times more resistant than parental Huh-7 cells to the BFA-induced toxicity. The EC50 for albumin secretion was only 1.5-1.8 fold higher in these cells than in Huh-7 cells. However their level of secretion in the presence of inhibitory doses of BFA was 5 to 15 times higher. Despite this partially effective secretory pathway in the presence of BFA, the HCV infection was almost as sensitive to BFA as in Huh-7 cells. This suggests that the function of GBF1 in HCV replication does not simply reflect its role of regulator of the secretory pathway of the host cell. Thus, our results confirm the involvement of GBF1 in HCV replication, and suggest that GBF1 might fulfill another function, in addition to the regulation of the secretory pathway, during HCV replication.

  14. Reactivity of chitosan derivatives and their interaction with guanine ...

    Indian Academy of Sciences (India)

    Density functional theory; hydrogen bonding; chitosan derivative; guanine; solvent effect. 1. Introduction .... Out of different models for accounting the solva- tion energies ..... Authors thank DST, New Delhi for financial support. (Grant No.

  15. Mutagenic and Cytotoxic Properties of 6-Thioguanine, S6-Methylthioguanine, and Guanine-S6-sulfonic Acid*S⃞

    OpenAIRE

    Yuan, Bifeng; Wang, Yinsheng

    2008-01-01

    Thiopurine drugs, including 6-thioguanine (SG), 6-mercaptopurine, and azathioprine, are widely employed anticancer agents and immunosuppressants. The formation of SG nucleotides from the thiopurine prodrugs and their subsequent incorporation into nucleic acids are important for the drugs to exert their cytotoxic effects. SG in DNA can be methylated by S-adenosyl-l-methionine to give S6-methylthioguanine (S6mG) and oxidized by UVA light to render guanine-S6-sulfonic acid ...

  16. [Triplet expansion cytosine-guanine-guanine: Three cases of OMIM syndrome in the same family].

    Science.gov (United States)

    González-Pérez, Jesús; Izquierdo-Álvarez, Silvia; Fuertes-Rodrigo, Cristina; Monge-Galindo, Lorena; Peña-Segura, José Luis; López-Pisón, Francisco Javier

    2016-04-01

    The dynamic increase in the number of triplet repeats of cytosine-guanine-guanine (CGG) in the FMR1 gene mutation is responsible for three OMIM syndromes with a distinct clinical phenotype: Fragile X syndrome (FXS) and two pathologies in adult carriers of the premutation (55-200 CGG repeats): Primary ovarian insufficiency (FXPOI) and tremor-ataxia syndrome (FXTAS) associated with FXS. CGG mutation dynamics of the FMR1 gene were studied in DNA samples from peripheral blood from the index case and other relatives of first, second and third degree by TP-PCR, and the percentage methylation. Diagnosis of FXS was confirmed in three patients (21.4%), eight patients (57.1%) were confirmed in the premutation range transmitters, one male patient with full mutation/permutation mosaicism (7.1%) and two patients (14.3%) with normal study. Of the eight permutated patients, three had FXPOI and one male patient had FXTAS. Our study suggests the importance of making an early diagnosis of SXF in order to carry out a family study and genetic counselling, which allow the identification of new cases or premutated patients with FMR1 gene- associated syndromes (FXTAS, FXPOI). Copyright © 2015 Elsevier España, S.L.U. All rights reserved.

  17. Characterization of oxidative guanine damage and repair in mammalian telomeres.

    Directory of Open Access Journals (Sweden)

    Zhilong Wang

    2010-05-01

    Full Text Available 8-oxo-7,8-dihydroguanine (8-oxoG and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG are among the most common oxidative DNA lesions and are substrates for 8-oxoguanine DNA glycosylase (OGG1-initiated DNA base excision repair (BER. Mammalian telomeres consist of triple guanine repeats and are subject to oxidative guanine damage. Here, we investigated the impact of oxidative guanine damage and its repair by OGG1 on telomere integrity in mice. The mouse cells were analyzed for telomere integrity by telomere quantitative fluorescence in situ hybridization (telomere-FISH, by chromosome orientation-FISH (CO-FISH, and by indirect immunofluorescence in combination with telomere-FISH and for oxidative base lesions by Fpg-incision/Southern blot assay. In comparison to the wild type, telomere lengthening was observed in Ogg1 null (Ogg1(-/- mouse tissues and primary embryonic fibroblasts (MEFs cultivated in hypoxia condition (3% oxygen, whereas telomere shortening was detected in Ogg1(-/- mouse hematopoietic cells and primary MEFs cultivated in normoxia condition (20% oxygen or in the presence of an oxidant. In addition, telomere length abnormalities were accompanied by altered telomere sister chromatid exchanges, increased telomere single- and double-strand breaks, and preferential telomere lagging- or G-strand losses in Ogg1(-/- mouse cells. Oxidative guanine lesions were increased in telomeres in Ogg1(-/- mice with aging and primary MEFs cultivated in 20% oxygen. Furthermore, oxidative guanine lesions persisted at high level in Ogg1(-/- MEFs after acute exposure to hydrogen peroxide, while they rapidly returned to basal level in wild-type MEFs. These findings indicate that oxidative guanine damage can arise in telomeres where it affects length homeostasis, recombination, DNA replication, and DNA breakage repair. Our studies demonstrate that BER pathway is required in repairing oxidative guanine damage in telomeres and maintaining telomere integrity

  18. Endogenous melatonin and oxidatively damaged guanine in DNA

    DEFF Research Database (Denmark)

    Davanipour, Zoreh; Poulsen, Henrik E; Weimann, Allan

    2009-01-01

    overnight guanine DNA damage. 8-oxodG and 8-oxoGua were measured using a high-performance liquid chromatography-electrospray ionization tandem mass spectrometry assay. The mother, father, and oldest sampled daughter were used for these analyses. Comparisons between the mothers, fathers, and daughters were...... attack and increase the rate of repair of that damage. This paper reports the results of a study relating the level of overnight melatonin production to the overnight excretion of the two primary urinary metabolites of the repair of oxidatively damaged guanine in DNA. METHODS: Mother...

  19. Effects of brefeldin A on the endomembrane system and germ tube formation of the tetraspore of Gelidium floridanum (Rhodophyta, Florideophyceae).

    Science.gov (United States)

    Simioni, Carmen; Rover, Ticiane; Schmidt, Éder C; de L Felix, Marthiellen R; Polo, Luz Karime; Santos, Rodrigo Dos; Costa, Giulia Burle; Kreusch, Marianne; Pereira, Debora T; Ouriques, Luciane C; Bouzon, Zenilda L

    2014-06-01

    Gelidium floridanum W.R. Taylor tetraspores are units of dispersal and are responsible for substrate attachment. This study aimed to examine evidence of direct interaction between germ tube formation and Golgi activity during tetraspore germination of G. floridanum. After release, the tetraspores were incubated with brefeldin A (BFA) in concentrations of 4 and 8 μM over a 6 h period. The controls and treatments were analyzed with light, fluorescence (FM4-64 dye) and transmission electron microscopy. In the control samples, the Golgi bodies were responsible for germ tube formation. In contrast, BFA-treated samples were observed to inhibit spore adhesion and germ tube formation. These tetraspores also showed an increase in volume (≥30 μm width). BFA treatment also resulted in the disassembly of Golgi cisternae and the formation of vesiculated areas of the cytoplasm, blocking the secretion of protein and amorphous matrix polysaccharides. When stained with FM4-64, the control samples showed fluorescence in the apical region of the germ tube, but the treated samples showed an intense fluorescence throughout the cytoplasm. From these results, we can conclude that the germ tube is formed by the incorporation of vesicles derived from Golgi. Thus, vesicle secretion and Golgi organization are basic processes and essential in adhesion and tube formation. By blocking the secretion of protein and amorphous matrix polysaccharides, BFA treatment precluded tetraspore germination. © 2014 Phycological Society of America.

  20. Endogenous melatonin and oxidatively damaged guanine in DNA

    Directory of Open Access Journals (Sweden)

    Poulsen Henrik E

    2009-10-01

    Full Text Available Abstract Background A significant body of literature indicates that melatonin, a hormone primarily produced nocturnally by the pineal gland, is an important scavenger of hydroxyl radicals and other reactive oxygen species. Melatonin may also lower the rate of DNA base damage resulting from hydroxyl radical attack and increase the rate of repair of that damage. This paper reports the results of a study relating the level of overnight melatonin production to the overnight excretion of the two primary urinary metabolites of the repair of oxidatively damaged guanine in DNA. Methods Mother-father-daughter(s families (n = 55 were recruited and provided complete overnight urine samples. Total overnight creatinine-adjusted 6-sulphatoxymelatonin (aMT6s/Cr has been shown to be highly correlated with total overnight melatonin production. Urinary 8-oxo-7,8-dihydro-guanine (8-oxoGua results from the repair of DNA or RNA guanine via the nucleobase excision repair pathway, while urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG may possibly result from the repair of DNA guanine via the nucleotide excision repair pathway. Total overnight urinary levels of 8-oxodG and 8-oxoGua are therefore a measure of total overnight guanine DNA damage. 8-oxodG and 8-oxoGua were measured using a high-performance liquid chromatography-electrospray ionization tandem mass spectrometry assay. The mother, father, and oldest sampled daughter were used for these analyses. Comparisons between the mothers, fathers, and daughters were calculated for aMT6s/Cr, 8-oxodG, and 8-oxoGua. Regression analyses of 8-oxodG and 8-oxoGua on aMT6s/Cr were conducted for mothers, fathers, and daughters separately, adjusting for age and BMI (or weight. Results Among the mothers, age range 42-80, lower melatonin production (as measured by aMT6s/CR was associated with significantly higher levels of 8-oxodG (p Conclusion Low levels of endogenous melatonin production among older individuals may lead to

  1. Guanine base stacking in G-quadruplex nucleic acids

    Science.gov (United States)

    Lech, Christopher Jacques; Heddi, Brahim; Phan, Anh Tuân

    2013-01-01

    G-quadruplexes constitute a class of nucleic acid structures defined by stacked guanine tetrads (or G-tetrads) with guanine bases from neighboring tetrads stacking with one another within the G-tetrad core. Individual G-quadruplexes can also stack with one another at their G-tetrad interface leading to higher-order structures as observed in telomeric repeat-containing DNA and RNA. In this study, we investigate how guanine base stacking influences the stability of G-quadruplexes and their stacked higher-order structures. A structural survey of the Protein Data Bank is conducted to characterize experimentally observed guanine base stacking geometries within the core of G-quadruplexes and at the interface between stacked G-quadruplex structures. We couple this survey with a systematic computational examination of stacked G-tetrad energy landscapes using quantum mechanical computations. Energy calculations of stacked G-tetrads reveal large energy differences of up to 12 kcal/mol between experimentally observed geometries at the interface of stacked G-quadruplexes. Energy landscapes are also computed using an AMBER molecular mechanics description of stacking energy and are shown to agree quite well with quantum mechanical calculated landscapes. Molecular dynamics simulations provide a structural explanation for the experimentally observed preference of parallel G-quadruplexes to stack in a 5′–5′ manner based on different accessible tetrad stacking modes at the stacking interfaces of 5′–5′ and 3′–3′ stacked G-quadruplexes. PMID:23268444

  2. Scambio, a novel guanine nucleotide exchange factor for Rho

    Directory of Open Access Journals (Sweden)

    Groffen John

    2004-04-01

    Full Text Available Abstract Background Small GTPases of the Rho family are critical regulators of various cellular functions including actin cytoskeleton organization, activation of kinase cascades and mitogenesis. For this reason, a major objective has been to understand the mechanisms of Rho GTPase regulation. Here, we examine the function of a novel protein, Scambio, which shares homology with the DH-PH domains of several known guanine nucleotide exchange factors for Rho family members. Results Scambio is located on human chromosome 14q11.1, encodes a protein of around 181 kDa, and is highly expressed in both heart and skeletal muscle. In contrast to most DH-PH-domain containing proteins, it binds the activated, GTP-bound forms of Rac and Cdc42. However, it fails to associate with V14RhoA. Immunofluorescence studies indicate that Scambio and activated Rac3 colocalize in membrane ruffles at the cell periphery. In accordance with these findings, Scambio does not activate either Rac or Cdc42 but rather, stimulates guanine nucleotide exchange on RhoA and its close relative, RhoC. Conclusion Scambio associates with Rac in its activated conformation and functions as a guanine nucleotide exchange factor for Rho.

  3. Electron detachment of the hydrogen-bonded amino acid side-chain guanine complexes

    Science.gov (United States)

    Wang, Jing; Gu, Jiande; Leszczynski, Jerzy

    2007-07-01

    The photoelectron spectra of the hydrogen-bonded amino acid side-chain-guanine complexes has been studied at the partial third order (P3) self-energy approximation of the electron propagator theory. The correlation between the vertical electron detachment energy and the charge distributions on the guanine moiety reveals that the vertical electron detachment energy (VDE) increases as the positive charge distribution on the guanine increases. The low VDE values determined for the negatively charged complexes of the guanine-side-chain-group of Asp/Glu suggest that the influence of the H-bonded anionic groups on the VDE of guanine could be more important than that of the anionic backbone structure. The even lower vertical electron detachment energy for guanine is thus can be expected in the H-bonded protein-DNA systems.

  4. Quantum molecular modeling of the interaction between guanine and alkylating agents--2--nitrogen mustard.

    Science.gov (United States)

    Hamza, A; Broch, H; Vasilescu, D

    1996-06-01

    The alkylation mechanism of guanine by nitrogen mustard (HN2) was studied by using a supermolecular modeling at the ab initio 6-31G level. Our computations show that interaction of guanine with the aziridinium form of HN2 necessitates a transition state for the N7 alkylation route. The pathway of N7-guanine alkylation by nitrogen and sulfur mustards is discussed on the basis of the Molecular Electrostatic Potential and HOMO-LUMO properties of these molecules.

  5. Using positive-ion electrospray ionization mass spectrometry and H/D exchange study phosphoryl group transfer reactions involved in amino acid ester isopropyl phosphoramidates of Brefeldin A

    International Nuclear Information System (INIS)

    Fang, Mei-Juan; Zhang, He; Liao, Chao; Qiu, Ying-Kun; Fang, Hua; Zheng, Zhen-Yu; Gao, Xiang; Zhao, Yu-Fen; Wu, Zhen

    2015-01-01

    Highlights: • ESI-MS n , HRMS and H/D exchange were used. • The fragmentation pathways of NPAAE-BFA in ESI-MS n were described. • Fragment ions involved in phosphorus group’s rearrangement reactions were observed. • Two rearrangement mechanisms about phosphorylation–dephosphorylation were proposed. - Abstract: As mini-chemical models, amino acid ester isopropyl phosphoramidates of Brefeldin A (compounds 2a–2d) were synthesized and investigated by electrospray ionization tandem mass spectrometry in combination with H/D exchange. To further confirm the fragments’s structures, off-line Fourier transform resonance tandem mass spectrometry (FT-ICR-MS/MS) was also performed. The fragmentation rules of compounds 2a–2d have been summarized and the plausible schemes for the fragmentation pathways were proposed. In this study, one dephosphorylated ion and two phosphorylated ions were observed in ESI-MS 2 spectra of [M + Na] + ions for compounds 2a–2d. The possible mechanisms about phosphorylation and dephosphorylation were proposed and confirmed by H/D exchange. For the “dephosphorylation” rearrangement, a nitrogen atom was migrated from the phosphoryl group to the carbon atom of Brefeldin A’s backbone with losing a molecule of C 3 H 7 PO 3 (122 Da). For the “phosphorylation” rearrangement, an oxygen atom of one phosphoryl group attacked the sideward phosphorus atom to form a nine-member ring intermediate, then two steps of C-H covalent bond cleavage with consecutive migration of hydrogen atom to lose a molecule of C 16 H 20 O 2 (244 Da). The two proposed rearrangement mechanisms about phosphoryl group transfer might be valuable for the structure analysis of other analogs and provide insights into elucidating the dynamic process of the phosphorylation–dephosphorylation of proteins

  6. Using positive-ion electrospray ionization mass spectrometry and H/D exchange study phosphoryl group transfer reactions involved in amino acid ester isopropyl phosphoramidates of Brefeldin A

    Energy Technology Data Exchange (ETDEWEB)

    Fang, Mei-Juan; Zhang, He; Liao, Chao; Qiu, Ying-Kun [School of Pharmaceutical Sciences and the Key Laboratory for Chemical Biology of Fujian Province, Xiamen University, Xiang-An South Road, Xiamen 361102 (China); Fang, Hua [The Third Institute of Oceanography of the State Oceanic Administration, Xiamen 361005 (China); Zheng, Zhen-Yu [College of Chemistry and Chemical Engineering, Department of Chemistry, Xiamen University, Xiamen 361005 (China); Gao, Xiang [School of Pharmaceutical Sciences and the Key Laboratory for Chemical Biology of Fujian Province, Xiamen University, Xiang-An South Road, Xiamen 361102 (China); Zhao, Yu-Fen [School of Pharmaceutical Sciences and the Key Laboratory for Chemical Biology of Fujian Province, Xiamen University, Xiang-An South Road, Xiamen 361102 (China); College of Chemistry and Chemical Engineering, Department of Chemistry, Xiamen University, Xiamen 361005 (China); Wu, Zhen, E-mail: wuzhen@xmu.edu.cn [School of Pharmaceutical Sciences and the Key Laboratory for Chemical Biology of Fujian Province, Xiamen University, Xiang-An South Road, Xiamen 361102 (China)

    2015-01-01

    Highlights: • ESI-MS{sup n}, HRMS and H/D exchange were used. • The fragmentation pathways of NPAAE-BFA in ESI-MS{sup n} were described. • Fragment ions involved in phosphorus group’s rearrangement reactions were observed. • Two rearrangement mechanisms about phosphorylation–dephosphorylation were proposed. - Abstract: As mini-chemical models, amino acid ester isopropyl phosphoramidates of Brefeldin A (compounds 2a–2d) were synthesized and investigated by electrospray ionization tandem mass spectrometry in combination with H/D exchange. To further confirm the fragments’s structures, off-line Fourier transform resonance tandem mass spectrometry (FT-ICR-MS/MS) was also performed. The fragmentation rules of compounds 2a–2d have been summarized and the plausible schemes for the fragmentation pathways were proposed. In this study, one dephosphorylated ion and two phosphorylated ions were observed in ESI-MS{sup 2} spectra of [M + Na]{sup +} ions for compounds 2a–2d. The possible mechanisms about phosphorylation and dephosphorylation were proposed and confirmed by H/D exchange. For the “dephosphorylation” rearrangement, a nitrogen atom was migrated from the phosphoryl group to the carbon atom of Brefeldin A’s backbone with losing a molecule of C{sub 3}H{sub 7}PO{sub 3} (122 Da). For the “phosphorylation” rearrangement, an oxygen atom of one phosphoryl group attacked the sideward phosphorus atom to form a nine-member ring intermediate, then two steps of C-H covalent bond cleavage with consecutive migration of hydrogen atom to lose a molecule of C{sub 16}H{sub 20}O{sub 2} (244 Da). The two proposed rearrangement mechanisms about phosphoryl group transfer might be valuable for the structure analysis of other analogs and provide insights into elucidating the dynamic process of the phosphorylation–dephosphorylation of proteins.

  7. Mutagenic and cytotoxic properties of 6-thioguanine, S6-methylthioguanine, and guanine-S6-sulfonic acid.

    Science.gov (United States)

    Yuan, Bifeng; Wang, Yinsheng

    2008-08-29

    Thiopurine drugs, including 6-thioguanine ((S)G), 6-mercaptopurine, and azathioprine, are widely employed anticancer agents and immunosuppressants. The formation of (S)G nucleotides from the thiopurine prodrugs and their subsequent incorporation into nucleic acids are important for the drugs to exert their cytotoxic effects. (S)G in DNA can be methylated by S-adenosyl-l-methionine to give S(6)-methylthioguanine (S(6)mG) and oxidized by UVA light to render guanine-S(6)-sulfonic acid ((SO3H)G). Here, we constructed single-stranded M13 shuttle vectors carrying a (S)G, S(6)mG, or (SO3H)G at a unique site and allowed the vectors to propagate in wild-type and bypass polymerase-deficient Escherichia coli cells. Analysis of the replication products by using the competitive replication and adduct bypass and a slightly modified restriction enzyme digestion and post-labeling assays revealed that, although none of the three thionucleosides considerably blocked DNA replication in all transfected E. coli cells, both S(6)mG and (SO3H)G were highly mutagenic, which resulted in G-->A mutation at frequencies of 94 and 77%, respectively, in wild-type E. coli cells. Deficiency in bypass polymerases does not result in alteration of mutation frequencies of these two lesions. In contrast to what was found from previous steady-state kinetic analysis, our data demonstrated that 6-thioguanine is mutagenic, with G-->A transition occurring at a frequency of approximately 10%. The mutagenic properties of 6-thioguanine and its derivatives revealed in the present study offered important knowledge about the biological implications of these thionucleosides.

  8. Quantum molecular modeling of the interaction between guanine and alkylating agents--1--sulfur mustard.

    Science.gov (United States)

    Broch, H; Hamza, A; Vasilescu, D

    1996-06-01

    Interaction between Guanine and the episulfonium form of Sulfur mustard (HD) was studied using the ab initio LCAO-MO method at the HF/6-31G level. The alkylation mechanism on guanine-N7 was analyzed by using a supermolecular modeling. Our stereostructural results associated with the molecular electrostatic potentials and HOMO-LUMO properties, show that in vacuum the alkylation of the N7 of guanine by HD in the aggressive episulfonium form is a direct process without transition state and of which the pathway is determined.

  9. Application of Ammonium Persulfate for Selective Oxidation of Guanines for Nucleic Acid Sequencing

    Directory of Open Access Journals (Sweden)

    Yafen Wang

    2017-07-01

    Full Text Available Nucleic acids can be sequenced by a chemical procedure that partially damages the nucleotide positions at their base repetition. Many methods have been reported for the selective recognition of guanine. The accurate identification of guanine in both single and double regions of DNA and RNA remains a challenging task. Herein, we present a new, non-toxic and simple method for the selective recognition of guanine in both DNA and RNA sequences via ammonium persulfate modification. This strategy can be further successfully applied to the detection of 5-methylcytosine by using PCR.

  10. Purine nucleotide synthesis from exogenous adenine and guanine in rodent small intestine

    International Nuclear Information System (INIS)

    Gross, C.J.; Karlberg, P.K.; Savaiano, D.A.

    1986-01-01

    14 C-Adenine and 14 C-guanine uptake was studied in isolated guinea pig enterocytes. Cells were incubated in Hank's buffer and separated from the medium by centrifugation through silicone oil into 1M PCA. Uptake was temperature and concentration dependent. Both compounds were incorporated into nucleotides as measured by HPLC and HVE. Adenine was more extensively incorporated into nucleotides than was guanine. Adenine nucleotides accounted for about 70% of the intracellular label after 30 min with a majority being ADP and ATP (medium concentration = 10 μM). Guanine nucleotides accounted for only 30% of the intracellular label after 30 min. Labeled intracellular free adenine or guanine were not detected. Significantly more guanine vs. adenine was converted to uric acid. After 30 min, 11.5 +/- 3.9% (n=3) and 83.0 +/- 8.4% (n=4) of the label was present as uric acid in the medium when adenine and guanine, respectively, were the substrate. After 1 min, 34.8 +/- 3.4% (n=4) of the label in the medium was present as uric acid when guanine was the substrate. Decreasing the concentration of adenine resulted in an increase in the percent of uric acid in the medium. 14 C-adenine (75 nmol) was injected into 1 gm segments of rat jejunum. After 5 min., segments were quickly flushed and the tissue homogenized in 1M PCA. Only uric acid was present after 5 min (n=6). In contrast, in animals fasted 3 to 5 days, less conversion to uric acid was observed in the intestinal content (50-80% of the same dose was still present as adenine after 5 min) and nucleotide formation was observed in the tissue. The results indicate that uric acid and nucleotide synthesis from exogenous adenine and guanine are concentration dependent and affected by nutritional state

  11. Guanine nucleotides stimulate hydrolysis of phosphatidyl inositol bis phosphate in human myelin membranes

    International Nuclear Information System (INIS)

    Boulias, C.; Moscarello, M.A.

    1989-01-01

    Phosphodiesterase activity was stimulated in myelin membranes in the presence of guanine nucleotide analogues. This activity was reduced in myelin membranes which had been adenosine diphosphate ribosylated in the presence of cholera toxin which ADP-ribosylated three proteins of Mr 46,000, 43,000 and 18,500. Aluminum fluoride treatment of myelin had the same stimulatory effects on phosphodiesterase activity as did the guanine nucleotides

  12. Charge splitters and charge transport junctions based on guanine quadruplexes

    Science.gov (United States)

    Sha, Ruojie; Xiang, Limin; Liu, Chaoren; Balaeff, Alexander; Zhang, Yuqi; Zhang, Peng; Li, Yueqi; Beratan, David N.; Tao, Nongjian; Seeman, Nadrian C.

    2018-04-01

    Self-assembling circuit elements, such as current splitters or combiners at the molecular scale, require the design of building blocks with three or more terminals. A promising material for such building blocks is DNA, wherein multiple strands can self-assemble into multi-ended junctions, and nucleobase stacks can transport charge over long distances. However, nucleobase stacking is often disrupted at junction points, hindering electric charge transport between the two terminals of the junction. Here, we show that a guanine-quadruplex (G4) motif can be used as a connector element for a multi-ended DNA junction. By attaching specific terminal groups to the motif, we demonstrate that charges can enter the structure from one terminal at one end of a three-way G4 motif, and can exit from one of two terminals at the other end with minimal carrier transport attenuation. Moreover, we study four-way G4 junction structures by performing theoretical calculations to assist in the design and optimization of these connectors.

  13. Myristoylated α subunits of guanine nucleotide-binding regulatory proteins

    International Nuclear Information System (INIS)

    Buss, J.E.; Mumby, S.M.; Casey, P.J.; Gilman, A.G.; Sefton, B.M.

    1987-01-01

    Antisera directed against specific subunits of guanine nucleotide-binding regulatory proteins (G proteins) were used to immunoprecipitate these polypeptides from metabolically labeled cells. This technique detects, in extracts of a human astrocytoma cell line, the α subunits of G/sub s/ (stimulatory) (α 45 and α 52 ), a 41-kDa subunit of G/sub i/ (inhibitory) (α 41 ), a 40-kDa protein (α 40 ), and the 36-kDa β subunit. No protein that comigrated with the α subunit of G 0 (unknown function) (α 39 ) was detected. In cells grown in the presence of [ 3 H]myristic acid, α 41 and α 40 contained 3 H label, while the β subunit did not. Chemical analysis of lipids attached covalently to purified α 41 and α 39 from bovine brain also revealed myristic acid. Similar analysis of brain G protein β and γ subunits and of G/sub t/ (Transducin) subunits (α, β, and γ) failed to reveal fatty acids. The fatty acid associated with α 41 , α 40 , and α 39 was stable to treatment with base, suggesting that the lipid is linked to the polypeptide via an amide bond. These GTP binding proteins are thus identified as members of a select group of proteins that contains myristic acid covalently attached to the peptide backbone. Myristate may play an important role in stabilizing interactions of G proteins with phospholipid or with membrane-bound proteins

  14. Active site labeling of the guanine-7-methyltransferase

    International Nuclear Information System (INIS)

    Streaker, E.; Sitz, T.O.

    1992-01-01

    Studies on the guanine-7-methyltransferase have defined three domains in the active site: the S-adenosylmethionine (SAM) region, the cap region (GpppG), and the RNA binding domain (--NpNpNpNpNp---). The authors attempted to label the SAM binding domain by a photoaffinity label using 8-azido-SAM and another method using 3 H-SAM and long exposures to uv-light. Neither method was successful. The next approach was to attempt to label the cap-RNA binding domain (GpppGpNpNpNpNpN) by synthesizing RNA containing 8-azido-Ap using an in vitro transcription system and T7 RNA polymerase. The 8-azido-ATP inhibited the T7 RNA polymerase preventing the synthesis of RNA. As they were unable to synthesize the photoaffinity label, they next tried to synthesize an end labeled RNA and directly label by long exposures to uv-light. When the enzyme was incubated with 32 P-labeled RNA for 15 min at 37 degrees and then exposed to a germicidal lamp for various times at O degrees, optimal labeling occurred after 45 min. Various enzyme preparations were labeled by this method and two polypeptides were found to specifically bind the non-methylated mRNA analog. This labeling method should allow characterization of the subunit structure and generate information about the nature of the RNA binding domain

  15. Chlamydial entry involves TARP binding of guanine nucleotide exchange factors.

    Directory of Open Access Journals (Sweden)

    B Josh Lane

    2008-03-01

    Full Text Available Chlamydia trachomatis attachment to cells induces the secretion of the elementary body-associated protein TARP (Translocated Actin Recruiting Protein. TARP crosses the plasma membrane where it is immediately phosphorylated at tyrosine residues by unknown host kinases. The Rac GTPase is also activated, resulting in WAVE2 and Arp2/3-dependent recruitment of actin to the sites of chlamydia attachment. We show that TARP participates directly in chlamydial invasion activating the Rac-dependent signaling cascade to recruit actin. TARP functions by binding two distinct Rac guanine nucleotide exchange factors (GEFs, Sos1 and Vav2, in a phosphotyrosine-dependent manner. The tyrosine phosphorylation profile of the sequence YEPISTENIYESI within TARP, as well as the transient activation of the phosphatidylinositol 3-kinase (PI3-K, appears to determine which GEF is utilized to activate Rac. The first and second tyrosine residues, when phosphorylated, are utilized by the Sos1/Abi1/Eps8 and Vav2, respectively, with the latter requiring the lipid phosphatidylinositol 3,4,5-triphosphate. Depletion of these critical signaling molecules by siRNA resulted in inhibition of chlamydial invasion to varying degrees, owing to a possible functional redundancy of the two pathways. Collectively, these data implicate TARP in signaling to the actin cytoskeleton remodeling machinery, demonstrating a mechanism by which C.trachomatis invades non-phagocytic cells.

  16. Fluorescence enhancement of DNA-silver nanoclusters from guanine proximity

    Energy Technology Data Exchange (ETDEWEB)

    Yeh, Hsin-chih [Los Alamos National Laboratory; Sharma, Jaswinder [Los Alamos National Laboratory; Yoo, Hyojong [Los Alamos National Laboratory; Martinez, Jennifer S [Los Alamos National Laboratory

    2010-01-01

    Oligonucleotide-templated, silver nanoclusters (DNA/Ag NCs) are a versatile set of fluorophores and have already been used for live cell imaging, detection of specific metal ions, and single-nucleotide variation identification. Compared to commonly used organic dyes, these fluorescent nanoclusters have much better photostability and are often a few times brighter. Owing to their small size, simple preparation, and biocompatibility (i.e. made of nontoxic metals), DNA/Ag NCs should find more applications in biological imaging and chemical detection in the years to come. While clearly promising as new fluorophores, DNA/Ag NCs possess a unique and poorly understood dynamic process not shared by organic dyes or photoluminescent nanocrystals - the conversion among different NC species due to silver oxidation/reduction or NC regrouping. While this environmental sensitivity can be viewed as a drawback, in the appropriate context, it can be used as a sensor or reporter. Often reversible, conversions among different NC species have been found to depend upon a number of factors, including time, temperature, oxygen and salt content. In this communication, we report significant fluorescence enhancement of DNA/Ag NCs via interactions with guanine-rich DNA sequences. Moreover, we demonstrated this property can be used for sensitive detection of specific target DNA from a human oncogene (i.e. Braf gene).

  17. cis-Golgi proteins accumulate near the ER exit sites and act as the scaffold for Golgi regeneration after brefeldin A treatment in tobacco BY-2 cells.

    Science.gov (United States)

    Ito, Yoko; Uemura, Tomohiro; Shoda, Keiko; Fujimoto, Masaru; Ueda, Takashi; Nakano, Akihiko

    2012-08-01

    The Golgi apparatus forms stacks of cisternae in many eukaryotic cells. However, little is known about how such a stacked structure is formed and maintained. To address this question, plant cells provide a system suitable for live-imaging approaches because individual Golgi stacks are well separated in the cytoplasm. We established tobacco BY-2 cell lines expressing multiple Golgi markers tagged by different fluorescent proteins and observed their responses to brefeldin A (BFA) treatment and BFA removal. BFA treatment disrupted cis, medial, and trans cisternae but caused distinct relocalization patterns depending on the proteins examined. Medial- and trans-Golgi proteins, as well as one cis-Golgi protein, were absorbed into the endoplasmic reticulum (ER), but two other cis-Golgi proteins formed small punctate structures. After BFA removal, these puncta coalesced first, and then the Golgi stacks regenerated from them in the cis-to-trans order. We suggest that these structures have a property similar to the ER-Golgi intermediate compartment and function as the scaffold of Golgi regeneration.

  18. The trans-Golgi Network and the Golgi Stacks Behave Independently During Regeneration After Brefeldin A Treatment in Tobacco BY-2 Cells.

    Science.gov (United States)

    Ito, Yoko; Toyooka, Kiminori; Fujimoto, Masaru; Ueda, Takashi; Uemura, Tomohiro; Nakano, Akihiko

    2017-04-01

    The trans-Golgi network (TGN) plays an essential role in intracellular membrane trafficking. In plant cells, recent live-cell imaging studies have revealed the dynamic behavior of the TGN independent from the Golgi apparatus. In order to better understand the relationships between the two organelles, we examined their dynamic responses to the reagent brefeldin A (BFA) and their recovery after BFA removal. Golgi markers responded to BFA similarly over a range of concentrations, whereas the behavior of the TGN was BFA concentration dependent. The TGN formed aggregates at high concentrations of BFA; however, TGN proteins relocalized to numerous small vesicular structures dispersed throughout the cytoplasm at lower BFA concentrations. During recovery from weak BFA treatment, the TGN started to regenerate earlier than the completion of the Golgi. The regeneration of the two organelles proceeded independently of each other for a while, and eventually was completed by their association. Our data suggest that there is some degree of autonomy for the regeneration of the TGN and the Golgi in tobacco BY-2 cells. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  19. Fission Yeast SCYL1/2 Homologue Ppk32: A Novel Regulator of TOR Signalling That Governs Survival during Brefeldin A Induced Stress to Protein Trafficking.

    Science.gov (United States)

    Kowalczyk, Katarzyna M; Petersen, Janni

    2016-05-01

    Target of Rapamycin (TOR) signalling allows eukaryotic cells to adjust cell growth in response to changes in their nutritional and environmental context. The two distinct TOR complexes (TORC1/2) localise to the cell's internal membrane compartments; the endoplasmic reticulum (ER), Golgi apparatus and lysosomes/vacuoles. Here, we show that Ppk32, a SCYL family pseudo-kinase, is a novel regulator of TOR signalling. The absence of ppk32 expression confers resistance to TOR inhibition. Ppk32 inhibition of TORC1 is critical for cell survival following Brefeldin A (BFA) induced stress. Treatment of wild type cells with either the TORC1 specific inhibitor rapamycin or the general TOR inhibitor Torin1 confirmed that a reduction in TORC1 activity promoted recovery from BFA induced stress. Phosphorylation of Ppk32 on two residues that are conserved within the SCYL pseudo-kinase family are required for this TOR inhibition. Phosphorylation on these sites controls Ppk32 protein levels and sensitivity to BFA. BFA induced ER stress does not account for the response to BFA that we report here, however BFA is also known to induce Golgi stress and impair traffic to lysosomes. In summary, Ppk32 reduce TOR signalling in response to BFA induced stress to support cell survival.

  20. Guanine is indispensable for immunoglobulin switch region RNA-DNA hybrid formation

    International Nuclear Information System (INIS)

    Mizuta, Ryushin; Mizuta, Midori; Kitamura, Daisuke

    2005-01-01

    It is suggested that the formation of the switch (S) region RNA-DNA hybrid and the subsequent generation of higher-order chromatin structures including R-loop initiate a class switch recombination of the immunoglobulin gene. The primary factor of this recombination is the S-region derived noncoding RNA. However, the biochemical character of this guanine-rich (G-rich) transcript is poorly understood. The present study was performed to analyze the structure of this G-rich RNA using atomic force microscope (AFM). The in vitro transcribed S-region RNA was spread on a mica plate, air-dried and observed by non-contact mode AFM in air. The G-rich transcripts tend to aggregate on the template DNA and to generate a higher-order RNA-DNA complex. However, the transcripts that incorporated guanine analogues as substitutes for guanine neither aggregated nor generated higher-order structures. Incorporation of guanine analogues in transcribes RNA partially disrupts hydrogen bonds related to guanine, such as Watson-Crick GC-base pair and Hoogsteen bond GG-base pair. Thus, aggregation of S-region RNA and generation of the higher-order RNA-DNA complex are attributed to hydrogen bonds of guanine. (author)

  1. High pressure {sup 31}P NMR spectroscopy on guanine nucleotides

    Energy Technology Data Exchange (ETDEWEB)

    Spoerner, Michael; Karl, Matthias; Lopes, Pedro; Hoering, Marcus; Loeffel, Karoline; Nuehs, Andrea; Adelsberger, Joseph; Kremer, Werner; Kalbitzer, Hans Robert, E-mail: hans-robert.kalbitzer@ur.de [University of Regensburg, Centre of Magnetic Resonance in Chemistry and Biomedicine, Institute of Biophysics and Physical Biochemistry (Germany)

    2017-01-15

    The {sup 31}P NMR pressure response of guanine nucleotides bound to proteins has been studied in the past for characterizing the pressure perturbation of conformational equilibria. The pressure response of the {sup 31}P NMR chemical shifts of the phosphate groups of GMP, GDP, and GTP as well as the commonly used GTP analogs GppNHp, GppCH{sub 2}p and GTPγS was measured in the absence and presence of Mg{sup 2+}-ions within a pressure range up to 200 MPa. The pressure dependence of chemical shifts is clearly non-linear. For all nucleotides a negative first order pressure coefficient B{sub 1} was determined indicating an upfield shift of the resonances with pressure. With exception of the α-phosphate group of Mg{sup 2+}·GMP and Mg{sup 2+}·GppNHp the second order pressure coefficients are positive. To describe the data of Mg{sup 2+}·GppCH{sub 2}p and GTPγS a Taylor expansion of 3rd order is required. For distinguishing pH effects from pressure effects a complete pH titration set is presented for GMP, as well as GDP and GTP in absence and presence of Mg{sup 2+} ions using indirect referencing to DSS under identical experimental conditions. By a comparison between high pressure {sup 31}P NMR data on free Mg{sup 2+}-GDP and Mg{sup 2+}-GDP in complex with the proto-oncogene Ras we demonstrate that pressure induced changes in chemical shift are clearly different between both forms.

  2. Behavior of the guanine base in G-quadruplexes probed by the fluorescent guanine analog, 6-methyl isozanthopterin

    Energy Technology Data Exchange (ETDEWEB)

    Han, Ji Hoon; Chitrapriya, Nataraj; Lee, Hyun Suk; Lee, Young Ae; Kim, Seog K. [Dept. of Chemistry, Yeungnam University, Gyeongsan (Korea, Republic of); Jung, Maeng Joon [Dept. of Chemistry, Kyungpook National University, Daegu (Korea, Republic of)

    2017-02-15

    In this study, circular dichroism (CD) spectrum and fluorescence techniques were used to examine the dynamic properties and microenvironment of the guanine base (G) at the central loop and at the middle of the G-stem of the G-quadruplex formed from the G{sub 3}T{sub 2}G{sub 3}TGTG{sub 3}T{sub 2}G{sub 3} sequence (G-quadruplex 1), in which the G base at the 10th and 13th position were replaced with a fluorescent G analog, 6-methyl isoxanthopterin (6MI) (G-quadruplex 2 and 3, respectively). For all G-quadruplexes, the CD spectrum revealed a positive band at 263 nm and a shoulder at 298 nm, and the thermal melting profiles were the sum of at least two sigmoidal curves. These observations indicated the presence of two conformers in the G-quadruplex. The fluorescence intensity of G-quadruplex 2 was greater than 3, as expected from the extent of stacking interaction, which is larger in the G(6MI)G sequence than the T(6MI)T sequence. The efficiency of fluorescence quenching by the polar acrylamide quencher and negatively charged I− quencher were larger for G-quadruplex 3, suggesting that 6MI in the G(6MI)G stem is exposed more to the aqueous environment compared to that in the T(6MI)T central loop. In the latter case, 6MI may direct to the center of the top G-quartet layer. The possibility of hydrogen bond formation between the carbonyl group of 6MI and the acrylamide of the G-quadruplex 3 was proposed.

  3. Live-cell imaging of dual-labeled Golgi stacks in tobacco BY-2 cells reveals similar behaviors for different cisternae during movement and brefeldin A treatment.

    Science.gov (United States)

    Madison, Stephanie L; Nebenführ, Andreas

    2011-09-01

    In plant cells, the Golgi apparatus consists of numerous stacks that, in turn, are composed of several flattened cisternae with a clear cis-to-trans polarity. During normal functioning within living cells, this unusual organelle displays a wide range of dynamic behaviors such as whole stack motility, constant membrane flux through the cisternae, and Golgi enzyme recycling through the ER. In order to further investigate various aspects of Golgi stack dynamics and integrity, we co-expressed pairs of established Golgi markers in tobacco BY-2 cells to distinguish sub-compartments of the Golgi during monensin treatments, movement, and brefeldin A (BFA)-induced disassembly. A combination of cis and trans markers revealed that Golgi stacks remain intact as they move through the cytoplasm. The Golgi stack orientation during these movements showed a slight preference for the cis side moving ahead, but trans cisternae were also found at the leading edge. During BFA treatments, the different sub-compartments of about half of the observed stacks fused with the ER sequentially; however, no consistent order could be detected. In contrast, the ionophore monensin resulted in swelling of trans cisternae while medial and particularly cis cisternae were mostly unaffected. Our results thus demonstrate a remarkable equivalence of the different cisternae with respect to movement and BFA-induced fusion with the ER. In addition, we propose that a combination of dual-label fluorescence microscopy and drug treatments can provide a simple alternative approach to the determination of protein localization to specific Golgi sub-compartments.

  4. Highly sensitive and selective fluorescent assay for guanine based on the Cu(2+)/eosin Y system.

    Science.gov (United States)

    Shi, Huimin; Cui, Yi; Gong, Yijun; Feng, Suling

    2016-05-15

    A fluorescent probe has been developed for the determination of guanine based on the quenched fluorescence signal of Cu(2+)/eosin Y. Cu(2+) interacted with eosin Y, resulting in fluorescence quenching. Subsequently, with the addition of guanine to the Cu(2+)/eosin Y system, guanine reacted with Cu(2+) to form 1:1 chelate cation, which further combined with eosin Y to form a 1:1 ternary ion-association complex by electrostatic attraction and hydrophobic interaction, resulting in significant decrease of the fluorescence. Hence, a fluorescent system was constructed for rapid, sensitive and selective detection of guanine with a detection limit as low as 1.5 nmol L(-1) and a linear range of 3.3-116 nmol L(-1). The method has been applied satisfactorily to the determination of guanine in DNA and urine samples with the recoveries from 98.7% to 105%. This study significantly expands the realm of application of ternary ion-association complex in fluorescence probe. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Highly sensitive and selective fluorescent assay for guanine based on the Cu2 +/eosin Y system

    Science.gov (United States)

    Shi, Huimin; Cui, Yi; Gong, Yijun; Feng, Suling

    2016-05-01

    A fluorescent probe has been developed for the determination of guanine based on the quenched fluorescence signal of Cu2 +/eosin Y. Cu2 + interacted with eosin Y, resulting in fluorescence quenching. Subsequently, with the addition of guanine to the Cu2 +/eosin Y system, guanine reacted with Cu2 + to form 1:1 chelate cation, which further combined with eosin Y to form a 1:1 ternary ion-association complex by electrostatic attraction and hydrophobic interaction, resulting in significant decrease of the fluorescence. Hence, a fluorescent system was constructed for rapid, sensitive and selective detection of guanine with a detection limit as low as 1.5 nmol L- 1 and a linear range of 3.3-116 nmol L- 1. The method has been applied satisfactorily to the determination of guanine in DNA and urine samples with the recoveries from 98.7% to 105%. This study significantly expands the realm of application of ternary ion-association complex in fluorescence probe.

  6. Preparation and bioevaluation of 99mTc-carbonyl complex of guanine

    International Nuclear Information System (INIS)

    Cigdem Ichedef; Serap Teksoez; Kamile Senocak; Eser Ucar; Ayfer Yurt Kilcar

    2011-01-01

    The aim of this study is to prepare radiolabeled guanine with 99m Tc(CO) 3 + core. For this purpose, guanine has been radiolabeled with 99m Tc(CO) 3 + core. Quality control study of radiolabeled guanine molecule with 99m Tc(CO) 3 + core was performed by thin layer radio chromatography (TLRC) and high performance liquid radio chromatography (HPLRC). The results showed that the radiolabeling yield was quite high (94 ± 3%). Beside that 99m Tc(CO) 3 -Gua complex has showed good in vitro stability during the 24 h period. Radiopharmaceutical potential of this complex was evaluated in Wistar Albino Rats. It was concluded that 99m Tc(CO) 3 -Gua could be used as a nucleotide radiopharmaceutical for in vivo applications. (author)

  7. Synthesis of a Pseudodisaccharide α-C-Glycosidically Linked to an 8-Alkylated Guanine

    Directory of Open Access Journals (Sweden)

    Jan Duchek

    2013-04-01

    Full Text Available The synthesis of stable guanofosfocin analogues has attracted considerable attention in the past 15 years. Several guanofosfocin analogues mimicking the three constitutional elements of mannose, ribose, and guanine were designed and synthesized. Interest in ether-linked pseudodisaccharides and 8-alkylated guanines is increasing, due to their potential applications in life science. In this article, a novel guanofosfocin analogue 6, an ether-linked pseudodisaccharide connected α-C-glycosidically to an 8-alkylated guanine, was synthesized in a 10-longest linear step sequence from known diol 13, resulting in an overall yield of 26%. The key steps involve the ring-opening of cyclic sulfate 8 by alkoxide generated from 7 and a reductive cyclization of 4-N-acyl-2,4-diamino-5-nitrosopyrimidine 19 to form compound 6.

  8. A DFT investigation on interactions between asymmetric derivatives of cisplatin and nucleobase guanine

    Science.gov (United States)

    Tai, Truong Ba; Nhat, Pham Vu

    2017-07-01

    The interactions of hydrolysis products of cisplatin and its asymmetric derivatives cis- and trans-[PtCl2(iPram)(Mepz)] with guanine were studied using DFT methods. These interactions are dominated by electrostatic effects, namely hydrogen bond contributions and there exists a charge flow from H-atoms of ligands to the O-atoms of guanine. The replacement of NH3 moieties by larger functional groups accompanies with a moderate reaction between PtII and guanine molecule, diminishing the cytotoxicity of the drug. The asymmetric and symmetric NH2 stretching modes of complexes having strong hydrogen bond interactions are red shifted importantly as compared to complexes without presence of hydrogen bond interactions.

  9. Guanine as a hygienic index for allergological relevant mite infestation in mattress dust

    NARCIS (Netherlands)

    Bronswijk, van J.E.M.H.

    1986-01-01

    Since guanine is not only an essential constituent of vital nucleic acids, but also the main end product of nitrogenous waste excretion in arachnids, it is a potential candidate for a hygienic index for mite activity in house dust. The public health significance of these mites is based on their

  10. Solubilization and reconstitution of the formylmethionylleucylphenylalanine receptor coupled to guanine nucleotide regulatory protein

    International Nuclear Information System (INIS)

    Williamson, K.; Dickey, B.F.; Pyun, H.Y.; Navarro, J.

    1988-01-01

    The authors describe the solubilization, resolution, and reconstitution of the formylmethionylleucylphenylalanine (fMet-Leu-Phe) receptor and guanine nucleotide regulatory proteins (G-proteins). The receptor was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Guanine nucleotides decreased the number of high-affinity binding sites and accelerated the rate of dissociation of the receptor-ligand complex, suggesting that the solubilized receptor remained coupled to endogenous G-proteins. The solubilized receptor was resolved from endogenous G-proteins by fractionation on a wheat germ agglutinin (WGA)-Sepharose 4B column. High-affinity [ 3 H]fMet-Leu-Phe binding to the WGA-purified receptor was diminished and exhibited reduced guanine nucleotide sensitivity. High-affinity [ 3 H]fMET-Leu-Phe binding and guanine nucleotide sensitivity were reconstituted upon the addition of purified brain G-proteins. Similar results were obtained when the receptor was reconstituted with brain G-proteins into phospholipid vesicles by gel filtration chromatography. In addition, they also demonstrated fMET-Leu-Phe-dependent GTP hydrolysis in the reconstituted vesicles. The results of this work indicate that coupling of the fMet-Leu-Phe receptor to G-proteins converts the receptor to a high-affinity binding state and that agonist produces activation of G-proteins. The resolution and functional reconstitution of this receptor should provide an important step toward the elucidation of the molecular mechanism of the fMet-Leu-Phe transduction system in neutrophils

  11. Circular dichroism spectroscopy of conformers of (guanine + adenine) repeat strands of DNA

    Czech Academy of Sciences Publication Activity Database

    Kejnovská, Iva; Kypr, Jaroslav; Vorlíčková, Michaela

    2003-01-01

    Roč. 15, č. 7 (2003), s. 584-592 ISSN 0899-0042 R&D Projects: GA AV ČR IAA4004201; GA ČR GA204/01/0561 Institutional research plan: CEZ:AV0Z5004920 Keywords : DNA conformation * (guanine + adenine) repeats * homoduplexes Subject RIV: BO - Biophysics Impact factor: 1.793, year: 2003

  12. Primary overproduction of urate caused by a partial deficiency of hypoxanthine-guanine phosphoribosyl transferase

    International Nuclear Information System (INIS)

    Cassidy, M.; Gregory, M.C.; Harley, E.H.

    1980-01-01

    Inherited enzyme deficiencies are found in a small proportion of patients with gout who produce an excess of uric acid. The clinical, biochemical and therapeutic aspects of a case of hyperuricaemia caused by an atypical mutant hypoxanthine-guanine phophoribosyl transferase are presented. Urate overproduction was moderate and controlled by allopurinol therapy

  13. The Study of Adsorption of Patulin by Nanocellulose Conjugated with Poly Guanine in Contaminated Apple juice

    Directory of Open Access Journals (Sweden)

    M Ghafori Bidakhavidi

    2016-07-01

    Full Text Available Abstract Introdction: Patulin is a dangerous toxin produced by various fungi. Hence, the current study aimed to evaluate adsorption of Patulin by nanocellulose conjugated with Poly-guanine in contaminated apple juice. Methods: Firstly, nanocellulose was synthesized, and then it was bonded to poly-guanine by a cross-linker. Then, concentration serial of Patulin was prepared in the apple juice, conjugated nanoparticles were added to them, and all were incubated at 37 ºC. After incubation, the Patulin concentration was measured by HPLC, and finally the adsorption percentage was calculated for each tube. Regarding molecular simulation, the initial structures of Patulin and nanocellulose conjugated with Poly-guanine were inserted into Hyperchem software, and their intermolecular energy was calculated during 50 picoseconds. Results: The results of the present study demonstrated that there was a significant direct correlation between the initial concentration of Patulin and the adsorption percentage of toxin. In addition, the adsorption maximum was reported 70±5 %, and the intermolecular energy between two structures was -20.3 Kcal/mol based on the computational simulation. Conclusions: It can be concluded that nanocellulose conjugated with Poly-guanine seems to be a good adsorbent for Patulin, which is demanded to be used in the future studies in regard with its application.

  14. Coupling of guanine nucleotide inhibitory protein to somatostatin receptors on pancreatic acinar membranes

    International Nuclear Information System (INIS)

    Sakamoto, C.; Matozaki, T.; Nagao, M.; Baba, S.

    1987-01-01

    Guanine nucleotides and pertussis toxin were used to investigate whether somatostatin receptors interact with the guanine nucleotide inhibitory protein (NI) on pancreatic acinar membranes in the rat. Guanine nucleotides reduced 125 I-[Tyr 1 ]somatostatin binding to acinar membranes up to 80%, with rank order of potency being 5'-guanylyl imidodiphosphate [Gpp(NH)p]>GTP>TDP>GMP. Scatchard analysis revealed that the decrease in somatostatin binding caused by Gpp(NH)p was due to the decrease in the maximum binding capacity without a significant change in the binding affinity. The inhibitory effect of Gpp(NH)p was partially abolished in the absence of Mg 2+ . When pancreatic acini were treated with 1 μg/ml pertussis toxin for 4 h, subsequent 125 I-[Tyr 1 ]somatostatin binding to acinar membranes was reduced. Pertussis toxin treatment also abolished the inhibitory effect of somatostatin on vasoactive intestinal peptide-stimulated increase in cellular content of adenosine 3',5'-cyclic monophosphate (cAMP) in the acini. The present results suggest that 1) somatostatin probably functions in the pancreas to regulate adenylate cyclase enzyme system via Ni, 2) the extent of modification of Ni is correlated with the ability of somatostatin to inhibit cAMP accumulation in acini, and 3) guanine nucleotides also inhibit somatostatin binding to its receptor

  15. Scaffold-hopping from xanthines to tricyclic guanines: A case study of dipeptidyl peptidase 4 (DPP4) inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Pissarnitski, Dmitri A.; Zhao, Zhiqiang; Cole, David; Wu, Wen-Lian; Domalski, Martin; Clader, John W.; Scapin, Giovanna; Voigt, Johannes; Soriano, Aileen; Kelly, Theresa; Powles, Mary Ann; Yao, Zuliang; Burnett, Duane A. (Merck)

    2016-11-01

    Molecular modeling of unbound tricyclic guanine scaffolds indicated that they can serve as effective bioisosteric replacements of xanthines. This notion was further confirmed by a combination of X-ray crystallography and SAR studies, indicating that tricyclic guanine DPP4 inhibitors mimic the binding mode of xanthine inhibitors, exemplified by linagliptin. Realization of the bioisosteric relationship between these scaffolds potentially will lead to a wider application of cyclic guanines as xanthine replacements in drug discovery programs for a variety of biological targets. Newly designed DPP4 inhibitors achieved sub-nanomolar potency range and demonstrated oral activity in vivo in mouse glucose tolerance test.

  16. Guanine nucleotide binding to the Bateman domain mediates the allosteric inhibition of eukaryotic IMP dehydrogenases

    Science.gov (United States)

    Buey, Rubén M.; Ledesma-Amaro, Rodrigo; Velázquez-Campoy, Adrián; Balsera, Mónica; Chagoyen, Mónica; de Pereda, José M.; Revuelta, José L.

    2015-11-01

    Inosine-5'-monophosphate dehydrogenase (IMPDH) plays key roles in purine nucleotide metabolism and cell proliferation. Although IMPDH is a widely studied therapeutic target, there is limited information about its physiological regulation. Using Ashbya gossypii as a model, we describe the molecular mechanism and the structural basis for the allosteric regulation of IMPDH by guanine nucleotides. We report that GTP and GDP bind to the regulatory Bateman domain, inducing octamers with compromised catalytic activity. Our data suggest that eukaryotic and prokaryotic IMPDHs might have developed different regulatory mechanisms, with GTP/GDP inhibiting only eukaryotic IMPDHs. Interestingly, mutations associated with human retinopathies map into the guanine nucleotide-binding sites including a previously undescribed non-canonical site and disrupt allosteric inhibition. Together, our results shed light on the mechanisms of the allosteric regulation of enzymes mediated by Bateman domains and provide a molecular basis for certain retinopathies, opening the door to new therapeutic approaches.

  17. Absence of hypoxanthine:guanine phosphoribosyltransferase activity in murine Dunn osteosarcoma

    International Nuclear Information System (INIS)

    Abelson, H.T.; Gorka, C.

    1983-01-01

    The transplantable murine Dunn osteosarcoma has no detectable hypoxanthine:guanine phosphoribosyltransferase (EC 2.4.2.8) activity. This was established from the tumors directly and from tissue culture cell lines derived from the tumor using a variety of assays: e.g., no [3H]hypoxanthine uptake into tumor or tissue culture cells, no conversion of [3H]hypoxanthine to [3H]IMP by cell extracts from tumors or tissue culture cells, no growth of tissue culture cells in hypoxanthine:aminopterin:thymidine medium, and normal growth of these cells in 10 microM 6-mercaptopurine. Ten human osteosarcomas have been assayed, and two have no apparent hypoxanthine:guanine phosphoribosyltransferase enzyme activity. After high-dose methotrexate treatment in vivo, murine tumors could be selectively killed and normal tissues could be spared by using a rescue regimen of hypoxanthine-thymidine-allopurinol

  18. Structure-wise discrimination of adenine and guanine by proteins on the basis of their nonbonded interactions.

    Science.gov (United States)

    Usha, S; Selvaraj, S

    2015-01-01

    We have analyzed the nonbonded interactions of the structurally similar moieties, adenine and guanine forming complexes with proteins. The results comprise (a) the amino acid-ligand atom preferences, (b) solvent accessibility of ligand atoms before and after complex formation with proteins, and (c) preferred amino acid residue atoms involved in the interactions. We have observed that the amino acid preferences involved in the hydrogen bonding interactions vary for adenine and guanine. The structural variation between the purine atoms is clearly reflected by their burial tendency in the solvent environment. Correlation of the mean amino acid preference values show the variation that exists between adenine and guanine preferences of all the amino acid residues. All our observations provide evidence for the discriminating nature of the proteins in recognizing adenine and guanine.

  19. Guanine nucleotide regulation of α1-adrenergic receptors of muscle and kidney eptihelial cells

    International Nuclear Information System (INIS)

    Terman, B.I.; Hughes, R.J.; Slivka, S.R.; Insel, P.A.

    1986-01-01

    The authors have examined the effect of guanine nucleotides on the interaction of adrenergic agents with α 1 -adrenergic receptors of two cell lines, the Madin-Darby Canine Kidney (MDCK) and BC3H-1 muscle cells. While gaunylylimidodiphosphoate (Gpp(NH)p) had no effect on the affinity or the total number of [ -3 H]prazosin binding sites in membranes prepared from these cells, the nucleotide decreased the apparent affinity of the agonist epinephrine in competing for [ 3 H]prazosin binding sites in both cell types. The EC 50 of Gpp(NH)p was ∼100 μM, and a maximal effect was seen at 500 μM. In contrast, 100 μM Gpp(NH)p yielding maximal shifts in binding of epinephrine to β-adrenergic receptors in BC3H-1 cell membranes. Guanine nucleotides were significantly more effective than adenine nucleotides in shifting agonist affinity for the α 1 -receptor and Mg ++ was required to observe a maximal effect. α 1 -receptor agonists activated phosphatidylinositol (PI) hydrolysis in both cell types, but have no direct effect on membrane adenylate cyclase activity. In intact BC3H-1 cells, α 1 -agonists inhibited β-adrenergic cAMP production, an effect which appears in preliminary studies not to result from enhanced phosphodieterase activity. These results show that agonist binding to α 1 -adrenergic receptors in mammalian kidney and muscle cells is regulated by guanine nucleotides. This regulation and inturn transmembrane signalling (PI hydrolysis) by these receptors appear to involve a guanine nucleotide binding (G) protein, which may be different than G/sub s/ and G/sub i/

  20. Benchmark Theoretical and Experimental Study on N-15 NMR Shifts of Oxidatively Damaged Guanine

    Czech Academy of Sciences Publication Activity Database

    Dračínský, Martin; Šála, Michal; Klepetářová, Blanka; Šebera, Jakub; Fukal, Jiří; Holečková, Veronika; Tanaka, Y.; Nencka, Radim; Sychrovský, Vladimír

    2016-01-01

    Roč. 120, č. 5 (2016), s. 915-925 ISSN 1520-6106 R&D Projects: GA ČR GA13-27676S; GA ČR GA15-11223S Institutional support: RVO:61388963 Keywords : NMR spectroscopy * DFT calculations * oxidatively damaged guanine * hOGG1 Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.177, year: 2016

  1. Voltammetric Determination of Guanine on the Electrode Modified by Gold Deposit and Nafion Film

    Directory of Open Access Journals (Sweden)

    L.G. Shaidarova

    2016-09-01

    Full Text Available Electrodeposited gold and Nafion-gold composite on the surface of glassy carbon electrodes (GCE have shown electrocatalytic activity during guanine oxidation. In comparison with the unmodified electrode, decreasing of the oxidation potential by 100 mV and increasing of the current of organic compound oxidation have been observed. When the Nafion (NF film is applied to the surface of the glassy carbon electrode with electrodeposited gold, a five-fold increase of guanine oxidation current has been achieved compared to its oxidation on the modified electrode without the NF film. Conditions have been found for electrodeposition of gold on the surface of the glassy carbon electrode, including that one covered with the NF film, as well as for registration of the maximum catalytic current on these electrodes. Linear dependence of the electrocatalytic response of the modified electrode from the guanine concentration has been observed in the range from 5·10–6 to 5·10–3 mol·L–1 (for Au GCE and from 5·10–7 to 5·10–3 mol·L–1 (for NF-Au GCE.

  2. Covalent Bonding of Pyrrolobenzodiazepines (PBDs) to Terminal Guanine Residues within Duplex and Hairpin DNA Fragments

    Science.gov (United States)

    Mantaj, Julia; Jackson, Paul J. M.; Karu, Kersti; Rahman, Khondaker M.; Thurston, David E.

    2016-01-01

    Pyrrolobenzodiazepines (PBDs) are covalent-binding DNA-interactive agents with growing importance as payloads in Antibody Drug Conjugates (ADCs). Until now, PBDs were thought to covalently bond to C2-NH2 groups of guanines in the DNA-minor groove across a three-base-pair recognition sequence. Using HPLC/MS methodology with designed hairpin and duplex oligonucleotides, we have now demonstrated that the PBD Dimer SJG-136 and the C8-conjugated PBD Monomer GWL-78 can covalently bond to a terminal guanine of DNA, with the PBD skeleton spanning only two base pairs. Control experiments with the non-C8-conjugated anthramycin along with molecular dynamics simulations suggest that the C8-substituent of a PBD Monomer, or one-half of a PBD Dimer, may provide stability for the adduct. This observation highlights the importance of PBD C8-substituents, and also suggests that PBDs may bind to terminal guanines within stretches of DNA in cells, thus representing a potentially novel mechanism of action at the end of DNA strand breaks. PMID:27055050

  3. Photoluminescence properties of a novel conjugate of water-soluble CdTe quantum dots to guanine

    Energy Technology Data Exchange (ETDEWEB)

    Feng Xuejiao [North-East Normal University, Changchun 130024 (China); Shang, Qingkun, E-mail: shangqk995@nenu.edu.c [North-East Normal University, Changchun 130024 (China); Liu Hongjian [Relia Diagnostic Systems, Burlingame, CA 94010 (United States); Wang Wenlan; Wang Zhidan; Liu Junyu [North-East Normal University, Changchun 130024 (China)

    2010-04-15

    A novel conjugate of water-soluble CdTe quantum dots to a small biomolecule guanine has been obtained in aqueous phase. The photoluminescence property and the stability of the conjugate increased comparing to CdTe QDs. The interaction between CdTe QDs and guanine was studied by TEM, fluorescence microscope and photoluminescence (PL), IR, UV-Vis spectra. The effects of reflux time, pH value, ionic strength, and the ratio of CdTe QDs to guanine on the photoluminescence properties of conjugate were investigated in detail. The results show that guanine has a great influence on both the photoluminescence property and stability of thioglycolic acid-stabilized CdTe QDs. The formation of coordination and hydrogen bond between guanine molecules and CdTe including thioglycolic acid on its surface may effectively enhance the PL intensity and stability of CdTe QDs. The maximum PL intensity of the conjugate was obtained on the condition with lower ionic strength, less than 30 min reflux time, neutral pH value and 6/1 as molar ratio of guanine to CdTe.

  4. Increased mobility and on/off ratio in organic field-effect transistors using low-cost guanine-pentacene multilayers

    Science.gov (United States)

    Shi, Wei; Zheng, Yifan; Taylor, André D.; Yu, Junsheng; Katz, Howard E.

    2017-07-01

    Layer-by-layer deposited guanine and pentacene in organic field-effect transistors (OFETs) is introduced. Through adjusting the layer thickness ratio of guanine and pentacene, the tradeoff of two electronic parameters in OFETs, charge carrier mobility and current on/off ratio, was controlled. The charge mobility was enhanced by depositing pentacene over and between guanine layers and by increasing the proportion of pentacene in the layer-by-layer system, while the current on/off ratio was increased via the decreased off current induced by the guanine layers. The tunable device performance was mainly ascribed to the trap and dopant neutralizing properties of the guanine layers, which would decrease the density of free hydroxyl groups in the OFETs. Furthermore, the cost of the devices could be reduced remarkably via the adoption of low-cost guanine.

  5. Effect O6-guanine alkylation on DNA flexibility studied by comparative molecular dynamics simulations.

    Science.gov (United States)

    Kara, Mahmut; Drsata, Tomas; Lankas, Filip; Zacharias, Martin

    2015-01-01

    Alkylation of guanine at the O6 atom is a highly mutagenic DNA lesion because it alters the coding specificity of the base causing G:C to A:T transversion mutations. Specific DNA repair enzymes, e.g. O(6)-alkylguanin-DNA-Transferases (AGT), recognize and repair such damage after looping out the damaged base to transfer it into the enzyme active site. The exact mechanism how the repair enzyme identifies a damaged site within a large surplus of undamaged DNA is not fully understood. The O(6)-alkylation of guanine may change the deformability of DNA which may facilitate the initial binding of a repair enzyme at the damaged site. In order to characterize the effect of O(6)-methyl-guanine (O(6)-MeG) containing base pairs on the DNA deformability extensive comparative molecular dynamics (MD) simulations on duplex DNA with central G:C, O(6)-MeG:C or O(6)-MeG:T base pairs were performed. The simulations indicate significant differences in the helical deformability due to the presence of O(6)-MeG compared to regular undamaged DNA. This includes enhanced base pair opening, shear and stagger motions and alterations in the backbone fine structure caused in part by transient rupture of the base pairing at the damaged site and transient insertion of water molecules. It is likely that the increased opening motions of O(6)-MeG:C or O(6)-MeG:T base pairs play a decisive role for the induced fit recognition or for the looping out of the damaged base by repair enzymes. © 2014 Wiley Periodicals, Inc.

  6. Adenine and guanine nucleotide metabolism during platelet storage at 22 degree C

    International Nuclear Information System (INIS)

    Edenbrandt, C.M.; Murphy, S.

    1990-01-01

    Adenine and guanine nucleotide metabolism of platelet concentrates (PCs) was studied during storage for transfusion at 22 +/- 2 degrees C over a 7-day period using high-pressure liquid chromatography. There was a steady decrease in platelet adenosine triphosphate (ATP) and adenosine diphosphate (ADP), which was balanced quantitatively by an increase in plasma hypoxanthine. As expected, ammonia accumulated along with hypoxanthine but at a far greater rate. A fall in platelet guanosine triphosphate (GTP) and guanosine diphosphate (GDP) paralleled the fall in ATP + ADP. When adenine was present in the primary anticoagulant, it was carried over into the PC and metabolized. ATP, GTP, total adenine nucleotides, and total guanine nucleotides declined more slowly in the presence of adenine than in its absence. With adenine, the increase in hypoxanthine concentration was more rapid and quantitatively balanced the decrease in adenine and platelet ATP + ADP. Plasma xanthine rose during storage but at a rate that exceeded the decline in GTP + GDP. When platelet ATP + ADP was labeled with 14C-adenine at the initiation of storage, half of the radioactivity was transferred to hypoxanthine (45%) and GTP + GDP + xanthine (5%) by the time storage was completed. The isotopic data were consistent with the presence of a radioactive (metabolic) and a nonradioactive (storage) pool of ATP + ADP at the initiation of storage with each pool contributing approximately equally to the decline in ATP + ADP during storage. The results suggested a continuing synthesis of GTP + GDP from ATP + ADP, explaining the slower rate of fall of GTP + GDP relative to the rate of rise of plasma xanthine. Throughout storage, platelets were able to incorporate 14C-hypoxanthine into both adenine and guanine nucleotides but at a rate that was only one fourth the rate of hypoxanthine accumulation

  7. Silver (I) as DNA glue: Ag+-mediated guanine pairing revealed by removing Watson-Crick constraints

    Science.gov (United States)

    Swasey, Steven M.; Leal, Leonardo Espinosa; Lopez-Acevedo, Olga; Pavlovich, James; Gwinn, Elisabeth G.

    2015-01-01

    Metal ion interactions with DNA have far-reaching implications in biochemistry and DNA nanotechnology. Ag+ is uniquely interesting because it binds exclusively to the bases rather than the backbone of DNA, without the toxicity of Hg2+. In contrast to prior studies of Ag+ incorporation into double-stranded DNA, we remove the constraints of Watson-Crick pairing by focusing on homo-base DNA oligomers of the canonical bases. High resolution electro-spray ionization mass spectrometry reveals an unanticipated Ag+-mediated pairing of guanine homo-base strands, with higher stability than canonical guanine-cytosine pairing. By exploring unrestricted binding geometries, quantum chemical calculations find that Ag+ bridges between non-canonical sites on guanine bases. Circular dichroism spectroscopy shows that the Ag+-mediated structuring of guanine homobase strands persists to at least 90 °C under conditions for which canonical guanine-cytosine duplexes melt below 20 °C. These findings are promising for DNA nanotechnology and metal-ion based biomedical science. PMID:25973536

  8. Silver (I) as DNA glue: Ag(+)-mediated guanine pairing revealed by removing Watson-Crick constraints.

    Science.gov (United States)

    Swasey, Steven M; Leal, Leonardo Espinosa; Lopez-Acevedo, Olga; Pavlovich, James; Gwinn, Elisabeth G

    2015-05-14

    Metal ion interactions with DNA have far-reaching implications in biochemistry and DNA nanotechnology. Ag(+) is uniquely interesting because it binds exclusively to the bases rather than the backbone of DNA, without the toxicity of Hg(2+). In contrast to prior studies of Ag(+) incorporation into double-stranded DNA, we remove the constraints of Watson-Crick pairing by focusing on homo-base DNA oligomers of the canonical bases. High resolution electro-spray ionization mass spectrometry reveals an unanticipated Ag(+)-mediated pairing of guanine homo-base strands, with higher stability than canonical guanine-cytosine pairing. By exploring unrestricted binding geometries, quantum chemical calculations find that Ag(+) bridges between non-canonical sites on guanine bases. Circular dichroism spectroscopy shows that the Ag(+)-mediated structuring of guanine homobase strands persists to at least 90 °C under conditions for which canonical guanine-cytosine duplexes melt below 20 °C. These findings are promising for DNA nanotechnology and metal-ion based biomedical science.

  9. Silver (I) as DNA glue: Ag+-mediated guanine pairing revealed by removing Watson-Crick constraints

    Science.gov (United States)

    Swasey, Steven M.; Leal, Leonardo Espinosa; Lopez-Acevedo, Olga; Pavlovich, James; Gwinn, Elisabeth G.

    2015-05-01

    Metal ion interactions with DNA have far-reaching implications in biochemistry and DNA nanotechnology. Ag+ is uniquely interesting because it binds exclusively to the bases rather than the backbone of DNA, without the toxicity of Hg2+. In contrast to prior studies of Ag+ incorporation into double-stranded DNA, we remove the constraints of Watson-Crick pairing by focusing on homo-base DNA oligomers of the canonical bases. High resolution electro-spray ionization mass spectrometry reveals an unanticipated Ag+-mediated pairing of guanine homo-base strands, with higher stability than canonical guanine-cytosine pairing. By exploring unrestricted binding geometries, quantum chemical calculations find that Ag+ bridges between non-canonical sites on guanine bases. Circular dichroism spectroscopy shows that the Ag+-mediated structuring of guanine homobase strands persists to at least 90 °C under conditions for which canonical guanine-cytosine duplexes melt below 20 °C. These findings are promising for DNA nanotechnology and metal-ion based biomedical science.

  10. CeO{sub 2} nanoparticles decorated multi-walled carbon nanotubes for electrochemical determination of guanine and adenine

    Energy Technology Data Exchange (ETDEWEB)

    Wei Yan [College of Chemistry and Materials Sciences, Anhui Normal University, Wuhu 241000 (China); Department of Chemistry, Wannan Medical College, Wuhu 241002 (China); Huang Qinan [Department of Chemistry, Wannan Medical College, Wuhu 241002 (China); Li Maoguo [College of Chemistry and Materials Sciences, Anhui Normal University, Wuhu 241000 (China); Huang Xingjiu [Institute of Intelligent Machines, Chinese Academy of Sciences, Hefei 230031 (China); Fang Bin, E-mail: binfang_47@yahoo.com.cn [College of Chemistry and Materials Sciences, Anhui Normal University, Wuhu 241000 (China); Wang Lun, E-mail: wanglun@mail.ahnu.edu.cn [College of Chemistry and Materials Sciences, Anhui Normal University, Wuhu 241000 (China)

    2011-10-01

    Sub-10 nm CeO{sub 2} nanoparticles decorated multi-walled carbon nanotubes has been constructed for electrochemial determination of guanine and adenine. Transmission electron microscopy (TEM) and X-ray diffraction (XRD) were used to characterize the nanoparticles CeO{sub 2}/MWCNTs. Electrochemical impedance spectroscopy (EIS) was used to characterize the electrode modifying process. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were used to study the electrocatalytic activity toward the electrochemical oxidation of guanine and adenine. The detection limit (S/N = 3) for adenine and guanine was found to be 20 and 10 nM, respectively. The obtained sensitivity toward guanine and adenine was 1.26 and 1.13 {mu}A/{mu}M in the linear concentration range 5-50 {mu}M and 5-35 {mu}M, respectively. These results demonstrate that the carbon nanotubes could provide huge locations and facilitate the adsorptive accumulation of the guanine and adenine, and the CeO{sub 2} nanoparticles are promising substrates for the development of high-performance electrocatalysts for biosensing.

  11. First-Principles Vibrational Electron Energy Loss Spectroscopy of β -Guanine

    Science.gov (United States)

    Radtke, G.; Taverna, D.; Lazzeri, M.; Balan, E.

    2017-07-01

    A general approach to model vibrational electron energy loss spectra obtained using an electron beam positioned away from the specimen is presented. The energy-loss probability of the fast electron is evaluated using first-principles quantum mechanical calculations (density functional theory) of the dielectric response of the specimen. The validity of the method is assessed using recently measured anhydrous β -guanine, an important molecular solid used by animals to produce structural colors. The good agreement between theory and experiments lays the basis for a quantitative interpretation of this spectroscopy in complex systems.

  12. The Emerging Role of Guanine Exchange Factors in ALS and other neurodegenerative diseases

    Directory of Open Access Journals (Sweden)

    Cristian eDroppelmann

    2014-09-01

    Full Text Available Small GTPases participate in a broad range of cellular processes such as proliferation, differentiation and migration. The exchange of GDP for GTP resulting in the activation of these GTPases is catalyzed by a group of enzymes called guanine nucleotide exchange factors (GEFs, of which two classes: Dbl-related exchange factors and the more recently described Dock family exchange factors. Increasingly, deregulation of normal GEF activity or function has been associated with a broad range of disease states, including neurodegeneration and neurodevelopmental disorders. In this review, we examine this evidence with special emphasis on the novel role of Rho guanine nucleotide exchange factor (RGNEF/p190RhoGEF in the pathogenesis of amyotrophic lateral sclerosis (ALS. RGNEF is the first neurodegeneration-linked GEF that regulates not only RhoA GTPase activation but also functions as an RNA binding protein that directly acts with low molecular weight neurofilament (NEFL mRNA 3’UTR to regulate its stability. This dual role for RGNEF, coupled with the increasing understanding of the key role for GEFs in modulating the GTPase function in cell survival suggests a prominent role for GEFs in mediating a critical balance between cytotoxicity and neuroprotection which, when disturbed, contributes to neuronal loss.

  13. New investigations of the guanine trichloro cuprate(II) complex crystal

    Science.gov (United States)

    Fabijanić, Ivana; Matković-Čalogović, Dubravka; Pilepić, Viktor; Ivanišević, Irena; Mohaček-Grošev, Vlasta; Sanković, Krešimir

    2017-01-01

    Crystals of the guanine trichloro cuprate(II) complex, (HGua)2[Cu2Cl6]·2H2O (HGua = protonated guanine), were prepared and analysed by spectroscopic (IR, Raman) and computational methods. A new single-crystal X-ray diffraction analysis was conducted to obtain data with lower standard uncertainties than those in the previously published structure. Raman and IR spectroscopy and quantum-mechanical analysis gave us new insight into the vibrational states of the (HGua)2[Cu2Cl6]·2H2O crystal. The vibrational spectra of the crystal were assigned by performing a normal coordinate analysis for a free dimer with a centre of inversion as the only symmetry element. The stretching vibration observed at 279 cm-1 in the infrared spectrum corresponds to the N-Cu bond. The noncovalent interaction (NCI) plots and quantum theory of atoms in molecules (QTAIM) analysis of the electron density obtained from periodic DFT calculations elucidated the interactions that exist within the crystal structure. Closed-shell ionic attractions, as well as weak and medium strength hydrogen bonds, prevailed in the crystal packing.

  14. A multi-functional guanine derivative for studying the DNA G-quadruplex structure.

    Science.gov (United States)

    Ishizuka, Takumi; Zhao, Pei-Yan; Bao, Hong-Liang; Xu, Yan

    2017-10-23

    In the present study, we developed a multi-functional guanine derivative, 8F G, as a G-quadruplex stabilizer, a fluorescent probe for the detection of G-quadruplex formation, and a 19 F sensor for the observation of the G-quadruplex. We demonstrate that the functional nucleoside bearing a 3,5-bis(trifluoromethyl)benzene group at the 8-position of guanine stabilizes the DNA G-quadruplex structure and fluoresces following the G-quadruplex formation. Furthermore, we show that the functional sensor can be used to directly observe DNA G-quadruplexes by 19 F-NMR in living cells. To our knowledge, this is the first study showing that the nucleoside derivative simultaneously allows for three kinds of functions at a single G-quadruplex DNA. Our results suggest that the multi-functional nucleoside derivative can be broadly used for studying the G-quadruplex structure and serves as a powerful tool for examining the molecular basis of G-quadruplex formation in vitro and in living cells.

  15. Guanine nucleotide regulatory protein co-purifies with the D2-dopamine receptor

    International Nuclear Information System (INIS)

    Senogles, S.E.; Caron, M.G.

    1986-01-01

    The D 2 -dopamine receptor from bovine anterior pituitary was purified ∼1000 fold by affinity chromatography on CMOS-Sepharose. Reconstitution of the affinity-purified receptor into phospholipid vesicles revealed the presence of high and low affinity agonist sites as detected by N-n-propylnorapomorphine (NPA) competition experiments with 3 H-spiperone. High affinity agonist binding could be converted to the low affinity form by guanine nucleotides, indicating the presence of an endogenous guanine nucleotide binding protein (N protein) in the affinity-purified D 2 receptor preparations. Furthermore, this preparation contained an agonist-sensitive GTPase activity which was stimulated 2-3 fold over basal by 10 μM NPA. 35 S-GTPγS binding to these preparations revealed a stoichiometry of 0.4-0.7 mole N protein/mole receptor, suggesting the N protein may be specifically coupled with the purified D 2 -dopamine receptor and not present as a contaminant. Pertussis toxin treatment of the affinity purified receptor preparations prevented high affinity agonist binding, as well as agonist stimulation of the GTPase activity, presumably by inactivating the associated N protein. Pertussis toxin lead to the ADP-ribosylation of a protein of 39-40K on SDS-PAGE. These findings indicate that an endogenous N protein, N/sub i/ or N/sub o/, co-purifies with the D 2 -dopamine receptor which may reflect a precoupling of this receptor with an N protein within the membranes

  16. Polymerase recognition of 2-thio-iso-guanine·5-methyl-4-pyrimidinone (iGs·P)--A new DD/AA base pair.

    Science.gov (United States)

    Lee, Dong-Kye; Switzer, Christopher

    2016-02-15

    Polymerase specificity is reported for a previously unknown base pair with a non-standard DD/AA hydrogen bonding pattern: 2-thio-iso-guanine·5-methyl-4-pyrimidinone. Our findings suggest that atomic substitution may provide a solution for low fidelity previously associated with enzymatic copying of iso-guanine. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Electrochemical behaviors and simultaneous determination of guanine and adenine based on graphene–ionic liquid–chitosan composite film modified glassy carbon electrode

    International Nuclear Information System (INIS)

    Niu Xiuli; Yang Wu; Ren Jie; Guo Hao; Long Shijia; Chen Jiaojiao; Gao Jinzhang

    2012-01-01

    Highlights: ► This work developed a novel electrochemical biosensors for guanine and adenine detection simultaneously. ► A disposable electrode based on graphene sheets, ionic liquid and chitosan was proposed. ► The presented method was also applied to simultaneous determination of guanine and adenine in denatured DNA samples with satisfying results. ► Easy fabrication, high sensitivity, excellent reproducibility and long-term stability. - Abstract: A graphene sheets (GS), ionic liquid (IL) and chitosan (CS) modified electrode was fabricated and the modified electrode displayed excellent electrochemical catalytic activities toward guanine and adenine. The transfer electron number (n) and the charge transfer coefficient (α) were calculated with the result as n = 2, α = 0.58 for guanine, and n = 2, α = 0.51 for adenine, which indicated the electrochemical oxidation of guanine and adenine on GS/IL/CS modified electrode was a two-electron and two-proton process. The oxidation overpotentials of guanine and adenine were decreased significantly compared with those obtained at the bare glassy carbon electrode and multi-walled carbon nanotubes modified electrode. The modified electrode exhibited good analytical performance and was successfully applied for individual and simultaneous determination of guanine and adenine. Low detection limits of 0.75 μM for guanine and 0.45 μM for adenine were obtained, with the linear calibration curves over the concentration range 2.5–150 μM and 1.5–350 μM, respectively. At the same time, the proposed method was successfully applied for the determination of guanine and adenine in denatured DNA samples with satisfying results. Moreover, the GS/IL/CS modified electrode exhibited good sensitivity, long-term stability and reproducibility for the determination of guanine and adenine.

  18. Synthesis and Evaluation of Asymmetric Acyclic Nucleoside Bisphosphonates as Inhibitors of Plasmodium falciparum and Human Hypoxanthine-Guanine-(Xanthine) Phosphoribosyltransferase

    Czech Academy of Sciences Publication Activity Database

    Špaček, Petr; Keough, D. T.; Chavchich, M.; Dračínský, Martin; Janeba, Zlatko; Naesens, L.; Edstein, M. D.; Guddat, L. W.; Hocková, Dana

    2017-01-01

    Roč. 60, č. 17 (2017), s. 7539-7554 ISSN 0022-2623 R&D Projects: GA ČR(CZ) GA16-06049S Institutional support: RVO:61388963 Keywords : hypoxanthine-guanine phosphoribosyltransferase * 2nd phosphonate group * 6-oxopurine phosphoribosyltransferases Subject RIV: CC - Organic Chemistry OBOR OECD: Organic chemistry Impact factor: 6.259, year: 2016

  19. Nanoswitches based on DNA base pairs: why adenine-thymine is less suitable than guanine-cytosine

    NARCIS (Netherlands)

    Fonseca Guerra, C.; van der Wijst, T.; Bickelhaupt, F.M.

    2006-01-01

    Substituted Watson-Crick guanine-cytosine (GC) base pairs were recently shown to yield robust three-state nanoswitches. Here, we address the question: Can such supramolecular switches also be based on Watson-Crick adenine-thymine (AT) base pairs? We have theoretically analyzed AT pairs in which

  20. Higher order structural effects stabilizing the reverse watson-crick guanine-cytosine base pair in functional RNAs

    KAUST Repository

    Chawla, Mohit; Abdel-Azeim, Safwat; Oliva, Romina; Cavallo, Luigi

    2013-01-01

    of the Guanine can increase its stability. Herein, we extend our survey and report on other H-bonding interactions that can increase the stability of this base pair. To this aim, we performed a bioinformatics search of the PDB to locate all the occurencies of G

  1. Examination of the effect of the annealing cation on higher order structures containing guanine or isoguanine repeats

    Science.gov (United States)

    Pierce, Sarah E.; Wang, Junmei; Jayawickramarajah, Janarthanan; Hamilton, Andrew D.; Brodbelt, Jennifer S.

    2010-01-01

    Isoguanine (2-oxo-6-amino-guanine), a natural but non-standard base, exhibits unique self-association properties compared to its isomer, guanine, and results in formation of different higher order DNA structures. In this work, the higher order structures formed by oligonucleotides containing guanine repeats or isoguanine repeats after annealing in solutions containing various cations are evaluated by electrospray ionization mass spectrometry (ESI-MS) and circular dichroism (CD) spectroscopy. The guanine-containing strand (G9) consistently formed quadruplexes upon annealing, whereas the isoguanine strand (Ig9) formed both pentaplexes and quadruplexes depending on the annealing cation. Quadruplex formation with G9 showed some dependence on the identity of the cation present during annealing with high relative quadruplex formation detected with six of ten cations. Analogous annealing experiments with Ig9 resulted in complex formation with all ten cations, and the majority of the resulting complexes were pentaplexes. CD results indicated most of the original complexes survived the desalting process necessary for ESI-MS analysis. In addition, several complexes, especially the pentaplexes, were found to be capable of cation exchange with ammonium ions. Ab initio calculations were conducted for isoguanine tetrads and pentads coordinated with all ten cations to predict the most energetically stable structures of the complexes in the gas phase. The observed preference of forming quadruplexes versus pentaplexes as a function of the coordinated cation can be interpreted by the calculated reaction energies of both the tetrads and pentads in combination with the distortion energies of tetrads. PMID:19746468

  2. The role of alkali metal cations in the stabilization of guanine quadruplexes: why K(+) is the best.

    Science.gov (United States)

    Zaccaria, F; Paragi, G; Fonseca Guerra, C

    2016-08-21

    The alkali metal ion affinity of guanine quadruplexes has been studied using dispersion-corrected density functional theory (DFT-D). We have done computational investigations in aqueous solution that mimics artificial supramolecular conditions where guanine bases assemble into stacked quartets as well as biological environments in which telomeric quadruplexes are formed. In both cases, an alkali metal cation is needed to assist self-assembly. Our quantum chemical computations on these supramolecular systems are able to reproduce the experimental order of affinity of the guanine quadruplexes for the cations Li(+), Na(+), K(+), Rb(+), and Cs(+). The strongest binding is computed between the potassium cation and the quadruplex as it occurs in nature. The desolvation and the size of alkali metal cations are thought to be responsible for the order of affinity. Until now, the relative importance of these two factors has remained unclear and debated. By assessing the quantum chemical 'size' of the cation, determining the amount of deformation of the quadruplex needed to accommodate the cation and through the energy decomposition analysis (EDA) of the interaction energy between the cation and the guanines, we reveal that the desolvation and size of the alkali metal cation are both almost equally responsible for the order of affinity.

  3. Structural study and investigation of NMR tensors in interaction of dopamine with Adenine and guanine

    Directory of Open Access Journals (Sweden)

    Lingjia Xu

    2007-04-01

    Full Text Available The interaction of dopamine with adenine and guanine were studied at the Hartree-Fock level theory. The structural and vibrational properties of dopamine-4-N7GUA and dopamine-4-N3ADE were studied at level of HF/6-31G*. Interaction energies (ΔE were calculated to be -11.49 and -11.92 kcal/mol, respectively. Some of bond lengths, angels and tortions are compared. NBO studies were performed to the second-order and perturbative estimates of donor-acceptor interaction have been done. The procedures of gauge-invariant atomic orbital (GIAO and continuous-set-of-gauge-transformation (CSGT were employed to calculate isotropic shielding, chemical shifts anisotropy and chemical shifts anisotropy asymmetry and effective anisotropy using 6-31G* basis set. These calculations yielded molecular geometries in good agreement with available experimental data.

  4. INTRACELLULAR Leishmania amazonensis KILLING INDUCED BY THE GUANINE NUCLEOSIDE 8-BROMOGUANOSINE

    Directory of Open Access Journals (Sweden)

    GIORGIO Selma

    1998-01-01

    Full Text Available In this study we investigated the effect of 8-Bromoguanosine, an immunostimulatory compound, on the cytotoxicity of macrophages against Leishmania amazonensis in an in vitro system. The results showed that macrophages treated with 8-Bromoguanosine before or after infection are capable to reduce parasite load, as monitored by the number of amastigotes per macrophage and the percentage of infected cells (i.e. phagocytic index. Since 8-Bromoguanosine was not directly toxic to the promastigotes, it was concluded that the ribonucleoside induced macrophage activation. Presumably, 8-Bromoguanosine primed macrophages by inducing interferon alpha and beta which ultimately led to L. amazonensis amastigote killing. The results suggest that guanine ribonucleosides may be useful to treat infections with intracellular pathogens.

  5. Modified gold electrodes based on thiocytosine/guanine-gold nanoparticles for uric and ascorbic acid determination

    International Nuclear Information System (INIS)

    Vulcu, Adriana; Grosan, Camelia; Muresan, Liana Maria; Pruneanu, Stela; Olenic, Liliana

    2013-01-01

    The present paper describes the preparation of new modified surfaces for electrodes based on guanine/thiocytosine and gold nanoparticles. The gold nanoparticles were analyzed by UV–vis spectroscopy and transmission electron microscopy (TEM) and it was found that they have diameters between 30 and 40 nm. The layers were characterized by specular reflectance infrared spectroscopy (FTIR-RAS) and by atomic force microscopy (AFM). The thickness of layers was found to be approximately 30 nm for TC layers and 300 nm for GU layers. Every layer was characterized as electrochemical sensor (by cyclic voltammetry) both for uric acid and ascorbic acid determinations, separately and in their mixture. The modified sensors have good calibration functions with good sensitivity (between 1.145 and 1.406 mA cm −2 /decade), reproducibility ( t hiocytosine (Au T C) and gold g uanine (Au G U) layers

  6. Research Update: Density functional theory investigation of the interactions of silver nanoclusters with guanine

    Directory of Open Access Journals (Sweden)

    Brandon B. Dale

    2017-05-01

    Full Text Available Bare and guanine-complexed silver clusters Ag n z (n = 2-6; z = 0-2 are examined using density functional theory to elucidate the geometries and binding motifs that are present experimentally. Whereas the neutral systems remain planar in this size range, a 2D-3D transition occurs at Ag 5 + for the cationic system and at Ag 4 2 + for the dicationic system. Neutral silver clusters can bind with nitrogen 3 or with the pi system of the base. However, positively charged clusters interact with nitrogen 7 and the neighboring carbonyl group. Thus, the cationic silver-DNA clusters present experimentally may preferentially interact at these sites.

  7. Guanine holes are prominent targets for mutation in cancer and inherited disease.

    Directory of Open Access Journals (Sweden)

    Albino Bacolla

    Full Text Available Single base substitutions constitute the most frequent type of human gene mutation and are a leading cause of cancer and inherited disease. These alterations occur non-randomly in DNA, being strongly influenced by the local nucleotide sequence context. However, the molecular mechanisms underlying such sequence context-dependent mutagenesis are not fully understood. Using bioinformatics, computational and molecular modeling analyses, we have determined the frequencies of mutation at G • C bp in the context of all 64 5'-NGNN-3' motifs that contain the mutation at the second position. Twenty-four datasets were employed, comprising >530,000 somatic single base substitutions from 21 cancer genomes, >77,000 germline single-base substitutions causing or associated with human inherited disease and 16.7 million benign germline single-nucleotide variants. In several cancer types, the number of mutated motifs correlated both with the free energies of base stacking and the energies required for abstracting an electron from the target guanines (ionization potentials. Similar correlations were also evident for the pathological missense and nonsense germline mutations, but only when the target guanines were located on the non-transcribed DNA strand. Likewise, pathogenic splicing mutations predominantly affected positions in which a purine was located on the non-transcribed DNA strand. Novel candidate driver mutations and tissue-specific mutational patterns were also identified in the cancer datasets. We conclude that electron transfer reactions within the DNA molecule contribute to sequence context-dependent mutagenesis, involving both somatic driver and passenger mutations in cancer, as well as germline alterations causing or associated with inherited disease.

  8. Trichomonas vaginalis NTPDase and ecto-5'-nucleotidase hydrolyze guanine nucleotides and increase extracellular guanosine levels under serum restriction.

    Science.gov (United States)

    Menezes, Camila Braz; Durgante, Juliano; de Oliveira, Rafael Rodrigues; Dos Santos, Victor Hugo Jacks Mendes; Rodrigues, Luiz Frederico; Garcia, Solange Cristina; Dos Santos, Odelta; Tasca, Tiana

    2016-05-01

    Trichomonas vaginalis is the aethiologic agent of trichomoniasis, the most common non-viral sexually transmitted disease in the world. The purinergic signaling pathway is mediated by extracellular nucleotides and nucleosides that are involved in many biological effects as neurotransmission, immunomodulation and inflammation. Extracellular nucleotides can be hydrolyzed by a family of enzymes known as ectonucleotidases including the ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) family which hydrolyses nucleosides triphosphate and diphosphate as preferential substrates and ecto-5'-nucleotidase which catalyzes the conversion of monophosphates into nucleosides. In T. vaginalis the E-NTPDase and ecto-5'-nucleotidase activities upon adenine nucleotides have already been characterized in intact trophozoites but little is known concerning guanine nucleotides and nucleoside. These enzymes may exert a crucial role on nucleoside generation, providing the purine sources for the synthesis de novo of these essential nutrients, sustaining parasite growth and survival. In this study, we investigated the hydrolysis profile of guanine-related nucleotides and nucleoside in intact trophozoites from long-term-grown and fresh clinical isolates of T. vaginalis. Knowing that guanine nucleotides are also substrates for T. vaginalis ectoenzymes, we evaluated the profile of nucleotides consumption and guanosine uptake in trophozoites submitted to a serum limitation condition. Results show that guanine nucleotides (GTP, GDP, GMP) were substrates for T. vaginalis ectonucleotidases, with expected kinetic parameters for this enzyme family. Different T. vaginalis isolates (two from the ATCC and nine fresh clinical isolates) presented a heterogeneous hydrolysis profile. The serum culture condition increased E-NTPDase and ecto-5'-nucleotidase activities with high consumption of extracellular GTP generating enhanced GDP, GMP and guanosine levels as demonstrated by HPLC, with final

  9. Direct demonstration of guanine nucleotide sensitive receptors for vasoactive intestinal peptide in the anterior lobe of the rat pituitary gland

    International Nuclear Information System (INIS)

    Agui, T.; Matsumoto, K.

    1990-01-01

    The vasoactive intestinal peptide (VIP) receptors were identified on the membranes from the rat anterior pituitary gland with [ 125 I]VIP. The dissociation constant (Kd) and the maximal binding capacity (Bmax) values were estimated from the competitive inhibition data. The Kd and Bmax values were 1.05 +/- 0.75 nM and 103 +/- 11 fmol/mg protein, respectively. The order of molar potency of related peptides to inhibit [ 125 I]VIP binding was VIP greater than peptide histidine isoleucine (PHI) greater than secretin greater than glucagon. Glucagon was not effective to inhibit the binding. [ 125 I]VIP binding was effectively inhibited by the addition of guanine nucleotides. The order of molar potency to inhibit the binding was Gpp(NH)p greater than GTP greater than GDP greater than GMP greater than ATP. These results directly suggest the coupling of VIP receptors with guanine nucleotide binding proteins in the anterior pituitary gland

  10. Automated quantum chemistry based molecular dynamics simulations of electron ionization induced fragmentations of the nucleobases Uracil, Thymine, Cytosine, and Guanine.

    Science.gov (United States)

    Grimme, Stefan; Bauer, Christopher Alexander

    2015-01-01

    The gas-phase decomposition pathways of electron ionization (EI)-induced radical cations of the nucleobases uracil, thymine, cytosine, and guanine are investigated by means of mixed quantum-classical molecular dynamics. No preconceived fragmentation channels are used in the calculations. The results compare well to a plethora of experimental and theoretical data for these important biomolecules. With our combined stochastic and dynamic approach, one can access in an unbiased way the energetically available decomposition mechanisms. Additionally, we are able to separate the EI mass spectra of different tautomers of cytosine and guanine. Our method (previously termed quantum chemistry electron ionization mass spectra) reproduces free nucleobase experimental mass spectra well and provides detailed mechanistic in-sight into high-energy unimolecular decomposition processes.

  11. Structure of Radicals from X-irradiated Guanine Derivatives: An Experimental and Computational Study of Sodium Guanosine Dihydrate Single Crystals

    Science.gov (United States)

    Jayatilaka, Nayana; Nelson, William H.

    2008-01-01

    In sodium guanosine dihydrate single crystals, the guanine moiety is deprotonated at N1 due to growth from high-pH (>12) solutions. EPR and ENDOR study of crystals x-irradiated at 10 K detected evidence for three radical forms. Radical R1,characterized by two proton and two nitrogen hyperfine interactions, was identified as the product of net hydrogenation at N7 of the N1-deprotonated guanine unit. R1 exhibited an unusually distorted structure leading to net positive isotropic components of the hydrogen couplings. Radical R2, characterized by one proton and one nitrogen hyperfine coupling was identified as the primary electron loss product. This product is equivalent to that of deprotonation at N1 by the guanine cation and represents the first ENDOR characterization of that product. Radical R3, characterized by a single hydrogen hyperfine coupling, was identified as the product of net dehydrogenation at C1 of the ribose moiety. The identification of radicals R1-R3 was supported by DFT calculations on several possible structures using the B3LYP/6-311G(2df,p)//6-31G(d,p) approach. Radical R4, detected after warming the crystals to room temperature, was identified as the well-known product of net hydrogenation of C8 of the (N1-deprotonated) guanine component. Radical R1, evidently formed by protonation of the primary electron addition product, was present as roughly 60% of the total radicals detected at 10 K. Radical R2 was present as roughly 27% of the total yield, and the concentration of R3 contributed the remaining 13%. R3 is evidently the product of oneelectron oxidation followed by deprotonation; thus, the balance of oxidation and reduction products is approximately equal within experimental uncertainty. PMID:17249824

  12. Ball with hair: modular functionalization of highly stable G-quadruplex DNA nano-scaffolds through N2-guanine modification.

    Science.gov (United States)

    Lech, Christopher Jacques; Phan, Anh Tuân

    2017-06-20

    Functionalized nanoparticles have seen valuable applications, particularly in the delivery of therapeutic and diagnostic agents in biological systems. However, the manufacturing of such nano-scale systems with the consistency required for biological application can be challenging, as variation in size and shape have large influences in nanoparticle behavior in vivo. We report on the development of a versatile nano-scaffold based on the modular functionalization of a DNA G-quadruplex. DNA sequences are functionalized in a modular fashion using well-established phosphoramidite chemical synthesis with nucleotides containing modification of the amino (N2) position of the guanine base. In physiological conditions, these sequences fold into well-defined G-quadruplex structures. The resulting DNA nano-scaffolds are thermally stable, consistent in size, and functionalized in a manner that allows for control over the density and relative orientation of functional chemistries on the nano-scaffold surface. Various chemistries including small modifications (N2-methyl-guanine), bulky aromatic modifications (N2-benzyl-guanine), and long chain-like modifications (N2-6-amino-hexyl-guanine) are tested and are found to be generally compatible with G-quadruplex formation. Furthermore, these modifications stabilize the G-quadruplex scaffold by 2.0-13.3 °C per modification in the melting temperature, with concurrent modifications producing extremely stable nano-scaffolds. We demonstrate the potential of this approach by functionalizing nano-scaffolds for use within the biotin-avidin conjugation approach. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. QuadBase2: web server for multiplexed guanine quadruplex mining and visualization

    Science.gov (United States)

    Dhapola, Parashar; Chowdhury, Shantanu

    2016-01-01

    DNA guanine quadruplexes or G4s are non-canonical DNA secondary structures which affect genomic processes like replication, transcription and recombination. G4s are computationally identified by specific nucleotide motifs which are also called putative G4 (PG4) motifs. Despite the general relevance of these structures, there is currently no tool available that can allow batch queries and genome-wide analysis of these motifs in a user-friendly interface. QuadBase2 (quadbase.igib.res.in) presents a completely reinvented web server version of previously published QuadBase database. QuadBase2 enables users to mine PG4 motifs in up to 178 eukaryotes through the EuQuad module. This module interfaces with Ensembl Compara database, to allow users mine PG4 motifs in the orthologues of genes of interest across eukaryotes. PG4 motifs can be mined across genes and their promoter sequences in 1719 prokaryotes through ProQuad module. This module includes a feature that allows genome-wide mining of PG4 motifs and their visualization as circular histograms. TetraplexFinder, the module for mining PG4 motifs in user-provided sequences is now capable of handling up to 20 MB of data. QuadBase2 is a comprehensive PG4 motif mining tool that further expands the configurations and algorithms for mining PG4 motifs in a user-friendly way. PMID:27185890

  14. WBSCR16 Is a Guanine Nucleotide Exchange Factor Important for Mitochondrial Fusion

    Directory of Open Access Journals (Sweden)

    Guorui Huang

    2017-07-01

    Full Text Available Regulated inter-mitochondrial fusion/fission is essential for maintaining optimal mitochondrial respiration and control of apoptosis and autophagy. In mammals, mitochondrial fusion is controlled by outer membrane GTPases MFN1 and MFN2 and by inner membrane (IM GTPase OPA1. Disordered mitochondrial fusion/fission contributes to various pathologies, and MFN2 or OPA1 mutations underlie neurodegenerative diseases. Here, we show that the WBSCR16 protein is primarily associated with the outer face of the inner mitochondrial membrane and is important for mitochondrial fusion. We provide evidence of a WBSCR16/OPA1 physical interaction in the intact cell and of a WBSCR16 function as an OPA1-specific guanine nucleotide exchange factor (GEF. Homozygosity for a Wbscr16 mutation causes early embryonic lethality, whereas neurons of mice heterozygous for the mutation have mitochondria with reduced membrane potential and increased susceptibility to fragmentation upon exposure to stress, suggesting roles for WBSCR16 deficits in neuronal pathologies.

  15. Proteomic analysis of Rac1 signaling regulation by guanine nucleotide exchange factors.

    Science.gov (United States)

    Marei, Hadir; Carpy, Alejandro; Macek, Boris; Malliri, Angeliki

    2016-08-02

    The small GTPase Rac1 is implicated in various cellular processes that are essential for normal cell function. Deregulation of Rac1 signaling has also been linked to a number of diseases, including cancer. The diversity of Rac1 functioning in cells is mainly attributed to its ability to bind to a multitude of downstream effectors following activation by Guanine nucleotide Exchange Factors (GEFs). Despite the identification of a large number of Rac1 binding partners, factors influencing downstream specificity are poorly defined, thus hindering the detailed understanding of both Rac1's normal and pathological functions. In a recent study, we demonstrated a role for 2 Rac-specific GEFs, Tiam1 and P-Rex1, in mediating Rac1 anti- versus pro-migratory effects, respectively. Importantly, via conducting a quantitative proteomic screen, we identified distinct changes in the Rac1 interactome following activation by either GEF, indicating that these opposing effects are mediated through GEF modulation of the Rac1 interactome. Here, we present the full list of identified Rac1 interactors together with functional annotation of the differentially regulated Rac1 binding partners. In light of this data, we also provide additional insights into known and novel signaling cascades that might account for the GEF-mediated Rac1-driven cellular effects.

  16. The selective phosphorylation of a guanine nucleotide-binding regulatory protein

    International Nuclear Information System (INIS)

    Carlson, K.E.

    1989-01-01

    Receptor-activated signal transduction pathways regulate the responsiveness of cells to external stimuli. These transduction pathways themselves are subject to regulation, most commonly by phosphorylation. Guanine nucleotide-binding regulatory proteins (G Proteins), as requisite signal transducing elements for many plasma membrane receptors, are considered likely targets for regulation by phosphorylation. Protein kinase C (PKC) has been shown to phosphorylate the α subunit of G i and other G proteins in solution. However, the occurrence of the phosphorylation of G 1 within intact cells in response to activation of PKC has not been rigorously demonstrated. In this thesis, the extent to which the α subunits of G i undergo phosphorylation within human platelets in response to activation of PKC was examined by means of radiolabeling and immunoprecipitation. Incubation of platelets with phorbol-12-myristate-13-acetate (PMA), a potent activator of PKC, promoted the phosphorylation of several proteins within saponin-permeabilized and intact platelets incubated with [γ 32 P]ATP and [ 32 P]H 3 PO 4 , respectively. None of the phosphoproteins, however, were precipitated by either of two antisera containing antibodies differing in specificities for epitopes within G iα -despite precipitation of a substantial fraction of the subunit itself. In contrast, other antisera, containing antibodies specific for the recently describe G zα , or antibodies for both G zα and G iα , precipitated a 40-kDa phosphoprotein

  17. In vitro guanine nucleotide exchange activity of DHR-2/DOCKER/CZH2 domains.

    Science.gov (United States)

    Côté, Jean-François; Vuori, Kristiina

    2006-01-01

    Rho family GTPases regulate a large variety of biological processes, including the reorganization of the actin cytoskeleton. Like other members of the Ras superfamily of small GTP-binding proteins, Rho GTPases cycle between a GDP-bound (inactive) and a GTP-bound (active) state, and, when active, the GTPases relay extracellular signals to a large number of downstream effectors. Guanine nucleotide exchange factors (GEFs) promote the exchange of GDP for GTP on Rho GTPases, thereby activating them. Most Rho-GEFs mediate their effects through their signature domain known as the Dbl Homology-Pleckstrin Homology (DH-PH) module. Recently, we and others identified a family of evolutionarily conserved, DOCK180-related proteins that also display GEF activity toward Rho GTPases. The DOCK180-family of proteins lacks the canonical DH-PH module. Instead, they rely on a novel domain, termed DHR-2, DOCKER, or CZH2, to exchange GDP for GTP on Rho targets. In this chapter, the experimental approach that we used to uncover the exchange activity of the DHR-2 domain of DOCK180-related proteins will be described.

  18. Quenching of light flickering in synthetic guanine crystals in aqueous solutions under strong static magnetic fields

    Science.gov (United States)

    Mootha, A.; Takanezawa, Y.; Iwasaka, M.

    2018-05-01

    The present study focused on the vibration of micro crystal particles of guanine due to Brownian motion. The organic particle has a refractive index of 1.83 and caused a flickering of light. To test the possibility of using magnetic properties under wet conditions, changes in the frequency of particle vibration by applying magnetic fields were investigated. At first, we found that the exposure at 5 T inhibited the flickering light intensities and the particle vibration slightly decreased. Next, we carried out a high speed camera measurement of the Brownian motion of the particle with a time resolution of 100 flame per second (fps) with and without magnetic field exposures. It was revealed that the vibrational speed of synthetic particles was enhanced at 500 mT. Detailed analyses of the particle vibration by changing the direction of magnetic fields versus the light source revealed that the Brownian motion's vibrational frequency was entrained under magnetic fields at 500 mT, and an increase in vibration speed to 20Hz was observed. Additional measurements of light scattering fluctuation using photo-detector and analyses on auto-correlation also confirmed this speculation. The studied Brownian vibration may be influenced by the change in mechanical interactions between the vibration particles and surrounding medium. The discovered phenomena can be applied for molecular and biological interactions in future studies.

  19. Guanine nucleotide binding protein-like 3 is a potential prognosis indicator of gastric cancer.

    Science.gov (United States)

    Chen, Jing; Dong, Shuang; Hu, Jiangfeng; Duan, Bensong; Yao, Jian; Zhang, Ruiyun; Zhou, Hongmei; Sheng, Haihui; Gao, Hengjun; Li, Shunlong; Zhang, Xianwen

    2015-01-01

    Guanine nucleotide binding protein-like 3 (GNL3) is a GIP-binding nuclear protein that has been reported to be involved in various biological processes, including cell proliferation, cellular senescence and tumorigenesis. This study aimed to investigate the expression level of GNL3 in gastric cancer and to evaluate the relationship between its expression and clinical variables and overall survival of gastric cancer patients. The expression level of GNL3 was examined in 89 human gastric cancer samples using immunohistochemistry (IHC) staining. GNL3 in gastric cancer tissues was significantly upregulated compared with paracancerous tissues. GNL3 expression in adjacent non-cancerous tissues was associated with sex and tumor size. Survival analyses showed that GNL3 expression in both gastric cancer and adjacent non-cancerous tissues were not related to overall survival. However, in the subgroup of patients with larger tumor size (≥ 6 cm), a close association was found between GNL3 expression in gastric cancer tissues and overall survival. GNL3-positive patients had a shorter survival than GNL3-negative patients. Our study suggests that GNL3 might play an important role in the progression of gastric cancer and serve as a biomarker for poor prognosis in gastric cancer patients.

  20. Expression of a Rho guanine nucleotide exchange factor, Ect2, in the developing mouse pituitary.

    Science.gov (United States)

    Islam, M S; Tsuji, T; Higashida, C; Takahashi, M; Higashida, H; Koizumi, K

    2010-05-01

    The pituitary gland is a highly mitotically active tissue after birth. Various cell types are known to undergo proliferation in the anterior pituitary. However, little is known about the mechanisms regulating mitotic activity in this tissue. When searching for genes specifically expressed in the pituitary gland among those that we previously screened in Drosophila, we found epithelial cell-transforming gene 2 (Ect2). Ect2 is a guanine nucleotide exchange factor for Rho GTPases, which is known to play an essential role in cytokinesis. Although there have been many cellular studies regarding the function of Ect2, the temporal and spatial expression patterns of Ect2 in vivo have not been determined. In the present study, we examined the postnatal developmental expression of Ect2 in the mouse pituitary. Enhanced Ect2 expression was detected in the mouse pituitary gland during the first 3 weeks after birth, which coincided well with the period of rapid pituitary expansion associated with increased growth rate. Immunostaining analysis showed that Ect2-expressing cells were distributed in the anterior and intermediate lobes, but not the posterior lobe, of the pituitary. These Ect2-expressing cells frequently incorporated the thymidine analogue, EdU (5-ethynyl-2'-deoxyuridine), indicating that these cells were mitotically active. Taken together, the results demonstrate the functional role of Ect2 in postnatal proliferating cells in the two lobes of the pituitary, thereby suggesting roles in developmental growth of the mammalian pituitary.

  1. Ric-8A, a Gα protein guanine nucleotide exchange factor potentiates taste receptor signaling

    Directory of Open Access Journals (Sweden)

    Claire J Fenech

    2009-10-01

    Full Text Available Taste receptors for sweet, bitter and umami tastants are G-protein coupled receptors (GPCRs. While much effort has been devoted to understanding G-protein-receptor interactions and identifying the components of the signalling cascade downstream of these receptors, at the level of the G-protein the modulation of receptor signal transduction remains relatively unexplored. In this regard a taste-specific regulator of G-protein signaling (RGS, RGS21, has recently been identified. To study whether guanine nucleotide exchange factors (GEFs are involved in the transduction of the signal downstream of the taste GPCRs we investigated the expression of Ric-8A and Ric-8B in mouse taste cells and their interaction with G-protein subunits found in taste buds. Mammalian Ric-8 proteins were initially identified as potent GEFs for a range of Gα subunits and Ric-8B has recently been shown to amplify olfactory signal transduction. We find that both Ric-8A and Ric-8B are expressed in a large portion of taste bud cells and that most of these cells contain IP3R-3 a marker for sweet, umami and bitter taste receptor cells. Ric-8A interacts with Gα-gustducin and Gαi2 through which it amplifies the signal transduction of hTas2R16, a receptor for bitter compounds. Overall, these findings are consistent with a role for Ric-8 in mammalian taste signal transduction.

  2. Characterization of a mimivirus RNA cap guanine-N2 methyltransferase.

    Science.gov (United States)

    Benarroch, Delphine; Qiu, Zhicheng R; Schwer, Beate; Shuman, Stewart

    2009-04-01

    A 2,2,7-trimethylguanosine (TMG) cap is a signature feature of eukaryal snRNAs, telomerase RNAs, and trans-spliced nematode mRNAs. TMG and 2,7-dimethylguanosine (DMG) caps are also present on mRNAs of two species of alphaviruses (positive strand RNA viruses of the Togaviridae family). It is presently not known how viral mRNAs might acquire a hypermethylated cap. Mimivirus, a giant DNA virus that infects amoeba, encodes many putative enzymes and proteins implicated in RNA transactions, including the synthesis and capping of viral mRNAs and the promotion of cap-dependent translation. Here we report the identification, purification, and characterization of a mimivirus cap-specific guanine-N2 methyltransferase (MimiTgs), a monomeric enzyme that catalyzes a single round of methyl transfer from AdoMet to an m(7)G cap substrate to form a DMG cap product. MimiTgs, is apparently unable to convert a DMG cap to a TMG cap, and is thereby distinguished from the structurally homologous yeast and human Tgs1 enzymes. Nonetheless, we show genetically that MimiTgs is a true ortholog of Saccharomyces cerevisiae Tgs1. Our results hint that DMG caps can satisfy many of the functions of TMG caps in vivo. We speculate that DMG capping of mimivirus mRNAs might favor viral protein synthesis in the infected host.

  3. Guanine limitation results in CodY-dependent and -independent alteration of Staphylococcus aureus physiology and gene expression.

    Science.gov (United States)

    King, Alyssa N; Borkar, Samiksha; Samuels, David J; Batz, Zachary; Bulock, Logan; Sadykov, Marat R; Bayles, Kenneth W; Brinsmade, Shaun R

    2018-04-30

    In Staphylococcus aureus , the global transcriptional regulator CodY modulates the expression of hundreds of genes in response to the availability of GTP and the branched-chain amino acids isoleucine, leucine, and valine (ILV). CodY DNA-binding activity is high when GTP and ILV are abundant. When GTP and ILV are limited, CodY's affinity for DNA drops, altering expression of CodY regulated targets. In this work, we investigated the impact of guanine nucleotides on S. aureus physiology and CodY activity by constructing a guaA null mutant (Δ guaA ). De novo biosynthesis of guanine monophosphate is abolished due to the guaA mutation; thus, the mutant cells require exogenous guanosine for growth. We also found that CodY activity was reduced when we knocked out guaA , activating the Agr two-component system and increasing secreted protease activity. Notably, in a rich, complex medium, we detected an increase in alternative sigma factor B activity in the Δ guaA mutant, which results in a 5-fold increase in production of the antioxidant pigment staphyloxanthin. Under biologically relevant flow conditions, Δ guaA cells failed to form robust biofilms when limited for guanine or guanosine. RNA-seq analysis of S. aureus transcriptome during growth in guanosine-limited chemostats revealed substantial CodY-dependent and -independent alteration of gene expression profiles. Importantly, these changes increase production of proteases and δ-toxin, suggesting that S. aureus exhibits a more invasive lifestyle when limited for guanosine. Further, gene-products upregulated under GN limitation, including those necessary for lipoic acid biosynthesis and sugar transport, may prove to be useful drug targets for treating Gram-positive infections. Importance Staphylococcus aureus infections impose a serious economic burden on healthcare facilities and patients because of the emergence of strains resistant to last-line antibiotics. Understanding the physiological processes governing

  4. Amyloid Precursor Protein Translation Is Regulated by a 3'UTR Guanine Quadruplex.

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    Ezekiel Crenshaw

    Full Text Available A central event in Alzheimer's disease is the accumulation of amyloid β (Aβ peptides generated by the proteolytic cleavage of the amyloid precursor protein (APP. APP overexpression leads to increased Aβ generation and Alzheimer's disease in humans and altered neuronal migration and increased long term depression in mice. Conversely, reduction of APP expression results in decreased Aβ levels in mice as well as impaired learning and memory and decreased numbers of dendritic spines. Together these findings indicate that therapeutic interventions that aim to restore APP and Aβ levels must do so within an ideal range. To better understand the effects of modulating APP levels, we explored the mechanisms regulating APP expression focusing on post-transcriptional regulation. Such regulation can be mediated by RNA regulatory elements such as guanine quadruplexes (G-quadruplexes, non-canonical structured RNA motifs that affect RNA stability and translation. Via a bioinformatics approach, we identified a candidate G-quadruplex within the APP mRNA in its 3'UTR (untranslated region at residues 3008-3027 (NM_201414.2. This sequence exhibited characteristics of a parallel G-quadruplex structure as revealed by circular dichroism spectrophotometry. Further, as with other G-quadruplexes, the formation of this structure was dependent on the presence of potassium ions. This G-quadruplex has no apparent role in regulating transcription or mRNA stability as wild type and mutant constructs exhibited equivalent mRNA levels as determined by real time PCR. Instead, we demonstrate that this G-quadruplex negatively regulates APP protein expression using dual luciferase reporter and Western blot analysis. Taken together, our studies reveal post-transcriptional regulation by a 3'UTR G-quadruplex as a novel mechanism regulating APP expression.

  5. Transduction proteins of olfactory receptor cells: identification of guanine nucleotide binding proteins and protein kinase C

    International Nuclear Information System (INIS)

    Anholt, R.R.H.; Mumby, S.M.; Stoffers, D.A.; Girard, P.R.; Kuo, J.F.; Snyder, S.H.

    1987-01-01

    The authors have analyzed guanine nucleotide binding proteins (G-proteins) in the olfactory epithelium of Rana catesbeiana using subunit-specific antisera. The olfactory epithelium contained the α subunits of three G-proteins, migrating on polyacrylamide gels in SDS with apparent molecular weights of 45,000, 42,000, and 40,000, corresponding to G/sub s/, G/sub i/, and G/sub o/, respectively. A single β subunit with an apparent molecular weight of 36,000 was detected. An antiserum against the α subunit of retinal transducin failed to detect immunoreactive proteins in olfactory cilia detached from the epithelium. The olfactory cilia appeared to be enriched in immunoreactive G/sub sα/ relative to G/sub ichemical bond/ and G/sub ochemical bond/ when compared to membranes prepared from the olfactory epithelium after detachment of the cilia. Bound antibody was detected by autoradiography after incubation with [ 125 I]protein. Immunohistochemical studies using an antiserum against the β subunit of G-proteins revealed intense staining of the ciliary surface of the olfactory epithelium and of the axon bundles in the lamina propria. In contrast, an antiserum against a common sequence of the α subunits preferentially stained the cell membranes of the olfactory receptor cells and the acinar cells of Bowman's glands and the deep submucosal glands. In addition to G-proteins, they have identified protein kinase C in olfactory cilia via a protein kinase C specific antiserum and via phorbol ester binding. However, in contrast to the G-proteins, protein kinase C occurred also in cilia isolated from respiratory epithelium

  6. Guanine nucleotide-binding protein regulation of melatonin receptors in lizard brain

    International Nuclear Information System (INIS)

    Rivkees, S.A.; Carlson, L.L.; Reppert, S.M.

    1989-01-01

    Melatonin receptors were identified and characterized in crude membrane preparations from lizard brain by using 125 I-labeled melatonin ( 125 I-Mel), a potent melatonin agonist. 125 I-Mel binding sites were saturable; Scatchard analysis revealed high-affinity and lower affinity binding sites, with apparent K d of 2.3 ± 1.0 x 10 -11 M and 2.06 ± 0.43 x 10 -10 M, respectively. Binding was reversible and inhibited by melatonin and closely related analogs but not by serotonin or norepinephrine. Treatment of crude membranes with the nonhydrolyzable GTP analog guanosine 5'-[γ-thio]triphosphate (GTP[γS]), significantly reduced the number of high-affinity receptors and increased the dissociation rate of 125 I-Mel from its receptor. Furthermore, GTP[γS] treatment of ligand-receptor complexes solubilized by Triton X-100 also led to a rapid dissociation of 125 I-Mel from solubilized ligand-receptor complexes. Gel filtration chromatography of solubilized ligand-receptor complexes revealed two major peaks of radioactivity corresponding to M r > 400,000 and M r ca. 110,000. This elution profile was markedly altered by pretreatment with GTP[γS] before solubilization; only the M r 110,000 peak was present in GTP[γS]-pretreated membranes. The results strongly suggest that 125 I-mel binding sites in lizard brain are melatonin receptors, with agonist-promoted guanine nucleotide-binding protein (G protein) coupling and that the apparent molecular size of receptors uncoupled from G proteins is about 110,000

  7. Expression Pattern and Localization Dynamics of Guanine Nucleotide Exchange Factor RIC8 during Mouse Oogenesis.

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    Merly Saare

    Full Text Available Targeting of G proteins to the cell cortex and their activation is one of the triggers of both asymmetric and symmetric cell division. Resistance to inhibitors of cholinesterase 8 (RIC8, a guanine nucleotide exchange factor, activates a certain subgroup of G protein α-subunits in a receptor independent manner. RIC8 controls the asymmetric cell division in Caenorhabditis elegans and Drosophila melanogaster, and symmetric cell division in cultured mammalian cells, where it regulates the mitotic spindle orientation. Although intensely studied in mitosis, the function of RIC8 in mammalian meiosis has remained unknown. Here we demonstrate that the expression and subcellular localization of RIC8 changes profoundly during mouse oogenesis. Immunofluorescence studies revealed that RIC8 expression is dependent on oocyte growth and cell cycle phase. During oocyte growth, RIC8 is abundantly present in cytoplasm of oocytes at primordial, primary and secondary preantral follicle stages. Later, upon oocyte maturation RIC8 also populates the germinal vesicle, its localization becomes cell cycle dependent, and it associates with chromatin and the meiotic spindle. After fertilization, RIC8 protein converges to the pronuclei and is also detectable at high levels in the nucleolus precursor bodies of both maternal and paternal pronucleus. During first cleavage of zygote RIC8 localizes in the mitotic spindle and cell cortex of forming blastomeres. In addition, we demonstrate that RIC8 co-localizes with its interaction partners Gαi1/2:GDP and LGN in meiotic/mitotic spindle, cell cortex and polar bodies of maturing oocytes and zygotes. Downregulation of Ric8 by siRNA leads to interferred translocation of Gαi1/2 to cortical region of maturing oocytes and reduction of its levels. RIC8 is also expressed at high level in female reproductive organs e.g. oviduct. Therefore we suggest a regulatory function for RIC8 in mammalian gametogenesis and fertility.

  8. The guanine nucleotide exchange factor RIC8 regulates conidial germination through Gα proteins in Neurospora crassa.

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    Carla J Eaton

    Full Text Available Heterotrimeric G protein signaling is essential for normal hyphal growth in the filamentous fungus Neurospora crassa. We have previously demonstrated that the non-receptor guanine nucleotide exchange factor RIC8 acts upstream of the Gα proteins GNA-1 and GNA-3 to regulate hyphal extension. Here we demonstrate that regulation of hyphal extension results at least in part, from an important role in control of asexual spore (conidia germination. Loss of GNA-3 leads to a drastic reduction in conidial germination, which is exacerbated in the absence of GNA-1. Mutation of RIC8 leads to a reduction in germination similar to that in the Δgna-1, Δgna-3 double mutant, suggesting that RIC8 regulates conidial germination through both GNA-1 and GNA-3. Support for a more significant role for GNA-3 is indicated by the observation that expression of a GTPase-deficient, constitutively active gna-3 allele in the Δric8 mutant leads to a significant increase in conidial germination. Localization of the three Gα proteins during conidial germination was probed through analysis of cells expressing fluorescently tagged proteins. Functional TagRFP fusions of each of the three Gα subunits were constructed through insertion of TagRFP in a conserved loop region of the Gα subunits. The results demonstrated that GNA-1 localizes to the plasma membrane and vacuoles, and also to septa throughout conidial germination. GNA-2 and GNA-3 localize to both the plasma membrane and vacuoles during early germination, but are then found in intracellular vacuoles later during hyphal outgrowth.

  9. Pre-thymic somatic mutation leads to high mutant frequency at hypoxanthine-guanine phosphoribosyltransferase gene

    Energy Technology Data Exchange (ETDEWEB)

    Jett, J. [Lawrence Livermore National Lab., CA (United States)

    1994-12-01

    While characterizing the background mutation spectrum of the Hypoxathine-guanine phosphoribosyltransferase (HPRT) gene in a healthy population, an outlier with a high mutant frequency of thioguanine resistant lymphocytes was found. When studied at the age of 46, this individual had been smoking 60 cigarettes per day for 38 years. His mutant frequency was calculated at 3.6 and 4.2x10{sup {minus}4} for two sampling periods eight months apart. Sequencing analysis of the HPRT gene in his mutant thioguanine resistant T lymphocytes was done to find whether the cells had a high rate of mutation, or if the mutation was due to a single occurrence of mutation and, if so, when in the T lymphocyte development the mutation occurred. By T-cell receptor analysis it has been found that out of 35 thioguanine resistant clones there was no dominant gamma T cell receptor gene rearrangement. During my appointment in the Science & Engineering Research Semester, I found that 34 of those clones have the same base substitution of G{yields}T at cDNA position 197. Due to the consistent mutant frequency from both sampling periods and the varying T cell receptors, the high mutant frequency cannot be due to recent proliferation of a mature mutant T lymphocyte. From the TCR and DNA sequence analysis we conclude that the G{yields}T mutation must have occurred in a T lymphocyte precursor before thymic differentiation so that the thioguanine resistant clones share the same base substitution but not the same gamma T cell receptor gene.

  10. The influence of N-7 guanine modifications on the strength of Watson-Crick base pairing and guanine N-1 acidity: Comparison of gas-phase and condensed-phase trends

    Czech Academy of Sciences Publication Activity Database

    Burda, J. V.; Šponer, Jiří; Hrabáková, J.; Zeizinger, M.; Leszczynski, J.

    2003-01-01

    Roč. 107, č. 22 (2003), s. 5349-5356 ISSN 1520-6106 R&D Projects: GA MŠk ME 517; GA MŠk LN00A016 Grant - others:Wellcome Trust(GB) GR067507MF; ONR(US) N00034-03-1-0116; National Science Foundation(US) CREST 9805465 Institutional research plan: CEZ:AV0Z5004920 Keywords : Watson-Crick base pairing * guanines * gas-phase and condensed-phase trends Subject RIV: BO - Biophysics Impact factor: 3.679, year: 2003

  11. Electron attachment to the guanine-cytosine nucleic acid base pair and the effects of monohydration and proton transfer.

    Science.gov (United States)

    Gupta, Ashutosh; Jaeger, Heather M; Compaan, Katherine R; Schaefer, Henry F

    2012-05-17

    The guanine-cytosine (GC) radical anion and its interaction with a single water molecule is studied using ab initio and density functional methods. Z-averaged second-order perturbation theory (ZAPT2) was applied to GC radical anion for the first time. Predicted spin densities show that the radical character is localized on cytosine. The Watson-Crick monohydrated GC anion is compared to neutral GC·H2O, as well as to the proton-transferred analogue on the basis of structural and energetic properties. In all three systems, local minima are identified that correspond to water positioned in the major and minor grooves of macromolecular DNA. On the anionic surface, two novel structures have water positioned above or below the GC plane. On the neutral and anionic surfaces, the global minimum can be described as water interacting with the minor groove. These structures are predicted to have hydration energies of 9.7 and 11.8 kcal mol(-1), respectively. Upon interbase proton-transfer (PT), the anionic global minimum has water positioned in the major groove, and the hydration energy increases to 13.4 kcal mol(-1). PT GC·H2O(•-) has distonic character; the radical character resides on cytosine, while the negative charge is localized on guanine. The effects of proton transfer are further investigated through the computed adiabatic electron affinities (AEA) of GC and monohydrated GC, and the vertical detachment energies (VDE) of the corresponding anions. Monohydration increases the AEAs and VDEs by only 0.1 eV, while proton-transfer increases the VDEs substantially (0.8 eV). The molecular charge distribution of monohydrated guanine-cytosine radical anion depends heavily on interbase proton transfer.

  12. The electrochemical reduction of the purines guanine and adenine at platinum electrodes in several room temperature ionic liquids

    International Nuclear Information System (INIS)

    Zanoni, Maria Valnice Boldrin; Rogers, Emma I.; Hardacre, Christopher; Compton, Richard G.

    2010-01-01

    The reduction of guanine was studied by microelectrode voltammetry in the room temperature ionic liquids (RTILs) N-hexyltriethylammonium bis (trifluoromethanesulfonyl) imide [N 6,2,2,2 ][N(Tf) 2 ], 1-butyl-3-methylimidazolium hexafluorosphosphate [C 4 mim][PF 6 ], N-butyl-N-methyl-pyrrolidinium bis(trifluoromethanesulfonyl)imide [C 4 mpyrr][N(Tf) 2 ], 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide [C 4 mim][N(Tf) 2 ], N-butyl-N-methyl-pyrrolidinium dicyanamide [C 4 mpyrr][N(NC) 2 ] and tris(P-hexyl)-tetradecylphosphonium trifluorotris(pentafluoroethyl)phosphate [P 14,6,6,6 ][FAP] on a platinum microelectrode. In [N 6,2,2,2 ][NTf 2 ] and [P 14,6,6,6 ][FAP], but not in the other ionic liquids studied, guanine reduction involves a one-electron, diffusion-controlled process at very negative potential to produce an unstable radical anion, which is thought to undergo a dimerization reaction, probably after proton abstraction from the cation of the ionic liquid. The rate of this subsequent reaction depends on the nature of the ionic liquid, and it is faster in the ionic liquid [P 14,6,6,6 ][FAP], in which the formation of the resulting dimer can be voltammetrically monitored at less negative potentials than required for the reduction of the parent molecule. Adenine showed similar behaviour to guanine but the pyrimidines thymine and cytosine did not; thymine was not reduced at potentials less negative than required for solvent (RTIL) decomposition while only a poorly defined wave was seen for cytosine. The possibility for proton abstraction from the cation in [N 6,2,2,2 ][NTf 2 ] and [P 14,6,6,6 ][FAP] is noted and this is thought to aid the electrochemical dimerization process. The resulting rapid reaction is thought to shift the reduction potentials for guanine and adenine to lower values than observed in RTILs where the scope for proton abstraction is not present. Such shifts are characteristic of so-called EC processes where reversible electron transfer

  13. Reactions of guanine with methyl chloride and methyl bromide: O6-methylation versus charge transfer complex formation

    Science.gov (United States)

    Shukla, P. K.; Mishra, P. C.; Suhai, S.

    Density functional theory (DFT) at the B3LYP/6-31+G* and B3LYP/AUG-cc-pVDZ levels was employed to study O6-methylation of guanine due to its reactions with methyl chloride and methyl bromide and to obtain explanation as to why the methyl halides cause genotoxicity and possess mutagenic and carcinogenic properties. Geometries of the various isolated species involved in the reactions, reactant complexes (RCs), and product complexes (PCs) were optimized in gas phase. Transition states connecting the reactant complexes with the product complexes were also optimized in gas phase at the same levels of theory. The reactant complexes, product complexes, and transition states were solvated in aqueous media using the polarizable continuum model (PCM) of the self-consistent reaction field theory. Zero-point energy (ZPE) correction to total energy and the corresponding thermal energy correction to enthalpy were made in each case. The reactant complexes of the keto form of guanine with methyl chloride and methyl bromide in water are appreciably more stable than the corresponding complexes involving the enol form of guanine. The nature of binding in the product complexes was found to be of the charge transfer type (O6mG+ · X-, X dbond Cl, Br). Binding of HCl, HBr, and H2O molecules to the PCs obtained with the keto form of guanine did not alter the positions of the halide anions in the PCs, and the charge transfer character of the PCs was also not modified due to this binding. Further, the complexes obtained due to the binding of HCl, HBr, and H2O molecules to the PCs had greater stability than the isolated PCs. The reaction barriers involved in the formation of PCs were found to be quite high (?50 kcal/mol). Mechanisms of genotoxicity, mutagenesis and carcinogenesis caused by the methyl halides appear to involve charge transfer-type complex formation. Thus the mechanisms of these processes involving the methyl halides appear to be quite different from those that involve the

  14. Kinetics of the interactions between yeast elongation factors 1A and 1Balpha, guanine nucleotides, and aminoacyl-tRNA

    DEFF Research Database (Denmark)

    Gromadski, Kirill B; Schümmer, Tobias; Strømgaard, Anne

    2007-01-01

    of guanine nucleotides. At the concentrations of nucleotides and factors prevailing in the cell, the overall exchange rate is expected to be in the range of 6 s(-1), which is compatible with the rate of protein synthesis in the cell. eEF1A.GTP binds Phe-tRNA(Phe) with a K(d) of 3 nm, whereas eEF1A.GDP shows...... no significant binding, indicating that eEF1A has similar tRNA binding properties as its prokaryotic homolog, EF-Tu. Udgivelsesdato: 2007-Dec-7...

  15. Regulation of mitotic spindle formation by the RhoA guanine nucleotide exchange factor ARHGEF10

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    Satoh Takaya

    2009-07-01

    Full Text Available Abstract Background The Dbl family guanine nucleotide exchange factor ARHGEF10 was originally identified as the product of the gene associated with slowed nerve-conduction velocities of peripheral nerves. However, the function of ARHGEF10 in mammalian cells is totally unknown at a molecular level. ARHGEF10 contains no distinctive functional domains except for tandem Dbl homology-pleckstrin homology and putative transmembrane domains. Results Here we show that RhoA is a substrate for ARHGEF10. In both G1/S and M phases, ARHGEF10 was localized in the centrosome in adenocarcinoma HeLa cells. Furthermore, RNA interference-based knockdown of ARHGEF10 resulted in multipolar spindle formation in M phase. Each spindle pole seems to contain a centrosome consisting of two centrioles and the pericentriolar material. Downregulation of RhoA elicited similar phenotypes, and aberrant mitotic spindle formation following ARHGEF10 knockdown was rescued by ectopic expression of constitutively activated RhoA. Multinucleated cells were not increased upon ARHGEF10 knockdown in contrast to treatment with Y-27632, a specific pharmacological inhibitor for the RhoA effector kinase ROCK, which induced not only multipolar spindle formation, but also multinucleation. Therefore, unregulated centrosome duplication rather than aberration in cytokinesis may be responsible for ARHGEF10 knockdown-dependent multipolar spindle formation. We further isolated the kinesin-like motor protein KIF3B as a binding partner of ARHGEF10. Knockdown of KIF3B again caused multipolar spindle phenotypes. The supernumerary centrosome phenotype was also observed in S phase-arrested osteosarcoma U2OS cells when the expression of ARHGEF10, RhoA or KIF3B was abrogated by RNA interference. Conclusion Collectively, our results suggest that a novel RhoA-dependent signaling pathway under the control of ARHGEF10 has a pivotal role in the regulation of the cell division cycle. This pathway is not involved in

  16. Simultaneous protection of organic p- and n-channels in complementary inverter from aging and bias-stress by DNA-base guanine/Al2O3 double layer.

    Science.gov (United States)

    Lee, Junyeong; Hwang, Hyuncheol; Min, Sung-Wook; Shin, Jae Min; Kim, Jin Sung; Jeon, Pyo Jin; Lee, Hee Sung; Im, Seongil

    2015-01-28

    Although organic field-effect transistors (OFETs) have various advantages of lightweight, low-cost, mechanical flexibility, and nowadays even higher mobility than amorphous Si-based FET, stability issue under bias and ambient condition critically hinder its practical application. One of the most detrimental effects on organic layer comes from penetrated atmospheric species such as oxygen and water. To solve such degradation problems, several molecular engineering tactics are introduced: forming a kinetic barrier, lowering the level of molecule orbitals, and increasing the band gap. However, direct passivation of organic channels, the most promising strategy, has not been reported as often as other methods. Here, we resolved the ambient stability issues of p-type (heptazole)/or n-type (PTCDI-C13) OFETs and their bias-stability issues at once, using DNA-base small molecule guanine (C5H5N5O)/Al2O3 bilayer. The guanine protects the organic channels as buffer/and H getter layer between the channels and capping Al2O3, whereas the oxide capping resists ambient molecules. As a result, both p-type and n-type OFETs are simultaneously protected from gate-bias stress and 30 days-long ambient aging, finally demonstrating a highly stable, high-gain complementary-type logic inverter.

  17. ARHGEF7 (Beta-PIX acts as guanine nucleotide exchange factor for leucine-rich repeat kinase 2.

    Directory of Open Access Journals (Sweden)

    Karina Haebig

    Full Text Available BACKGROUND: Mutations within the leucine-rich repeat kinase 2 (LRRK2 gene are a common cause of familial and sporadic Parkinson's disease. The multidomain protein LRRK2 exhibits overall low GTPase and kinase activity in vitro. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that the rho guanine nucleotide exchange factor ARHGEF7 and the small GTPase CDC42 are interacting with LRRK2 in vitro and in vivo. GTPase activity of full-length LRRK2 increases in the presence of recombinant ARHGEF7. Interestingly, LRRK2 phosphorylates ARHGEF7 in vitro at previously unknown phosphorylation sites. We provide evidence that ARHGEF7 might act as a guanine nucleotide exchange factor for LRRK2 and that R1441C mutant LRRK2 with reduced GTP hydrolysis activity also shows reduced binding to ARHGEF7. CONCLUSIONS/SIGNIFICANCE: Downstream effects of phosphorylation of ARHGEF7 through LRRK2 could be (i a feedback control mechanism for LRRK2 activity as well as (ii an impact of LRRK2 on actin cytoskeleton regulation. A newly identified familial mutation N1437S, localized within the GTPase domain of LRRK2, further underlines the importance of the GTPase domain of LRRK2 in Parkinson's disease pathogenesis.

  18. Molecular cloning, characterization, and expression of human ADP-ribosylation factors: Two guanine nucleotide-dependent activators of cholera toxin

    International Nuclear Information System (INIS)

    Bobak, D.A.; Nightingale, M.S.; Murtagh, J.J.; Price, S.R.; Moss, J.; Vaughan, M.

    1989-01-01

    ADP-ribosylation factors (ARFs) are small guanine nucleotide-binding proteins that enhance the enzymatic activities of cholera toxin. Two ARF cDNAs, ARF1 and ARF3, were cloned from a human cerebellum library. Based on deduced amino acid sequences and patterns of hybridization of cDNA and oligonucleotide probes with mammalian brain poly(A) + RNA, human ARF1 is the homologue of bovine ARF1. Human ARF3, which differs from bovine ARF1 and bovine ARF2, appears to represent a newly identified third type of ARF. Hybridization patterns of human ARF cDNA and clone-specific oligonucleotides with poly(A) + RNA are consistent with the presence of at least two, and perhaps four, separate ARF messages in human brain. In vitro translation of ARF1, ARF2, and ARF3 produced proteins that behaved, by SDS/PAGE, similar to a purified soluble brain ARF. Deduced amino acid sequences of human ARF1 and ARF3 contain regions, similar to those in other G proteins, that are believed to be involved in GTP binding and hydrolysis. ARFS also exhibit a modest degree of homology with a bovine phospholipase C. The observations reported here support the conclusion that the ARFs are members of a multigene family of small guanine nucleotide-binding proteins. Definition of the regulation of ARF mRNAs and of function(s) of recombinant ARF proteins will aid in the elucidation of the physiologic role(s) of ARFs

  19. Higher order structural effects stabilizing the reverse watson-crick guanine-cytosine base pair in functional RNAs

    KAUST Repository

    Chawla, Mohit

    2013-10-10

    The G:C reverse Watson-Crick (W:W trans) base pair, also known as Levitt base pair in the context of tRNAs, is a structurally and functionally important base pair that contributes to tertiary interactions joining distant domains in functional RNA molecules and also participates in metabolite binding in riboswitches. We previously indicated that the isolated G:C W:W trans base pair is a rather unstable geometry, and that dicationic metal binding to the Guanine base or posttranscriptional modification of the Guanine can increase its stability. Herein, we extend our survey and report on other H-bonding interactions that can increase the stability of this base pair. To this aim, we performed a bioinformatics search of the PDB to locate all the occurencies of G:C trans base pairs. Interestingly, 66% of the G:C trans base pairs in the PDB are engaged in additional H-bonding interactions with other bases, the RNA backbone or structured water molecules. High level quantum mechanical calculations on a data set of representative crystal structures were performed to shed light on the structural stability and energetics of the various crystallographic motifs. This analysis was extended to the binding of the preQ1 metabolite to a preQ1-II riboswitch. 2013 The Author(s).

  20. Multiscale QM/MM molecular dynamics study on the first steps of guanine damage by free hydroxyl radicals in solution.

    Science.gov (United States)

    Abolfath, Ramin M; Biswas, P K; Rajnarayanam, R; Brabec, Thomas; Kodym, Reinhard; Papiez, Lech

    2012-04-19

    Understanding the damage of DNA bases from hydrogen abstraction by free OH radicals is of particular importance to understanding the indirect effect of ionizing radiation. Previous studies address the problem with truncated DNA bases as ab initio quantum simulations required to study such electronic-spin-dependent processes are computationally expensive. Here, for the first time, we employ a multiscale and hybrid quantum mechanical-molecular mechanical simulation to study the interaction of OH radicals with a guanine-deoxyribose-phosphate DNA molecular unit in the presence of water, where all of the water molecules and the deoxyribose-phosphate fragment are treated with the simplistic classical molecular mechanical scheme. Our result illustrates that the presence of water strongly alters the hydrogen-abstraction reaction as the hydrogen bonding of OH radicals with water restricts the relative orientation of the OH radicals with respect to the DNA base (here, guanine). This results in an angular anisotropy in the chemical pathway and a lower efficiency in the hydrogen-abstraction mechanisms than previously anticipated for identical systems in vacuum. The method can easily be extended to single- and double-stranded DNA without any appreciable computational cost as these molecular units can be treated in the classical subsystem, as has been demonstrated here. © 2012 American Chemical Society

  1. Free terminal amines in DNA-binding peptides alter the product distribution from guanine radicals produced by single electron oxidation.

    Science.gov (United States)

    Konigsfeld, Katie M; Lee, Melissa; Urata, Sarah M; Aguilera, Joe A; Milligan, Jamie R

    2012-03-01

    Electron deficient guanine radical species are major intermediates produced in DNA by the direct effect of ionizing irradiation. There is evidence that they react with amine groups in closely bound ligands to form covalent crosslinks. Crosslink formation is very poorly characterized in terms of quantitative rate and yield data. We sought to address this issue by using oligo-arginine ligands to model the close association of DNA and its binding proteins in chromatin. Guanine radicals were prepared in plasmid DNA by single electron oxidation. The product distribution derived from them was assayed by strand break formation after four different post-irradiation incubations. We compared the yields of DNA damage produced in the presence of four ligands in which neither, one, or both of the amino and carboxylate termini were blocked with amides. Free carboxylate groups were unreactive. Significantly higher yields of heat labile sites were observed when the amino terminus was unblocked. The rate of the reaction was characterized by diluting the unblocked amino group with its amide blocked derivative. These observations provide a means to develop quantitative estimates for the yields in which these labile sites are formed in chromatin by exposure to ionizing irradiation.

  2. Sensitive detection of mercury and copper ions by fluorescent DNA/Ag nanoclusters in guanine-rich DNA hybridization.

    Science.gov (United States)

    Peng, Jun; Ling, Jian; Zhang, Xiu-Qing; Bai, Hui-Ping; Zheng, Liyan; Cao, Qiu-E; Ding, Zhong-Tao

    2015-02-25

    In this work, we designed a new fluorescent oligonucleotides-stabilized silver nanoclusters (DNA/AgNCs) probe for sensitive detection of mercury and copper ions. This probe contains two tailored DNA sequence. One is a signal probe contains a cytosine-rich sequence template for AgNCs synthesis and link sequence at both ends. The other is a guanine-rich sequence for signal enhancement and link sequence complementary to the link sequence of the signal probe. After hybridization, the fluorescence of hybridized double-strand DNA/AgNCs is 200-fold enhanced based on the fluorescence enhancement effect of DNA/AgNCs in proximity of guanine-rich DNA sequence. The double-strand DNA/AgNCs probe is brighter and stable than that of single-strand DNA/AgNCs, and more importantly, can be used as novel fluorescent probes for detecting mercury and copper ions. Mercury and copper ions in the range of 6.0-160.0 and 6-240 nM, can be linearly detected with the detection limits of 2.1 and 3.4 nM, respectively. Our results indicated that the analytical parameters of the method for mercury and copper ions detection are much better than which using a single-strand DNA/AgNCs. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. HIV1 V3 loop hypermutability is enhanced by the guanine usage bias in the part of env gene coding for it.

    Science.gov (United States)

    Khrustalev, Vladislav Victorovich

    2009-01-01

    Guanine is the most mutable nucleotide in HIV genes because of frequently occurring G to A transitions, which are caused by cytosine deamination in viral DNA minus strands catalyzed by APOBEC enzymes. Distribution of guanine between three codon positions should influence the probability for G to A mutation to be nonsynonymous (to occur in first or second codon position). We discovered that nucleotide sequences of env genes coding for third variable regions (V3 loops) of gp120 from HIV1 and HIV2 have different kinds of guanine usage biases. In the HIV1 reference strain and 100 additionally analyzed HIV1 strains the guanine usage bias in V3 loop coding regions (2G>1G>3G) should lead to elevated nonsynonymous G to A transitions occurrence rates. In the HIV2 reference strain and 100 other HIV2 strains guanine usage bias in V3 loop coding regions (3G>2G>1G) should protect V3 loops from hypermutability. According to the HIV1 and HIV2 V3 alignment, insertion of the sequence enriched with 2G (21 codons in length) occurred during the evolution of HIV1 predecessor, while insertion of the different sequence enriched with 3G (19 codons in length) occurred during the evolution of HIV2 predecessor. The higher is the level of 3G in the V3 coding region, the lower should be the immune escaping mutation occurrence rates. This hypothesis was tested in this study by comparing the guanine usage in V3 loop coding regions from HIV1 fast and slow progressors. All calculations have been performed by our algorithms "VVK In length", "VVK Dinucleotides" and "VVK Consensus" (www.barkovsky.hotmail.ru).

  4. Fingerprints of Both Watson-Crick and Hoogsteen Isomers of the Isolated (Cytosine-Guanine)H+ Pair.

    Science.gov (United States)

    Cruz-Ortiz, Andrés F; Rossa, Maximiliano; Berthias, Francis; Berdakin, Matías; Maitre, Philippe; Pino, Gustavo A

    2017-11-16

     Gas phase protonated guanine-cytosine (CGH + ) pair was generated using an electrospray ionization source from solutions at two different pH (5.8 and 3.2). Consistent evidence from MS/MS fragmentation patterns and differential ion mobility spectra (DIMS) point toward the presence of two isomers of the CGH + pair, whose relative populations depend strongly on the pH of the solution. Gas phase infrared multiphoton dissociation (IRMPD) spectroscopy in the 900-1900 cm -1 spectral range further confirms that the Watson-Crick isomer is preferentially produced (91%) at pH = 5.8, while the Hoogsteen isomer predominates (66%) at pH = 3.2). These fingerprint signatures are expected to be useful for the development of new analytical methodologies and to trigger isomer selective photochemical studies of protonated DNA base pairs.

  5. Differential Rac1 signalling by guanine nucleotide exchange factors implicates FLII in regulating Rac1-driven cell migration

    Science.gov (United States)

    Marei, Hadir; Carpy, Alejandro; Woroniuk, Anna; Vennin, Claire; White, Gavin; Timpson, Paul; Macek, Boris; Malliri, Angeliki

    2016-01-01

    The small GTPase Rac1 has been implicated in the formation and dissemination of tumours. Upon activation by guanine nucleotide exchange factors (GEFs), Rac1 associates with a variety of proteins in the cell thereby regulating various functions, including cell migration. However, activation of Rac1 can lead to opposing migratory phenotypes raising the possibility of exacerbating tumour progression when targeting Rac1 in a clinical setting. This calls for the identification of factors that influence Rac1-driven cell motility. Here we show that Tiam1 and P-Rex1, two Rac GEFs, promote Rac1 anti- and pro-migratory signalling cascades, respectively, through regulating the Rac1 interactome. In particular, we demonstrate that P-Rex1 stimulates migration through enhancing the interaction between Rac1 and the actin-remodelling protein flightless-1 homologue, to modulate cell contraction in a RhoA-ROCK-independent manner. PMID:26887924

  6. Spectroscopic (UV/VIS, Raman) and Electrophoresis Study of Cytosine-Guanine Oligonucleotide DNA Influenced by Magnetic Field.

    Science.gov (United States)

    Banihashemian, Seyedeh Maryam; Periasamy, Vengadesh; Boon Tong, Goh; Abdul Rahman, Saadah

    2016-01-01

    Studying the effect of a magnetic field on oligonucleotide DNA can provide a novel DNA manipulation technique for potential application in bioengineering and medicine. In this work, the optical and electrochemical response of a 100 bases oligonucleotides DNA, cytosine-guanine (CG100), is investigated via exposure to different magnetic fields (250, 500, 750, and 1000 mT). As a result of the optical response of CG100 to the magnetic field, the ultra-violet-visible spectrum indicated a slight variation in the band gap of CG100 of about 0.3 eV. Raman spectroscopy showed a significant deviation in hydrogen and phosphate bonds' vibration after exposure to the magnetic field. Oligonucleotide DNA mobility was investigated in the external electric field using the gel electrophoresis technique, which revealed a small decrease in the migration of CG100 after exposure to the magnetic field.

  7. Spectroscopic (UV/VIS, Raman and Electrophoresis Study of Cytosine-Guanine Oligonucleotide DNA Influenced by Magnetic Field.

    Directory of Open Access Journals (Sweden)

    Seyedeh Maryam Banihashemian

    Full Text Available Studying the effect of a magnetic field on oligonucleotide DNA can provide a novel DNA manipulation technique for potential application in bioengineering and medicine. In this work, the optical and electrochemical response of a 100 bases oligonucleotides DNA, cytosine-guanine (CG100, is investigated via exposure to different magnetic fields (250, 500, 750, and 1000 mT. As a result of the optical response of CG100 to the magnetic field, the ultra-violet-visible spectrum indicated a slight variation in the band gap of CG100 of about 0.3 eV. Raman spectroscopy showed a significant deviation in hydrogen and phosphate bonds' vibration after exposure to the magnetic field. Oligonucleotide DNA mobility was investigated in the external electric field using the gel electrophoresis technique, which revealed a small decrease in the migration of CG100 after exposure to the magnetic field.

  8. Electron transfer from nucleobase electron adducts to 5-bromouracil. Is guanine an ultimate sink for the electron in irradiated DNA?

    International Nuclear Information System (INIS)

    Nese, C.; Yuan, Z.; Schuchmann, M.N.; Sonntag, C. von

    1992-01-01

    Electron transfer to 5-bromouracil (5-BrU) from nucleobase (N) electron adducts (and their protonated forms) has been studied by product analysis and pulse radiolysis. When an electron is transferred to 5-BrU, the ensuing 5-BrU radical anion rapidly loses a bromide ion; the uracilyl radical thus formed reacts with added t-butanol, yielding uracil. From the uracil yields measured as the function of [N]/[5-BrU] after γ-radiolysis of Ar-saturated solutions it is concluded that thymine and adenine electron adducts and their heteroatom-protonated forms transfer electrons quantitatively to 5-BrU. The data raise the question whether in DNA the guanine moiety may act as the ultimate sink of the electron in competition with other processes such as protonation at C(6) of the thymine electron adduct. (Author)

  9. The PDZ domain of the guanine nucleotide exchange factor PDZGEF directs binding to phosphatidic acid during brush border formation.

    Directory of Open Access Journals (Sweden)

    Sarah V Consonni

    Full Text Available PDZGEF is a guanine nucleotide exchange factor for the small G protein Rap. It was recently found that PDZGEF contributes to establishment of intestinal epithelial polarity downstream of the kinase Lkb1. By binding to phosphatidic acid enriched at the apical membrane, PDZGEF locally activates Rap2a resulting in induction of brush border formation via a pathway that includes the polarity players TNIK, Mst4 and Ezrin. Here we show that the PDZ domain of PDZGEF is essential and sufficient for targeting PDZGEF to the apical membrane of polarized intestinal epithelial cells. Inhibition of PLD and consequently production of phosphatidic acid inhibitis targeting of PDZGEF to the plasma membrane. Furthermore, localization requires specific positively charged residues within the PDZ domain. We conclude that local accumulation of PDZGEF at the apical membrane during establishment of epithelial polarity is mediated by electrostatic interactions between positively charged side chains in the PDZ domain and negatively charged phosphatidic acid.

  10. Estrogen Repression of MicroRNAs Is Associated with High Guanine Content in the Terminal Loop Sequences of Their Precursors

    Directory of Open Access Journals (Sweden)

    Amit Cohen

    2017-08-01

    Full Text Available Widespread microRNA (miRNA repression is a phenomenon observed in mammals after exposure to cigarette smoke and in many types of cancer. A comprehensive reduction in miRNA expression after treatment with the hormone estrogen has also previously been described. Here, we reveal a conserved association of miRNA downregulation after estrogen exposure in zebrafish, mouse, and human breast cancer cell line, with a high guanine content in the terminal loop sequences of their precursors, and offer a possible link between estrogen-related miRNA-adducts formation and carcinogenesis. We also show common gene expression patterns shared by breast cancer tumors and estrogen-treated zebrafish, suggesting that this organism can be used as a powerful model system for the study of human breast cancer.

  11. Protein Kinase A (PKA) Type I Interacts with P-Rex1, a Rac Guanine Nucleotide Exchange Factor

    Science.gov (United States)

    Chávez-Vargas, Lydia; Adame-García, Sendi Rafael; Cervantes-Villagrana, Rodolfo Daniel; Castillo-Kauil, Alejandro; Bruystens, Jessica G. H.; Fukuhara, Shigetomo; Taylor, Susan S.; Mochizuki, Naoki; Reyes-Cruz, Guadalupe; Vázquez-Prado, José

    2016-01-01

    Morphology of migrating cells is regulated by Rho GTPases and fine-tuned by protein interactions and phosphorylation. PKA affects cell migration potentially through spatiotemporal interactions with regulators of Rho GTPases. Here we show that the endogenous regulatory (R) subunit of type I PKA interacts with P-Rex1, a Rac guanine nucleotide exchange factor that integrates chemotactic signals. Type I PKA holoenzyme interacts with P-Rex1 PDZ domains via the CNB B domain of RIα, which when expressed by itself facilitates endothelial cell migration. P-Rex1 activation localizes PKA to the cell periphery, whereas stimulation of PKA phosphorylates P-Rex1 and prevents its activation in cells responding to SDF-1 (stromal cell-derived factor 1). The P-Rex1 DEP1 domain is phosphorylated at Ser-436, which inhibits the DH-PH catalytic cassette by direct interaction. In addition, the P-Rex1 C terminus is indirectly targeted by PKA, promoting inhibitory interactions independently of the DEP1-PDZ2 region. A P-Rex1 S436A mutant construct shows increased RacGEF activity and prevents the inhibitory effect of forskolin on sphingosine 1-phosphate-dependent endothelial cell migration. Altogether, these results support the idea that P-Rex1 contributes to the spatiotemporal localization of type I PKA, which tightly regulates this guanine exchange factor by a multistep mechanism, initiated by interaction with the PDZ domains of P-Rex1 followed by direct phosphorylation at the first DEP domain and putatively indirect regulation of the C terminus, thus promoting inhibitory intramolecular interactions. This reciprocal regulation between PKA and P-Rex1 might represent a key node of integration by which chemotactic signaling is fine-tuned by PKA. PMID:26797121

  12. Magnetically-assembled micro/mesopixels exhibiting light intensity enhancement in the (012) planes of fish guanine crystals

    Science.gov (United States)

    Chikashige, T.; Iwasaka, M.

    2018-05-01

    In this study, a new method was investigated to form light-reflecting dots at the micrometer scale using the magnetic orientations of biogenic guanine crystals obtained from fish skin and scales. The crystal platelets, possessing average dimensions of 5 μm×20 μm×100 nm, were dispersed in water and observed during exposure to vertical magnetic fields up to 5 T. The magnetic field direction was parallel to Earth's gravity, and allowed the narrowest edges of the crystals to be observed at the micrometer scale for the first time. The magnetic orientation process was initiated under conditions where the crystal platelets in water were laid on a glass substrate or where the platelets had random orientations. In the former case, the crystal platelets followed a two-stage magnetic orientation process where, in the first step, the platelet widths were aligned in the magnetic field direction. The second step required rotation of the ˜20-μm-long plates with respect to the Earth's gravity, where application of a 5 T magnetic field enabled their orientation. Real-time images of the magnetically aligning platelets provided new evidence that the crystal platelets also emitted reflected light from a very narrow window at two crystal planes (i.e., (0 1 ¯ 2 ¯ ) and (0 1 ¯ 2 )). In the latter case with random platelet orientation, spatially-condensed light-reflecting dots appeared while the guanine crystal platelets were floating and maintaining their orientation. The technique developed for controlling light-reflecting microscale objects in an aqueous medium can be applied to produce a type of microfluidic optical tool.

  13. Magnetically-assembled micro/mesopixels exhibiting light intensity enhancement in the (012 planes of fish guanine crystals

    Directory of Open Access Journals (Sweden)

    T. Chikashige

    2018-05-01

    Full Text Available In this study, a new method was investigated to form light-reflecting dots at the micrometer scale using the magnetic orientations of biogenic guanine crystals obtained from fish skin and scales. The crystal platelets, possessing average dimensions of 5 μm×20 μm×100 nm, were dispersed in water and observed during exposure to vertical magnetic fields up to 5 T. The magnetic field direction was parallel to Earth’s gravity, and allowed the narrowest edges of the crystals to be observed at the micrometer scale for the first time. The magnetic orientation process was initiated under conditions where the crystal platelets in water were laid on a glass substrate or where the platelets had random orientations. In the former case, the crystal platelets followed a two-stage magnetic orientation process where, in the first step, the platelet widths were aligned in the magnetic field direction. The second step required rotation of the ∼20-μm-long plates with respect to the Earth’s gravity, where application of a 5 T magnetic field enabled their orientation. Real-time images of the magnetically aligning platelets provided new evidence that the crystal platelets also emitted reflected light from a very narrow window at two crystal planes (i.e., (01¯2¯ and (01¯2. In the latter case with random platelet orientation, spatially-condensed light-reflecting dots appeared while the guanine crystal platelets were floating and maintaining their orientation. The technique developed for controlling light-reflecting microscale objects in an aqueous medium can be applied to produce a type of microfluidic optical tool.

  14. Stable isotope labeling-mass spectrometry analysis of methyl- and pyridyloxobutyl-guanine adducts of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in p53-derived DNA sequences.

    Science.gov (United States)

    Rajesh, Mathur; Wang, Gang; Jones, Roger; Tretyakova, Natalia

    2005-02-15

    The p53 tumor suppressor gene is a primary target in smoking-induced lung cancer. Interestingly, p53 mutations observed in lung tumors of smokers are concentrated at guanine bases within endogenously methylated (Me)CG dinucleotides, e.g., codons 157, 158, 245, 248, and 273 ((Me)C = 5-methylcytosine). One possible mechanism for the increased mutagenesis at these sites involves targeted binding of metabolically activated tobacco carcinogens to (Me)CG sequences. In the present work, a stable isotope labeling HPLC-ESI(+)-MS/MS approach was employed to analyze the formation of guanine lesions induced by the tobacco-specific lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) within DNA duplexes representing p53 mutational "hot spots" and surrounding sequences. Synthetic DNA duplexes containing p53 codons 153-159, 243-250, and 269-275 were prepared, where (Me)C was incorporated at all physiologically methylated CG sites. In each duplex, one of the guanine bases was replaced with [1,7,NH(2)-(15)N(3)-2-(13)C]-guanine, which served as an isotope "tag" to enable specific quantification of guanine lesions originating from that position. After incubation with NNK diazohydroxides, HPLC-ESI(+)-MS/MS analysis was used to determine the yields of NNK adducts at the isotopically labeled guanine and at unlabeled guanine bases elsewhere in the sequence. We found that N7-methyl-2'-deoxyguanosine and N7-[4-oxo-4-(3-pyridyl)but-1-yl]guanine lesions were overproduced at the 3'-guanine bases within polypurine runs, while the formation of O(6)-methyl-2'-deoxyguanosine and O(6)-[4-oxo-4-(3-pyridyl)but-1-yl]-2'-deoxyguanosine adducts was specifically preferred at the 3'-guanine base of 5'-GG and 5'-GGG sequences. In contrast, the presence of 5'-neighboring (Me)C inhibited O(6)-guanine adduct formation. These results indicate that the N7- and O(6)-guanine adducts of NNK are not overproduced at the endogenously methylated CG dinucleotides within the p53 tumor suppressor gene

  15. Human T cell recognition of the blood stage antigen Plasmodium hypoxanthine guanine xanthine phosphoribosyl transferase (HGXPRT in acute malaria

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    Woodberry Tonia

    2009-06-01

    Full Text Available Abstract Background The Plasmodium purine salvage enzyme, hypoxanthine guanine xanthine phosphoribosyl transferase (HGXPRT can protect mice against Plasmodium yoelii pRBC challenge in a T cell-dependent manner and has, therefore, been proposed as a novel vaccine candidate. It is not known whether natural exposure to Plasmodium falciparum stimulates HGXPRT T cell reactivity in humans. Methods PBMC and plasma collected from malaria-exposed Indonesians during infection and 7–28 days after anti-malarial therapy, were assessed for HGXPRT recognition using CFSE proliferation, IFNγ ELISPOT assay and ELISA. Results HGXPRT-specific T cell proliferation was found in 44% of patients during acute infection; in 80% of responders both CD4+ and CD8+ T cell subsets proliferated. Antigen-specific T cell proliferation was largely lost within 28 days of parasite clearance. HGXPRT-specific IFN-γ production was more frequent 28 days after treatment than during acute infection. HGXPRT-specific plasma IgG was undetectable even in individuals exposed to malaria for at least two years. Conclusion The prevalence of acute proliferative and convalescent IFNγ responses to HGXPRT demonstrates cellular immunogenicity in humans. Further studies to determine minimal HGXPRT epitopes, the specificity of responses for Plasmodia and associations with protection are required. Frequent and robust T cell proliferation, high sequence conservation among Plasmodium species and absent IgG responses distinguish HGXPRT from other malaria antigens.

  16. BLOC-3 mutated in Hermansky-Pudlak syndrome is a Rab32/38 guanine nucleotide exchange factor.

    Science.gov (United States)

    Gerondopoulos, Andreas; Langemeyer, Lars; Liang, Jin-Rui; Linford, Andrea; Barr, Francis A

    2012-11-20

    Hermansky-Pudlak syndrome (HPS) is a human disease characterized by partial loss of pigmentation and impaired blood clotting. These symptoms are caused by defects in the biogenesis of melanosomes and platelet dense granules, often referred to as lysosome-related organelles. Genes mutated in HPS encode subunits of the biogenesis of lysosome-related organelles complexes (BLOCs). BLOC-1 and BLOC-2, together with the AP-3 clathrin adaptor complex, act at early endosomes to sort components required for melanin formation and melanosome biogenesis away from the degradative lysosomal pathway toward early stage melanosomes. However the molecular functions of the Hps1-Hps4 complex BLOC-3 remain mysterious. Like other trafficking pathways, melanosome biogenesis and transport of enzymes involved in pigmentation involves specific Rab GTPases, in this instance Rab32 and Rab38. We now demonstrate that BLOC-3 is a Rab32 and Rab38 guanine nucleotide exchange factor (GEF). Silencing of the BLOC-3 subunits Hps1 and Hps4 results in the mislocalization of Rab32 and Rab38 and reduction in pigmentation. In addition, we show that BLOC-3 can promote specific membrane recruitment of Rab32/38. BLOC-3 therefore defines a novel Rab GEF family with a specific function in the biogenesis of lysosome-related organelles. Copyright © 2012 Elsevier Ltd. All rights reserved.

  17. A High-Throughput Assay for Rho Guanine Nucleotide Exchange Factors Based on the Transcreener GDP Assay.

    Science.gov (United States)

    Reichman, Melvin; Schabdach, Amanda; Kumar, Meera; Zielinski, Tom; Donover, Preston S; Laury-Kleintop, Lisa D; Lowery, Robert G

    2015-12-01

    Ras homologous (Rho) family GTPases act as molecular switches controlling cell growth, movement, and gene expression by cycling between inactive guanosine diphosphate (GDP)- and active guanosine triphosphate (GTP)-bound conformations. Guanine nucleotide exchange factors (GEFs) positively regulate Rho GTPases by accelerating GDP dissociation to allow formation of the active, GTP-bound complex. Rho proteins are directly involved in cancer pathways, especially cell migration and invasion, and inhibiting GEFs holds potential as a therapeutic strategy to diminish Rho-dependent oncogenesis. Methods for measuring GEF activity suitable for high-throughput screening (HTS) are limited. We developed a simple, generic biochemical assay method for measuring GEF activity based on the fact that GDP dissociation is generally the rate-limiting step in the Rho GTPase catalytic cycle, and thus addition of a GEF causes an increase in steady-state GTPase activity. We used the Transcreener GDP Assay, which relies on selective immunodetection of GDP, to measure the GEF-dependent stimulation of steady-state GTP hydrolysis by small GTPases using Dbs (Dbl's big sister) as a GEF for Cdc42, RhoA, and RhoB. The assay is well suited for HTS, with a homogenous format and far red fluorescence polarization (FP) readout, and it should be broadly applicable to diverse Rho GEF/GTPase pairs. © 2015 Society for Laboratory Automation and Screening.

  18. RINL, guanine nucleotide exchange factor Rab5-subfamily, is involved in the EphA8-degradation pathway with odin.

    Directory of Open Access Journals (Sweden)

    Hiroaki Kajiho

    Full Text Available The Rab family of small guanosine triphosphatases (GTPases plays a vital role in membrane trafficking. Its active GTP-bound state is driven by guanine nucleotide-exchange factors (GEFs. Ras and Rab interactor (or Ras interaction/interference-like (RINL, which contains a conserved VPS9 domain critical for GEF action, was recently identified as a new Rab5 subfamily GEF in vitro. However, its detailed function and interacting molecules have not yet been fully elucidated. Here we found that RINL has GEF activity for the Rab5 subfamily proteins by measuring their GTP-bound forms in cultured cells. We also found that RINL interacts with odin, a member of the ankyrin-repeat and sterile-alpha motif (SAM domain-containing (Anks protein family. In addition, the Eph tyrosine kinase receptor EphA8 formed a ternary complex with both RINL and odin. Interestingly, RINL expression in cultured cells reduced EphA8 levels in a manner dependent on both its GEF activity and interaction with odin. In addition, knockdown of RINL increased EphA8 level in HeLa cells. Our findings suggest that RINL, as a GEF for Rab5 subfamily, is implicated in the EphA8-degradation pathway via its interaction with odin.

  19. The Cdc42 guanine nucleotide exchange factor FGD6 coordinates cell polarity and endosomal membrane recycling in osteoclasts.

    Science.gov (United States)

    Steenblock, Charlotte; Heckel, Tobias; Czupalla, Cornelia; Espírito Santo, Ana Isabel; Niehage, Christian; Sztacho, Martin; Hoflack, Bernard

    2014-06-27

    The initial step of bone digestion is the adhesion of osteoclasts onto bone surfaces and the assembly of podosomal belts that segregate the bone-facing ruffled membrane from other membrane domains. During bone digestion, membrane components of the ruffled border also need to be recycled after macropinocytosis of digested bone materials. How osteoclast polarity and membrane recycling are coordinated remains unknown. Here, we show that the Cdc42-guanine nucleotide exchange factor FGD6 coordinates these events through its Src-dependent interaction with different actin-based protein networks. At the plasma membrane, FGD6 couples cell adhesion and actin dynamics by regulating podosome formation through the assembly of complexes comprising the Cdc42-interactor IQGAP1, the Rho GTPase-activating protein ARHGAP10, and the integrin interactors Talin-1/2 or Filamin A. On endosomes and transcytotic vesicles, FGD6 regulates retromer-dependent membrane recycling through its interaction with the actin nucleation-promoting factor WASH. These results provide a mechanism by which a single Cdc42-exchange factor controlling different actin-based processes coordinates cell adhesion, cell polarity, and membrane recycling during bone degradation. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Effect of ionic strength and cationic DNA affinity binders on the DNA sequence selective alkylation of guanine N7-positions by nitrogen mustards

    International Nuclear Information System (INIS)

    Hartley, J.A.; Forrow, S.M.; Souhami, R.L.

    1990-01-01

    Large variations in alkylation intensities exist among guanines in a DNA sequence following treatment with chemotherapeutic alkylating agents such as nitrogen mustards, and the substituent attached to the reactive group can impose a distinct sequence preference for reaction. In order to understand further the structural and electrostatic factors which determine the sequence selectivity of alkylation reactions, the effect of increase ionic strength, the intercalator ethidium bromide, AT-specific minor groove binders distamycin A and netropsin, and the polyamine spermine on guanine N7-alkylation by L-phenylalanine mustard (L-Pam), uracil mustard (UM), and quinacrine mustard (QM) was investigated with a modification of the guanine-specific chemical cleavage technique for DNA sequencing. The result differed with both the nitrogen mustard and the cationic agent used. The effect, which resulted in both enhancement and suppression of alkylation sites, was most striking in the case of netropsin and distamycin A, which differed from each other. DNA footprinting indicated that selective binding to AT sequences in the minor groove of DNA can have long-range effects on the alkylation pattern of DNA in the major groove

  1. Role of a guanine nucleotide-binding protein in α1-adrenergic receptor-mediated Ca2+ mobilization in DDT1 MF-2 cells

    International Nuclear Information System (INIS)

    Cornett, L.E.; Norris, J.S.

    1987-01-01

    In this study the mechanisms involved in α 1 -adrenergic receptor-mediated Ca 2+ mobilization at the level of the plasma membrane were investigated. Stimulation of 45 Ca 2+ efflux from saponin-permeabilized DDT 1 MF-2 cells was observed with the addition of either the α 1 -adrenergic agonist phenylephrine and guanosine-5'-triphosphate or the nonhydrolyzable guanine nucleotide guanylyl-imidodiphosphate. In the presence of [ 32 P] NAD, pertussis toxin was found to catalyze ADP-ribosylation of a M/sub r/ = 40,500 (n = 8) peptide in membranes prepared from DDT 1 , MF-2 cells, possibly the α-subunit of N/sub i/. However, stimulation of unidirectional 45 Ca 2+ efflux by phenylephrine was not affected by previous treatment of cells with 100 ng/ml pertussis toxin. These data suggest that the putative guanine nucleotide-binding protein which couples the α 1 -adrenergic receptor to Ca 2+ mobilization in DDT 1 MF-2 cells is not a pertussis toxin substrate and may possibly be an additional member of guanine nucleotide binding protein family

  2. Oxidative generation of guanine radicals by carbonate radicals and their reactions with nitrogen dioxide to form site specific 5-guanidino-4-nitroimidazole lesions in oligodeoxynucleotides.

    Science.gov (United States)

    Joffe, Avrum; Mock, Steven; Yun, Byeong Hwa; Kolbanovskiy, Alexander; Geacintov, Nicholas E; Shafirovich, Vladimir

    2003-08-01

    A simple photochemical approach is described for synthesizing site specific, stable 5-guanidino-4-nitroimidazole (NIm) adducts in single- and double-stranded oligodeoxynucleotides containing single and multiple guanine residues. The DNA sequences employed, 5'-d(ACC CG(1)C G(2)TC CG(3)C G(4)CC) and 5'-d(ACC CG(1)C G(2)TC C), were a portion of exon 5 of the p53 tumor suppressor gene, including the codons 157 (G(2)) and 158 (G(3)) mutation hot spots in the former sequence with four Gs and the codon 157 (G(2)) mutation hot spot in the latter sequence with two Gs. The nitration of oligodeoxynucleotides was initiated by the selective photodissociation of persulfate anions to sulfate radicals induced by UV laser pulses (308 nm). In aqueous solutions, of bicarbonate and nitrite anions, the sulfate radicals generate carbonate anion radicals and nitrogen dioxide radicals by one electron oxidation of the respective anions. The guanine residue in the oligodeoxynucleotide is oxidized by the carbonate anion radical to form the neutral guanine radical. While the nitrogen dioxide radicals do not react with any of the intact DNA bases, they readily combine with the guanine radicals at either the C8 or the C5 positions. The C8 addition generates the well-known 8-nitroguanine (8-nitro-G) lesions, whereas the C5 attack produces unstable adducts, which rapidly decompose to NIm lesions. The maximum yields of the nitro products (NIm + 8-nitro-G) were typically in the range of 20-40%, depending on the number of guanine residues in the sequence. The ratio of the NIm to 8-nitro-G lesions gradually decreases from 3.4 in the model compound, 2',3',5'-tri-O-acetylguanosine, to 2.1-2.6 in the single-stranded oligodeoxynucleotides and to 0.8-1.1 in the duplexes. The adduct of the 5'-d(ACC CG(1)C G(2)TC C) oligodeoxynucleotide containing the NIm lesion in codon 157 (G(2)) was isolated in HPLC-pure form. The integrity of this adduct was established by a detailed analysis of exonuclease digestion

  3. Formation of diastereomeric benzo[a]pyrene diol epoxide-guanine adducts in p53 gene-derived DNA sequences.

    Science.gov (United States)

    Matter, Brock; Wang, Gang; Jones, Roger; Tretyakova, Natalia

    2004-06-01

    G --> T transversion mutations in the p53 tumor suppressor gene are characteristic of smoking-related lung tumors, suggesting that these genetic changes may result from exposure to tobacco carcinogens. It has been previously demonstrated that the diol epoxide metabolites of bay region polycyclic aromatic hydrocarbons present in tobacco smoke, e.g., benzo[a]pyrene diol epoxide (BPDE), preferentially bind to the most frequently mutated guanine nucleotides within p53 codons 157, 158, 248, and 273 [Denissenko, M. F., Pao, A., Tang, M., and Pfeifer, G. P. (1996) Science 274, 430-432]. However, the methodology used in that work (ligation-mediated polymerase chain reaction in combination with the UvrABC endonuclease incision assay) cannot establish the chemical structures and stereochemical identities of BPDE-guanine lesions. In the present study, we employ a stable isotope-labeling HPLC-MS/MS approach [Tretyakova, N., Matter, B., Jones, R., and Shallop, A. (2002) Biochemistry 41, 9535-9544] to analyze the formation of diastereomeric N(2)-BPDE-dG lesions within double-stranded oligodeoxynucleotides representing p53 lung cancer mutational hotspots and their surrounding DNA sequences. (15)N-labeled dG was placed at defined positions within DNA duplexes containing 5-methylcytosine at all physiologically methylated sites, followed by (+/-)-anti-BPDE treatment and enzymatic hydrolysis of the adducted DNA to 2'-deoxynucleosides. Capillary HPLC-ESI(+)-MS/MS was used to establish the amounts of (-)-trans-N(2)-BPDE-dG, (+)-cis-N(2)-BPDE-dG, (-)-cis-N(2)-BPDE-dG, and (+)-trans-N(2)-BPDE-dG originating from the (15)N-labeled bases. We found that all four N(2)-BPDE-dG diastereomers were formed preferentially at the methylated CG dinucleotides, including the frequently mutated p53 codons 157, 158, 245, 248, and 273. The contributions of individual diastereomers to the total adducts number at a given site varied between 70.8 and 92.9% for (+)-trans-N(2)-BPDE-dG, 5.6 and 16.7% for

  4. Modulation of B-cell receptor and microenvironment signaling by a guanine exchange factor in B-cell malignancies

    International Nuclear Information System (INIS)

    Liao, Wei; Sharma, Sanjai

    2016-01-01

    Objective: Chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) cells over-express a guanine exchange factor (GEF), Rasgrf-1. This GEF increases active Ras as it catalyzes the removal of GDP from Ras so that GTP can bind and activate Ras. This study aims to study the mechanism of action of Rasgrf-1 in B-cell malignancies. Methods: N-terminus truncated Rasgrf-1 variants have a higher GEF activity as compared to the full-length transcript therefore a MCL cell line with stable over-expression of truncated Rasgrf-1 was established. The B-cell receptor (BCR) and chemokine signaling pathways were compared in the Rasgrf-1 over-expressing and a control transfected cell line. Results: Cells over-expressing truncated form of Rasgrf-1 have a higher proliferative rate as compared to control transfected cells. BCR was activated by lower concentrations of anti-IgM antibody in Rasgrf-1 over-expressing cells as compared to control cells indicating that these cells are more sensitive to BCR signaling. BCR signaling also phosphorylates Rasgrf-1 that further increases its GEF function and amplifies BCR signaling. This activation of Rasgrf-1 in over-expressing cells resulted in a higher expression of phospho-ERK, AKT, BTK and PKC-alpha as compared to control cells. Besides BCR, Rasgrf-1 over-expressing cells were also more sensitive to microenvironment stimuli as determined by resistance to apoptosis, chemotaxis and ERK pathway activation. Conclusions: This GEF protein sensitizes B-cells to BCR and chemokine mediated signaling and also upregulates a number of other signaling pathways which promotes growth and survival of these cells

  5. Localization of the 5-phospho-alpha-D-ribosyl-1-pyrophosphate binding site of human hypoxanthine-guanine phosphoribosyltransferase.

    Science.gov (United States)

    Keough, D T; Emmerson, B T; de Jersey, J

    1991-02-22

    Human erythrocyte hypoxanthine-guanine phosphoribosyltransferase (HPRT) is inactivated by iodoacetate in the absence, but not in the presence, of the substrate, 5-phospho-alpha-D-ribosyl-1-pyrophosphate (PRib-PP). Treatment of HPRT with [14C]iodoacetate followed by tryptic digestion, peptide separation and sequencing has shown that Cys-22 reacts with iodoacetate only in the absence of PRib-PP; this strongly suggests that Cys-22 is in or near the PRib-PP binding site. In contrast, Cys-105 reacts with [14C]iodoacetate both in the presence and absence of PRib-PP. Carboxymethylation of Cys-22 resulted in an increase in the Km for PRib-PP, but no change in Vmax. Storage of HPRT also resulted in an increase in the Km for PRib-PP and a decrease in its susceptibility to inactivation by iodoacetate. Dialysis of stored enzyme against 1 mM dithiothreitol resulted in a marked decrease in Km for PRib-PP. The stoichiometry of the reaction of [14C]iodoacetate with Cys-22 in HPRT leading to inactivation (approx. 1 residue modified per tetramer) showed that, in this preparation of HPRT purified from erythrocytes, only about 25% of the Cys-22 side chains were present as free and accessible thiols. Titration of thiol groups [with 5,5'-dithiobis(2-nitrobenzoic acid)] and the effect of dithiothreitol on Km for PRib-PP indicate that oxidation of thiol groups occurs on storage of HPRT, even in the presence of 1 mM beta-mercaptoethanol.

  6. The domain architecture of large guanine nucleotide exchange factors for the small GTP-binding protein Arf

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    Geldner Niko

    2005-02-01

    Full Text Available Abstract Background Small G proteins, which are essential regulators of multiple cellular functions, are activated by guanine nucleotide exchange factors (GEFs that stimulate the exchange of the tightly bound GDP nucleotide by GTP. The catalytic domain responsible for nucleotide exchange is in general associated with non-catalytic domains that define the spatio-temporal conditions of activation. In the case of small G proteins of the Arf subfamily, which are major regulators of membrane trafficking, GEFs form a heterogeneous family whose only common characteristic is the well-characterized Sec7 catalytic domain. In contrast, the function of non-catalytic domains and how they regulate/cooperate with the catalytic domain is essentially unknown. Results Based on Sec7-containing sequences from fully-annotated eukaryotic genomes, including our annotation of these sequences from Paramecium, we have investigated the domain architecture of large ArfGEFs of the BIG and GBF subfamilies, which are involved in Golgi traffic. Multiple sequence alignments combined with the analysis of predicted secondary structures, non-structured regions and splicing patterns, identifies five novel non-catalytic structural domains which are common to both subfamilies, revealing that they share a conserved modular organization. We also report a novel ArfGEF subfamily with a domain organization so far unique to alveolates, which we name TBS (TBC-Sec7. Conclusion Our analysis unifies the BIG and GBF subfamilies into a higher order subfamily, which, together with their being the only subfamilies common to all eukaryotes, suggests that they descend from a common ancestor from which species-specific ArfGEFs have subsequently evolved. Our identification of a conserved modular architecture provides a background for future functional investigation of non-catalytic domains.

  7. Guanine nucleotide regulation of muscarinic receptor-mediated inositol phosphate formation in permeabilized 1321N1 cells

    International Nuclear Information System (INIS)

    Orellana, S.A.; Trilivas, I.; Brown, J.H.

    1986-01-01

    Carbachol and guanine nucleotides stimulate formation of the ( 3 H)inositol phosphates IP, IP2, and IP3 in saponin-permeabilized monolayers labelled with ( 3 H) inositol. Carbachol alone has little effect on formation of the ( 3 H) inositol phosphates (IPs), but GTPγS causes synergistic accumulation of ( 3 H)IPs to levels similar to those seen in intact cells. GTP, GppNHp, and GTPγS all support formation of the ( 3 H)IPs, with or without hormone, but GTPγS is the most effective. In the presence of GTPγS, the effect of carbachol is dose-dependent. Half-maximal and maximal accumulation of the ( 3 H)IPs occur at ∼ 5 μM and ∼ 100 μM carbachol, respectively; values close to those seen in intact cells. GTPγS alone stimulates formation of the ( 3 H)IPs after a brief lag time. The combination of GTPγS and carbachol both increases the rate of, and decreases the lag in, formation of the ( 3 H)IPs. LiCl increases ( 3 H)IP and IP2, but not IP3, accumulation; while 2,3-diphosphoglycerate substantially increases that of ( 3 H)IP3. GTPγS and carbachol cause formation of ( 3 H)IPs in the absence of Ca ++ , but formation induced by GTPγS with or without carbachol is Ca ++ -sensitive over a range of physiological concentrations. Although carbachol, Ca ++ , and GTPγS all have effects on formation of ( 3 H)IPs, GTPγS appears to be a primary and obligatory regulator of phosphoinositide hydrolysis in the permeabilized 1321N1 astrocytoma cell

  8. Overexpression of GEFT, a Rho family guanine nucleotide exchange factor, predicts poor prognosis in patients with rhabdomyosarcoma.

    Science.gov (United States)

    Sun, Chao; Liu, Chunxia; Li, Shugang; Li, Hongan; Wang, Yuanyuan; Xie, Yuwen; Li, Bingcheng; Cui, Xiaobin; Chen, Yunzhao; Zhang, Wenjie; Li, Feng

    2014-01-01

    Rhabdomyosarcoma (RMS) is one of the most common soft-tissue sarcomas in children and adolescents with poor prognosis. Yet, there is lack of effective prognostic biomarkers for RMS. The present study, therefore, aimed to explore potential biomarkers for RMS based on our previous findings using array comparative genomic hybridization. We investigated guanine nucleotide exchange factor, GEFT, at expression level in 45 RMS patients and 36 normal striated muscle controls using immunohistochemistry using tissue microarrays. The expression rate of GEFT in RMS samples (42/45, 93.33%) was significantly higher (Prate of GEFT in RMS (31/45, 68.89%) was also significantly higher (P<0.05) than that in normal controls (0/36, 0.00%). Increased expression of GEFT correlated significantly with advanced disease stages (stages III/IV) (P=0.001), lymph node metastasis (P=0.019), and distant metastasis (P=0.004), respectively, in RMS patients. In addition, RMS patients having overexpressed GEFT experienced worse overall survival (OS) than those having low levels of GEFT (P=0.001). GEFT overexpression was determined to be an independent prognostic factor for poor OS in RMS patients (hazard ratio: 3.491, 95% confidence interval: 1.121-10.871, P=0.004). In conclusion, these observations provide the first evidence of GEFT overexpression in RMS and its correlations with disease aggressiveness and metastasis. These findings suggest that GEFT may serve as a promising biomarker predicting poor prognosis in RMS patients, thus implying its potential as a therapeutic target.

  9. Mutation analysis of inhibitory guanine nucleotide binding protein alpha (GNAI) loci in young and familial pituitary adenomas.

    Science.gov (United States)

    Demir, Hande; Donner, Iikki; Kivipelto, Leena; Kuismin, Outi; Schalin-Jäntti, Camilla; De Menis, Ernesto; Karhu, Auli

    2014-01-01

    Pituitary adenomas are neoplasms of the anterior pituitary lobe and account for 15-20% of all intracranial tumors. Although most pituitary tumors are benign they can cause severe symptoms related to tumor size as well as hypopituitarism and/or hypersecretion of one or more pituitary hormones. Most pituitary adenomas are sporadic, but it has been estimated that 5% of patients have a familial background. Germline mutations of the tumor suppressor gene aryl hydrocarbon receptor-interacting protein (AIP) predispose to hereditary pituitary neoplasia. Recently, it has been demonstrated that AIP mutations predispose to pituitary tumorigenesis through defective inhibitory GTP binding protein (Gαi) signaling. This finding prompted us to examine whether germline loss-of-function mutations in inhibitory guanine nucleotide (GTP) binding protein alpha (GNAI) loci are involved in genetic predisposition of pituitary tumors. To our knowledge, this is the first time GNAI genes are sequenced in order to examine the occurrence of inactivating germline mutations. Thus far, only somatic gain-of-function hot-spot mutations have been studied in these loci. Here, we have analyzed the coding regions of GNAI1, GNAI2, and GNAI3 in a set of young sporadic somatotropinoma patients (n = 32; mean age of diagnosis 32 years) and familial index cases (n = 14), thus in patients with a disease phenotype similar to that observed in AIP mutation carriers. In addition, expression of Gαi proteins was studied in human growth hormone (GH), prolactin (PRL), adrenocorticotropic hormone (ACTH)-secreting and non-functional pituitary tumors. No pathogenic germline mutations affecting the Gαi proteins were detected. The result suggests that loss-of-function mutations of GNAI loci are rare or nonexistent in familial pituitary adenomas.

  10. Increased PRPP synthetase activity in cultured rat hepatoma cells containing mutations in the hypoxanthine-guanine phosphoribosyltransferase gene.

    Science.gov (United States)

    Graf, L H; McRoberts, J A; Harrison, T M; Martin, D W

    1976-07-01

    Nine independently derived clones of mutagenized rat hepatoma cells selected for resistance to 6-mercaptopurine (6-MP) or 6-thioguanine (6-ThioG) have been isolated. Each has severely reduced catalytic activity of hypoxanthine-guanine phosphoribosyltransferase (HPRT) and seven of them possess significantly increased activities of phosphoribosylpyrophosphate (PRPP) synthetase. The degrees of elevations of PRPP synthetase activities do not correlate with the degrees of deficiencies of HPRT activities. The cells from one of these clones, 1020/12, posses 40% of the normal HPRT catalytic activity and overproduce purines. We have extensively examined the cells from this clone. Immunotration studies of 1020/12 cells indicate that there is a mutation in the structural gene for HPRT. Although they possess increased specific catalytic activities of the enzyme. PRPP synthetase, the catalytic parameters, heat stability, and isoelectric pH of PRPP synthetase from 1020/12 cells are indistinguishable from those of the enzyme from wild-type cells. The cause of purine overproduction by 1020/12 cells appears to be the elevated PRPP synthetase activity, rather than a PRPP "sparing" effect stemming from reduced HPRT activity. Support for this idea is provided by the observation that the complete loss of HPRT activity in a clone derived from 1020/12 cells does not further enhance the levels of PRPP synthetase or purine overproduction. We propose that the elevated levels of PRPP synthetase activity in these HPRT deficient cells result from a mutational event in the structural gene for HPRT, and that this causes the disruption of a previously undescribed regulatory function of this gene on the expression of the PRPP synthetase gene.

  11. Guanine nucleotide-binding protein subunit beta-2-like 1, a new Annexin A7 interacting protein

    International Nuclear Information System (INIS)

    Du, Yue; Meng, Jinyi; Huang, Yuhong; Wu, Jun; Wang, Bo; Ibrahim, Mohammed M.; Tang, Jianwu

    2014-01-01

    Highlights: • RACK1 formed a complex with Annexin A7. • Depletion of RACK1 inhibited the proliferation, migration and invasion. • RACK1 RNAi abolished RACK1-Annexin A7 interaction. • RACK1-Annexin A7 may play a role in regulating the metastatic potentials. - Abstract: We report for the first time that Guanine nucleotide-binding protein subunit beta-2-like 1 (RACK1) formed a complex with Annexin A7. Hca-F and Hca-P are a pair of syngeneic mouse hepatocarcinoma cell lines established and maintained in our laboratory. Our previous study showed that both Annexin A7 and RACK1 were expressed higher in Hca-F (lymph node metastasis >70%) than Hca-P (lymph node metastasis <30%). Suppression of Annexin A7 expression in Hca-F cells induced decreased migration and invasion ability. In this study, knockdown of RACK1 by RNA interference (RNAi) had the same impact on metastasis potential of Hca-F cells as Annexin A7 down-regulation. Furthermore, by co-immunoprecipitation and double immunofluorescence confocal imaging, we found that RACK1 was in complex with Annexin A7 in control cells, but not in the RACK1-down-regulated cells, indicating the abolishment of RACK1-Annexin A7 interaction in Hca-F cells by RACK1 RNAi. Taken together, these results suggest that RACK1-Annexin A7 interaction may be one of the means by which RACK1 and Annexin A7 influence the metastasis potential of mouse hepatocarcinoma cells in vitro

  12. Regulation of formyl peptide receptor binding to rabbit neutrophil plasma membranes. Use of monovalent cations, guanine nucleotides, and bacterial toxins to discriminate among different states of the receptor

    International Nuclear Information System (INIS)

    Feltner, D.E.; Marasco, W.A.

    1989-01-01

    The regulation by monovalent cations, guanine nucleotides, and bacterial toxins of [3H]FMLP binding to rabbit neutrophil plasma membranes was studied by using dissociation techniques to identify regulatory effects on separate receptor states. Under conditions of low receptor occupancy (1 nM [3H]FMLP) and in both Na+ and K+ buffers, dissociation is heterogenous, displaying two distinct, statistically significant off rates. [3H]FMLP binding was enhanced by substituting other monovalent cations for Na+. In particular, enhanced binding in the presence of K+ relative to Na+ was caused by additional binding to both rapidly and slowly dissociating receptors. Three receptor dissociation rates, two of which appear to correspond to the two affinity states detected in equilibrium binding studies, were defined by specific GTP and pertussis toxin (PT) treatments. Neither GTP, nor PT or cholera toxins (CT) had an effect on the rate of dissociation of [3H]FMLP from the rapidly dissociating form of the receptor. Both 100 microM GTP and PT treatments increased the percentage of rapidly dissociating receptors, correspondingly decreasing the percentage of slowly dissociating receptors. The observed changes in the rapidly and slowly dissociating receptors after GTP, PT, and CT treatments were caused by an absolute decrease in the amount of binding to the slowly dissociating receptors. However, complete inhibition of slowly dissociating receptor binding by GTP, PT, or both was never observed. Both GTP and PT treatments, but not CT treatment, increased by two-fold the rate of dissociation of 1 nM [3H]FMLP from the slowly dissociating form of the receptor, resulting in a third dissociation rate. Thus, slowly dissociating receptors comprise two different receptor states, a G protein-associated guanine nucleotide and PT-sensitive state and a guanine nucleotide-insensitive state

  13. Investigation of specificity determinants in bacterial tRNA-guanine transglycosylase reveals queuine, the substrate of its eucaryotic counterpart, as inhibitor.

    Directory of Open Access Journals (Sweden)

    Inna Biela

    Full Text Available Bacterial tRNA-guanine transglycosylase (Tgt catalyses the exchange of the genetically encoded guanine at the wobble position of tRNAs(His,Tyr,Asp,Asn by the premodified base preQ1, which is further converted to queuine at the tRNA level. As eucaryotes are not able to synthesise queuine de novo but acquire it through their diet, eucaryotic Tgt directly inserts the hypermodified base into the wobble position of the tRNAs mentioned above. Bacterial Tgt is required for the efficient pathogenicity of Shigella sp, the causative agent of bacillary dysentery and, hence, it constitutes a putative target for the rational design of anti-Shigellosis compounds. Since mammalian Tgt is known to be indirectly essential to the conversion of phenylalanine to tyrosine, it is necessary to create substances which only inhibit bacterial but not eucaryotic Tgt. Therefore, it seems of utmost importance to study selectivity-determining features within both types of proteins. Homology models of Caenorhabditis elegans Tgt and human Tgt suggest that the replacement of Cys158 and Val233 in bacterial Tgt (Zymomonas mobilis Tgt numbering by valine and accordingly glycine in eucaryotic Tgt largely accounts for the different substrate specificities. In the present study we have created mutated variants of Z. mobilis Tgt in order to investigate the impact of a Cys158Val and a Val233Gly exchange on catalytic activity and substrate specificity. Using enzyme kinetics and X-ray crystallography, we gained evidence that the Cys158Val mutation reduces the affinity to preQ1 while leaving the affinity to guanine unaffected. The Val233Gly exchange leads to an enlarged substrate binding pocket, that is necessary to accommodate queuine in a conformation compatible with the intermediately covalently bound tRNA molecule. Contrary to our expectations, we found that a priori queuine is recognised by the binding pocket of bacterial Tgt without, however, being used as a substrate.

  14. The effect of S-substitution at the O6-guanine site on the structure and dynamics of a DNA oligomer containing a G:T mismatch.

    Directory of Open Access Journals (Sweden)

    Elaine Ann Moore

    Full Text Available The effect of S-substitution on the O6 guanine site of a 13-mer DNA duplex containing a G:T mismatch is studied using molecular dynamics. The structure, dynamic evolution and hydration of the S-substituted duplex are compared with those of a normal duplex, a duplex with S-substitution on guanine, but no mismatch and a duplex with just a G:T mismatch. The S-substituted mismatch leads to cell death rather than repair. One suggestion is that the G:T mismatch recognition protein recognises the S-substituted mismatch (GS:T as G:T. This leads to a cycle of futile repair ending in DNA breakage and cell death. We find that some structural features of the helix are similar for the duplex with the G:T mismatch and that with the S-substituted mismatch, but differ from the normal duplex, notably the helical twist. These differences arise from the change in the hydrogen-bonding pattern of the base pair. However a marked feature of the S-substituted G:T mismatch duplex is a very large opening. This showed considerable variability. It is suggested that this enlarged opening would lend support to an alternative model of cell death in which the mismatch protein attaches to thioguanine and activates downstream damage-response pathways. Attack on the sulphur by reactive oxygen species, also leading to cell death, would also be aided by the large, variable opening.

  15. Decrease in Survival Rate of Colorectal Cancer Patients Due to Insertion of a Single Guanine Base in Promoter Sequences of Matrix Metalloproteinase-1 Gene (in Tehran Population

    Directory of Open Access Journals (Sweden)

    Z Hojati

    2009-01-01

    Full Text Available Introduction: Insertion or deletion of a guanine in -1607 at promoter region of matrix metalloproteinase-1 enzyme creates two allelic types for this gene in the population: 2G and 1G, respectively. 2G allele contains an extra binding site for ETS transcription factors that this may increase the level of gene expression. Therefore, aim of this study was investigation of the single Guanine insertion in the promoter gene and its association with colorectal cancer patient survival rate and tumor progression. Methods: Blood samples from 150 colorectal patients and 100 cases were extracted. The mean follow-up was 25 months (12-36 months. Cases and patients were genotyped using genomic DNA extraction and PCR-RFLP. Results: Colorectal cancer patients were divided in two groups; with activity of metastasis (M+ and without activity of metastasis (M-. 2G allele in metastasis group (55% showed more frequency rather than controls (23%. Survival analyses showed that 3 years survival patients rate in the patients without metastasis activity carrying 1G allele (homo and heterozygote was 81% and for 2G homozygote is 66% (p=0.04. The survival rate dependent to cancer was 90% and 71%, respectively (P=0.01. Conclusion: According to the results, it seems that patients carrying 1G allele show a better survival rate dependent on cancer as compared to patients who do not carry this allele.

  16. Causes and consequences of plant radio-resistance. Formation of DNA basis lesions and self-repairing activity of one of them, the 8-oxo-7,8-dihydro-guanine in Arabidopsis thaliana

    International Nuclear Information System (INIS)

    Dany, A.L.

    2001-01-01

    In this research thesis, the author first explains how and why DNA is injured when it is submitted to an oxidizing stress, and describes precisely the formation and the biological consequences of lesions of DNA bases, the 8-oxo-7,8-dihydro-guanine (8-oxoGua). She describes the repairing activities of the oxidized DNA, and more particularly the repairing of 8-oxoGua, in prokaryotes as well as in yeast, mammals and plants. Methodologies used are described, together with the repair activities of the 8-oxo-7,8-dihydro-guanine following a biochemical type approach and a molecular biology approach

  17. Identification of the Structural Features of Guanine Derivatives as MGMT Inhibitors Using 3D-QSAR Modeling Combined with Molecular Docking

    Directory of Open Access Journals (Sweden)

    Guohui Sun

    2016-06-01

    Full Text Available DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT, which plays an important role in inducing drug resistance against alkylating agents that modify the O6 position of guanine in DNA, is an attractive target for anti-tumor chemotherapy. A series of MGMT inhibitors have been synthesized over the past decades to improve the chemotherapeutic effects of O6-alkylating agents. In the present study, we performed a three-dimensional quantitative structure activity relationship (3D-QSAR study on 97 guanine derivatives as MGMT inhibitors using comparative molecular field analysis (CoMFA and comparative molecular similarity indices analysis (CoMSIA methods. Three different alignment methods (ligand-based, DFT optimization-based and docking-based alignment were employed to develop reliable 3D-QSAR models. Statistical parameters derived from the models using the above three alignment methods showed that the ligand-based CoMFA (Qcv2 = 0.672 and Rncv2 = 0.997 and CoMSIA (Qcv2 = 0.703 and Rncv2 = 0.946 models were better than the other two alignment methods-based CoMFA and CoMSIA models. The two ligand-based models were further confirmed by an external test-set validation and a Y-randomization examination. The ligand-based CoMFA model (Qext2 = 0.691, Rpred2 = 0.738 and slope k = 0.91 was observed with acceptable external test-set validation values rather than the CoMSIA model (Qext2 = 0.307, Rpred2 = 0.4 and slope k = 0.719. Docking studies were carried out to predict the binding modes of the inhibitors with MGMT. The results indicated that the obtained binding interactions were consistent with the 3D contour maps. Overall, the combined results of the 3D-QSAR and the docking obtained in this study provide an insight into the understanding of the interactions between guanine derivatives and MGMT protein, which will assist in designing novel MGMT inhibitors with desired activity.

  18. From lin-Benzoguanines to lin-Benzohypoxanthines as Ligands for Zymomonas mobilis tRNA-Guanine Transglycosylase: Replacement of Protein-Ligand Hydrogen Bonding by Importing Water Clusters.

    NARCIS (Netherlands)

    Barandun, L.J.; Immekus, F.; Kohler, P.C.; Tonazzi, S.; Wagner, B.; Wendelspiess, S.; Ritschel, T.; Heine, A.; Kansy, M.; Klebe, G.; Diederich, F.

    2012-01-01

    The foodborne illness shigellosis is caused by Shigella bacteria that secrete the highly cytotoxic Shiga toxin, which is also formed by the closely related enterohemorrhagic Escherichia coli (EHEC). It has been shown that tRNA-guanine transglycosylase (TGT) is essential for the pathogenicity of

  19. Cyclosporine a inhibits apoptosis of rat gingival epithelium.

    Science.gov (United States)

    Ma, Su; Liu, Peihong; Li, Yanwu; Hou, Lin; Chen, Li; Qin, Chunlin

    2014-08-01

    The use of cyclosporine A (CsA) induces hyperplasia of the gingival epithelium in a site-specific response manner, but the molecular mechanism via which the lesion occurs is unclear. The present research aims to investigate the site-specific effect of CsA on the apoptosis of gingival epithelium associated with gingival hyperplasia. Forty Wistar rats were divided into CsA-treated and non-treated groups. Paraffin-embedded sections of mandibular first molars were selected for hematoxylin and eosin staining, immunohistochemistry analyses of bcl-2 and caspase-3, and the staining of terminal deoxynucleotidyl transfer-mediated dUTP nick-end labeling (TUNEL). The area of the whole gingival epithelium and the length of rete pegs were measured, and the number of bcl-2- and caspase-3-positive cells in the longest rete peg were counted. The analysis of variance for factorial designs and Fisher least significant difference test for post hoc analysis were used to determine the significance levels. In CsA-treated rats, bcl-2 expression was significantly upregulated, whereas caspase-3 expression was downregulated, along with a reduced number of TUNEL-positive cells. The site-specific distribution of bcl-2 was consistent with the site-specific hyperplasia of the gingival epithelium in CsA-treated rats. CsA inhibited gingival epithelial apoptosis via the mitochondrial pathway and common pathway. The antiapoptotic protein bcl-2 might play a critical role in the pathogenesis of the site-specific hyperplasia of gingival epithelium induced by CsA. There were mechanistic differences in the regulation of apoptosis for cells in the attached gingival epithelium, free gingival epithelium, and junctional epithelium.

  20. Studies on the energy metabolism of opossum (Didelphis virginiana) erythrocytes: V. Utilization of hypoxanthine for the synthesis of adenine and guanine nucleotides in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Bethlenfalvay, N.C.; White, J.C.; Chadwick, E.; Lima, J.E. (Fitzsimons Army Medical Center, Aurora, CO (USA))

    1990-06-01

    High pressure liquid radiochromatography was used to test the ability of opossum erythrocytes to incorporate tracer amounts of (G-{sup 3}H) hypoxanthine (Hy) into ({sup 3}H) labelled triphosphates of adenine and guanine. In the presence of supraphysiologic (30 mM) phosphate which is optimal for PRPP synthesis, both ATP and GTP are extensively labelled. When physiologic (1 mM) medium phosphate is used, red cells incubated under an atmosphere of nitrogen accumulate ({sup 3}H) ATP in a linear fashion suggesting ongoing PRPP synthesis in red cells whose hemoglobin is deoxygenated. In contrast, a lesser increase of labelled ATP is observed in cells incubated under oxygen, suggesting that conditions for purine nucleotide formation from ambient Hy are more favorable in the venous circulation.

  1. Specificities and pH profiles of adenine and hypoxanthine-guanine-xanthine phosphoribosyltransferases (nucleotide synthases) of the thermoacidophile archaeon Sulfolobus solfataricus

    DEFF Research Database (Denmark)

    Hansen, Michael Riis; Jensen, Kristine Steen; Rasmussen, Mads Skytte

    2014-01-01

    Two open reading frames in the genome of Sulfolobus solfataricus (SSO2341 and SSO2424) were cloned and expressed in E. coli. The protein products were purified and their enzymatic activity characterized. Although SSO2341 was annotated as a gene (gpT-1) encoding a 6-oxopurine...... phosphoribosyltransferase (PRTase), the protein product turned out to be a PRTase highly specific for adenine and we suggest that the reading frame should be renamed apT. The other reading frame SSO2424 (gpT-2) proved to be a true 6-oxopurine PRTase active with hypoxanthine, xanthine and guanine as substrates, and we.......5, while maximal activity with xanthine was observed at pH 7.5. We discuss likely reasons why SSO2341 in S. solfataricus and similar open reading frames in other Crenarchaeota could not be identified as genes encoding APRTase....

  2. Guanine nucleotide exchange factor αPIX leads to activation of the Rac 1 GTPase/glycogen phosphorylase pathway in interleukin (IL)-2-stimulated T cells

    DEFF Research Database (Denmark)

    Llavero, Francisco; Urzelai, Bakarne; Osinalde, Nerea

    2015-01-01

    Recently, we have reported that the active form of Rac 1 GTPase binds to the glycogen phosphorylase muscle isoform (PYGM) and modulates its enzymatic activity leading to T cell proliferation. In the lymphoid system, Rac 1 and in general other small GTPases of the Rho family participate...... in the signaling cascades that are activated after engagement of the T cell antigen receptor. However, little is known about the IL-2-dependent Rac 1 activator molecules. For the first time, a signaling pathway leading to the activation of Rac 1/PYGM in response to IL-2-stimulated T cell proliferation is described....... More specifically, αPIX, a known guanine nucleotide exchange factor for the small GTPases of the Rho family, preferentially Rac 1, mediates PYGM activation in Kit 225 T cells stimulated with IL-2. Using directed mutagenesis, phosphorylation of αPIX Rho-GEF serines 225 and 488 is required for activation...

  3. Relative Stability of the La and Lb Excited States in Adenine and Guanine: Direct Evidence from TD-DFT Calculations of MCD Spectra.

    Science.gov (United States)

    Santoro, Fabrizio; Improta, Roberto; Fahleson, Tobias; Kauczor, Joanna; Norman, Patrick; Coriani, Sonia

    2014-06-05

    The relative position of La and Lb ππ* electronic states in purine nucleobases is a much debated topic, since it can strongly affect our understanding of their photoexcited dynamics. To assess this point, we calculated the absorption and magnetic circular dichroism (MCD) spectra of adenine, guanine, and their nucleosides in gas-phase and aqueous solution, exploiting recent developments in MCD computational technology within time-dependent density functional theory. MCD spectroscopy allows us to resolve the intense S0→ La transition from the weak S0→ Lb transition. The spectra obtained in water solution, by using B3LYP and CAM-B3LYP functionals and describing solvent effect by cluster models and by the polarizable continuum model (PCM), are in very good agreement with the experimental counterparts, thus providing direct and unambiguous evidence that the energy ordering predicted by TD-DFT, La < Lb, is the correct one.

  4. Studies on the energy metabolism of opossum (Didelphis virginiana) erythrocytes: V. Utilization of hypoxanthine for the synthesis of adenine and guanine nucleotides in vitro

    International Nuclear Information System (INIS)

    Bethlenfalvay, N.C.; White, J.C.; Chadwick, E.; Lima, J.E.

    1990-01-01

    High pressure liquid radiochromatography was used to test the ability of opossum erythrocytes to incorporate tracer amounts of [G- 3 H] hypoxanthine (Hy) into [ 3 H] labelled triphosphates of adenine and guanine. In the presence of supraphysiologic (30 mM) phosphate which is optimal for PRPP synthesis, both ATP and GTP are extensively labelled. When physiologic (1 mM) medium phosphate is used, red cells incubated under an atmosphere of nitrogen accumulate [ 3 H] ATP in a linear fashion suggesting ongoing PRPP synthesis in red cells whose hemoglobin is deoxygenated. In contrast, a lesser increase of labelled ATP is observed in cells incubated under oxygen, suggesting that conditions for purine nucleotide formation from ambient Hy are more favorable in the venous circulation

  5. Protein Kinase A (PKA) Type I Interacts with P-Rex1, a Rac Guanine Nucleotide Exchange Factor: EFFECT ON PKA LOCALIZATION AND P-Rex1 SIGNALING.

    Science.gov (United States)

    Chávez-Vargas, Lydia; Adame-García, Sendi Rafael; Cervantes-Villagrana, Rodolfo Daniel; Castillo-Kauil, Alejandro; Bruystens, Jessica G H; Fukuhara, Shigetomo; Taylor, Susan S; Mochizuki, Naoki; Reyes-Cruz, Guadalupe; Vázquez-Prado, José

    2016-03-18

    Morphology of migrating cells is regulated by Rho GTPases and fine-tuned by protein interactions and phosphorylation. PKA affects cell migration potentially through spatiotemporal interactions with regulators of Rho GTPases. Here we show that the endogenous regulatory (R) subunit of type I PKA interacts with P-Rex1, a Rac guanine nucleotide exchange factor that integrates chemotactic signals. Type I PKA holoenzyme interacts with P-Rex1 PDZ domains via the CNB B domain of RIα, which when expressed by itself facilitates endothelial cell migration. P-Rex1 activation localizes PKA to the cell periphery, whereas stimulation of PKA phosphorylates P-Rex1 and prevents its activation in cells responding to SDF-1 (stromal cell-derived factor 1). The P-Rex1 DEP1 domain is phosphorylated at Ser-436, which inhibits the DH-PH catalytic cassette by direct interaction. In addition, the P-Rex1 C terminus is indirectly targeted by PKA, promoting inhibitory interactions independently of the DEP1-PDZ2 region. A P-Rex1 S436A mutant construct shows increased RacGEF activity and prevents the inhibitory effect of forskolin on sphingosine 1-phosphate-dependent endothelial cell migration. Altogether, these results support the idea that P-Rex1 contributes to the spatiotemporal localization of type I PKA, which tightly regulates this guanine exchange factor by a multistep mechanism, initiated by interaction with the PDZ domains of P-Rex1 followed by direct phosphorylation at the first DEP domain and putatively indirect regulation of the C terminus, thus promoting inhibitory intramolecular interactions. This reciprocal regulation between PKA and P-Rex1 might represent a key node of integration by which chemotactic signaling is fine-tuned by PKA. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Guanine nucleotide-dependent, pertussis toxin-insensitive, stimulation of inositol phosphate formation by carbachol in a membrane preparation from astrocytoma cells

    International Nuclear Information System (INIS)

    Hepler, J.R.; Harden, T.K.

    1986-01-01

    Formation of the inositol phosphates (InsP), InsP 3 , InsP 2 , and InsP 1 was increased in a concentration dependent manner (K/sub 0.5/ ∼ 5 μM) by GTPΣS in washed membranes prepared from 3 H-inositol-prelabelled 1321N1 human astrocytoma cells. Both GTPγS and GppNHp stimulated InsP formation by 2-3 fold over control; GTP and GDP were much less efficacious and GMP had no effect. Although the muscarinic cholinergic receptor agonist carbachol had no effect in the absence of guanine nucleotide, in the presence of 10 μM GTPγS, carbachol stimulated (K/sub 0.5/ ∼ 10 μ M) the formation of InsP above the level achieved with GTPγS alone. The effect of carbachol was completely blocked by atropine. The order of potency for a series of nucleotides for stimulation of InsP formation in the presence of 500 μM carbachol was GTPγS > GppNHp > GTP = GDP. Pertussis toxin, at concentrations that fully ADP-ribosylate and functionally inactivate G/sub i/, had no effect on InsP formation in the presence of GTPγS or GTPγS plus carbachol. Histamine and bradykinin also stimulated InsP formation in the presence of GTPγS in washed membranes from 1321N1 cells. These data are consistent with the idea that a guanine nucleotide regulatory protein that is not G/sub i/ is involved in receptor-mediated stimulation of InsP formation in 1321N1 human astrocytoma cells

  7. A simplified one-pot synthesis of 9-[(3-[{sup 18}F]Fluoro-1-hydroxy-2-propoxy)methyl]guanine([{sup 18}F]FHPG) and 9-(4-[{sup 18}F]Fluoro-3-hydroxymethylbutyl)guanine ([{sup 18}F]FHBG) for gene therapy

    Energy Technology Data Exchange (ETDEWEB)

    Shiue, Grace G.; Shiue, Chyng-Yann E-mail: Shiue@rad.upenn.edu; Lee, Roland L.; MacDonald, Douglas; Hustinx, Roland; Eck, Stephen L.; Alavi, Abass A

    2001-10-01

    9-[(3-[{sup 18}F]Fluoro-1-hydroxy-2-propoxy)methyl]guanine ([{sup 18}F]FHPG, 2) has been synthesized by nucleophilic substitution of N{sup 2}-(p-anisyldiphenylmethyl)-9-{l_brace}[1-(p-anisyldiphenylmethoxy)-3 -toluenesulfonyloxy-2-propoxy]methyl{r_brace}guanine (1) with potassium [{sup 18}F]fluoride/Kryptofix 2.2.2 followed by deprotection with 1 N HCl and purification with different methods in variable yields. When both the nucleophilic substitution and deprotection were carried out at 90 deg. C and the product was purified by HPLC (method A), the yield of compound 2 was 5-10% and the synthesis time was 90 min from EOB. However, if both the nucleophilic substitution and deprotection were carried out at 120 deg. C and the product was purified by HPLC, the yield of compound 2 decreased to 2%. When compound 2 was synthesized at 90 deg. C and purified by Silica Sep-Pak (method B), the yield increased to 10-15% and the synthesis time was 60 min from EOB. Similarly, 9-(4-[{sup 18}F]fluoro-3-hydroxymethylbutyl)guanine ([{sup 18}F]FHBG, 4) was synthesized with method A and method B in 9% and 10-15% yield, respectively, in a synthesis time of 90 and 60 min, respectively, from EOB. Compound 2 was relatively unstable in acidic medium at 120 deg. C while compound 4 was stable under the same condition. Both compound 2 and compound 4 had low lipid/water partition coefficient (0.126{+-}0.022, n=5 and 0.165{+-}0.023, n=5, respectively). Although it contains non-radioactive ganciclovir ({approx}5-30 {mu}g) as a chemical by-product, compound 2 synthesized by method B has a similar uptake in 9L glioma cells as that synthesized by method A, and is a potential tracer for imaging herpes simplex virus thymidine kinase gene expression in tumors using PET. Similarly, compound 4 synthesized by method B contains {approx}10-25 {mu}g of penciclovir as a chemical by-product. Thus, the simplified one pot synthesis (method B) is a useful method for synthesizing both compound 2 and compound 4 in

  8. A simplified one-pot synthesis of 9-[(3-[18F]Fluoro-1-hydroxy-2-propoxy)methyl]guanine([18F]FHPG) and 9-(4-[18F]Fluoro-3-hydroxymethylbutyl)guanine ([18F]FHBG) for gene therapy

    International Nuclear Information System (INIS)

    Shiue, Grace G.; Shiue, Chyng-Yann; Lee, Roland L.; MacDonald, Douglas; Hustinx, Roland; Eck, Stephen L.; Alavi, Abass A.

    2001-01-01

    9-[(3-[ 18 F]Fluoro-1-hydroxy-2-propoxy)methyl]guanine ([ 18 F]FHPG, 2) has been synthesized by nucleophilic substitution of N 2 -(p-anisyldiphenylmethyl)-9-{[1-(p-anisyldiphenylmethoxy)-3 -toluenesulfonyloxy-2-propoxy]methyl}guanine (1) with potassium [ 18 F]fluoride/Kryptofix 2.2.2 followed by deprotection with 1 N HCl and purification with different methods in variable yields. When both the nucleophilic substitution and deprotection were carried out at 90 deg. C and the product was purified by HPLC (method A), the yield of compound 2 was 5-10% and the synthesis time was 90 min from EOB. However, if both the nucleophilic substitution and deprotection were carried out at 120 deg. C and the product was purified by HPLC, the yield of compound 2 decreased to 2%. When compound 2 was synthesized at 90 deg. C and purified by Silica Sep-Pak (method B), the yield increased to 10-15% and the synthesis time was 60 min from EOB. Similarly, 9-(4-[ 18 F]fluoro-3-hydroxymethylbutyl)guanine ([ 18 F]FHBG, 4) was synthesized with method A and method B in 9% and 10-15% yield, respectively, in a synthesis time of 90 and 60 min, respectively, from EOB. Compound 2 was relatively unstable in acidic medium at 120 deg. C while compound 4 was stable under the same condition. Both compound 2 and compound 4 had low lipid/water partition coefficient (0.126±0.022, n=5 and 0.165±0.023, n=5, respectively). Although it contains non-radioactive ganciclovir (∼5-30 μg) as a chemical by-product, compound 2 synthesized by method B has a similar uptake in 9L glioma cells as that synthesized by method A, and is a potential tracer for imaging herpes simplex virus thymidine kinase gene expression in tumors using PET. Similarly, compound 4 synthesized by method B contains ∼10-25 μg of penciclovir as a chemical by-product. Thus, the simplified one pot synthesis (method B) is a useful method for synthesizing both compound 2 and compound 4 in good yield for routine clinical use, and the method is

  9. Multi-level Quantum Mechanics and Molecular Mechanics Study of Ring Opening Process of Guanine Damage by Hydroxyl Radical in Aqueous Solution.

    Science.gov (United States)

    Liu, Peng; Wang, Qiong; Niu, Meixing; Wang, Dunyou

    2017-08-10

    Combining multi-level quantum mechanics theories and molecular mechanics with an explicit water model, we investigated the ring opening process of guanine damage by hydroxyl radical in aqueous solution. The detailed, atomic-level ring-opening mechanism along the reaction pathway was revealed in aqueous solution at the CCSD(T)/MM levels of theory. The potentials of mean force in aqueous solution were calculated at both the DFT/MM and CCSD(T)/MM levels of the theory. Our study found that the aqueous solution has a significant effect on this reaction in solution. In particular, by comparing the geometries of the stationary points between in gas phase and in aqueous solution, we found that the aqueous solution has a tremendous impact on the torsion angles much more than on the bond lengths and bending angles. Our calculated free-energy barrier height 31.6 kcal/mol at the CCSD(T)/MM level of theory agrees well with the one obtained based on gas-phase reaction profile and free energies of solvation. In addition, the reaction path in gas phase was also mapped using multi-level quantum mechanics theories, which shows a reaction barrier at 19.2 kcal/mol at the CCSD(T) level of theory, agreeing very well with a recent ab initio calculation result at 20.8 kcal/mol.

  10. The G-BHQ synergistic effect: Improved double quenching molecular beacons based on guanine and Black Hole Quencher for sensitive simultaneous detection of two DNAs.

    Science.gov (United States)

    Xiang, Dongshan; Li, Fengquan; Wu, Chenyi; Shi, Boan; Zhai, Kun

    2017-11-01

    We designed two double quenching molecular beacons (MBs) with simple structure based on guanine (G base) and Black Hole Quencher (BHQ), and developed a new analytical method for sensitive simultaneous detection of two DNAs by synchronous fluorescence analysis. In this analytical method, carboxyl fluorescein (FAM) and tetramethyl-6-carboxyrhodamine (TAMRA) were respectively selected as fluorophore of two MBs, Black Hole Quencher 1 (BHQ-1) and Black Hole Quencher 2 (BHQ-2) were respectively selected as organic quencher, and three continuous nucleotides with G base were connected to organic quencher (BHQ-1 and BHQ-2). In the presence of target DNAs, the two MBs hybridize with the corresponding target DNAs, the fluorophores are separated from organic quenchers and G bases, leading to recovery of fluorescence of FAM and TAMRA. Under a certain conditions, the fluorescence intensities of FAM and TAMRA all exhibited good linear dependence on their concentration of target DNAs (T1 and T2) in the range from 4 × 10 -10 to 4 × 10 -8 molL -1 (M). The detection limit (3σ, n = 13) of T1 was 3 × 10 -10 M and that of T2 was 2×10 -10 M, respectively. Compared with the existing analysis methods for multiplex DNA with MBs, this proposed method based on double quenching MBs is not only low fluorescence background, short analytical time and low detection cost, but also easy synthesis and good stability of MB probes. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Solvent effect on the intermolecular proton transfer of the Watson and Crick guanine-cytosine and adenine-thymine base pairs: a polarizable continuum model study.

    Science.gov (United States)

    Romero, Eduardo E; Hernandez, Florencio E

    2018-01-03

    Herein we present our results on the study of the double proton transfer (DPT) mechanism in the adenine-thymine (AT) and guanine-cytosine (GC) base pairs, both in gas phase and in solution. The latter was modeled using the polarizable continuum method (PCM) in different solvents. According to our DFT calculations, the DPT may occur for both complexes in a stepwise mechanism in condensate phase. In gas phase only the GC base pair exhibits a concerted DPT mechanism. Using the Wigner's tunneling corrections to the transition state theory we demonstrate that such corrections are important for the prediction of the rate constants of both systems in gas and in condensate phase. We also show that (i) as the polarity of the medium decreases the equilibrium constant of the DPT reaction increases in both complexes, and (ii) that the equilibrium constant in the GC complex is four orders of magnitude larger than in AT. This observation suggests that the spontaneous mutations in DNA base pairs are more probable in GC than in AT.

  12. The influence of anharmonic and solvent effects on the theoretical vibrational spectra of the guanine-cytosine base pairs in Watson-Crick and Hoogsteen configurations.

    Science.gov (United States)

    Bende, Attila; Muntean, Cristina M

    2014-03-01

    The theoretical IR and Raman spectra of the guanine-cytosine DNA base pairs in Watson-Crick and Hoogsteen configurations were computed using DFT method with M06-2X meta-hybrid GGA exchange-correlation functional, including the anharmonic corrections and solvent effects. The results for harmonic frequencies and their anharmonic corrections were compared with our previously calculated values obtained with the B3PW91 hybrid GGA functional. Significant differences were obtained for the anharmonic corrections calculated with the two different DFT functionals, especially for the stretching modes, while the corresponding harmonic frequencies did not differ considerable. For the Hoogtseen case the H⁺ vibration between the G-C base pair can be characterized as an asymmetric Duffing oscillator and therefore unrealistic anharmonic corrections for normal modes where this proton vibration is involved have been obtained. The spectral modification due to the anharmonic corrections, solvent effects and the influence of sugar-phosphate group for the Watson-Crick and Hoogsteen base pair configurations, respectively, were also discussed. For the Watson-Crick case also the influence of the stacking interaction on the theoretical IR and Raman spectra was analyzed. Including the anharmonic correction in our normal mode analysis is essential if one wants to obtain correct assignments of the theoretical frequency values as compared with the experimental spectra.

  13. A Histidine pH sensor regulates activation of the Ras-specific guanine nucleotide exchange factor RasGRP1.

    Science.gov (United States)

    Vercoulen, Yvonne; Kondo, Yasushi; Iwig, Jeffrey S; Janssen, Axel B; White, Katharine A; Amini, Mojtaba; Barber, Diane L; Kuriyan, John; Roose, Jeroen P

    2017-09-27

    RasGRPs are guanine nucleotide exchange factors that are specific for Ras or Rap, and are important regulators of cellular signaling. Aberrant expression or mutation of RasGRPs results in disease. An analysis of RasGRP1 SNP variants led to the conclusion that the charge of His 212 in RasGRP1 alters signaling activity and plasma membrane recruitment, indicating that His 212 is a pH sensor that alters the balance between the inactive and active forms of RasGRP1. To understand the structural basis for this effect we compared the structure of autoinhibited RasGRP1, determined previously, to those of active RasGRP4:H-Ras and RasGRP2:Rap1b complexes. The transition from the autoinhibited to the active form of RasGRP1 involves the rearrangement of an inter-domain linker that displaces inhibitory inter-domain interactions. His 212 is located at the fulcrum of these conformational changes, and structural features in its vicinity are consistent with its function as a pH-dependent switch.

  14. Purine salvage in the apicomplexan Sarcocystis neurona, and generation of hypoxanthine-xanthine-guanine phosphoribosyltransferase-deficient clones for positive-negative selection of transgenic parasites.

    Science.gov (United States)

    Dangoudoubiyam, Sriveny; Zhang, Zijing; Howe, Daniel K

    2014-09-01

    Sarcocystis neurona is an apicomplexan parasite that causes severe neurological disease in horses and marine mammals. The Apicomplexa are all obligate intracellular parasites that lack purine biosynthesis pathways and rely on the host cell for their purine requirements. Hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) and adenosine kinase (AK) are key enzymes that function in two complementary purine salvage pathways in apicomplexans. Bioinformatic searches of the S. neurona genome revealed genes encoding HXGPRT, AK and all of the major purine salvage enzymes except purine nucleoside phosphorylase. Wild-type S. neurona were able to grow in the presence of mycophenolic acid (MPA) but were inhibited by 6-thioxanthine (6-TX), suggesting that the pathways involving either HXGPRT or AK are functional in this parasite. Prior work with Toxoplasma gondii demonstrated the utility of HXGPRT as a positive-negative selection marker. To enable the use of HXGPRT in S. neurona, the SnHXGPRT gene sequence was determined and a gene-targeting plasmid was transfected into S. neurona. SnHXGPRT-deficient mutants were selected with 6-TX, and single-cell clones were obtained. These Sn∆HXG parasites were susceptible to MPA and could be complemented using the heterologous T. gondii HXGPRT gene. In summary, S. neurona possesses both purine salvage pathways described in apicomplexans, thus allowing the use of HXGPRT as a positive-negative drug selection marker in this parasite.

  15. GDP-bound and nucleotide-free intermediates of the guanine nucleotide exchange in the Rab5·Vps9 system.

    Science.gov (United States)

    Uejima, Tamami; Ihara, Kentaro; Goh, Tatsuaki; Ito, Emi; Sunada, Mariko; Ueda, Takashi; Nakano, Akihiko; Wakatsuki, Soichi

    2010-11-19

    Many GTPases regulate intracellular transport and signaling in eukaryotes. Guanine nucleotide exchange factors (GEFs) activate GTPases by catalyzing the exchange of their GDP for GTP. Here we present crystallographic and biochemical studies of a GEF reaction with four crystal structures of Arabidopsis thaliana ARA7, a plant homolog of Rab5 GTPase, in complex with its GEF, VPS9a, in the nucleotide-free and GDP-bound forms, as well as a complex with aminophosphonic acid-guanylate ester and ARA7·VPS9a(D185N) with GDP. Upon complex formation with ARA7, VPS9 wedges into the interswitch region of ARA7, inhibiting the coordination of Mg(2+) and decreasing the stability of GDP binding. The aspartate finger of VPS9a recognizes GDP β-phosphate directly and pulls the P-loop lysine of ARA7 away from GDP β-phosphate toward switch II to further destabilize GDP for its release during the transition from the GDP-bound to nucleotide-free intermediates in the nucleotide exchange reaction.

  16. Effect of Six Sessions of High Intensity Interval Training on Levels of Hypoxanthine, Xanthine, Hypoxanthine-Guanine phosphoribosyltransferase (HGPRT and Serum Uric Acid in active young men

    Directory of Open Access Journals (Sweden)

    ROHOULLAH HAGHSHENAS GATABI

    2017-01-01

    Full Text Available Abstract Introduction and objectives: long-term sport and physical activity results in compatibility in maintaining purine derivatives but the compatibility achieved within a few sessions is not well investigated. This study aimed to investigate the effect of a 30-seconds high intensity interval training on Hypoxanthine, xanthine, hypoxanthine-guanine phosphoribosyltransferase (HGPRT and serum uric acid in young college men. Methods: In this study, 18 untrained healthy men were divided into two control and training groups after homogenization based on their personal characteristics. Training included six sessions (every other day for two weeks with different intervals (4, 7, 6, 6, 5 & 4, respectively with a fixed four-minute rest between each interval, and with a constant load of .6 on the cycle-ergometer. Blood samples were taken before and 48 hours after the last training session, and were used to analyze hypoxanthine, xanthine, uric acid, and serum HGPRT. Statistical analysis was performed using analysis of covariance (ANCOVA. Results: The results showed that high-intensity interval training for two weeks did not cause significant changes in serum HGPRT (P = .73; likewise, the increase in serum hypoxanthine (P = .170 and serum xanthine (P = .170 was not statistically significant but significant reduction was observed in serum uric acid (P = .025. Discussion and conclusion: The results of this study indicated that two-week HIIT training is likely to enhance athletic performance and recovery of purine nucleotide cycle.

  17. An adenine-to-guanine nucleotide change in the IRES SL-IV domain of picornavirus/hepatitis C chimeric viruses leads to a nonviable phenotype

    International Nuclear Information System (INIS)

    McKnight, Kevin L.; Sandefur, Stephanie; Phipps, Krista M.; Heinz, Beverly A.

    2003-01-01

    The inability for the internal ribosomal entry site (IRES) of hepatitis C virus (HCV) to be readily studied in the context of viral replication has been circumvented by constructing chimeras such as with poliovirus (PV), in which translation of the genome polyprotein is under control of the HCV IRES. During our attempts to configure the PV/HCV chimera for our drug discovery efforts, we discovered that an adenine- (A) to-guanine (G) change at nt 350 in domain IV of the HCV IRES resulted in a nonviable phenotype. Similarly, a mengovirus (MV)/HCV chimera using the same configuration with a G at nt 350 (G-350) was found to be nonviable. In contrast, a bovine viral diarrhea virus (BVDV)/HCV chimera remained viable with G-350 in the HCV IRES insert. Second-site, resuscitating mutations were identified from the G-350 PV/HCV and MV/HCV viruses after blind passaging. For both viruses, the resuscitating mutations involved destabilization of domain IV in the HCV IRES. The nonviability of G-350 in the picornavirus/HCV chimeric background might be linked to translation efficiency as indicated by analyses with dual reporter and PV/HCV replicon constructs

  18. Identification of a negative regulatory region for the exchange activity and characterization of T332I mutant of Rho guanine nucleotide exchange factor 10 (ARHGEF10).

    Science.gov (United States)

    Chaya, Taro; Shibata, Satoshi; Tokuhara, Yasunori; Yamaguchi, Wataru; Matsumoto, Hiroshi; Kawahara, Ichiro; Kogo, Mikihiko; Ohoka, Yoshiharu; Inagaki, Shinobu

    2011-08-26

    The T332I mutation in Rho guanine nucleotide exchange factor 10 (ARHGEF10) was previously found in persons with slowed nerve conduction velocities and thin myelination of peripheral nerves. However, the molecular and cellular basis of the T332I mutant is not understood. Here, we show that ARHGEF10 has a negative regulatory region in the N terminus, in which residue 332 is located, and the T332I mutant is constitutively active. An N-terminal truncated ARHGEF10 mutant, ARHGEF10 ΔN (lacking amino acids 1-332), induced cell contraction that was inhibited by a Rho kinase inhibitor Y27632 and had higher GEF activity for RhoA than the wild type. The T332I mutant also showed the phenotype similar to the N-terminal truncated mutant. These data suggest that the ARHGEF10 T332I mutation-associated phenotype observed in the peripheral nerves is due to activated GEF activity of the ARHGEF10 T332I mutant.

  19. Identification of a Negative Regulatory Region for the Exchange Activity and Characterization of T332I Mutant of Rho Guanine Nucleotide Exchange Factor 10 (ARHGEF10)*

    Science.gov (United States)

    Chaya, Taro; Shibata, Satoshi; Tokuhara, Yasunori; Yamaguchi, Wataru; Matsumoto, Hiroshi; Kawahara, Ichiro; Kogo, Mikihiko; Ohoka, Yoshiharu; Inagaki, Shinobu

    2011-01-01

    The T332I mutation in Rho guanine nucleotide exchange factor 10 (ARHGEF10) was previously found in persons with slowed nerve conduction velocities and thin myelination of peripheral nerves. However, the molecular and cellular basis of the T332I mutant is not understood. Here, we show that ARHGEF10 has a negative regulatory region in the N terminus, in which residue 332 is located, and the T332I mutant is constitutively active. An N-terminal truncated ARHGEF10 mutant, ARHGEF10 ΔN (lacking amino acids 1–332), induced cell contraction that was inhibited by a Rho kinase inhibitor Y27632 and had higher GEF activity for RhoA than the wild type. The T332I mutant also showed the phenotype similar to the N-terminal truncated mutant. These data suggest that the ARHGEF10 T332I mutation-associated phenotype observed in the peripheral nerves is due to activated GEF activity of the ARHGEF10 T332I mutant. PMID:21719701

  20. Endogenous 5-methylcytosine protects neighboring guanines from N7 and O6-methylation and O6-pyridyloxobutylation by the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

    Science.gov (United States)

    Ziegel, Rebecca; Shallop, Anthony; Upadhyaya, Pramod; Jones, Roger; Tretyakova, Natalia

    2004-01-20

    All CG dinucleotides along exons 5-8 of the p53 tumor suppressor gene contain endogenous 5-methylcytosine (MeC). These same sites (e.g., codons 157, 158, 245, 248, and 273) are mutational hot spots in smoking-induced lung cancer. Several groups used the UvrABC endonuclease incision assay to demonstrate that methylated CG dinucleotides of the p53 gene are the preferred binding sites for the diol epoxides of bay region polycyclic aromatic hydrocarbons (PAH). In contrast, effects of endogenous cytosine methylation on the distribution of DNA lesions induced by tobacco-specific nitrosamines, e.g., 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), have not been elucidated. In the work presented here, a stable isotope labeling HPLC-ESI-MS/MS approach was employed to analyze the reactivity of the N7 and O6 positions of guanines within hemimethylated and fully methylated CG dinucleotides toward NNK-derived methylating and pyridyloxobutylating species. 15N3-labeled guanine bases were placed within synthetic DNA sequences representing endogenously methylated p53 codons 154, 157, and 248, followed by treatment with acetylated precursors to NNK diazohydroxides. HPLC-ESI-MS/MS analysis was used to determine the relative yields of N7- and O6-guanine adducts at the 15N3-labeled position. In all cases, the presence of MeC inhibited the formation of N7-methylguanine, O6-methylguanine, and O6-pyridyloxobutylguanine at a neighboring G, with the greatest decrease observed in fully methylated dinucleotides and at guanines preceded by MeC. Furthermore, the O6-Me-dG/N7-Me-G molar ratios were decreased in the presence of the 5'-neighboring MeC, suggesting that the observed decline in O6-alkylguanine adduct yields is, at least partially, a result of an altered reactivity pattern in methylated CG dinucleotides. These results indicate that, unlike N2-guanine adducts of PAH diol epoxides, NNK-induced N7- and O6-alkylguanine adducts are not preferentially formed at the endogenously

  1. Immunostimulation by cytosine-phosphate-guanine oligodeoxynucleotides in combination with IL-2 can improve the success rate of karyotype analysis in chronic lymphocytic leukaemia.

    Science.gov (United States)

    Lin, Xiaolan; Chen, Jiadi; Huang, Huifang

    2016-07-01

    To assess whether immunostimulatory cytosine-phosphate-guanine oligodeoxynucleotides (CpG-ODN) combined with interleukin-2 (IL-2) improves the number of mitotic metaphases and the detection rate of chromosomal abnormalities in chronic lymphocytic leukaemia (CLL). Bone marrow specimens were collected from 36 patients with CLL. CLL cells were cultured with CpG-ODN type DSP30 plus IL-2 for 72 h, following which R-banding analysis was conducted. Conventional culture without the immunostimulant served as the control group. The incidence of genetic abnormalities was measured by fluorescence in situ hybridisation (FISH) using a panel of five specific probes: D13S25 (13q14.3), RB1 (13q14), P53 (17p13), ATM (11q22.3) and CSP12 (trisomy 12, +12). In the control group, chromosome analysis achieved a success rate of only 22.2, and 11.1% of abnormal karyotypes were detected. After immunostimulation with DSP30 plus IL-2, chromosome analysis achieved a success rate of up to 91.6, and 41.6% of abnormal karyotypes were detected. FISH analysis detected 77.7% of abnormalities. FISH combined with CpG-ODN DSP30 plus IL-2 improved the detection rate of chromosomal abnormalities in CLL to 83.3%. CpG-ODN DSP30 combined with IL-2 is effective in improving the detection rate of chromosomal abnormalities in CLL cells. This combination with FISH analysis is conducive to increasing the detection rate of genetic abnormalities in CLL.

  2. Solo, a RhoA-targeting guanine nucleotide exchange factor, is critical for hemidesmosome formation and acinar development in epithelial cells.

    Science.gov (United States)

    Fujiwara, Sachiko; Matsui, Tsubasa S; Ohashi, Kazumasa; Deguchi, Shinji; Mizuno, Kensaku

    2018-01-01

    Cell-substrate adhesions are essential for various physiological processes, including embryonic development and maintenance of organ functions. Hemidesmosomes (HDs) are multiprotein complexes that attach epithelial cells to the basement membrane. Formation and remodeling of HDs are dependent on the surrounding mechanical environment; however, the upstream signaling mechanisms are not well understood. We recently reported that Solo (also known as ARHGEF40), a guanine nucleotide exchange factor targeting RhoA, binds to keratin8/18 (K8/K18) intermediate filaments, and that their interaction is important for force-induced actin and keratin cytoskeletal reorganization. In this study, we show that Solo co-precipitates with an HD protein, β4-integrin. Co-precipitation assays revealed that the central region (amino acids 330-1057) of Solo binds to the C-terminal region (1451-1752) of β4-integrin. Knockdown of Solo significantly suppressed HD formation in MCF10A mammary epithelial cells. Similarly, knockdown of K18 or treatment with Y-27632, a specific inhibitor of Rho-associated kinase (ROCK), suppressed HD formation. As Solo knockdown or Y-27632 treatment is known to disorganize K8/K18 filaments, these results suggest that Solo is involved in HD formation by regulating K8/K18 filament organization via the RhoA-ROCK signaling pathway. We also showed that knockdown of Solo impairs acinar formation in MCF10A cells cultured in 3D Matrigel. In addition, Solo accumulated at the site of traction force generation in 2D-cultured MCF10A cells. Taken together, these results suggest that Solo plays a crucial role in HD formation and acinar development in epithelial cells by regulating mechanical force-induced RhoA activation and keratin filament organization.

  3. Rac1 Activation Caused by Membrane Translocation of a Guanine Nucleotide Exchange Factor in Akt2-Mediated Insulin Signaling in Mouse Skeletal Muscle.

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    Nobuyuki Takenaka

    Full Text Available Insulin-stimulated glucose uptake in skeletal muscle is mediated by the glucose transporter GLUT4, which is translocated to the plasma membrane following insulin stimulation. Several lines of evidence suggested that the protein kinase Akt2 plays a key role in this insulin action. The small GTPase Rac1 has also been implicated as a regulator of insulin-stimulated GLUT4 translocation, acting downstream of Akt2. However, the mechanisms whereby Akt2 regulates Rac1 activity remain obscure. The guanine nucleotide exchange factor FLJ00068 has been identified as a direct regulator of Rac1 in Akt2-mediated signaling, but its characterization was performed mostly in cultured myoblasts. Here, we provide in vivo evidence that FLJ00068 indeed acts downstream of Akt2 as a Rac1 regulator by using mouse skeletal muscle. Small interfering RNA knockdown of FLJ00068 markedly diminished GLUT4 translocation to the sarcolemma following insulin administration or ectopic expression of a constitutively activated mutant of either phosphoinositide 3-kinase or Akt2. Additionally, insulin and these constitutively activated mutants caused the activation of Rac1 as shown by immunofluorescent microscopy using a polypeptide probe specific to activated Rac1 in isolated gastrocnemius muscle fibers and frozen sections of gastrocnemius muscle. This Rac1 activation was also abrogated by FLJ00068 knockdown. Furthermore, we observed translocation of FLJ00068 to the cell periphery following insulin stimulation in cultured myoblasts. Localization of FLJ00068 in the plasma membrane in insulin-stimulated, but not unstimulated, myoblasts and mouse gastrocnemius muscle was further affirmed by subcellular fractionation and subsequent immunoblotting. Collectively, these results strongly support a critical role of FLJ00068 in Akt2-mediated Rac1 activation in mouse skeletal muscle insulin signaling.

  4. The radioprotector WR-2721 reduces neutron-induced mutations at the hypoxanthine-guanine phosphoribosyl transferase locus in mouse splenocytes when administered prior to or following irradiation

    International Nuclear Information System (INIS)

    Grdina, D.J.; Basic, I.

    1992-01-01

    An in vitro T-lymphocyte cloning technique has been applied to study the effects of JANUS fission-spectrum neutron irradiation and the radioprotector S-2-(3-aminopropylamino) ethylphosphorothioic acid (WR-2721) on the subsequent development of somatic mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in hybrid B6CF1 male mice. In control studies performed to establish an in vitro cloning technique, the mutant frequencies of splenic T-lymphocytes, as a result of exposure to a 100 cGy dose of neutrons, increased with time from a control level of 9 x 10 -7 to a maximum value of 1.7 x 10 -5 at 56 days following irradiation. Between 56 and 150 days after irradiation, mutant frequencies were observed to plateau and remain stable. All subsequent determinations were performed at 56 days following the experimental treatment of animals. WR-2721 at a dose of 400 mg/kg was effective in protecting against the induction of hprt mutants (i.e. a mutant frequency reduction factor, MFRF) following the largest dose of neutrons used (i.e. 150 cGy). The antimutagenic effectiveness of WR-2721 administered 30 min prior to irradiation was unaffected, even when the dose was reduced to 200 mg/kg. These findings confirm our earlier report using the radioprotector N-(2-mercaptoethyl)-1,2-diaminopropane (WR-1065) under in vitro conditions, and demonstrate that these agents can be used as effective antimutagens even when they are administered up to 3 h following radiation exposure. (Author)

  5. Hypoxanthine-guanine phosphoribosyltransferase and inosine 5’-monophosphate dehydrogenase activities in three mammalian species: aquatic (Mirounga angustirostris, semiaquatic (Lontra longicaudis annectens and terrestrial (Sus scrofa

    Directory of Open Access Journals (Sweden)

    Myrna eBarjau Perez-Milicua

    2015-07-01

    Full Text Available Aquatic and semiaquatic mammals have the capacity of breath hold (apnea diving. Northern elephant seals (Mirounga angustirostris have the ability to perform deep and long duration dives; during a routine dive, adults can hold their breath for 25 min. Neotropical river otters (Lontra longicaudis annectens can hold their breath for about 30 sec. Such periods of apnea may result in reduced oxygen concentration (hypoxia and reduced blood supply (ischemia to tissues. Production of adenosine 5’-triphosphate (ATP requires oxygen, and most mammalian species, like the domestic pig (Sus scrofa, are not adapted to tolerate hypoxia and ischemia, conditions that result in ATP degradation. The objective of this study was to explore the differences in purine synthesis and recycling in erythrocytes and plasma of three mammalian species adapted to different environments: aquatic (northern elephant seal (n=11, semiaquatic (neotropical river otter (n=4 and terrestrial (domestic pig (n=11. Enzymatic activity of hypoxanthine-guanine phosphoribosyltransferase (HGPRT was determined by spectrophotometry, and activity of inosine 5’-monophosphate dehydrogenase (IMPDH and the concentration of hypoxanthine (HX, inosine 5’-monophosphate (IMP, adenosine 5’-monophosphate (AMP, adenosine 5’-diphosphate (ADP, ATP, guanosine 5’-diphosphate (GDP, guanosine 5’-triphosphate (GTP, and xanthosine 5’-monophosphate (XMP were determined by high-performance liquid chromatography (HPLC. The activities of HGPRT and IMPDH and the concentration of HX, IMP, AMP, ADP, ATP, GTP and XMP in erythrocytes of domestic pigs were higher than in erythrocytes of northern elephant seals and river otters. These results suggest that under basal conditions (no diving, sleep apnea or exercise, aquatic and semiaquatic mammals have less purine mobilization than their terrestrial counterparts.

  6. Simultaneous determination of adenine guanine and thymine at multi-walled carbon nanotubes incorporated with poly(new fuchsin) composite film

    Energy Technology Data Exchange (ETDEWEB)

    Tang Ching; Yogeswaran, Umasankar [Department of Chemical Engineering and Biotechnology, National Taipei University of Technology, No.1, Section 3, Chung-Hsiao East Road, Taipei 106, Taiwan (China); Chen, S.-M. [Department of Chemical Engineering and Biotechnology, National Taipei University of Technology, No.1, Section 3, Chung-Hsiao East Road, Taipei 106, Taiwan (China)], E-mail: smchen78@ms15.hinet.net

    2009-03-16

    A composite film (MWCNTs-PNF) which contains multi-walled carbon nanotubes (MWCNTs) along with the incorporation of poly(new fuchsin) (PNF) has been synthesized on glassy carbon electrode (GCE), gold (Au) and indium tin oxide (ITO) by potentiostatic methods. The presence of MWCNTs in the composite film enhances surface coverage concentration ({gamma}) of PNF to {approx}176.5%, and increases the electron transfer rate constant (k{sub s}) to {approx}346%. The composite film also exhibits promising enhanced electrocatalytic activity towards the mixture of biochemical compounds such as adenine (AD), guanine (GU) and thymine (THY). The surface morphology of the composite film deposited on ITO has been studied using scanning electron microscopy and atomic force microscopy. These two techniques reveal that the PNF incorporated on MWCNTs. Electrochemical quartz crystal microbalance study reveals the enhancement in the functional properties of MWCNTs and PNF. The electrocatalytic responses of analytes at MWCNTs and MWCNTs-PNF films were measured using both cyclic voltammetry (CV) and differential pulse voltammetry (DPV). From electrocatalysis studies, well separated voltammetric peaks have been obtained at the composite film for AD, GU and THY, with the peak separation of 320.3 and 132.7 mV between GU-AD and AD-THY respectively. The sensitivity of the composite film towards AD, GU and THY in DPV technique is 218.18, 12.62 and 78.22 mA M{sup -1} cm{sup -2} respectively, which are higher than MWCNTs film. Further, electroanalytical studies of AD, GU and THY present in single-strand deoxyribonucleic acid (ssDNA) have been carried out using semi-derivative CV and DPV.

  7. Regulation of follitropin-sensitive adenylate cyclase by stimulatory and inhibitory forms of the guanine nucleotide regulatory protein in immature rat Sertoli cells

    International Nuclear Information System (INIS)

    Johnson, G.P.

    1987-01-01

    Studies have been designed to examine the role of guanine nucleotides in mediating FSH-sensitive adenylate cyclase activity in Sertoli cell plasma membranes. Analysis of [ 3 H]GDP binding to plasma membranes suggested a single high affinity site with a K d = 0.24 uM. Competition studies indicated that GTP γ S was 7-fold more potent than GDP β S. Bound GDP could be released by FSH in the presence of GTP γ S, but not by FSH alone. Adenylate cyclase activity was enhanced 5-fold by FSH in the presence of GTP. Addition of GDP β S to the activated enzyme (FSH plus GTP) resulted in a time-dependent decay to basal activity within 20 sec. GDP β S competitively inhibited GTP γ S-stimulated adenylate cyclase activity with a K i = 0.18 uM. Adenylate cyclase activity was also demonstrated to be sensitive to the nucleotide bound state. In the presence of FSH, only the GTP γ S-bound form persisted even if GDP β S previously occupied all available binding sites. Two membrane proteins, M r = 43,000 and 48,000, were ADP·ribosylated using cholera toxin and labeling was enhanced 2 to 4-fold by GTP γ S but not by GDP β S. The M r = 43,000 and 48,000 proteins represented variant forms of G S . A single protein of M r = 40,000 (G i ) was ADP-ribosylated by pertussis toxin in vitro. GTP inhibited forskolin-stimulated adenylate cyclase activity with an IC 50 = 0.1 uM. The adenosine analog, N 6 ·phenylisopropyl adenosine enhanced GTP inhibition of forskolin-stimulated adenylate cyclase activity by an additional 15%. GTP-dependent inhibition of forskolin-sensitive adenylate cyclase activity was abolished in membranes prepared from Sertoli cells treated in culture with pertussis toxin

  8. Modeling of Toxicity-Relevant Electrophilic Reactivity for Guanine with Epoxides: Estimating the Hard and Soft Acids and Bases (HSAB) Parameter as a Predictor.

    Science.gov (United States)

    Zhang, Jing; Wang, Chenchen; Ji, Li; Liu, Weiping

    2016-05-16

    According to the electrophilic theory in toxicology, many chemical carcinogens in the environment and/or their active metabolites are electrophiles that exert their effects by forming covalent bonds with nucleophilic DNA centers. The theory of hard and soft acids and bases (HSAB), which states that a toxic electrophile reacts preferentially with a biological macromolecule that has a similar hardness or softness, clarifies the underlying chemistry involved in this critical event. Epoxides are hard electrophiles that are produced endogenously by the enzymatic oxidation of parent chemicals (e.g., alkenes and PAHs). Epoxide ring opening proceeds through a SN2-type mechanism with hard nucleophile DNA sites as the major facilitators of toxic effects. Thus, the quantitative prediction of chemical reactivity would enable a predictive assessment of the molecular potential to exert electrophile-mediated toxicity. In this study, we calculated the activation energies for reactions between epoxides and the guanine N7 site for a diverse set of epoxides, including aliphatic epoxides, substituted styrene oxides, and PAH epoxides, using a state-of-the-art density functional theory (DFT) method. It is worth noting that these activation energies for diverse epoxides can be further predicted by quantum chemically calculated nucleophilic indices from HSAB theory, which is a less computationally demanding method than the exacting procedure for locating the transition state. More importantly, the good qualitative/quantitative correlations between the chemical reactivity of epoxides and their bioactivity suggest that the developed model based on HSAB theory may aid in the predictive hazard evaluation of epoxides, enabling the early identification of mutagenicity/carcinogenicity-relevant SN2 reactivity.

  9. Coordinated regulation by two VPS9 domain-containing guanine nucleotide exchange factors in small GTPase Rab5 signaling pathways in fission yeast

    International Nuclear Information System (INIS)

    Tsukamoto, Yuta; Kagiwada, Satoshi; Shimazu, Sayuri; Takegawa, Kaoru; Noguchi, Tetsuko; Miyamoto, Masaaki

    2015-01-01

    The small GTPase Rab5 is reported to regulate various cellular functions, such as vesicular transport and endocytosis. VPS9 domain-containing proteins are thought to activate Rab5(s) by their guanine-nucleotide exchange activities. Numerous VPS9 proteins have been identified and are structurally conserved from yeast to mammalian cells. However, the functional relationships among VPS9 proteins in cells remain unclear. Only one Rab5 and two VPS9 proteins were identified in the Schizosaccharomyces pombe genome. Here, we examined the cellular function of two VPS9 proteins and the relationship between these proteins in cellular functions. Vps901-GFP and Vps902-GFP exhibited dotted signals in vegetative and differentiated cells. vps901 deletion mutant (Δvps901) cells exhibited a phenotype deficient in the mating process and responses to high concentrations of ions, such as calcium and metals, and Δvps901Δvps902 double mutant cells exhibited round cell shapes similar to ypt5-909 (Rab5 mutant allele) cells. Deletion of both vps901 and vps902 genes completely abolished the mating process and responses to various stresses. A lack of vacuole formation and aberrant inner cell membrane structures were also observed in Δvps901Δvps902 cells by electron microscopy. These data strongly suggest that Vps901 and Vps902 are cooperatively involved in the regulation of cellular functions, such as cell morphology, sexual development, response to ion stresses, and vacuole formation, via Rab5 signaling pathways in fission yeast cells. - Highlights: • Roles of Rab5 activator VPS9 proteins in cellular functions. • Cooperation between VPS9 proteins in Rab5 signaling pathway. • Roles of each VPS9 protein in Rab5 signaling pathway are discussed

  10. Coordinated regulation by two VPS9 domain-containing guanine nucleotide exchange factors in small GTPase Rab5 signaling pathways in fission yeast

    Energy Technology Data Exchange (ETDEWEB)

    Tsukamoto, Yuta [Department of Biology, Graduate School of Science, Kobe University, 1-1 Rokkodai-cho, Nada, Kobe 657-8501 (Japan); Kagiwada, Satoshi [Department of Biological Sciences, Faculty of Science, Nara Women' s University, Kitauoyanishi-machi, Nara 630-8506 (Japan); Shimazu, Sayuri [Center for Supports to Research and Education Activities, Kobe University, 1-1 Rokkodai-cho, Nada, Kobe 657-8501 (Japan); Takegawa, Kaoru [Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581 (Japan); Noguchi, Tetsuko [Department of Biological Sciences, Faculty of Science, Nara Women' s University, Kitauoyanishi-machi, Nara 630-8506 (Japan); Miyamoto, Masaaki, E-mail: miya@kobe-u.ac.jp [Department of Biology, Graduate School of Science, Kobe University, 1-1 Rokkodai-cho, Nada, Kobe 657-8501 (Japan); Center for Supports to Research and Education Activities, Kobe University, 1-1 Rokkodai-cho, Nada, Kobe 657-8501 (Japan)

    2015-03-20

    The small GTPase Rab5 is reported to regulate various cellular functions, such as vesicular transport and endocytosis. VPS9 domain-containing proteins are thought to activate Rab5(s) by their guanine-nucleotide exchange activities. Numerous VPS9 proteins have been identified and are structurally conserved from yeast to mammalian cells. However, the functional relationships among VPS9 proteins in cells remain unclear. Only one Rab5 and two VPS9 proteins were identified in the Schizosaccharomyces pombe genome. Here, we examined the cellular function of two VPS9 proteins and the relationship between these proteins in cellular functions. Vps901-GFP and Vps902-GFP exhibited dotted signals in vegetative and differentiated cells. vps901 deletion mutant (Δvps901) cells exhibited a phenotype deficient in the mating process and responses to high concentrations of ions, such as calcium and metals, and Δvps901Δvps902 double mutant cells exhibited round cell shapes similar to ypt5-909 (Rab5 mutant allele) cells. Deletion of both vps901 and vps902 genes completely abolished the mating process and responses to various stresses. A lack of vacuole formation and aberrant inner cell membrane structures were also observed in Δvps901Δvps902 cells by electron microscopy. These data strongly suggest that Vps901 and Vps902 are cooperatively involved in the regulation of cellular functions, such as cell morphology, sexual development, response to ion stresses, and vacuole formation, via Rab5 signaling pathways in fission yeast cells. - Highlights: • Roles of Rab5 activator VPS9 proteins in cellular functions. • Cooperation between VPS9 proteins in Rab5 signaling pathway. • Roles of each VPS9 protein in Rab5 signaling pathway are discussed.

  11. [3H]WB4101 labels the 5-HT1A serotonin receptor subtype in rat brain. Guanine nucleotide and divalent cation sensitivity

    International Nuclear Information System (INIS)

    Norman, A.B.; Battaglia, G.; Creese, I.

    1985-01-01

    In the presence of a 30 nM prazosin mask, [ 3 H]-2-(2,6-dimethoxyphenoxyethyl) aminomethyl-1,4-benzodioxane ([ 3 H]WB4101) can selectively label 5-HT1 serotonin receptors. Serotonin exhibits high affinity (Ki = 2.5 nM) and monophasic competition for [ 3 H] WB4101 binding in cerebral cortex. We have found a significant correlation (r = 0.96) between the affinities of a number of serotonergic and nonserotonergic compounds at [ 3 H]WB4101-binding sites in the presence of 30 nM prazosin and [ 3 H] lysergic acid diethylamide ([ 3 H]LSD)-labeled 5-HT1 serotonin receptors in homogenates of rat cerebral cortex. Despite similar pharmacological profiles, distribution studies indicate that, in the presence of 5 mM MgSO4, the Bmax of [ 3 H]WB4101 is significantly lower than the Bmax of [ 3 H]LSD in various brain regions. WB4101 competition for [ 3 H] LSD-labeled 5-HT1 receptors fits best to a computer-derived model assuming two binding sites, with the KH for WB4101 being similar to the KD of [ 3 H]WB4101 binding derived from saturation experiments. This suggests that [ 3 H]WB4101 labels only one of the subtypes of the 5-HT1 serotonin receptors labeled by [ 3 H]LSD. The selective 5-HT1A serotonin receptor antagonist, spiperone, and the selective 5-HT1A agonist, 8-hydroxy-2-(di-n-propylamino) tetraline, exhibit high affinity and monophasic competition for [ 3 H]WB4101 but compete for multiple [ 3 H]LSD 5-HT1 binding sites. These data indicate that [ 3 H]WB4101 selectively labels the 5-HT1A serotonin receptor, whereas [ 3 H] LSD appears to label both the 5-HT1A and the 5-HT1B serotonin receptor subtypes. The divalent cations, Mn2+, Mg2+, and Ca2+ were found to markedly increase the affinity and Bmax of [ 3 H]WB4101 binding in cerebral cortex. Conversely, the guanine nucleotides guanylylimidodiphosphate and GTP, but not the adenosine nucleotide ATP, markedly reduce the Bmax of [ 3 H]WB4101 binding

  12. The signaling pathway of Campylobacter jejuni-induced Cdc42 activation: Role of fibronectin, integrin beta1, tyrosine kinases and guanine exchange factor Vav2

    LENUS (Irish Health Repository)

    Krause-Gruszczynska, Malgorzata

    2011-12-28

    Abstract Background Host cell invasion by the foodborne pathogen Campylobacter jejuni is considered as one of the primary reasons of gut tissue damage, however, mechanisms and key factors involved in this process are widely unclear. It was reported that small Rho GTPases, including Cdc42, are activated and play a role during invasion, but the involved signaling cascades remained unknown. Here we utilised knockout cell lines derived from fibronectin-\\/-, integrin-beta1-\\/-, focal adhesion kinase (FAK)-\\/- and Src\\/Yes\\/Fyn-\\/- deficient mice, and wild-type control cells, to investigate C. jejuni-induced mechanisms leading to Cdc42 activation and bacterial uptake. Results Using high-resolution scanning electron microscopy, GTPase pulldowns, G-Lisa and gentamicin protection assays we found that each studied host factor is necessary for induction of Cdc42-GTP and efficient invasion. Interestingly, filopodia formation and associated membrane dynamics linked to invasion were only seen during infection of wild-type but not in knockout cells. Infection of cells stably expressing integrin-beta1 variants with well-known defects in fibronectin fibril formation or FAK signaling also exhibited severe deficiencies in Cdc42 activation and bacterial invasion. We further demonstrated that infection of wild-type cells induces increasing amounts of phosphorylated FAK and growth factor receptors (EGFR and PDGFR) during the course of infection, correlating with accumulating Cdc42-GTP levels and C. jejuni invasion over time. In studies using pharmacological inhibitors, silencing RNA (siRNA) and dominant-negative expression constructs, EGFR, PDGFR and PI3-kinase appeared to represent other crucial components upstream of Cdc42 and invasion. siRNA and the use of Vav1\\/2-\\/- knockout cells further showed that the guanine exchange factor Vav2 is required for Cdc42 activation and maximal bacterial invasion. Overexpression of certain mutant constructs indicated that Vav2 is a linker

  13. The signaling pathway of Campylobacter jejuni-induced Cdc42 activation: Role of fibronectin, integrin beta1, tyrosine kinases and guanine exchange factor Vav2

    Directory of Open Access Journals (Sweden)

    Krause-Gruszczynska Malgorzata

    2011-12-01

    Full Text Available Abstract Background Host cell invasion by the foodborne pathogen Campylobacter jejuni is considered as one of the primary reasons of gut tissue damage, however, mechanisms and key factors involved in this process are widely unclear. It was reported that small Rho GTPases, including Cdc42, are activated and play a role during invasion, but the involved signaling cascades remained unknown. Here we utilised knockout cell lines derived from fibronectin-/-, integrin-beta1-/-, focal adhesion kinase (FAK-/- and Src/Yes/Fyn-/- deficient mice, and wild-type control cells, to investigate C. jejuni-induced mechanisms leading to Cdc42 activation and bacterial uptake. Results Using high-resolution scanning electron microscopy, GTPase pulldowns, G-Lisa and gentamicin protection assays we found that each studied host factor is necessary for induction of Cdc42-GTP and efficient invasion. Interestingly, filopodia formation and associated membrane dynamics linked to invasion were only seen during infection of wild-type but not in knockout cells. Infection of cells stably expressing integrin-beta1 variants with well-known defects in fibronectin fibril formation or FAK signaling also exhibited severe deficiencies in Cdc42 activation and bacterial invasion. We further demonstrated that infection of wild-type cells induces increasing amounts of phosphorylated FAK and growth factor receptors (EGFR and PDGFR during the course of infection, correlating with accumulating Cdc42-GTP levels and C. jejuni invasion over time. In studies using pharmacological inhibitors, silencing RNA (siRNA and dominant-negative expression constructs, EGFR, PDGFR and PI3-kinase appeared to represent other crucial components upstream of Cdc42 and invasion. siRNA and the use of Vav1/2-/- knockout cells further showed that the guanine exchange factor Vav2 is required for Cdc42 activation and maximal bacterial invasion. Overexpression of certain mutant constructs indicated that Vav2 is a linker

  14. pH-Modulated Watson-Crick duplex-quadruplex equilibria of guanine-rich and cytosine-rich DNA sequences 140 base pairs upstream of the c-kit transcription initiation site.

    Science.gov (United States)

    Bucek, Pavel; Jaumot, Joaquim; Aviñó, Anna; Eritja, Ramon; Gargallo, Raimundo

    2009-11-23

    Guanine-rich regions of DNA are sequences capable of forming G-quadruplex structures. The formation of a G-quadruplex structure in a region 140 base pairs (bp) upstream of the c-kit transcription initiation site was recently proposed (Fernando et al., Biochemistry, 2006, 45, 7854). In the present study, the acid-base equilibria and the thermally induced unfolding of the structures formed by a guanine-rich region and by its complementary cytosine-rich strand in c-kit were studied by means of circular dichroism and molecular absorption spectroscopies. In addition, competition between the Watson-Crick duplex and the isolated structures was studied as a function of pH value and temperature. Multivariate data analysis methods based on both hard and soft modeling were used to allow accurate quantification of the various acid-base species present in the mixtures. Results showed that the G-quadruplex and i-motif coexist with the Watson-Crick duplex over the pH range from 3.0 to 6.5, approximately, under the experimental conditions tested in this study. At pH 7.0, the duplex is practically the only species present.

  15. Platelet cytosolic 44-kDa protein is a substrate of cholera toxin-induced ADP-ribosylation and is not recognized by antisera against the α subunit of the stimulatory guanine nucleotide-binding regulatory protein

    International Nuclear Information System (INIS)

    Molina Y Vedia, L.M.; Reep, B.R.; Lapetina, E.G.

    1988-01-01

    ADP-ribosylation induced by cholera toxin and pertussis toxin was studied in particulate and cytosolic fractions of human platelets. Platelets were disrupted by a cycle of freezing and thawing in the presence of a hyposmotic buffer containing protease inhibitors. In both fractions, the A subunit of cholera toxin ADP-ribosylates two proteins with molecular masses of 42 and 44 kDa, whereas pertussis toxin ADP-ribosylates a 41-kDa polypeptide. Two antisera against the α subunit of the stimulatory guanine nucleotide-binding regulatory protein recognize only the 42-kDa polypeptide. Cholera toxin-induced ADP-ribosylation of the 42- and 44-kDa proteins is reduced by pretreatment of platelets with iloprost, a prostacyclin analog. The 44-kDa protein, which is substrate of cholera toxin, could be extracted completely from the membrane and recovered in the cytosolic fraction when the cells were disrupted by Dounce homogenization and the pellet was extensively washed. A 44-kDa protein can also be labeled with 8-azidoguanosine 5'-[α- 32 P]triphosphate in the cytosol and membranes. These finding indicate that cholera and pertussis toxins produced covalent modifications of proteins present in particulate and cytosolic platelet fractions. Moreover, the 44-kDa protein might be an α subunit of a guanine nucleotide-binding regulatory protein that is not recognized by available antisera

  16. DNA sequence polymorphisms within the bovine guanine nucleotide-binding protein Gs subunit alpha (Gsα-encoding (GNAS genomic imprinting domain are associated with performance traits

    Directory of Open Access Journals (Sweden)

    Mullen Michael P

    2011-01-01

    Full Text Available Abstract Background Genes which are epigenetically regulated via genomic imprinting can be potential targets for artificial selection during animal breeding. Indeed, imprinted loci have been shown to underlie some important quantitative traits in domestic mammals, most notably muscle mass and fat deposition. In this candidate gene study, we have identified novel associations between six validated single nucleotide polymorphisms (SNPs spanning a 97.6 kb region within the bovine guanine nucleotide-binding protein Gs subunit alpha gene (GNAS domain on bovine chromosome 13 and genetic merit for a range of performance traits in 848 progeny-tested Holstein-Friesian sires. The mammalian GNAS domain consists of a number of reciprocally-imprinted, alternatively-spliced genes which can play a major role in growth, development and disease in mice and humans. Based on the current annotation of the bovine GNAS domain, four of the SNPs analysed (rs43101491, rs43101493, rs43101485 and rs43101486 were located upstream of the GNAS gene, while one SNP (rs41694646 was located in the second intron of the GNAS gene. The final SNP (rs41694656 was located in the first exon of transcripts encoding the putative bovine neuroendocrine-specific protein NESP55, resulting in an aspartic acid-to-asparagine amino acid substitution at amino acid position 192. Results SNP genotype-phenotype association analyses indicate that the single intronic GNAS SNP (rs41694646 is associated (P ≤ 0.05 with a range of performance traits including milk yield, milk protein yield, the content of fat and protein in milk, culled cow carcass weight and progeny carcass conformation, measures of animal body size, direct calving difficulty (i.e. difficulty in calving due to the size of the calf and gestation length. Association (P ≤ 0.01 with direct calving difficulty (i.e. due to calf size and maternal calving difficulty (i.e. due to the maternal pelvic width size was also observed at the rs

  17. NMR solution structure of an N2-guanine DNA adduct derived from the potent tumorigen dibenzo[a,l]pyrene: Intercalation from the minor groove with ruptured Watson-Crick base pairing

    Science.gov (United States)

    Tang, Yijin; Liu, Zhi; Ding, Shuang; Lin, Chin H.; Cai, Yuqin; Rodriguez, Fabian A.; Sayer, Jane M.; Jerina, Donald M.; Amin, Shantu; Broyde, Suse; Geacintov, Nicholas E.

    2012-01-01

    The most potent tumorigen identified among the polycyclic aromatic hydrocarbons (PAH) is the non-planar fjord region dibenzo[a,l]pyrene (DB[a,l]P). It is metabolically activated in vivo through the widely-studied diol epoxide (DE) pathway to form covalent adducts with DNA bases, predominantly guanine and adenine. The (+)-11S,12R,13R,14S DE enantiomer forms adducts via its C14-position with the exocyclic amino group of guanine. Here, we present the first NMR solution structure of a DB[a,l]P-derived adduct, the 14R (+)-trans-anti-DB[a,l]P–N2-dG (DB[a,l]P-dG) lesion in double-stranded DNA. In contrast to the stereochemically identical benzo[a]pyrene-derived N2-dG adduct (B[a]P-dG) in which the B[a]P rings reside in the B-DNA minor groove on the 3’-side of the modifed deoxyguanosine, in the DB[a,l]P-derived adduct the DB[a,l]P rings intercalate into the duplex on the 3’-side of the modified base from the sterically crowded minor groove. Watson-Crick base pairing of the modified guanine with the partner cytosine is broken, but these bases retain some stacking with the bulky DB[a,l]P ring system. This new theme in PAH DE - DNA adduct conformation differs from: (1) the classical intercalation motif where Watson-Crick base-pairing is intact at the lesion site, and (2) the base-displaced intercalation motif in which the damaged base and its partner are extruded from the helix . The structural considerations that lead to the intercalated conformation of the DB[a,l]P-dG lesion in contrast to the minor groove alignment of the B[a]P-dG adduct, and the implications of the DB[a,l]P-dG conformational motif for the recognition of such DNA lesions by the human nucleotide excision repair apparatus, are discussed. PMID:23121427

  18. Nuclear magnetic resonance solution structure of an N(2)-guanine DNA adduct derived from the potent tumorigen dibenzo[a,l]pyrene: intercalation from the minor groove with ruptured Watson-Crick base pairing.

    Science.gov (United States)

    Tang, Yijin; Liu, Zhi; Ding, Shuang; Lin, Chin H; Cai, Yuqin; Rodriguez, Fabian A; Sayer, Jane M; Jerina, Donald M; Amin, Shantu; Broyde, Suse; Geacintov, Nicholas E

    2012-12-04

    The most potent tumorigen identified among the polycyclic aromatic hydrocarbons (PAH) is the nonplanar fjord region dibenzo[a,l]pyrene (DB[a,l]P). It is metabolically activated in vivo through the widely studied diol epoxide (DE) pathway to form covalent adducts with DNA bases, predominantly guanine and adenine. The (+)-11S,12R,13R,14S DE enantiomer forms adducts via its C14 position with the exocyclic amino group of guanine. Here, we present the first nuclear magnetic resonance solution structure of a DB[a,l]P-derived adduct, the 14R-(+)-trans-anti-DB[a,l]P-N(2)-dG (DB[a,l]P-dG) lesion in double-stranded DNA. In contrast to the stereochemically identical benzo[a]pyrene-derived N(2)-dG adduct (B[a]P-dG) in which the B[a]P rings reside in the B-DNA minor groove on the 3'-side of the modifed deoxyguanosine, in the DB[a,l]P-derived adduct the DB[a,l]P rings intercalate into the duplex on the 3'-side of the modified base from the sterically crowded minor groove. Watson-Crick base pairing of the modified guanine with the partner cytosine is broken, but these bases retain some stacking with the bulky DB[a,l]P ring system. This new theme in PAH DE-DNA adduct conformation differs from (1) the classical intercalation motif in which Watson-Crick base pairing is intact at the lesion site and (2) the base-displaced intercalation motif in which the damaged base and its partner are extruded from the helix. The structural considerations that lead to the intercalated conformation of the DB[a,l]P-dG lesion in contrast to the minor groove alignment of the B[a]P-dG adduct, and the implications of the DB[a,l]P-dG conformational motif for the recognition of such DNA lesions by the human nucleotide excision repair apparatus, are discussed.

  19. On the Formation and Properties of Interstrand DNA-DNA Cross-links Forged by Reaction of an Abasic Site With the Opposing Guanine Residue of 5′-CAp Sequences in Duplex DNA

    Science.gov (United States)

    Johnson, Kevin M.; Price, Nathan E.; Wang, Jin; Fekry, Mostafa I.; Dutta, Sanjay; Seiner, Derrick R.; Wang, Yinsheng; Gates, Kent S.

    2014-01-01

    We recently reported that the aldehyde residue of an abasic (Ap) site in duplex DNA can generate an interstrand cross-link via reaction with a guanine residue on the opposing strand. This finding is intriguing because the highly deleterious nature of interstrand cross-links suggests that even small amounts of Ap-derived cross-links could make a significant contribution to the biological consequences stemming from the generation of Ap sites in cellular DNA. Incubation of 21-bp duplexes containing a central 5′-CAp sequence under conditions of reductive amination (NaCNBH3, pH 5.2) generated much higher yields of cross-linked DNA than reported previously. At pH 7, in the absence of reducing agents, these Ap-containing duplexes also produced cross-linked duplexes that were readily detected on denaturing polyacrylamide gels. Cross-link formation was not highly sensitive to reaction conditions and, once formed, the cross-link was stable to a variety of work-up conditions. Results of multiple experiments including MALDI-TOF mass spectrometry, gel mobility, methoxyamine capping of the Ap aldehyde, inosine-for-guanine replacement, hydroxyl radical footprinting, and LCMS/MS were consistent with a cross-linking mechanism involving reversible reaction of the Ap aldehyde residue with the N2-amino group of the opposing guanine residue in 5′-CAp sequences to generate hemiaminal, imine, or cyclic hemiaminal cross-links (7-10) that were irreversibly converted under conditions of reductive amination (NaCNBH3/pH 5.2) to a stable amine linkage. Further support for the importance of the exocyclic N2-amino group in this reaction was provided by an experiment showing that installation of a 2-aminopurine-thymine base pair at the cross-linking site produced high yields (15-30%) of a cross-linked duplex at neutral pH, in the absence of NaCNBH3. PMID:23215239

  20. High-performance liquid chromatography/electrospray mass spectrometry for the analysis of modified bases in DNA: 7-(2-hydroxyethyl)guanine, the major ethylene oxide-DNA adduct.

    Science.gov (United States)

    Leclercq, L; Laurent, C; De Pauw, E

    1997-05-15

    A method was developed for the analysis of 7-(2-hydroxyethyl)guanine (7HEG), the major DNA adduct formed after exposure to ethylene oxide (EO). The method is based on DNA neutral thermal hydrolysis, adduct micro-concentration, and final characterization and quantification by HPLC coupled to single-ion monitoring electrospray mass spectrometry (HPLC/SIR-ESMS). The method was found to be selective, sensitive, and easy to handle with no need for enzymatic digestion or previous sample derivatization. Detection limit was found to be close to 1 fmol of adduct injected (10(-10) M), thus allowing the detection of approximately three modified bases on 10(8) intact nucleotides in blood sample analysis. Quantification results are shown for 7HEG after calf thymus DNA and blood exposure to various doses of EO, in both cases obtaining clear dose-response relationships.

  1. PDE1A inhibition elicits cGMP-dependent relaxation of rat mesenteric arteries

    DEFF Research Database (Denmark)

    Khammy, Makhala Michell; Dalsgaard, Thomas; Larsen, Peter Hjorringgaard

    2017-01-01

    (EC50 = 32 nM). Inhibition of NOS with L-NAME, soluble GC with ODQ, or PKG with Rp-8-Br-PET-cGMP all attenuated PDE1 inhibition-induced relaxation, whereas PKA inhibition with H89 had no effect. CONCLUSION AND IMPLICATIONS: Pde1a was the dominant PDE1 isoform present in VSMC and relaxation mediated...... by PDE1A-inhibition was predominantly driven by enhanced cGMP signalling. These results imply that isoform-selective PDE1 inhibitors are powerful investigative tools allowing examination of physiological and pathological roles of PDE1 isoforms....

  2. Mixed adenine/guanine quartets with three trans-a2 Pt(II) (a=NH(3) or MeNH(2)) cross-links: linkage and rotational isomerism, base pairing, and loss of NH(3).

    Science.gov (United States)

    Albertí, Francisca M; Rodríguez-Santiago, Luis; Sodupe, Mariona; Mirats, Andrea; Kaitsiotou, Helena; Sanz Miguel, Pablo J; Lippert, Bernhard

    2014-03-17

    Of the numerous ways in which two adenine and two guanines (N9 positions blocked in each) can be cross-linked by three linear metal moieties such as trans-a2 Pt(II) (with a=NH3 or MeNH2 ) to produce open metalated purine quartets with exclusive metal coordination through N1 and N7 sites, one linkage isomer was studied in detail. The isomer trans,trans,trans-[{Pt(NH3 )2 (N7-9-EtA-N1)2 }{Pt(MeNH2 )2 (N7-9-MeGH)}2 ][(ClO4 )6 ]⋅3H2 O (1) (with 9-EtA=9-ethyladenine and 9-MeGH=9-methylguanine) was crystallized from water and found to adopt a flat Z-shape in the solid state as far as the trinuclear cation is concerned. In the presence of excess 9-MeGH, a meander-like construct, trans,trans,trans-[{Pt(NH3 )2 (N7-9-EtA-N1)2 }{Pt(MeNH2 )2 (N7-9-MeGH)2 }][(ClO4 )6 ]⋅[(9-MeGH)2 ]⋅7 H2 O (2) is formed, in which the two extra 9-MeGH nucleobases are hydrogen bonded to the two terminal platinated guanine ligands of 1. Compound 1, and likewise the analogous complex 1 a (with NH3 ligands only), undergo loss of an ammonia ligand and formation of NH4 (+) when dissolved in [D6 ]DMSO. From the analogy between the behavior of 1 and 1 a it is concluded that a NH3 ligand from the central Pt atom is lost. Addition of 1-methylcytosine (1-MeC) to such a DMSO solution reveals coordination of 1-MeC to the central Pt. In an analogous manner, 9-MeGH can coordinate to the central Pt in [D6 ]DMSO. It is proposed that the proton responsible for formation of NH4 (+) is from one of the exocyclic amino groups of the two adenine bases, and furthermore, that this process is accompanied by a conformational change of the cation from Z-form to U-form. DFT calculations confirm the proposed mechanism and shed light on possible pathways of this process. Calculations show that rotational isomerism is not kinetically hindered and that it would preferably occur previous to the displacement of NH3 by DMSO. This displacement is the most energetically costly step, but it is compensated by the proton

  3. Isotope Dilution nanoLC/ESI+-HRMS3 Quantitation of Urinary N7-(1-Hydroxy-3-buten-2-yl) Guanine Adducts in Humans and Their Use as Biomarkers of Exposure to 1,3-Butadiene.

    Science.gov (United States)

    Sangaraju, Dewakar; Boldry, Emily J; Patel, Yesha M; Walker, Vernon; Stepanov, Irina; Stram, Daniel; Hatsukami, Dorothy; Tretyakova, Natalia

    2017-02-20

    1,3-Butadiene (BD) is an important industrial and environmental chemical classified as a known human carcinogen. Occupational exposure to BD in the polymer and monomer industries is associated with an increased incidence of lymphoma. BD is present in automobile exhaust, cigarette smoke, and forest fires, raising concern about potential exposure of the general population to this carcinogen. Following inhalation exposure, BD is bioactivated to 3,4-epoxy-1-butene (EB). If not detoxified, EB is capable of modifying guanine and adenine bases of DNA to form nucleobase adducts, which interfere with accurate DNA replication and cause cancer-initiating mutations. We have developed a nanoLC/ESI + -HRMS 3 methodology for N7-(1-hydroxy-3-buten-2-yl) guanine (EB-GII) adducts in human urine (limit of detection: 0.25 fmol/mL urine; limit of quantitation: 1.0 fmol/mL urine). This new method was successfully used to quantify EB-GII in urine of F344 rats treated with 0-200 ppm of BD, occupationally exposed workers, and smokers belonging to two different ethnic groups. EB-GII amounts increased in a dose-dependent manner in urine of laboratory rats exposed to 0, 62.5, or 200 ppm of BD. Urinary EB-GII levels were significantly increased in workers occupationally exposed to 0.1-2.2 ppm of BD (1.25 ± 0.51 pg/mg of creatinine) as compared to administrative controls exposed to <0.01 ppm of BD (0.22 ± 0.08 and pg/mg of creatinine) (p = 0.0024), validating the use of EB-GII as a biomarker of human exposure to BD. EB-GII was also detected in smokers' urine with European American smokers excreting significantly higher amounts of EB-GII than African American smokers (0.48 ± 0.09 vs 0.12 ± 0.02 pg/mg of creatinine, p = 3.1 × 10 -7 ). Interestingly, small amounts of EB-GII were observed in animals and humans with no known exposure to BD, providing preliminary evidence for its endogenous formation. Urinary EB-GII adduct levels and urinary mercapturic acids of BD (MHBMA, DHBMA) were compared

  4. Synthesis and preliminary evaluation of 9-(4-[{sup 18}F]fluoro-3-hydroxymethylbutyl) guanine ([{sup 18}F]FHBG) in HSV1-tk gene transduced hepatoma cell

    Energy Technology Data Exchange (ETDEWEB)

    Moon, Byung Seok; Lee, Tae Sup [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of); Lee, Myoung Keun [Yonsei University, Wonju (Korea, Republic of)] (and others)

    2006-08-15

    The HSV1-tk reporter gene system is the most widely used system because of its advantage that direct monitoring is possible without the introduction of a separate reporter gene in case of HSV1-tk suicide gene therapy. In this study, we investigate the usefulness of the reporter probe (substrate), 9-(4-[{sup 18}F]fluoro-3-hydroxymethylbutyl) guanine ([{sup 18}F]FHBG) for non-invasive reporter gene imaging using PET in HSV1-tk expressing hepatoma model. Radiolabeled FHBG was prepared in 8 steps from a commercially available triester. The labeling reaction was carried out by NCA nucleophilic substitution with K[{sup 18}F]/K2.2.2. in acetonitrile using N2-monomethoxytrityl-9-[4-(tosly)-3-monomethoxytritylmethylbutl] guanine as a precursor, followed by deprotection with 1 N HCI. Preliminary biological properties of the probe were evaluated with MCA cells and MCA-tk cells transduced with HSV1-tk reporter gene. In vitro uptake and release-out studies of [{sup 18}F]FHBG were performed, and was analyzed correlation between [{sup 18}F]FHBG uptake ratio according to increasing numeric count of MCA-tk cells and degree of gene expression. MicroPET scan image was obtained with MCA and MCA-tk tumor beating Balb/c-nude mouse model. [{sup 18}F]FHBG was purified by reverse phase semi-HPLC system and collected at around 16-18 min. Radiochemical yield was about 20-25% (corrected for decay), radiochemical purity was > 95% and specific activity was around > 55.5 GBq/ {mu} mol. Specific accumulation of [{sup 18}F]FHBG was observed in HSV1-tk gene transduced MCA-tk cells but not MCA cells, and consecutive 1 hour release-out results showed more than 86% of uptaked [{sup 18}F]FHBG was retained inside of cells. The uptake of [{sup 18}F]FHBG was showed a highly significant linear correlation (R{sup 2} = 0.995) with increasing percentage of MCA-tk numeric cell count. In microPET scan images, remarkable difference of accumulation was observed for the two type of tumors. [{sup 18}F]FHBG appears

  5. Increased GABA(A) inhibition of the RVLM after hindlimb unloading in rats

    Science.gov (United States)

    Moffitt, Julia A.; Heesch, Cheryl M.; Hasser, Eileen M.

    2002-01-01

    Attenuated baroreflex-mediated increases in renal sympathetic nerve activity (RSNA) in hindlimb unloaded (HU) rats apparently are due to changes within the central nervous system. We hypothesized that GABA(A) receptor-mediated inhibition of the rostral ventrolateral medulla (RVLM) is increased after hindlimb unloading. Responses to bilateral microinjection of the GABA(A) antagonist (-)-bicuculline methiodide (BIC) into the RVLM were examined before and during caudal ventrolateral medulla (CVLM) inhibition in Inactin-anesthetized control and HU rats. Increases in mean arterial pressure (MAP), heart rate (HR), and RSNA in response to BIC in the RVLM were significantly enhanced in HU rats. Responses to bilateral CVLM blockade were not different. When remaining GABA(A) inhibition in the RVLM was blocked by BIC during CVLM inhibition, the additional increases in MAP and RSNA were significantly greater in HU rats. These data indicate that GABA(A) receptor-mediated inhibition of RVLM neurons is augmented after hindlimb unloading. Effects of input from the CVLM were unaltered. Thus, after cardiovascular deconditioning in rodents, the attenuated increase in sympathetic nerve activity in response to hypotension is associated with greater GABA(A) receptor-mediated inhibition of RVLM neurons originating at least in part from sources other than the CVLM.

  6. Monoamine Oxidase-A Inhibition and Associated Antioxidant Activity in Plant Extracts with Potential Antidepressant Actions

    Directory of Open Access Journals (Sweden)

    Tomás Herraiz

    2018-01-01

    Full Text Available Monoamine oxidase (MAO catalyzes the oxidative deamination of amines and neurotransmitters and is involved in mood disorders, depression, oxidative stress, and adverse pharmacological reactions. This work studies the inhibition of human MAO-A by Hypericum perforatum, Peganum harmala, and Lepidium meyenii, which are reported to improve and affect mood and mental conditions. Subsequently, the antioxidant activity associated with the inhibition of MAO is determined in plant extracts for the first time. H. perforatum inhibited human MAO-A, and extracts from flowers gave the highest inhibition (IC50 of 63.6 μg/mL. Plant extracts were analyzed by HPLC-DAD-MS and contained pseudohypericin, hypericin, hyperforin, adhyperforin, hyperfirin, and flavonoids. Hyperforin did not inhibit human MAO-A and hypericin was a poor inhibitor of this isoenzyme. Quercetin and flavonoids significantly contributed to MAO-A inhibition. P. harmala seed extracts highly inhibited MAO-A (IC50 of 49.9 μg/L, being a thousand times more potent than H. perforatum extracts owing to its content of β-carboline alkaloids (harmaline and harmine. L. meyenii root (maca extracts did not inhibit MAO-A. These plants may exert protective actions related to antioxidant effects. Results in this work show that P. harmala and H. perforatum extracts exhibit antioxidant activity associated with the inhibition of MAO (i.e., lower production of H2O2.

  7. EVA1A inhibits GBM cell proliferation by inducing autophagy and apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Shen, Xue; Kan, Shifeng; Liu, Zhen [Department of Cell Biology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191 (China); Lu, Guang [Department of Cell Biology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191 (China); Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597 (Singapore); Zhang, Xiaoyan [Department of Cell Biology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191 (China); Chen, Yingyu [Department of Immunology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191 (China); Peking University Center for Human Disease Genomics, Beijing 100191 (China); Bai, Yun, E-mail: baiyun@bjmu.edu.cn [Department of Cell Biology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191 (China)

    2017-03-01

    Eva-1 homolog A (EVA1A) is a novel lysosome and endoplasmic reticulum-associated protein involved in autophagy and apoptosis. In this study, we constructed a recombinant adenovirus 5-EVA1A vector (Ad5-EVA1A) to overexpress EVA1A in glioblastoma (GBM) cell lines and evaluated its anti-tumor activities in vitro and in vivo. We found that overexpression of EVA1A in three GBM cell lines (U251, U87 and SHG44) resulted in a suppression of tumor cell growth via activation of autophagy and induction of cell apoptosis in a dose- and time-dependent manner. EVA1A-mediated autophagy was associated with inactivation of the mTOR/RPS6KB1 signaling pathway. Furthermore in vivo, overexpression of EVA1A successfully inhibited tumor growth in NOD/SCID mice. Our data suggest that EVA1A-induced autophagy and apoptosis play a role in suppressing the development of GBM and their up-regulation may be an effective method for treating this form of cancer. - Highlights: • Overexpression of EVA1A suppresses GBM cell growth. • EVA1A induces autophagy through the mTOR/RPS6KB1 pathway. • EVA1A induces GBM cell apoptosis. • EVA1A inhibits the development of GBM in vivo.

  8. Vitisin A inhibits adipocyte differentiation through cell cycle arrest in 3T3-L1 cells

    International Nuclear Information System (INIS)

    Kim, Soon-hee; Park, Hee-Sook; Lee, Myoung-su; Cho, Yong-Jin; Kim, Young-Sup; Hwang, Jin-Taek; Sung, Mi Jeong; Kim, Myung Sunny; Kwon, Dae Young

    2008-01-01

    Inhibition of adipocyte differentiation is one approach among the anti-obesity strategies. This study demonstrates that vitisin A, a resveratrol tetramer, inhibits adipocyte differentiation most effectively of 18 stilbenes tested. Fat accumulation and PPARγ expression were decreased by vitisin A in a dose-dependent manner. Vitisin A significantly inhibited preadipocyte proliferation and consequent differentiation within the first 2 days of treatment, indicating that the anti-adipogenic effect of vitisin A was derived from anti-proliferation. Based on cell cycle analysis, vitisin A blocked the cell cycle at the G1-S phase transition, causing cells to remain in the preadipocyte state. Vitisin A increased p21 expression, while the Rb phosphorylation level was reduced. Therefore, vitisin A seems to induce G1 arrest through p21- and consequent Rb-dependent suppression of transcription. On the other hand, ERK and Akt signaling pathways were not involved in the anti-mitotic regulation by vitisin A. Taken together, these results suggest that vitisin A inhibits adipocyte differentiation through preadipocyte cell cycle arrest

  9. Surface amplification of pencil graphite electrode with polypyrrole and reduced graphene oxide for fabrication of a guanine/adenine DNA based electrochemical biosensors for determination of didanosine anticancer drug

    Science.gov (United States)

    Karimi-Maleh, Hassan; Bananezhad, Asma; Ganjali, Mohammad R.; Norouzi, Parviz; Sadrnia, Abdolhossein

    2018-05-01

    Didanosine is nucleoside analog reverse transcriptase inhibitors with many side effects such as nausea and vomiting, stomach pain, tingling, burning and numbness and determination of this drug is very important in biological samples. This paper presents a DNA biosensor for determination of didanosine (DDI) in pharmaceutical samples. A pencil graphite electrode modified with conductive materials such as polypyrrole (PPy) and reduced graphene oxide (rGO) (PGE/PPy/rGO) was used for this goal. The double-stranded DNA was successfully immobilized on PGE/PPy/rGO. The PGE/PPy/rGO was characterized by microscopic and electrochemical methods. Then, the interaction of DDI with DNA was identified by decreases in the oxidation currents of guanine and adenine by differential pulse voltammetric (DPV) method. The dynamic range of DDI identified in the range of 0.02-50.0 μM and this electrode provided a low limit of detection (LOD = 8.0 nM) for DDI. The PGE/PPy/rGO loaded with ds-DNA was utilized for the measurement of DDI in real samples and obtained data were compared with HPLC method. The statistical tests such as F-test and t-test were used for confirming ability of PGE/PPy/rGO loaded with ds-DNA for analysis of DDI in real samples.

  10. WNT5A inhibits metastasis and alters splicing of Cd44 in breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Wen Jiang

    Full Text Available Wnt5a is a non-canonical signaling Wnt. Low expression of WNT5A is correlated with poor prognosis in breast cancer patients. The highly invasive breast cancer cell lines, MDA-MB-231 and 4T1, express very low levels of WNT5A. To determine if enhanced expression of WNT5A would affect metastatic behavior, we generated WNT5A expressing cells from the 4T1 and MDA-MB-231 parental cell lines. WNT5A expressing cells demonstrated cobblestone morphology and reduced in vitro migration relative to controls. Cell growth was not altered. Metastasis to the lung via tail vein injection was reduced in the 4T1-WNT5A expressing cells relative to 4T1-vector controls. To determine the mechanism of WNT5A action on metastasis, we performed microarray and whole-transcriptome sequence analysis (RNA-seq to compare gene expression in 4T1-WNT5A and 4T1-vector cells. Analysis indicated highly significant alterations in expression of genes associated with cellular movement. Down-regulation of a subset of these genes, Mmp13, Nos2, Il1a, Cxcl2, and Lamb3, in WNT5A expressing cells was verified by semi-quantitative RT-PCR. Significant differences in transcript splicing were also detected in cell movement associated genes including Cd44. Cd44 is an adhesion molecule with a complex genome structure. Variable exon usage is associated with metastatic phenotype. Alternative spicing of Cd44 in WNT5A expressing cells was confirmed using RT-PCR. We conclude that WNT5A inhibits metastasis through down-regulation of multiple cell movement pathways by regulating transcript levels and splicing of key genes like Cd44.

  11. Hypoxia-activated chemotherapeutic TH-302 enhances the effects of VEGF-A inhibition and radiation on sarcomas.

    Science.gov (United States)

    Yoon, C; Lee, H-J; Park, D J; Lee, Y-J; Tap, W D; Eisinger-Mathason, T S K; Hart, C P; Choy, E; Simon, M C; Yoon, S S

    2015-06-30

    Human sarcomas with a poor response to vascular endothelial growth factor-A (VEGF-A) inhibition and radiation therapy (RT) have upregulation of hypoxia-inducible factor 1α (HIF-1α) and HIF-1α target genes. This study examines the addition of the hypoxia-activated chemotherapy TH-302 to VEGF-A inhibition and RT (a.k.a. trimodality therapy). Trimodality therapy was examined in two xenograft models and in vitro in tumour endothelial cells and sarcoma cell lines. In both mouse models, VEGF-A inhibition and radiation showed greater efficacy than either therapy alone in slowing sarcoma growth. When TH-302 was added, this trimodality therapy completely blocked tumour growth with tumours remaining dormant for over 3 months after cessation of therapy. Trimodality therapy caused 2.6- to 6.2-fold more endothelial cell-specific apoptosis than bimodality therapies, and microvessel density and HIF-1α activity were reduced to 11-13% and 13-20% of control, respectively. When trimodality therapy was examined in vitro, increases in DNA damage and apoptosis were much more pronounced in tumour endothelial cells compared with that in sarcoma cells, especially under hypoxia. The combination of TH-302, VEGF-A inhibition, and RT is highly effective in preclinical models of sarcoma and is associated with increased DNA damage and apoptosis in endothelial cells and decreased HIF-1α activity.

  12. A novel missense variant (Gln220Arg) of GNB4 encoding guanine nucleotide-binding protein, subunit beta-4 in a Japanese family with autosomal dominant motor and sensory neuropathy.

    Science.gov (United States)

    Miura, Shiroh; Morikawa, Takuya; Fujioka, Ryuta; Noda, Kazuhito; Kosaka, Kengo; Taniwaki, Takayuki; Shibata, Hiroki

    2017-09-01

    Dominant intermediate Charcot-Marie-Tooth disease F (CMTDIF) is an autosomal dominant hereditary form of Charcot-Marie-Tooth disease (CMT) caused by variations in the guanine nucleotide-binding protein, subunit beta-4 gene (GNB4). We examined two Japanese familial cases with CMT. Case 1 was a 49-year-old male whose chief complaint was slowly progressive gait disturbance and limb dysesthesia that appeared at the age of 47. On neurological examination, he showed hyporeflexia or areflexia, distal limb muscle weakness, and distal sensory impairment with lower dominancy. Nerve conduction studies demonstrated demyelinating sensorimotor neuropathy with reduced action potentials in the lower limbs. Case 2 was an 80-year-old man, Case 1's father, who reported difficulty in riding a bicycle at the age of 76. On neurological examination, he showed areflexia in the upper and lower limbs. Distal sensory impairment in the lower limbs was also observed. Nerve conduction studies revealed mainly axonal involvement. Exome sequencing identified a novel heterozygous nonsynonymous variant (NM_021629.3:c.659T > C [p.Gln220Arg]) in GNB4 exon 8, which is known to be responsible for CMT. Sanger sequencing confirmed that both patients are heterozygous for the variation, which causes an amino acid substitution, Gln220Arg, in the highly conserved region of the WD40 domain of GNB4. The frequency of this variant in the Exome Aggregation Consortium Database was 0.000008247, and we confirmed its absence in 502 Japanese control subjects. We conclude that this novel GNB4 variant is causative for CMTDIF in these patients, who represent the first record of the disease in the Japanese population. Copyright © 2017. Published by Elsevier Masson SAS.

  13. Comprehensive behavioral analysis of mice deficient in Rapgef2 and Rapgef6, a subfamily of guanine nucleotide exchange factors for Rap small GTPases possessing the Ras/Rap-associating domain.

    Science.gov (United States)

    Maeta, Kazuhiro; Hattori, Satoko; Ikutomo, Junji; Edamatsu, Hironori; Bilasy, Shymaa E; Miyakawa, Tsuyoshi; Kataoka, Tohru

    2018-05-10

    Rapgef2 and Rapgef6 define a subfamily of guanine nucleotide exchange factors for Rap small GTPases, characterized by the possession of the Ras/Rap-associating domain. Previous genomic analyses suggested their possible involvement in the etiology of schizophrenia. We recently demonstrated the development of an ectopic cortical mass (ECM), which resembles the human subcortical band heterotopia, in the dorsal telencephalon-specific Rapgef2 conditional knockout (Rapgef2-cKO) brains. Additional knockout of Rapgef6 in Rapgef2-cKO mice resulted in gross enlargement of the ECM whereas knockout of Rapgef6 alone (Rapgef6-KO) had no discernible effect on the brain morphology. Here, we performed a battery of behavioral tests to examine the effects of Rapgef2 or Rapgef6 deficiency on higher brain functions. Rapgef2-cKO mice exhibited hyperlocomotion phenotypes. They showed decreased anxiety-like behavior in the elevated plus maze and the open-field tests as well as increased depression-like behavior in the Porsolt forced swim and tail suspension tests. They also exhibited increased sociability especially in novel environments. They showed defects in cognitive function as evidenced by reduced learning ability in the Barnes circular maze test and by impaired working memory in the T maze tests. In contrast, although Rapgef6 and Rapgef2 share similarities in biochemical roles, Rapgef6-KO mice exhibited mild behavioral abnormalities detected with a number of behavioral tests, such as hyperlocomotion phenotype in the open-field test and the social interaction test with a novel environment and working-memory defects in the T-maze test. In conclusion, although there were differences in their brain morphology and the magnitude of the behavioral abnormalities, Rapgef2-cKO mice and Rapgef6-KO mice exhibited hyperlocomotion phenotype and working-memory defect, both of which could be recognized as schizophrenia-like behavior.

  14. How does the long G·G* Watson-Crick DNA base mispair comprising keto and enol tautomers of the guanine tautomerise? The results of a QM/QTAIM investigation.

    Science.gov (United States)

    Brovarets', Ol'ha O; Hovorun, Dmytro M

    2014-08-14

    The double proton transfer (DPT) in the long G·G* Watson-Crick base mispair (|C6N1(G*)N1C6(G)| = 36.4°; C1 symmetry), involving keto and enol tautomers of the guanine (G) nucleobase, along two intermolecular neighboring O6H···O6 (8.39) and N1···HN1 (6.14 kcal mol(-1)) H-bonds that were established to be slightly anti-cooperative, leads to its transformation into the G*·G base mispair through a single transition state (|C6N1N1C6| = 37.1°; C1), namely to the interconversion into itself. It was shown that the G·G* ↔ G*·G tautomerisation via the DPT is assisted by the third specific contact, that sequentially switches along the intrinsic reaction coordinate (IRC) in an original way: (G)N2H···N2(G*) H-bond (-25.13 to -10.37) → N2···N2 van der Waals contact (-10.37 to -9.23) → (G)N2···HN2(G*) H-bond (-9.23 to 0.79) → (G*)N2···HN2(G) H-bond (0.79 to 7.35 Bohr). The DPT tautomerisation was found to proceed through the asynchronous concerted mechanism by employing the QM/QTAIM approach and the methodology of the scans of the geometric, electron-topological, energetic, polar and NBO properties along the IRC. Nine key points, that can be considered as part of the tautomerisation repertoire, have been established and analyzed in detail. Furthermore, it was shown that the G·G* or G*·G base mispair is a thermodynamically and dynamically stable structure with a lifetime of 8.22 × 10(-10) s and all 6 low-frequency intermolecular vibrations are able to develop during this time span. Lastly, our results highlight the importance of the G·G* ↔ G*·G DPT tautomerisation, which can have implications for biological and chemical sensing applications.

  15. Follicle-stimulating hormone receptor-mediated uptake of 45Ca2+ by cultured rat Sertoli cells does not require activation of cholera toxin- or pertussis toxin-sensitive guanine nucleotide binding proteins or adenylate cyclase

    International Nuclear Information System (INIS)

    Grasso, P.; Reichert, L.E. Jr.

    1990-01-01

    We have previously reported that FSH stimulates flux of 45Ca2+ into cultured Sertoli cells from immature rats via voltage-sensitive and voltage-independent calcium channels. In the present study, we show that this effect of FSH does not require cholera toxin (CT)- or pertussis toxin (PT)-sensitive guanine nucleotide binding (G) protein or activation of adenylate cyclase (AC). Significant stimulation of 45Ca2+ influx was observed within 1 min, and maximal response (3.2-fold over basal levels) was achieved within 2 min after exposure to FSH. FSH-stimulated elevations in cellular cAMP paralleled increases in 45Ca2+ uptake, suggesting a possible coupling of AC activation to 45Ca2+ influx. (Bu)2cAMP, however, was not able to enhance 45Ca2+ uptake over basal levels at a final concentration of 1000 microM, although a concentration-related increase in androstenedione conversion to estradiol was evident. Exposure of Sertoli cells to CT (10 ng/ml) consistently stimulated basal levels of androstenedione conversion to estradiol but had no effect on basal levels of 45Ca2+ uptake. Similarly, CT had no effect on FSH-induced 45Ca2+ uptake, but potentiated FSH-stimulated estradiol synthesis. PT (10 ng/ml) augmented basal and FSH-stimulated estradiol secretion without affecting 45Ca2+ influx. The adenosine analog N6-phenylisopropyladenosine, which binds to Gi-coupled adenosine receptors on Sertoli cells, inhibited FSH-stimulated androgen conversion to estradiol in a dose-related (1-1000 nM) manner, but FSH-stimulated 45Ca2+ influx remained unchanged. Our results show that in contrast to FSH-stimulated estradiol synthesis, the flux of 45Ca2+ into Sertoli cells in response to FSH is not mediated either directly or indirectly by CT- or PT-sensitive G protein, nor does it require activation of AC. Our data further suggest that the FSH receptor itself may function as a calcium channel

  16. Follicle-stimulating hormone receptor-mediated uptake of sup 45 Ca sup 2+ by cultured rat Sertoli cells does not require activation of cholera toxin- or pertussis toxin-sensitive guanine nucleotide binding proteins or adenylate cyclase

    Energy Technology Data Exchange (ETDEWEB)

    Grasso, P.; Reichert, L.E. Jr. (Albany Medical College, NY (USA))

    1990-08-01

    We have previously reported that FSH stimulates flux of 45Ca2+ into cultured Sertoli cells from immature rats via voltage-sensitive and voltage-independent calcium channels. In the present study, we show that this effect of FSH does not require cholera toxin (CT)- or pertussis toxin (PT)-sensitive guanine nucleotide binding (G) protein or activation of adenylate cyclase (AC). Significant stimulation of 45Ca2+ influx was observed within 1 min, and maximal response (3.2-fold over basal levels) was achieved within 2 min after exposure to FSH. FSH-stimulated elevations in cellular cAMP paralleled increases in 45Ca2+ uptake, suggesting a possible coupling of AC activation to 45Ca2+ influx. (Bu)2cAMP, however, was not able to enhance 45Ca2+ uptake over basal levels at a final concentration of 1000 microM, although a concentration-related increase in androstenedione conversion to estradiol was evident. Exposure of Sertoli cells to CT (10 ng/ml) consistently stimulated basal levels of androstenedione conversion to estradiol but had no effect on basal levels of 45Ca2+ uptake. Similarly, CT had no effect on FSH-induced 45Ca2+ uptake, but potentiated FSH-stimulated estradiol synthesis. PT (10 ng/ml) augmented basal and FSH-stimulated estradiol secretion without affecting 45Ca2+ influx. The adenosine analog N6-phenylisopropyladenosine, which binds to Gi-coupled adenosine receptors on Sertoli cells, inhibited FSH-stimulated androgen conversion to estradiol in a dose-related (1-1000 nM) manner, but FSH-stimulated 45Ca2+ influx remained unchanged. Our results show that in contrast to FSH-stimulated estradiol synthesis, the flux of 45Ca2+ into Sertoli cells in response to FSH is not mediated either directly or indirectly by CT- or PT-sensitive G protein, nor does it require activation of AC. Our data further suggest that the FSH receptor itself may function as a calcium channel.

  17. Topoisomerase IB of Deinococcus radiodurans resolves guanine ...

    Indian Academy of Sciences (India)

    2015-11-28

    Nov 28, 2015 ... All the oligonucleotides men- tioned here were ... further reactions with DraTopoIB were carried out at 37°C ... of G4 DNA moves faster than unfolded and intermolecular .... for its action on intramolecular G4 DNA structure was.

  18. MiR-34a inhibits colon cancer proliferation and metastasis by inhibiting platelet-derived growth factor receptor α.

    Science.gov (United States)

    Li, Chunyan; Wang, Yulin; Lu, Shuming; Zhang, Zhuqing; Meng, Hua; Liang, Lina; Zhang, Yan; Song, Bo

    2015-11-01

    The microRNA (miRNA), miR‑34a is significant in colon cancer progression. In the present study, the role of miR‑34a in colon cancer cell proliferation and metastasis was investigated. It was found that the expression of miR‑34a in colon cancer tissues and cell lines was lower when compared with that of normal tissues and cells. Further research demonstrated that miR‑34a inhibited cell proliferation, induced G1 phase arrest, and suppressed metastasis and epithelial mesenchymal transition in colon cancer cells. Bioinformatic prediction indicated that platelet‑derived growth factor receptor α (PDGFRA) was a potential target gene of miR‑34a and a luciferase assay identified that PDGFRA was a novel direct target gene of miR‑34a. In addition, assays of western blot analyses and quantitative reverse‑transcription polymerase chain reaction confirmed that miR‑34a decreased PDGFRA mRNA expression and protein levels in colon cancer cells. Assessment of cellular function indicated that miR‑34a inhibited colon cancer progression via PDGFRA. These findings demonstrate that miR‑34a may act as a negative regulator in colon cancer by targeting PDGFRA.

  19. Cyclosporin A inhibits the propagation of influenza virus by interfering with a late event in the virus life cycle.

    Science.gov (United States)

    Hamamoto, Itsuki; Harazaki, Kazuhiro; Inase, Naohiko; Takaku, Hiroshi; Tashiro, Masato; Yamamoto, Norio

    2013-01-01

    Influenza is a global public health problem that causes a serious respiratory disease. Influenza virus frequently undergoes amino acid substitutions, which result in the emergence of drug-resistant viruses. To control influenza viruses that are resistant to currently available drugs, it is essential to develop new antiviral drugs with a novel molecular target. Here, we report that cyclosporin A (CsA) inhibits the propagation of influenza virus in A549 cells by interfering with a late event in the virus life cycle. CsA did not affect adsorption, internalization, viral RNA replication, or synthesis of viral proteins in A549 cells, but inhibited the step(s) after viral protein synthesis, such as assembly or budding. In addition, siRNA-mediated knockdown of the expression of the major CsA targets, namely cyclophilin A (CypA), cyclophilin B (CypB), and P-glycoprotein (Pgp), did not inhibit influenza virus propagation. These results suggest that CsA inhibits virus propagation by mechanism(s) independent of the inhibition of the function of CypA, CypB, and Pgp. CsA may target an unknown molecule that works as a positive regulator in the propagation of influenza virus. Our findings would contribute to the development of a novel anti-influenza virus therapy and clarification of the regulatory mechanism of influenza virus multiplication.

  20. Foot-and-mouth disease virus non-structural protein 3A inhibits the interferon-β signaling pathway

    Science.gov (United States)

    Li, Dan; Lei, Caoqi; Xu, Zhisheng; Yang, Fan; Liu, Huanan; Zhu, Zixiang; Li, Shu; Liu, Xiangtao; Shu, Hongbing; Zheng, Haixue

    2016-01-01

    Foot-and-mouth disease virus (FMDV) is the etiological agent of FMD, which affects cloven-hoofed animals. The pathophysiology of FMDV has not been fully understood and the evasion of host innate immune system is still unclear. Here, the FMDV non-structural protein 3A was identified as a negative regulator of virus-triggered IFN-β signaling pathway. Overexpression of the FMDV 3A inhibited Sendai virus-triggered activation of IRF3 and the expressions of RIG-I/MDA5. Transient transfection and co-immunoprecipitation experiments suggested that FMDV 3A interacts with RIG-I, MDA5 and VISA, which is dependent on the N-terminal 51 amino acids of 3A. Furthermore, 3A also inhibited the expressions of RIG-I, MDA5, and VISA by disrupting their mRNA levels. These results demonstrated that 3A inhibits the RLR-mediated IFN-β induction and uncovered a novel mechanism by which the FMDV 3A protein evades the host innate immune system. PMID:26883855

  1. Is the DPT tautomerization of the long A·G Watson-Crick DNA base mispair a source of the adenine and guanine mutagenic tautomers? A QM and QTAIM response to the biologically important question.

    Science.gov (United States)

    Brovarets', Ol'ha O; Zhurakivsky, Roman O; Hovorun, Dmytro M

    2014-03-05

    Herein, we first address the question posed in the title by establishing the tautomerization trajectory via the double proton transfer of the adenine·guanine (A·G) DNA base mispair formed by the canonical tautomers of the A and G bases into the A*·G* DNA base mispair, involving mutagenic tautomers, with the use of the quantum-mechanical calculations and quantum theory of atoms in molecules (QTAIM). It was detected that the A·G ↔ A*·G* tautomerization proceeds through the asynchronous concerted mechanism. It was revealed that the A·G base mispair is stabilized by the N6H···O6 (5.68) and N1H···N1 (6.51) hydrogen bonds (H-bonds) and the N2H···HC2 dihydrogen bond (DH-bond) (0.68 kcal·mol(-1) ), whereas the A*·G* base mispair-by the O6H···N6 (10.88), N1H···N1 (7.01) and C2H···N2 H-bonds (0.42 kcal·mol(-1) ). The N2H···HC2 DH-bond smoothly and without bifurcation transforms into the C2H···N2 H-bond at the IRC = -10.07 Bohr in the course of the A·G ↔ A*·G* tautomerization. Using the sweeps of the energies of the intermolecular H-bonds, it was observed that the N6H···O6 H-bond is anticooperative to the two others-N1H···N1 and N2H···HC2 in the A·G base mispair, while the latters are significantly cooperative, mutually strengthening each other. In opposite, all three O6H···N6, N1H···N1, and C2H···N2 H-bonds are cooperative in the A*·G* base mispair. All in all, we established the dynamical instability of the А*·G* base mispair with a short lifetime (4.83·10(-14) s), enabling it not to be deemed feasible source of the A* and G* mutagenic tautomers of the DNA bases. The small lifetime of the А*·G* base mispair is predetermined by the negative value of the Gibbs free energy for the A*·G* → A·G transition. Moreover, all of the six low-frequency intermolecular vibrations cannot develop during this lifetime that additionally confirms the aforementioned results. Thus, the A*·G* base mispair cannot be

  2. eIF4A inhibition allows translational regulation of mRNAs encoding proteins involved in Alzheimer's disease.

    Directory of Open Access Journals (Sweden)

    Andrew Bottley

    2010-09-01

    Full Text Available Alzheimer's disease (AD is the main cause of dementia in our increasingly aging population. The debilitating cognitive and behavioral symptoms characteristic of AD make it an extremely distressing illness for patients and carers. Although drugs have been developed to treat AD symptoms and to slow disease progression, there is currently no cure. The incidence of AD is predicted to increase to over one hundred million by 2050, placing a heavy burden on communities and economies, and making the development of effective therapies an urgent priority. Two proteins are thought to have major contributory roles in AD: the microtubule associated protein tau, also known as MAPT; and the amyloid-beta peptide (A-beta, a cleavage product of amyloid precursor protein (APP. Oxidative stress is also implicated in AD pathology from an early stage. By targeting eIF4A, an RNA helicase involved in translation initiation, the synthesis of APP and tau, but not neuroprotective proteins, can be simultaneously and specifically reduced, representing a novel avenue for AD intervention. We also show that protection from oxidative stress is increased upon eIF4A inhibition. We demonstrate that the reduction of these proteins is not due to changes in mRNA levels or increased protein degradation, but is a consequence of translational repression conferred by inhibition of the helicase activity of eIF4A. Inhibition of eIF4A selectively and simultaneously modulates the synthesis of proteins involved in Alzheimer's disease: reducing A-beta and tau synthesis, while increasing proteins predicted to be neuroprotective.

  3. Cyclosporin A Inhibits Rotavirus Replication and Restores Interferon-Beta Signaling Pathway In Vitro and In Vivo

    Science.gov (United States)

    He, Haiyang; Wu, Yuzhang

    2013-01-01

    Rotavirus (RV) is the most common cause of severe diarrhea among infants and young children. Currently, there is no specific drug available against rotavirus, largely due to the lack of an ideal target molecule which has hampered drug development. Our previous studies have revealed that cyclosporin A (CsA) might be potentially useful as an anti-RV drug. We therefore used both cellular and mouse models to study the immunological safety and effectiveness of CsA as an anti-RV drug. We found that CsA treatment of HT-29 cells before, during, and after viral infection efficiently inhibited Wa strain RV replication and restored IFN-β expression in a HT-29 cell line model. Exploring the underlying mechanisms showed that CsA promoted Interferon Regulatory Factor-5 (IRF-5) expression (a key positive regulator of the type I IFN signaling pathway), but not IRF-1, IRF-3, or IRF-7. Additionally, CsA inhibited SOCS-1 expression (the key negative regulator of IFN-α/β), but not SOCS-2 or SOCS-3. The antiviral effect of CsA was confirmed in an RV-infected neonatal mouse model by evaluation of antigen clearance and assessment of changes in intestinal tissue pathology. Also, no differences in T cell frequency or proliferation between the CsA- and vehicle-treated groups were observed. Thus, both our in vitro and in vivo findings suggest that CsA, through modulating the expression of key regulators in IFN signaling pathway, promote type I IFN-based intracellular innate immunity in RV host cells. These findings suggest that CsA may be a useful candidate to develop a new anti-RV strategy, although further evaluation and characterization of CsA on RV-induced diarrhea are warranted. PMID:23990993

  4. Protein phosphatase 2A inhibition and circumvention of cisplatin cross-resistance by novel TCM-platinum anticancer agents containing demethylcantharidin.

    Science.gov (United States)

    To, Kenneth K W; Wang, Xinning; Yu, Chun Wing; Ho, Yee-Ping; Au-Yeung, Steve C F

    2004-09-01

    Novel TCM-platinum compounds [Pt(C(8)H(8)O(5))(NH(2)R)(2)] 1-5, derived from integrating demethylcantharidin, a modified component from a traditional Chinese medicine (TCM) with a platinum moiety, possess anticancer and protein phosphatase 2A inhibition properties. The compounds are able to circumvent cisplatin resistance by apparently targeting the DNA repair mechanism. Novel isosteric analogues [Pt(C(9)H(10)O(4))(NH(2)R)(2)] A and B, devoid of PP2A-inhibitory activity, were found to suffer from an enhanced DNA repair and were cross-resistant to cisplatin. The results advocate a well-defined structure-activity requirement associating the PP2A-inhibiting demethylcantharidin with the circumvention of cisplatin cross-resistance demonstrated by TCM-Pt compounds 1-5.

  5. The immunosuppressives FK 506 and cyclosporin A inhibit the generation of protein factors binding to the two purine boxes of the interleukin 2 enhancer.

    Science.gov (United States)

    Brabletz, T; Pietrowski, I; Serfling, E

    1991-01-11

    Like Cyclosporin A (CsA), the macrolide FK 506 is a potent immunosuppressive that inhibits early steps of T cell activation, including the synthesis of Interleukin 2 (II-2) and numerous other lymphokines. The block of II-2 synthesis occurs at the transcriptional level. At concentrations that block T cell activation, FK 506 and CsA inhibit the proto-enhancer activity of Purine boxes of the II-2 promoter and the generation of lymphocyte-specific factors binding to the Purine boxes. Under the same conditions, the DNA binding of other II-2 enhancer factors remains unaffected by both compounds. These results support the view that FK 506 and CsA, which both inhibit the activity of peptidylprolyl cis/trans isomerases, suppress T cell activation by a similar, if not identical mechanism.

  6. Application of HPLC to study the kinetics of a branched bi-enzyme system consisting of hypoxanthine-guanine phosphoribosyltransferase and xanthine oxidase--an important biochemical system to evaluate the efficiency of the anticancer drug 6-mercaptopurine in ALL cell line.

    Science.gov (United States)

    Kalra, Sukirti; Paul, Manash K; Balaram, Hemalatha; Mukhopadhyay, Anup Kumar

    2007-05-01

    The thiopurine antimetabolite 6-mercaptopurine (6MP) is an important chemotherapeutic drug in the conventional treatment of childhood acute lymphoblastic leukemia (ALL). 6MP is mainly catabolized by both hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and xanthine oxidase (XOD) to form thioinosinic monophosphate (TIMP) (therapeutically active metabolite) and 6-thiouric acid (6TUA) (inactive metabolite), respectively. The activity of both the enzymes varies among ALL patients governing the active and the inactive metabolite profile within the immature lymphocytes. Therefore, an attempt was made to study the kinetic nature of the branched bi-enzyme system acting on 6MP and to quantitate TIMP and 6TUA formed when the two enzymes are present in equal and variable ratios. The quantification of the branched kinetics using spectrophotometric method presents problem due to the closely apposed lambda(max) of the substrates and products. Hence, employing an HPLC method, the quantification of the products was done with the progress of time. The limit of quantification (LOQ) of substrate was found to be 10nM and for products as 50 nM. The limit of detection (LOD) was found to be 1 nM for the substrate and the products. The method exhibited linearity in the range of 0.01-100 microM for 6MP and 0.05-100 microM for both 6TUA and TIMP. The amount of TIMP formed was higher than that of 6TUA in the bi-enzyme system when both the enzymes were present in equivalent enzymatic ratio. It was further found that enzymatic ratios play an important role in determining the amounts of TIMP and 6TUA. This method was further validated using actively growing T-ALL cell line (Jurkat) to study the branched kinetics, wherein it was observed that treatment of 50 microM 6MP led to the generation of 12 microM TIMP and 0.8 microM 6TUA in 6 h at 37 degrees C.

  7. Photochemical selectivity in guanine-cytosine base-pair structures

    Czech Academy of Sciences Publication Activity Database

    Abo-Riziq, A.; Grace, L.; Nir, E.; Kabeláč, Martin; Hobza, Pavel; Vries de, M. S.

    2005-01-01

    Roč. 102, č. 1 (2005), s. 20-23 ISSN 0027-8424 R&D Projects: GA ČR(CZ) GA203/05/0009 Grant - others:NSF(US) CHE-0244341 Institutional research plan: CEZ:AV0Z40550506 Keywords : DNA base pairs * IR-UV spectroscopy * phytochemistry Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 10.231, year: 2005

  8. Oligomeric state of hypoxanthine-guanine phosphoribosyltransferase from Mycobacterium tuberculosis

    Czech Academy of Sciences Publication Activity Database

    Eng, W. S.; Keough, D. T.; Hocková, Dana; Winzor, D. J.; Guddat, L. W.

    2017-01-01

    Roč. 135, Apr (2017), s. 6-14 ISSN 0300-9084 R&D Projects: GA ČR(CZ) GA16-06049S Institutional support: RVO:61388963 Keywords : enzyme inhibitors * acyclic nucleoside phosphonates * 6-oxopurine phosphoribosyltransferase Subject RIV: CC - Organic Chemistry OBOR OECD: Organic chemistry Impact factor: 3.112, year: 2016

  9. Towards understanding of poly-guanine activated fluorescent silver nanoclusters

    International Nuclear Information System (INIS)

    Walczak, Sylwia; Morishita, Kiyoshi; Ahmed, Moin; Liu, Juewen

    2014-01-01

    It has been recently reported that the fluorescence of some DNA-templated silver nanoclusters (AgNCs) can be significantly enhanced upon by hybridizing with a partially complementary DNA containing a G-rich overhang near the AgNCs. This discovery has found a number of analytical applications but many fundamental questions remain to be answered. In this work, the photostability of these activated AgNCs is reported. After adding the G-rich DNA activator, the fluorescence intensity peaks in ∼1 h and then starts to decay, where the decaying rate is much faster with light exposure. The lost fluorescence is recovered by adding NaBH 4 , suggesting that the bleaching is an oxidative process. Once activated, the G-rich activator can be removed while the AgNCs still maintain most of their fluorescence intensity. UV–vis spectroscopy suggests that new AgNC species are generated upon hybridization with the activator. The base sequence and length of the template DNA have also been varied, leading to different emission colors and color change after hybridization. G-rich aptamers can also serve as activators. Our results indicate that activation of the fluorescence by G-rich DNA could be a convenient method for biosensor development since the unstable NaBH 4 is not required for the activation step. (paper)

  10. Next generation bone tissue engineering: non-viral miR-133a inhibition using collagen-nanohydroxyapatite scaffolds rapidly enhances osteogenesis

    Science.gov (United States)

    Mencía Castaño, Irene; Curtin, Caroline M.; Duffy, Garry P.; O'Brien, Fergal J.

    2016-06-01

    Bone grafts are the second most transplanted materials worldwide at a global cost to healthcare systems valued over $30 billion every year. The influence of microRNAs in the regenerative capacity of stem cells offers vast therapeutic potential towards bone grafting; however their efficient delivery to the target site remains a major challenge. This study describes how the functionalisation of porous collagen-nanohydroxyapatite (nHA) scaffolds with miR-133a inhibiting complexes, delivered using non-viral nHA particles, enhanced human mesenchymal stem cell-mediated osteogenesis through the novel focus on a key activator of osteogenesis, Runx2. This study showed enhanced Runx2 and osteocalcin expression, as well as increased alkaline phosphatase activity and calcium deposition, thus demonstrating a further enhanced therapeutic potential of a biomaterial previously optimised for bone repair applications. The promising features of this platform offer potential for a myriad of applications beyond bone repair and tissue engineering, thus presenting a new paradigm for microRNA-based therapeutics.

  11. Tributyltin induces a G2/M cell cycle arrest in human amniotic cells via PP2A inhibition-mediated inactivation of the ERK1/2 cascades.

    Science.gov (United States)

    Zhang, Yali; Guo, Zonglou; Xu, Lihong

    2014-03-01

    The molecular mechanisms underlying the cell cycle alterations induced by tributyltin (TBT), a highly toxic environmental contaminant, remain elusive. In this study, cell cycle progression and some key regulators in G2/M phase were investigated in human amniotic cells treated with TBT. Furthermore, protein phosphatase (PP) 2A and the ERK cascades were examined. The results showed that TBT caused a G2/M cell cycle arrest that was accompanied by a decrease in the total cdc25C protein level and an increase in the p-cdc2 level in the nucleus. TBT caused a decrease in PP2A activity and inhibited the ERK cascade by inactivating Raf-1, resulting in the dephosphorylation of MEK1/2, ERK1/2, and c-Myc. Taken together, TBT leads to a G2/M cell cycle arrest in FL cells, an increase in p-cdc2 and a decrease in the levels of total cdc25C protein, which may be caused by the PP2A inhibition-mediated inactivation of the ERK1/2 cascades. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. miR-20a inhibits TCR-mediated signaling and cytokine production in human naïve CD4+ T cells.

    Directory of Open Access Journals (Sweden)

    Amarendra V Reddycherla

    Full Text Available Upon TCR stimulation by peptide-MHC complexes, CD4+ T cells undergo activation and proliferation. This process will ultimately culminate in T-cell differentiation and the acquisition of effector functions. The production of specific cytokines by differentiated CD4+ T cells is crucial for the generation of the appropriate immune response. Altered CD4+ T-cell activation and cytokine production result in chronic inflammatory conditions and autoimmune disorders. miRNAs have been shown to be important regulators of T-cell biology. In this study, we have focused our investigation on miR-20a, a member of the miR-17-92 cluster, whose expression is decreased in patients suffering from multiple sclerosis. We have found that miR-20a is rapidly induced upon TCR-triggering in primary human naïve CD4+ T cells and that its transcription is regulated in a Erk-, NF-κB-, and Ca++-dependent manner. We have further shown that overexpression of miR-20a inhibits TCR-mediated signaling but not the proliferation of primary human naïve CD4+ T cells. However, miR-20a overexpression strongly suppresses IL-10 secretion and moderately decreases IL-2, IL-6 and IL8 production, which are crucial regulators of inflammatory responses. Our study suggests that miR-20a is a new player in the regulation of TCR signaling strength and cytokine production.

  13. Cyclosporin A inhibits nucleotide excision repair via downregulation of the xeroderma pigmentosum group A and G proteins, which is mediated by calcineurin inhibition.

    Science.gov (United States)

    Kuschal, Christiane; Thoms, Kai-Martin; Boeckmann, Lars; Laspe, Petra; Apel, Antje; Schön, Michael P; Emmert, Steffen

    2011-10-01

    Cyclosporin A (CsA) inhibits nucleotide excision repair (NER) in human cells, a process that contributes to the skin cancer proneness in organ transplant patients. We investigated the mechanisms of CsA-induced NER reduction by assessing all xeroderma pigmentosum (XP) genes (XPA-XPG). Western blot analyses revealed that XPA and XPG protein expression was reduced in normal human GM00637 fibroblasts exposed to 0.1 and 0.5 μm CsA. Interestingly, the CsA treatment reduced XPG, but not XPA, mRNA expression. Calcineurin knockdown in GM00637 fibroblasts using RNAi led to similar results suggesting that calcineurin-dependent signalling is involved in XPA and XPG protein regulation. CsA-induced reduction in NER could be complemented by the overexpression of either XPA or XPG protein. Likewise, XPA-deficient fibroblasts with stable overexpression of XPA (XP2OS-pCAH19WS) did not show the inhibitory effect of CsA on NER. In contrast, XPC-deficient fibroblasts overexpressing XPC showed CsA-reduced NER. Our data indicate that the CsA-induced inhibition of NER is a result of downregulation of XPA and XPG protein in a calcineurin-dependent manner. © 2011 John Wiley & Sons A/S.

  14. Bioactivity-guided identification and cell signaling technology to delineate the lactate dehydrogenase A inhibition effects of Spatholobus suberectus on breast cancer.

    Directory of Open Access Journals (Sweden)

    Zhiyu Wang

    Full Text Available Aerobic glycolysis is an important feature of cancer cells. In recent years, lactate dehydrogenase A (LDH-A is emerging as a novel therapeutic target for cancer treatment. Seeking LDH-A inhibitors from natural resources has been paid much attention for drug discovery. Spatholobus suberectus (SS is a common herbal medicine used in China for treating blood-stasis related diseases such as cancer. This study aims to explore the potential medicinal application of SS for LDH-A inhibition on breast cancer and to determine its bioactive compounds. We found that SS manifested apoptosis-inducing, cell cycle arresting and anti-LDH-A activities in both estrogen-dependent human MCF-7 cells and estrogen-independent MDA-MB-231 cell. Oral herbal extracts (1 g/kg/d administration attenuated tumor growth and LDH-A expression in both breast cancer xenografts. Bioactivity-guided fractionation finally identified epigallocatechin as a key compound in SS inhibiting LDH-A activity. Further studies revealed that LDH-A plays a critical role in mediating the apoptosis-induction effects of epigallocatechin. The inhibited LDH-A activities by epigallocatechin is attributed to disassociation of Hsp90 from HIF-1α and subsequent accelerated HIF-1α proteasome degradation. In vivo study also demonstrated that epigallocatechin could significantly inhibit breast cancer growth, HIF-1α/LDH-A expression and trigger apoptosis without bringing toxic effects. The preclinical study thus suggests that the potential medicinal application of SS for inhibiting cancer LDH-A activity and the possibility to consider epigallocatechin as a lead compound to develop LDH-A inhibitors. Future studies of SS for chemoprevention or chemosensitization against breast cancer are thus warranted.

  15. MicroRNA-20a inhibits autophagic process by targeting ATG7 and ATG16L1 and favors mycobacterial survival in macrophage cells.

    Directory of Open Access Journals (Sweden)

    Le Guo

    2016-10-01

    Full Text Available Autophagy plays important roles in the host immune response against mycobacterial infection. Mycobacterium tuberculosis (M. tuberculosis can live in macrophages owing to its ability to evade attacks by regulating autophagic response. MicroRNAs (miRNAs are small noncoding, endogenously encoded RNA which plays critical roles in precise regulation of macrophage functions. Whether miRNAs specifically influence the activation of macrophage autophagy during M. tuberculosis infection are largely unknown. In this study, we demonstrate that BCG infection of macrophages resulted in enhanced expression of miRNA-20a, which inhibits autophagic process by targeting ATG7 and ATG16L1 and promotes BCG survival in macrophages. Forced overexpression of miR-20a decreased the expression levels of LC3-II and the number of LC3 puncta in macrophages, and promoted BCG survival in macrophages, while transfection with miR-20a inhibitor had the opposite effect. Moreover, the inhibitory effect of miR-20a on autophagy was further confimed by transmission electron microscopy (TEM analysis. Quantification of autophagosomes per cellular cross-section revealed a significant reduction upon transfection with miR-20a mimic, but transfection with miR-20a inhibitor increased the number of autophagosomes per cellular cross-section. Moreover, silencing of ATG7 significantly inhibited autophagic response, and transfection with ATG7 siRNA plus miR-20a mimic could further decrease autophagic response. Collectively, our data reveal that miR-20a inhibits autophagic response and promotes BCG survival in macrophages by targeting ATG7 and ATG16L1, which may have implications for a better understanding of pathogenesis of M. tuberculosis infection.

  16. microRNA-20a Inhibits Autophagic Process by Targeting ATG7 and ATG16L1 and Favors Mycobacterial Survival in Macrophage Cells.

    Science.gov (United States)

    Guo, Le; Zhao, Jin; Qu, Yuliang; Yin, Runting; Gao, Qian; Ding, Shuqin; Zhang, Ying; Wei, Jun; Xu, Guangxian

    2016-01-01

    Autophagy plays important roles in the host immune response against mycobacterial infection. Mycobacterium tuberculosis ( M. tuberculosis ) can live in macrophages owing to its ability to evade attacks by regulating autophagic response. MicroRNAs (miRNAs) are small noncoding, endogenously encoded RNA which plays critical roles in precise regulation of macrophage functions. Whether miRNAs specifically influence the activation of macrophage autophagy during M. tuberculosis infection are largely unknown. In this study, we demonstrate that BCG infection of macrophages resulted in enhanced expression of miRNA-20a, which inhibits autophagic process by targeting ATG7 and ATG16L1 and promotes BCG survival in macrophages. Forced overexpression of miR-20a decreased the expression levels of LC3-II and the number of LC3 puncta in macrophages, and promoted BCG survival in macrophages, while transfection with miR-20a inhibitor had the opposite effect. Moreover, the inhibitory effect of miR-20a on autophagy was further confirmed by transmission electron microscopy (TEM) analysis. Quantification of autophagosomes per cellular cross-section revealed a significant reduction upon transfection with miR-20a mimic, but transfection with miR-20a inhibitor increased the number of autophagosomes per cellular cross-section. Moreover, silencing of ATG7 significantly inhibited autophagic response, and transfection with ATG7 siRNA plus miR-20a mimic could further decrease autophagic response. Collectively, our data reveal that miR-20a inhibits autophagic response and promotes BCG survival in macrophages by targeting ATG7 and ATG16L1, which may have implications for a better understanding of pathogenesis of M. tuberculosis infection.

  17. Modulation of mGlu2 Receptors, but Not PDE10A Inhibition Normalizes Pharmacologically-Induced Deviance in Auditory Evoked Potentials and Oscillations in Conscious Rats.

    Directory of Open Access Journals (Sweden)

    Abdallah Ahnaou

    Full Text Available Improvement of cognitive impairments represents a high medical need in the development of new antipsychotics. Aberrant EEG gamma oscillations and reductions in the P1/N1 complex peak amplitude of the auditory evoked potential (AEP are neurophysiological biomarkers for schizophrenia that indicate disruption in sensory information processing. Inhibition of phosphodiesterase (i.e. PDE10A and activation of metabotropic glutamate receptor (mGluR2 signaling are believed to provide antipsychotic efficacy in schizophrenia, but it is unclear whether this occurs with cognition-enhancing potential. The present study used the auditory paired click paradigm in passive awake Sprague Dawley rats to 1 model disruption of AEP waveforms and oscillations as observed in schizophrenia by peripheral administration of amphetamine and the N-methyl-D-aspartate (NMDA antagonist phencyclidine (PCP; 2 confirm the potential of the antipsychotics risperidone and olanzapine to attenuate these disruptions; 3 evaluate the potential of mGluR2 agonist LY404039 and PDE10 inhibitor PQ-10 to improve AEP deficits in both the amphetamine and PCP models. PCP and amphetamine disrupted auditory information processing to the first click, associated with suppression of the P1/N1 complex peak amplitude, and increased cortical gamma oscillations. Risperidone and olanzapine normalized PCP and amphetamine-induced abnormalities in AEP waveforms and aberrant gamma/alpha oscillations, respectively. LY404039 increased P1/N1 complex peak amplitudes and potently attenuated the disruptive effects of both PCP and amphetamine on AEPs amplitudes and oscillations. However, PQ-10 failed to show such effect in either models. These outcomes indicate that modulation of the mGluR2 results in effective restoration of abnormalities in AEP components in two widely used animal models of psychosis, whereas PDE10A inhibition does not.

  18. Protein phosphatase 2A inhibition and subsequent cytoskeleton reorganization contributes to cell migration caused by microcystin-LR in human laryngeal epithelial cells (Hep-2).

    Science.gov (United States)

    Wang, Beilei; Liu, Jinghui; Huang, Pu; Xu, Kailun; Wang, Hanying; Wang, Xiaofeng; Guo, Zonglou; Xu, Lihong

    2017-03-01

    The major toxic mechanism of Microcystin-LR is inhibition of the activity of protein phosphatase 2A (PP2A), resulting in a series of cytotoxic effects. Our previous studies have demonstrated that microcystin-LR (MCLR) induced very different molecular effects in normal cells and the tumor cell line SMMC7721. To further explore the MCLR toxicity mechanism in tumor cells, human laryngeal epithelial cells (Hep-2) was examined in this study. Western blot, immunofluorescence, immunoprecipitation, and transwell migration assay were used to detect the effects of MCLR on PP2A activity, PP2A substrates, cytoskeleton, and cell migration. The results showed that the protein level of PP2A subunits and the posttranslational modification of the catalytic subunit were altered and that the binding of the AC core enzyme as well as the binding of PP2A/C and α4, was also affected. As PP2A substrates, the phosphorylation of MAPK pathway members, p38, ERK1/2, and the cytoskeleton-associated proteins, Hsp27, VASP, Tau, and Ezrin were increased. Furthermore, MCLR induced reorganization of the cytoskeleton and promoted cell migration. Taken together, direct covalent binding to PP2A/C, alteration of the protein levels and posttranslational modification, as well as the binding of subunits, are the main pattern for the effects of MCLR on PP2A in Hep-2. A dose-dependent change in p-Tau and p-Ezrin due to PP2A inhibition may contribute to the changes in the cytoskeleton and be related to the cell migration in Hep-2. Our data provide a comprehensive exposition of the MCLR mechanism on tumor cells. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 890-903, 2017. © 2016 Wiley Periodicals, Inc.

  19. Histone Demethylase JMJD2A Inhibition Attenuates Neointimal Hyperplasia in the Carotid Arteries of Balloon-Injured Diabetic Rats via Transcriptional Silencing: Inflammatory Gene Expression in Vascular Smooth Muscle Cells

    Directory of Open Access Journals (Sweden)

    Hu Qi

    2015-09-01

    Full Text Available Background/Aims: Diabetic patients suffer from severe neointimal hyperplasia following angioplasty. The epigenetic abnormalities are increasingly considered to be relevant to the pathogenesis of diabetic cardiovascular complications. But the epigenetic mechanisms linking diabetes and coronary restenosis have not been fully elucidated. In this study, we explored the protective effect and underlying mechanisms of demethylases JMJD2A inhibition in balloon-injury induced neointimal formation in diabetic rats. Methods: JMJD2A inhibition was achieved by the chemical inhibitor 2,4-pyridinedicarboxylic acid (2,4-PDCA and small interfering RNA (siRNA. In vitro, we investigated the proliferation, migration and inflammation of rat vascular smooth muscle cells (VSMCs in response to high glucose (HG. In vivo, diabetic rats induced using high-fat diet and low-dose streptozotocin (35mg/kg underwent carotid artery balloon injury. Morphometric analysis was performed using hematein eosin and immumohistochemical staining. Chromatin Immunoprecipitation (ChIP was conducted to detect modification of H3K9me3 at inflammatory genes promoters. Results: The global JMJD2A was increased in HG-stimulated VSMCs and balloon-injured arteries of diabetic rats, accompanied by decreased H3K9me3. The inhibition of JMJD2A suppressed VSMCs proliferation, migration and inflammation induced by high glucose (HG in vitro. And JMJDA2A inhibition attenuated neointimal formation in balloon-injured diabetic rats. The underlying mechanisms were relevant to the restoration of H3K9me3 levels at the promoters of MCP-1 and IL-6, and then the suppressed expression of MCP-1 and IL-6. Conclusion: The JMJD2A inhibition significantly attenuated neointimal formation in balloon injured diabetic rats via the suppression of VSMCs proliferation, migration, and inflammation by restoring H3K9me3.

  20. Proinflammatory Cytokines IL-6 and TNF-α Increased Telomerase Activity through NF-κB/STAT1/STAT3 Activation, and Withaferin A Inhibited the Signaling in Colorectal Cancer Cells

    Directory of Open Access Journals (Sweden)

    Seyung S. Chung

    2017-01-01

    Full Text Available There are increasing evidences of proinflammatory cytokine involvement in cancer development. Here, we found that two cytokines, IL-6 and TNF-α, activated colorectal cancer cells to be more invasive and stem-like. Combined treatment of IL-6 and TNF-α phosphorylated transcription factors STAT3 in a synergistic manner. STAT3, STAT1, and NF-κB physically interacted upon the cytokine stimulation. STAT3 was bound to the promoter region of human telomerase reverse transcriptase (hTERT. IL-6 and TNF-α stimulation further enhanced STAT3 binding affinity. Stem cell marker Oct-4 was upregulated in colorectal cancer cells upon IL-6 and TNF-α stimulation. Withaferin A, an anti-inflammatory steroidal lactone, inhibited the IL-6- and TNF-α-induced cancer cell invasion and decreased colonosphere formation. Notably, withaferin A inhibited STAT3 phosphorylation and abolished the STAT3, STAT1, and NF-κB interactions. Oct-4 expression was also downregulated by withaferin A inhibition. The binding of STAT3 to the hTERT promoter region and telomerase activity showed reduction with withaferin A treatments. Proinflammatory cytokine-induced cancer cell invasiveness is mediated by a STAT3-regulated mechanism in colorectal cancer cells. Our data suggest that withaferin A could be a promising anticancer agent that effectively inhibits the progression of colorectal cancer.

  1. Chemical suppressors of mlo-mediated powdery mildew resistance

    Science.gov (United States)

    Wu, Hongpo; Kwaaitaal, Mark; Strugala, Roxana; Schaffrath, Ulrich; Bednarek, Paweł

    2017-01-01

    Loss-of-function of barley mildew locus o (Mlo) confers durable broad-spectrum penetration resistance to the barley powdery mildew pathogen, Blumeria graminis f. sp. hordei (Bgh). Given the importance of mlo mutants in agriculture, surprisingly few molecular components have been identified to be required for this type of resistance in barley. With the aim to identify novel cellular factors contributing to mlo-based resistance, we devised a pharmacological inhibitor screen. Of the 41 rationally chosen compounds tested, five caused a partial suppression of mlo resistance in barley, indicated by increased levels of Bgh host cell entry. These chemicals comprise brefeldin A (BFA), 2′,3′-dideoxyadenosine (DDA), 2-deoxy-d-glucose, spermidine, and 1-aminobenzotriazole. Further inhibitor analysis corroborated a key role for both anterograde and retrograde endomembrane trafficking in mlo resistance. In addition, all four ribonucleosides, some ribonucleoside derivatives, two of the five nucleobases (guanine and uracil), some guanine derivatives as well as various polyamines partially suppress mlo resistance in barley via yet unknown mechanisms. Most of the chemicals identified to be effective in partially relieving mlo resistance in barley also to some extent compromised powdery mildew resistance in an Arabidopsis mlo2 mlo6 double mutant. In summary, our study identified novel suppressors of mlo resistance that may serve as valuable probes to unravel further the molecular processes underlying this unusual type of disease resistance. PMID:29127104

  2. MCLR-induced PP2A inhibition and subsequent Rac1 inactivation and hyperphosphorylation of cytoskeleton-associated proteins are involved in cytoskeleton rearrangement in SMMC-7721 human liver cancer cell line.

    Science.gov (United States)

    Wang, Hao; Liu, Jinghui; Lin, Shuyan; Wang, Beilei; Xing, Mingluan; Guo, Zonglou; Xu, Lihong

    2014-10-01

    Cyanobacteria-derived toxin microcystin-LR (MCLR) has been widely investigated in its effects on normal cells, there is little information concerning its effects on cancer cells. In the present study, the SMMC-7721 human liver cancer cell line treated with MCLR was used to investigate the change of PP2A, cytoskeleton rearrangement, phosphorylation levels of PP2A substrates that related with cytoskeleton stability and explored underlying mechanisms. Here, we confirmed that MCLR entered into SMMC-7721 cells, bound to PP2A/C subunit and inhibited the activity of PP2A. The upregulation of phosphorylation of the PP2A/C subunit and PP2A regulation protein α4, as well as the change in the association of PP2A/C with α4, were responsible for the decrease in PP2A activity. Another novel finding is that the rearrangement of filamentous actin and microtubules led by MCLR may attribute to the increased phosphorylation of HSP27, VASP and cofilin due to PP2A inhibition. As a result of weakened interactions with PP2A and alterations in its subcellular localization, Rac1 may contribute to the cytoskeletal rearrangement induced by MCLR in SMMC-7721 cells. The current paper presents the first report demonstrating the characteristic of PP2A in MCLR exposed cancer cells, which were more susceptible to MCLR compared with the normal cell lines we previously found, which may be owing to the absence of some type of compensatory mechanisms. The hyperphosphorylation of cytoskeleton-associated proteins and Rac1 inactivation which were induced by inhibition of PP2A are shown to be involved in cytoskeleton rearrangement. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. Repair of O6-(2-chloroethyl)guanine mediates the biological effects of chloroethylnitrosoureas.

    OpenAIRE

    Bodell, W J; Aida, T; Berger, M S; Rosenblum, M L

    1985-01-01

    Chloroethylnitrosoureas (CENUs) are alkylating and crosslinking agents used for the treatment of human cancer; they are both mutagenic and carcinogenic. We compared the levels of induction of sister chromatid exchanges (SCEs) and the cytotoxicity of nitrosoureas that alkylate only with CENUs. CENUs are 200-fold more cytotoxic and induce SCEs with 45-fold greater efficiency than agents that do not crosslink; therefore, crosslinking is probably the most important molecular event that leads to c...

  4. A Theoretical Study of the Mechanism of the Alkylation of Guanine by N- Nitroso Compounds.

    Science.gov (United States)

    1992-01-01

    these chemical agents alkylate DNA, but, as yet, the precise mechanism is unknown. What is known is that the result is a DNA-mutagen adduct with an alkyl ... nitrosoureas , Singer et. al. found that about 25% of the alkylation caused by MNU was on the DNA phospate backbone while, for ENU, phosphate...sites. 1.3 Mutagenicity of N-Nitroso Compounds In early experimental work with agents which alkylate DNA, comparisons of ultraviolet absorption

  5. Theoretical study of the guanine → 6-thioguanine substitution in duplexes, triplexes, and tetraplexes

    Czech Academy of Sciences Publication Activity Database

    Špačková, Naďa; Cubero, E.; Šponer, Jiří; Orozco, M.

    2004-01-01

    Roč. 126, č. 44 (2004), s. 14642-14650 ISSN 0002-7863 R&D Projects: GA MŠk LN00A016 Grant - others:European Training Mobility Program(XE) HPRI-CT-1999-00071; Ministry of Science and Technology (ES) BIO2003-06848; Wellcome Trust(GB) GR067507MF Institutional research plan: CEZ:AV0Z5004920 Keywords : 6-thioguanine * molecular dynamics simulations * thermodynamic integration Subject RIV: BO - Biophysics Impact factor: 6.903, year: 2004

  6. The regulatory G4 motif of the Kirsten ras (KRAS) gene is sensitive to guanine oxidation

    DEFF Research Database (Denmark)

    Cogoi, Susanna; Ferino, Annalisa; Miglietta, Giulia

    2018-01-01

    KRAS is one of the most mutated genes in human cancer. It is controlled by a G4 motif located upstream of the transcription start site. In this paper, we demonstrate that 8-oxoguanine (8-oxoG), being more abundant in G4 than in non-G4 regions, is a new player in the regulation of this oncogene. W...

  7. Guanine nucleotide binding proteins in zucchini seedlings: Characterization and interactions with the NPA receptor

    International Nuclear Information System (INIS)

    Lindeberg, M.; Jacobs, M.

    1989-01-01

    A microsomal membrane preparation from hypocotyls of dark-grown Cucurbita pepo L. seedlings contains specific high-affinity binding sites for the non-hydrolyzable GTP analog guanosine 5'-[γ-thio] triphosphate (GTP-γ-S). Both the binding affinity and the pattern of binding specificity for GTP and GTP analogs are similar to animal G-proteins, and two zucchini membrane proteins are recognized in western blots by antiserum specific for the σ subunit of platelet G s protein. GTP-γ-S can increase specific naphthylphthalamic acid (NPA) binding in zucchini microsomal membrane preparations, with its stimulation increasing with large tissue age. Al +3 and F - agents known to activate G-proteins - decreased NPA specific binding by ca. 15%. In tests of in vitro auxin transport employing zucchini plasma membrane vesicles, AlF - 4 strongly inhibited 3 H-indoleacetic acid nor accumulation; GTP-γ-S effects on this system will be discussed

  8. Voltametric Determination of Adenine, Guanine and DNA Using Liquid Mercury Free Polished Silver Solid Amalgam Electrode

    Czech Academy of Sciences Publication Activity Database

    Fadrná, Renata; Josypčuk, Bohdan; Fojta, Miroslav; Navrátil, Tomáš; Novotný, Ladislav

    2004-01-01

    Roč. 37, č. 3 (2004), s. 399-413 ISSN 0003-2719 R&D Projects: GA AV ČR KSK4040110 Grant - others:GIT(AR) 101/02/U111/CZ Keywords : voltammetry * DNA * polished silver solid amalgam electrode Subject RIV: CG - Electrochemistry Impact factor: 1.165, year: 2004

  9. Guanine nucleotide regulation of dopamine receptor agonist affinity states in rat estradiol-induced pituitary tumors

    Energy Technology Data Exchange (ETDEWEB)

    Di Paolo, T.; Falardeau, P.

    1987-08-31

    The authors have investigated dopamine (DA) receptor agonist high- and low-affinity states in female rate estradiol-induced prolactin (PRL)-secreting pituitary tumors and intact pituitary tissue. Estradiol treatment increased the anterior pituitary weight 9-fold and plasma prolactin levels 74-fold and these measures are correlated (R = 0.745, n = 73, p < 0.001). Competition for (/sup 3/H)-spiperone binding to the DA receptor by apomorphine was compared in normal and adenomatous pituitary tissue. The inhibition constants (Ki) and the proportions of the two apomorphine sites are unchanged in tumors compared to intact pituitary tissue. Guanosine 5'-(..beta..-..gamma..-imino)triphosphate (Gpp(NH)p) causes complete conversion of the high into low affinity dopaminergic agonist site in normal pituitary and in tumors. These results suggest that rats with primary estradiol-induced pituitary tumors have normal and functional DA receptors. 9 references, 2 tables.

  10. Guanine nucleotide regulation of dopamine receptor agonist affinity states in rat estradiol-induced pituitary tumors

    International Nuclear Information System (INIS)

    Di Paolo, T.; Falardeau, P.

    1987-01-01

    The authors have investigated dopamine (DA) receptor agonist high- and low-affinity states in female rate estradiol-induced prolactin (PRL)-secreting pituitary tumors and intact pituitary tissue. Estradiol treatment increased the anterior pituitary weight 9-fold and plasma prolactin levels 74-fold and these measures are correlated (R = 0.745, n = 73, p 3 H]-spiperone binding to the DA receptor by apomorphine was compared in normal and adenomatous pituitary tissue. The inhibition constants (Ki) and the proportions of the two apomorphine sites are unchanged in tumors compared to intact pituitary tissue. Guanosine 5'-[β-γ-imino]triphosphate (Gpp(NH)p) causes complete conversion of the high into low affinity dopaminergic agonist site in normal pituitary and in tumors. These results suggest that rats with primary estradiol-induced pituitary tumors have normal and functional DA receptors. 9 references, 2 tables

  11. Theoretical study on the detailed repair of O6-methyl guanine to ...

    Indian Academy of Sciences (India)

    Living cells are constantly exposed to both exogenous. (chemical pollutants and UV radiation) and endoge- nous (the normal metabolic byproducts, especially in the oxidative deamination process) sources of DNA damaging agents.1–6 Methylating agents which lead to a wide range of DNA damage such as aging, chronic.

  12. Biocatalytic separation of N-7/N-9 guanine nucleosides

    DEFF Research Database (Denmark)

    Singh, Sunil K.; Sharma, Vivek K.; Olsen, Carl Erik

    2010-01-01

    Vorbrüggen coupling of trimethylsilylated 2-N-isobutanoylguanine with peracetylated pentofuranose derivatives generally gives inseparable N-7/N-9 glycosyl mixtures. We have shown that the two isomers can be separated biocatalytically by Novozyme-435-mediated selective deacetylation of the 5'-O-a...

  13. Functional reconstitution of prostaglandin E receptor from bovine adrenal medulla with guanine nucleotide binding proteins

    International Nuclear Information System (INIS)

    Negishi, M.; Ito, S.; Yokohama, H.; Hayashi, H.; Katada, T.; Ui, M.; Hayaishi, O.

    1988-01-01

    Prostaglandin E 2 (PEG 2 ) was found to bind specifically to a 100,000 x g pellet prepared from bovine adrenal medulla. The PGE receptor was associated with a GTP-binding protein (G-protein) and could be covalently cross-linked with this G-protein by dithiobis(succinimidyl propionate) in the 100,000 x g pellet. In order to characterize the G-protein associated with the PGE receptor and reconstitute these proteins in phospholipid vesicles, the authors purified the G-protein to apparent homogeneity from the 100,000 x g pellet. The G-protein served as a substrate of pertussis toxin but differed in its α subunit from two known pertussis toxin substrate G-proteins (G/sub i/ and G 0 ) purified from bovine brain. The molecular weight of the α subunit was 40,000, which is between those of G/sub i/ and G 0 . The purified protein was also distinguished immunologically from G/sub i/ and G 0 and was referred to as G/sub am/. Reconstitution of the PGE receptor with pure C/sub am/, G/sub i/, or G 0 in phospholipid vesicles resulted in a remarkable restoration of [ 3 H]PGE 2 binding activity in a GTP-dependent manner. The efficiency of these three G-proteins in this capacity was roughly equal. When pertussis toxin- or N-ethylmaleimide-treated G-proteins, instead of the native ones, were reconstituted into vesicles, the restoration of binding activity was no longer observed. These results indicate that the PGE receptor can couple functionally with G/sub am/, G/sub i/, or G 0 in phospholipid vesicles and suggest that G/sub am/ may be involved in signal transduction of the PGE receptor in bovine adrenal medulla

  14. Effect O6-Guanine Alkylation on DNA Flexibility Studied by Comparative Molecular Dynamics Simulations

    Czech Academy of Sciences Publication Activity Database

    Kara, M.; Dršata, Tomáš; Lankaš, Filip; Zacharias, M.

    2015-01-01

    Roč. 103, č. 1 (2015), s. 23-32 ISSN 0006-3525 R&D Projects: GA ČR(CZ) GA14-21893S Institutional support: RVO:61388963 Keywords : DNA damage * DNA alkylation * DNA repair * molecular simulation * molecular dynamics simulation Subject RIV: BO - Biophysics Impact factor: 2.248, year: 2015

  15. Different effects of guanine nucleotides (GDP and GTP) on protein-mediated mitochondrial proton leak.

    Science.gov (United States)

    Woyda-Ploszczyca, Andrzej M; Jarmuszkiewicz, Wieslawa

    2014-01-01

    In this study, we compared the influence of GDP and GTP on isolated mitochondria respiring under conditions favoring oxidative phosphorylation (OXPHOS) and under conditions excluding this process, i.e., in the presence of carboxyatractyloside, an adenine nucleotide translocase inhibitor, and/or oligomycin, an FOF1-ATP synthase inhibitor. Using mitochondria isolated from rat kidney and human endothelial cells, we found that the action of GDP and GTP can differ diametrically depending on the conditions. Namely, under conditions favoring OXPHOS, both in the absence and presence of linoleic acid, an activator of uncoupling proteins (UCPs), the addition of 1 mM GDP resulted in the state 4 (non-phosphorylating respiration)-state 3 (phosphorylating respiration) transition, which is characteristic of ADP oxidative phosphorylation. In contrast, the addition of 1 mM GTP resulted in a decrease in the respiratory rate and an increase in the membrane potential, which is characteristic of UCP inhibition. The stimulatory effect of GDP, but not GTP, was also observed in inside-out submitochondrial particles prepared from rat kidney mitochondria. However, the effects of GDP and GTP were more similar in the presence of OXPHOS inhibitors. The importance of these observations in connection with the action of UCPs, adenine nucleotide translocase (or other carboxyatractyloside-sensitive carriers), carboxyatractyloside- and purine nucleotide-insensitive carriers, as well as nucleoside-diphosphate kinase (NDPK) are considered. Because the measurements favoring oxidative phosphorylation better reflect in vivo conditions, our study strongly supports the idea that GDP cannot be considered a significant physiological inhibitor of UCP. Moreover, it appears that, under native conditions, GTP functions as a more efficient UCP inhibitor than GDP and ATP.

  16. Molecular dynamics simulations of guanine quadruplex loops: Advances and force field limitations

    Czech Academy of Sciences Publication Activity Database

    Fadrná, E.; Špačková, Naďa; Štefl, R.; Koča, J.; Cheatham III, T. E.; Šponer, Jiří

    2004-01-01

    Roč. 87, č. 1 (2004), s. 227-242 ISSN 0006-3495 R&D Projects: GA MŠk LN00A016 Grant - others:Wellcome Trust(GB) GR067507MF Institutional research plan: CEZ:AV0Z5004920 Keywords : quanine quadruplex * four-thymidine loop * locally enhanced sampling Subject RIV: BO - Biophysics Impact factor: 4.585, year: 2004

  17. Site-Specific Covalent Conjugation of Modified mRNA by tRNA Guanine Transglycosylase.

    Science.gov (United States)

    Ehret, Fabian; Zhou, Cun Yu; Alexander, Seth C; Zhang, Dongyang; Devaraj, Neal K

    2018-03-05

    Modified mRNA (mod-mRNA) has recently been widely studied as the form of RNA useful for therapeutic applications due to its high stability and lowered immune response. Herein, we extend the scope of the recently established RNA-TAG (transglycosylation at guanosine) methodology, a novel approach for genetically encoded site-specific labeling of large mRNA transcripts, by employing mod-mRNA as substrate. As a proof of concept, we covalently attached a fluorescent probe to mCherry encoding mod-mRNA transcripts bearing 5-methylcytidine and/or pseudouridine substitutions with high labeling efficiencies. To provide a versatile labeling methodology with a wide range of possible applications, we employed a two-step strategy for functionalization of the mod-mRNA to highlight the therapeutic potential of this new methodology. We envision that this novel and facile labeling methodology of mod-RNA will have great potential in decorating both coding and noncoding therapeutic RNAs with a variety of diagnostic and functional moieties.

  18. Study of Adenine and Guanine Oxidation Mechanism by Surface-Enhanced Raman Spectroelectrochemistry

    Czech Academy of Sciences Publication Activity Database

    Ibanez, D.; Santidrian, Ana; Heras, A.; Kalbáč, Martin; Colina, A.

    2015-01-01

    Roč. 119, č. 15 (2015), s. 8191-8198 ISSN 1932-7447 R&D Projects: GA MŠk LL1301 Institutional support: RVO:61388955 Keywords : nucleic- acid bases * electrochemical oxidation * silver electrode Subject RIV: CG - Electrochemistry Impact factor: 4.509, year: 2015

  19. Inhibition of hypoxanthine-guanine phosphoribosyltransferase by acyclic nucleoside phosphonates: A new class of antimalarial therapeutics

    Czech Academy of Sciences Publication Activity Database

    Keough, D. T.; Hocková, Dana; Holý, Antonín; Naesens, L.; Skinner-Adams, T. S.; de Jersey, J.; Guddat, L. W.

    2009-01-01

    Roč. 52, č. 14 (2009), s. 4391-4399 ISSN 0022-2623 R&D Projects: GA MŠk 1M0508; GA AV ČR 1QS400550501 Institutional research plan: CEZ:AV0Z40550506 Keywords : acyclic nucleoside phosphonates * phosphoribosyltransferase * enzyme inhibitors * Plasmodium falciparum Subject RIV: CC - Organic Chemistry Impact factor: 4.802, year: 2009

  20. Oxidative damage to guanine nucleosides following combination chemotherapy with 5-fluorouracil and oxaliplatin

    DEFF Research Database (Denmark)

    Afzal, Shoaib; Jensen, Søren Astrup; Sørensen, Jens Benn

    2011-01-01

    PURPOSE: Recent in vitro and animal studies have suggested that the cytotoxicity of 5-fluorouracil and oxaliplatin is linked to increased formation of reactive oxygen species (ROS). This prospective study was undertaken to examine the generation of oxidative stress, in 106 colorectal cancer patie...... concentrations of 8-oxoGuo and 8-oxodG and the treatment effect and the other variables. RESULTS: The analysis showed that chemotherapy increased the excretion of 8-oxoGuo and 8-oxodG around 15% (P ...

  1. Guanine nucleotide-binding regulatory proteins in retinal pigment epithelial cells.

    OpenAIRE

    Jiang, M; Pandey, S; Tran, V T; Fong, H K

    1991-01-01

    The expression of GTP-binding regulatory proteins (G proteins) in retinal pigment epithelial (RPE) cells was analyzed by RNA blot hybridization and cDNA amplification. Both adult and fetal human RPE cells contain mRNA for multiple G protein alpha subunits (G alpha) including Gs alpha, Gi-1 alpha, Gi-2 alpha, Gi-3 alpha, and Gz alpha (or Gx alpha), where Gs and Gi are proteins that stimulate or inhibit adenylyl cyclase, respectively, and Gz is a protein that may mediate pertussis toxin-insensi...

  2. Crystal structures and inhibition of Trypanosoma brucei hypoxanthine-guanine phosphoribosyltransferase

    Czech Academy of Sciences Publication Activity Database

    Terán, D.; Hocková, Dana; Česnek, Michal; Zíková, Alena; Naesens, L.; Keough, D. T.; Guddat, L. W.

    2016-01-01

    Roč. 6, Oct 27 (2016), č. článku 35894. ISSN 2045-2322 R&D Projects: GA ČR(CZ) GA16-06049S; GA MŠk LL1205 Institutional support: RVO:61388963 ; RVO:60077344 Keywords : enzyme inhibitors * acyclic nucleoside phosphonates * HGPRT Subject RIV: CC - Organic Chemistry; EE - Microbiology, Virology (BC-A) Impact factor: 4.259, year: 2016 http://www.nature.com/articles/srep35894

  3. Design of Plasmodium vivax Hypoxanthine-Guanine Phosphoribosyltransferase Inhibitors as Potential Antimalarial Therapeutics

    Czech Academy of Sciences Publication Activity Database

    Keough, D. T.; Rejman, Dominik; Pohl, Radek; Zborníková, Eva; Hocková, Dana; Croll, T.; Edstein, M. D.; Birrell, G. W.; Chavchich, M.; Naesens, L. M. J.; Pierens, G. K.; Brereton, I. M.; Guddat, L. W.

    2018-01-01

    Roč. 13, č. 1 (2018), s. 82-90 ISSN 1554-8929 R&D Projects: GA ČR(CZ) GA16-06049S; GA ČR GA15-11711S Institutional support: RVO:61388963 Keywords : plasmodium vivax * inhibitor * pyrrolidine nucleotide bisphosphonate * HXGPRT Subject RIV: CC - Organic Chemistry OBOR OECD: Organic chemistry Impact factor: 4.995, year: 2016

  4. Guanine nucleotide-binding regulatory proteins in retinal pigment epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, Meisheng; Tran, V.T.; Fong, H.K.W. (Univ. of Southern California, Los Angeles (United States)); Pandey, S. (Doheny Eye Inst., Los Angeles, CA (United States))

    1991-05-01

    The expression of GTP-binding regulatory proteins (G proteins) in retinal pigment epithelial (RPE) cells was analyzed by RNA blot hybridization and cDNA amplification. Both adult and fetal human RPE cells contain mRNA for multiple G protein {alpha} subunits (G{alpha}) including G{sub s}{alpha}, G{sub i-1}{alpha}, G{sub i-2}{alpha}, G{sub i-3}{alpha}, and G{sub z}{alpha} (or G{sub x}{alpha}), where G{sub s} and G{sub i} are proteins that stimulate or inhibit adenylyl cyclase, respectively, and G{sub z} is a protein that may mediate pertussis toxin-insensitive events. Other G{alpha}-related mRNA transcripts were detected in fetal RPE cells by low-stringency hybridization to G{sub i-2}{alpha} and G{sub s}{alpha} protein-coding cDNA probes. The diversity of G proteins in RPE cells was further studied by cDNA amplification with reverse transcriptase and the polymerase chain reaction. This approach revealed that, besides the above mentioned members of the G{alpha} gene family, at least two other G{alpha} subunits are expressed in RPE cells. Human retinal cDNA clones that encode one of the additional G{alpha} subunits were isolated and characterized. The results indicate that this G{alpha} subunit belongs to a separate subfamily of G proteins that may be insensitive to inhibition by pertussis toxin.

  5. The guanine-rich fragile X chromosome repeats are reluctant to form tetraplexes

    Czech Academy of Sciences Publication Activity Database

    Fojtík, Petr; Kejnovská, Iva; Vorlíčková, Michaela

    2004-01-01

    Roč. 32, č. 1 (2004), s. 298-306 ISSN 0305-1048 R&D Projects: GA ČR GA204/01/0561; GA AV ČR IAA4004201 Institutional research plan: CEZ:AV0Z5004920 Keywords : fragile X chromosome syndrom * trinucleotide repeats * DNA polymorphism Subject RIV: BO - Biophysics Impact factor: 7.260, year: 2004

  6. Rebaudioside A inhibits pentylenetetrazol-induced convulsions in rats

    Directory of Open Access Journals (Sweden)

    Yigit Uyanikgil

    2016-09-01

    Full Text Available The safety of patients with epilepsy consuming sweetening agents, which is becoming increasingly prevalent for various reasons, is a topic that should be emphasized as sensitively as it is for other diseases. Patients with epilepsy consume sweetening agents for different reasons such being diabetic or overweight. They can occasionally be exposed to sweetening agents unrestrainedly through consuming convenience food, primarily beverages. This study aimed to investigate the effects of rebaudioside A (Reb-A, which is a steviol glycoside produced from the herb Stevia rebaudiana (Bertoni, on epileptic seizures and convulsions induced by pentylenetetrazole (PTZ. Forty-eight male rats were used. Twenty-four rats were administered 35 mg/kg PTZ to trigger epileptiform activity; the remaining 24 rats were administered 70 mg/kg PTZ to trigger the convulsion model. The epileptiform activity was evaluated by spike percentage, whereas convulsion was evaluated by Racine's Convulsion Scale and the onset time of the first myoclonic jerk. Statistical analysis revealed a statistically significant decrease in the Racine's Convulsion Scale score and increase in the latency of first myoclonic jerk in a dose-dependent manner for the rat groups in which PTZ epilepsy had been induced and Reb-A had been administered. For the groups that were administered Reb-A, the spike decrease was apparent in a dose-dependent manner, based on the spike percentage calculation. These results indicated that Reb-A has positive effects on PTZ-induced convulsions.

  7. A critical role of a cellular membrane traffic protein in poliovirus RNA replication.

    Directory of Open Access Journals (Sweden)

    George A Belov

    2008-11-01

    Full Text Available Replication of many RNA viruses is accompanied by extensive remodeling of intracellular membranes. In poliovirus-infected cells, ER and Golgi stacks disappear, while new clusters of vesicle-like structures form sites for viral RNA synthesis. Virus replication is inhibited by brefeldin A (BFA, implicating some components(s of the cellular secretory pathway in virus growth. Formation of characteristic vesicles induced by expression of viral proteins was not inhibited by BFA, but they were functionally deficient. GBF1, a guanine nucleotide exchange factor for the small cellular GTPases, Arf, is responsible for the sensitivity of virus infection to BFA, and is required for virus replication. Knockdown of GBF1 expression inhibited virus replication, which was rescued by catalytically active protein with an intact N-terminal sequence. We identified a mutation in GBF1 that allows growth of poliovirus in the presence of BFA. Interaction between GBF1 and viral protein 3A determined the outcome of infection in the presence of BFA.

  8. Interplay between toxin transport and flotillin localization

    DEFF Research Database (Denmark)

    Pust, Sascha; Dyve, Anne Berit; Torgersen, Maria L

    2010-01-01

    The flotillin proteins are localized in lipid domains at the plasma membrane as well as in intracellular compartments. In the present study, we examined the importance of flotillin-1 and flotillin-2 for the uptake and transport of the bacterial Shiga toxin (Stx) and the plant toxin ricin and we...... for flotillin-1 or -2. However, the Golgi-dependent sulfation of both toxins was significantly reduced in flotillin knockdown cells. Interestingly, when the transport of ricin to the ER was investigated, we obtained an increased mannosylation of ricin in flotillin-1 and flotillin-2 knockdown cells. The toxicity...... of both toxins was twofold increased in flotillin-depleted cells. Since BFA (Brefeldin A) inhibits the toxicity even in flotillin knockdown cells, the retrograde toxin transport is apparently still Golgi-dependent. Thus, flotillin proteins regulate and facilitate the retrograde transport of Stx and ricin....

  9. Combined theoretical and computational study of interstrand DNA guanine-guanine cross-linking by trans-[Pt(pyridine)2] derived from the photoactivated prodrug trans,trans,trans-[Pt(N3)2(OH)2(pyridine)2

    Czech Academy of Sciences Publication Activity Database

    Tai, H.-Ch.; Brodbeck, R.; Kašpárková, Jana; Farrer, N.J.; Brabec, Viktor; Sadler, P.J.; Deeth, R.J.

    2012-01-01

    Roč. 51, č. 12 (2012), s. 6830-6841 ISSN 0020-1669 R&D Projects: GA ČR(CZ) GAP301/10/0598 Institutional research plan: CEZ:AV0Z50040702 Keywords : platinum * photoactivation * DNA Subject RIV: BO - Biophysics Impact factor: 4.593, year: 2012

  10. Liquid chromatography-tandem mass spectrometry quantification of 6-thioguanine in DNA using endogenous guanine as internal standard

    DEFF Research Database (Denmark)

    Jacobsen, Jack Hummeland; Schmiegelow, Kjeld; Nersting, Jakob

    2012-01-01

    was estimated at 63% (RSD 26%), which is corrected for by the internal standard resulting in stable quantification. The TG levels found were above the LOQ in 18 out of 18 childhood leukemia patients on 6-mercaptopurine/methotrexate maintenance therapy (median 377, range 45-1190 fmol/μg DNA) with intra...

  11. Relative Stability of Different DNA Guanine Quadruplex Stem Topologies Derived Using Large-Scale Quantum-Chemical Computations

    Czech Academy of Sciences Publication Activity Database

    Šponer, Jiří (ed.); Mládek, Arnošt; Špačková, Naďa; Cang, X.; Cheatham III, Thomas E.; Grimme, S.

    2013-01-01

    Roč. 135, č. 26 (2013), s. 9785-9796 ISSN 0002-7863 R&D Projects: GA MŠk(CZ) ED1.1.00/02.0068; GA ČR(CZ) GAP208/11/1822 Institutional research plan: CEZ:AV0Z50040702 Institutional support: RVO:68081707 Keywords : DENSITY -FUNCTIONAL THEORY * MOLECULAR-DYNAMICS SIMULATIONS * SUGAR-PHOSPHATE BACKBONE Subject RIV: BO - Biophysics Impact factor: 11.444, year: 2013

  12. Repair of ultraviolet-light damaged ColE1 factor carrying Escherichia coli genes for guanine synthesis

    International Nuclear Information System (INIS)

    Kibe, A.; Shimada, K.; Tagaki, Y.

    1979-01-01

    Hybrid ColE1 plasmids called ColE1-cos lambda-guaA or ColE1-cos lambda-gal can be efficiently transduced into various E.coli K-12 cells through packaging into lambda phage particles. Using these plasmids, repair of ultraviolet-light (UV) damaged ColE1 DNAs was studied in various UV sensitive E.coli K-12 mutants. The host mutations uvrA and uvrB markedly reduced host-cell reactivation of UV-irradiated ColE1-cos lambda-guaA. Pre-existing hybrid ColE1 plasmids had no effect on the frequency of lambda phage-mediated transduction of another differentially marked hybrid ColE1 DNAs. ColE1-cos lambda-guaA and ColE1-cos lambda-gal DNAs could temporarily but not stably co-exist in E.coli K-12 recA cells. The presence of ColE1-cos lambda-gal in uvrB cells promoted the repair of super-infected UV-irradiated ColE1-cos lambda-guaA about 7-fold. The same ColE1-cos lambda-gal plasmid in a uvrB recA double mutant did not have this promoting effect. These results indicate that the effect of resident hybrid ColE1 plasmids is manifested by the host recA + gene function(s) and suggest that ColE1 plasmit itself provides no recA + -like functions. (orig.) [de

  13. [Quantum-chemical investigation of tautomerization ways of Watson-Crick DNA base pair guanine-cytosine].

    Science.gov (United States)

    Brovarets', O O; Hovorun, D M

    2010-01-01

    A novel physico-chemical mechanism of the Watson-Crick DNA base pair Gua.Cyt tautomerization Gua.Cyt*Gua.CytGua*.Cyt (mutagenic tautomers of bases are marked by asterisks) have been revealed and realized in a pathway of single proton transfer through two mutual isoenergetic transition states with Gibbs free energy of activation 30.4 and 30.6 kcal/mol and they are ion pairs stabilized by three (N2H...N3, N1H...N4- and O6+H...N4-) and five (N2H...O2, N1H...O2, N1H...N3, O6+H...N4- and 06+H...N4-) H-bonds accordingly. Stable base pairs Gua-Cyt* and Gua*.Cyt which dissociate comparably easy into monomers have acceptable relative Gibbs energies--12.9 and 14.3 kcal/mol--for the explanation of the nature of the spontaneous transitions of DNA replication. Results are obtained at the MP2/6-311++G(2df,pd)//B3LYP/6-31 1++G(d,p) level of theory in vacuum approach.

  14. Methyltransferase That Modifies Guanine 966 of the 16 S rRNA: FUNCTIONAL IDENTIFICATION AND TERTIARY STRUCTURE*

    Science.gov (United States)

    Lesnyak, Dmitry V.; Osipiuk, Jerzy; Skarina, Tatiana; Sergiev, Petr V.; Bogdanov, Alexey A.; Edwards, Aled; Savchenko, Alexei; Joachimiak, Andrzej; Dontsova, Olga A.

    2010-01-01

    N2-Methylguanine 966 is located in the loop of Escherichia coli 16 S rRNA helix 31, forming a part of the P-site tRNA-binding pocket. We found yhhF to be a gene encoding for m2G966 specific 16 S rRNA methyltransferase. Disruption of the yhhF gene by kanamycin resistance marker leads to a loss of modification at G966. The modification could be rescued by expression of recombinant protein from the plasmid carrying the yhhF gene. Moreover, purified m2G966 methyltransferase, in the presence of S-adenosylomethionine (AdoMet), is able to methylate 30 S ribosomal subunits that were purified from yhhF knock-out strain in vitro. The methylation is specific for G966 base of the 16 S rRNA. The m2G966 methyltransferase was crystallized, and its structure has been determined and refined to 2.05 Å. The structure closely resembles RsmC rRNA methyltransferase, specific for m2G1207 of the 16 S rRNA. Structural comparisons and analysis of the enzyme active site suggest modes for binding AdoMet and rRNA to m2G966 methyltransferase. Based on the experimental data and current nomenclature the protein expressed from the yhhF gene was renamed to RsmD. A model for interaction of RsmD with ribosome has been proposed. PMID:17189261

  15. Methyltransferase that modifies guanine 966 of the 16 S rRNA: functional identification and tertiary structure.

    Science.gov (United States)

    Lesnyak, Dmitry V; Osipiuk, Jerzy; Skarina, Tatiana; Sergiev, Petr V; Bogdanov, Alexey A; Edwards, Aled; Savchenko, Alexei; Joachimiak, Andrzej; Dontsova, Olga A

    2007-02-23

    N(2)-Methylguanine 966 is located in the loop of Escherichia coli 16 S rRNA helix 31, forming a part of the P-site tRNA-binding pocket. We found yhhF to be a gene encoding for m(2)G966 specific 16 S rRNA methyltransferase. Disruption of the yhhF gene by kanamycin resistance marker leads to a loss of modification at G966. The modification could be rescued by expression of recombinant protein from the plasmid carrying the yhhF gene. Moreover, purified m(2)G966 methyltransferase, in the presence of S-adenosylomethionine (AdoMet), is able to methylate 30 S ribosomal subunits that were purified from yhhF knock-out strain in vitro. The methylation is specific for G966 base of the 16 S rRNA. The m(2)G966 methyltransferase was crystallized, and its structure has been determined and refined to 2.05A(.) The structure closely resembles RsmC rRNA methyltransferase, specific for m(2)G1207 of the 16 S rRNA. Structural comparisons and analysis of the enzyme active site suggest modes for binding AdoMet and rRNA to m(2)G966 methyltransferase. Based on the experimental data and current nomenclature the protein expressed from the yhhF gene was renamed to RsmD. A model for interaction of RsmD with ribosome has been proposed.

  16. Folding of guanine quadruplex molecules-funnel-like mechanism or kinetic partitioning? An overview from MD simulation studies

    Czech Academy of Sciences Publication Activity Database

    Šponer, Jiří; Bussi, G.; Stadlbauer, Petr; Kührová, P.; Banáš, P.; Islam, Barira; Haider, S.; Neidle, S.; Otyepka, M.

    2017-01-01

    Roč. 1861, č. 5 (2017), s. 1246-1263 ISSN 0304-4165 R&D Projects: GA ČR(CZ) GA16-13721S Institutional support: RVO:68081707 Keywords : telomeric g-quadruplex * parallel g-quadruplex Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 4.702, year: 2016

  17. VEGF-induced Rac1 activation in endothelial cells is regulated by the guanine nucleotide exchange factor Vav2

    NARCIS (Netherlands)

    Garrett, Tiana A.; van Buul, Jaap D.; Burridge, Keith

    2007-01-01

    Vascular endothelial growth factor (VEGF) signaling is critical for both normal and disease-associated vascular development. Dysregulated VEGF signaling has been implicated in ischemic stroke, tumor angiogenesis, and many other vascular diseases. VEGF signals through several effectors, including the

  18. Are genes destiny? Have adenine, cytosine, guanine and thymine replaced Lachesis, Clotho and Atropos as the weavers of our fate?

    Science.gov (United States)

    Eisenberg, Leon

    2005-02-01

    It is as futile to ask how much of the phenotype of an organism is due to nature and how much to its nurture as it is to determine how much of the area of a rectangle is due to its length and how much to its height. Phenotype and area are joint products. The spectacular success of genomics, unfortunately, threatens to re-awaken belief in genes as the principal determinants of human behavior. This paper develops the thesis that gene expression is modified by environmental inputs and that the impact of the environment on a given organism is modified by its genome. Genes set the boundaries of the possible; environments parse out the actual.

  19. Effect of Guanine to Inosine Substitution on Stability of Canonical DNA and RNA Duplexes: Molecular Dynamics Thermodynamics Integration Study

    Czech Academy of Sciences Publication Activity Database

    Krepl, Miroslav; Otyepka, M.; Banáš, Pavel; Šponer, Jiří

    2013-01-01

    Roč. 117, č. 6 (2013), s. 1872-1879 ISSN 1520-6106 R&D Projects: GA ČR(CZ) GBP305/12/G034 Grant - others:GA ČR(CZ) GPPP301/11/P558; GA MŠk(CZ) ED1.1.00/02.0068 Program:ED Institutional research plan: CEZ:AV0Z50040702 Institutional support: RVO:68081707 Keywords : FREE-ENERGY CALCULATIONS * PARTICLE MESH EWALD * ACID BASE-PAIRS Subject RIV: BO - Biophysics Impact factor: 3.377, year: 2013

  20. Can We Execute Reliable MM-PBSA Free Energy Computations of Relative Stabilities of Different Guanine Quadruplex Folds?

    Czech Academy of Sciences Publication Activity Database

    Islam, B.; Stadlbauer, Petr; Neidle, S.; Haider, S.; Šponer, Jiří

    2016-01-01

    Roč. 120, č. 11 (2016), s. 2899-2912 ISSN 1520-6106 R&D Projects: GA ČR(CZ) GA16-13721S Institutional support: RVO:68081707 Keywords : MOLECULAR-DYNAMICS SIMULATIONS * TELOMERIC G-QUADRUPLEX * AMBER FORCE-FIELD Subject RIV: BO - Biophysics Impact factor: 3.177, year: 2016

  1. Strikingly Different Effects of Hydrogen Bonding on the Photodynamics of Individual Nucleobases in DNA: Comparison of Guanine and Cytosine

    Czech Academy of Sciences Publication Activity Database

    Zelený, T.; Ruckenbauer, M.; Aquino, A. J. A.; Müller, T.; Lankaš, Filip; Dršata, Tomáš; Hase, W. L.; Nachtigallová, Dana; Lischka, H.

    2012-01-01

    Roč. 134, č. 33 (2012), s. 13662-13669 ISSN 0002-7863 R&D Projects: GA ČR GAP208/12/1318; GA MŠk(CZ) LH11021 Grant - others:GA ČR(CZ) GD203/09/H046 Program:GD Institutional support: RVO:61388963 Keywords : excited-state dynamics * ultrafast internal-conversion * photoinduced nonadiabatic dynamics * nonradiative decay mechanisms * MR-CI level * ab initio Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 10.677, year: 2012

  2. Tyrosine Phosphorylation of the Guanine Nucleotide Exchange Factor GIV Promotes Activation of PI3K During Cell Migration

    Science.gov (United States)

    Lin, Changsheng; Ear, Jason; Pavlova, Yelena; Mittal, Yash; Kufareva, Irina; Ghassemian, Majid; Abagyan, Ruben; Garcia-Marcos, Mikel; Ghosh, Pradipta

    2014-01-01

    GIV (Gα-interacting vesicle-associated protein; also known as Girdin), enhances Akt activation downstream of multiple growth factor– and G-protein–coupled receptors to trigger cell migration and cancer invasion. Here we demonstrate that GIV is a tyrosine phosphoprotein that directly binds to and activates phosphoinositide 3-kinase (PI3K). Upon ligand stimulation of various receptors, GIV was phosphorylated at Tyr1764 and Tyr1798 by both receptor and non-receptor tyrosine kinases. These phosphorylation events enabled direct binding of GIV to the N- and C-terminal SH2 domains of p85α, a regulatory subunit of PI3K, stabilized receptor association with PI3K, and enhanced PI3K activity at the plasma membrane to trigger cell migration. Tyrosine phosphorylation of GIV and its association with p85α increased during metastatic progression of a breast carcinoma. These results suggest a mechanism by which multiple receptors activate PI3K through tyrosine phosphorylation of GIV, thereby making the GIVPI3K interaction a potential therapeutic target within the PI3K-Akt pathway. PMID:21954290

  3. Repair of ultraviolet-light damaged ColE1 factor carrying Escherichia coli genes for guanine synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Kibe, A; Shimada, K; Tagaki, Y [Kyushu Univ., Fukuoka (Japan). Dept. of Biochemistry

    1979-01-01

    Hybrid ColE1 plasmids called ColE1-cos lambda-guaA or ColE1-cos lambda-gal can be efficiently transduced into various E.coli K-12 cells through packaging into lambda phage particles. Using these plasmids, repair of ultraviolet-light (UV) damaged ColE1 DNAs was studied in various UV sensitive E.coli K-12 mutants. The host mutations uvrA and uvrB markedly reduced host-cell reactivation of UV-irradiated ColE1-cos lambda-guaA. Pre-existing hybrid ColE1 plasmids had no effect on the frequency of lambda phage-mediated transduction of another differentially marked hybrid ColE1 DNAs. ColE1-cos lambda-guaA and ColE1-cos lambda-gal DNAs could temporarily but not stably co-exist in E.coli K-12 recA cells. The presence of ColE1-cos lambda-gal in uvrB cells promoted the repair of super-infected UV-irradiated ColE1-cos lambda-guaA about 7-fold. The same ColE1-cos lambda-gal plasmid in a uvrB recA double mutant did not have this promoting effect. These results indicate that the effect of resident hybrid ColE1 plasmids is manifested by the host recA/sup +/ gene function(s) and suggest that ColE1 plasmit itself provides no recA/sup +/-like functions.

  4. Hydrogen Evolution Facilitates Reduction of DNA Guanine Residues at the Hanging Mercury Drop Electrode: Evidence for a Chemical Mechanism

    Czech Academy of Sciences Publication Activity Database

    Daňhel, Aleš; Havran, Luděk; Trnková, L.; Fojta, Miroslav

    2016-01-01

    Roč. 28, č. 11 (2016), s. 2785-2790 ISSN 1040-0397 R&D Projects: GA ČR GAP206/11/1638; GA ČR(CZ) GA16-01625S Institutional support: RVO:68081707 Keywords : central trinucleotide sequences * cyclic voltammetry * cisplatin Subject RIV: BO - Biophysics Impact factor: 2.851, year: 2016

  5. Cell-Specific Establishment of Poliovirus Resistance to an Inhibitor Targeting a Cellular Protein

    Science.gov (United States)

    Viktorova, Ekaterina G.; Nchoutmboube, Jules; Ford-Siltz, Lauren A.

    2015-01-01

    ABSTRACT It is hypothesized that targeting stable cellular factors involved in viral replication instead of virus-specific proteins may raise the barrier for development of resistant mutants, which is especially important for highly adaptable small (+)RNA viruses. However, contrary to this assumption, the accumulated evidence shows that these viruses easily generate mutants resistant to the inhibitors of cellular proteins at least in some systems. We investigated here the development of poliovirus resistance to brefeldin A (BFA), an inhibitor of the cellular protein GBF1, a guanine nucleotide exchange factor for the small cellular GTPase Arf1. We found that while resistant viruses can be easily selected in HeLa cells, they do not emerge in Vero cells, in spite that in the absence of the drug both cultures support robust virus replication. Our data show that the viral replication is much more resilient to BFA than functioning of the cellular secretory pathway, suggesting that the role of GBF1 in the viral replication is independent of its Arf activating function. We demonstrate that the level of recruitment of GBF1 to the replication complexes limits the establishment and expression of a BFA resistance phenotype in both HeLa and Vero cells. Moreover, the BFA resistance phenotype of poliovirus mutants is also cell type dependent in different cells of human origin and results in a fitness loss in the form of reduced efficiency of RNA replication in the absence of the drug. Thus, a rational approach to the development of host-targeting antivirals may overcome the superior adaptability of (+)RNA viruses. IMPORTANCE Compared to the number of viral diseases, the number of available vaccines is miniscule. For some viruses vaccine development has not been successful after multiple attempts, and for many others vaccination is not a viable option. Antiviral drugs are needed for clinical practice and public health emergencies. However, viruses are highly adaptable and can

  6. Somatotropin has no effect on the quantity of guanine nucleotide binding proteins Gq alpha/G11 alpha in goat adipose tissue in vivo

    Czech Academy of Sciences Publication Activity Database

    Krbeček, Vlastimil; Kovářů, H.; Škarda, Josef; Barth, Tomislav; Velek, Jiří; Žižkovský, V.

    2000-01-01

    Roč. 49, - (2000), s. 673-678 ISSN 0862-8408 R&D Projects: GA ČR GA523/99/0843; GA AV ČR IAA7045608; GA AV ČR KSK2020602 Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.366, year: 2000

  7. Mono(ADP-ribosyl)ation of the N2 amino groups of guanine residues in DNA by pierisin-2, from the cabbage butterfly, Pieris brassicae

    International Nuclear Information System (INIS)

    Takamura-Enya, Takeji; Watanabe, Masahiko; Koyama, Kotaro; Sugimura, Takashi; Wakabayashi, Keiji

    2004-01-01

    Pierisin-2 is a cytotoxic and apoptosis-inducing protein present in Pieris brassicae with a 91% homology in the deduced amino acid sequences to pierisin-1 from Pieris rapae. We earlier showed pierisin-1 to catalyze mono(ADP-ribosyl)ation of 2'-deoxyguanosine (dG) in DNA to form N 2 -(ADP-ribos-1-yl)-2'-deoxyguanosine, this DNA modification appearing linked to its cytotoxicity and ability to induce apoptosis in mammalian cell lines. In this paper, we documented evidence that pierisin-2 also catalyzed ADP-ribosylation of dG in DNA to give the same reaction product as demonstrated for pierisin-1, with similar efficiency. With oligonucleotides as substrates, ADP-ribosylation by pierisin-2 was suggested to occur by one-side attack of the carbon atom at 1 position of the ribose moiety in NAD toward N 2 of dG. The presence of a unique ADP-ribosylation toxin targeting dG in DNA in two distinct species in a Pieris genus could be a quite important finding to better understand biological functions of pierisin-1 and -2 in Pieris butterflies and the generic evolution of these cabbage butterflies

  8. Hydration effects on the photoionization energy of 2‧-deoxyguanosine 5‧-phosphate and activation barriers for guanine methylation by carcinogenic methane diazonium ions

    Science.gov (United States)

    Eichler, Daniel R.; Hamann, Haley A.; Harte, Katherine A.; Papadantonakis, George A.

    2017-07-01

    Results from DFT calculations indicate that states originating from gas-phase ionization of the phosphate and the base are degenerate in syn-5‧-dGMP- and that bulk hydration lowers the base-localized ionization energy by diazonium ions are lower at the N7 than at the O6 sites and they are influenced by local ionization energy and steric interference.

  9. Methyl DNA adducts, DNA repair, and hypoxanthine-guanine phosphoribosyl transferase mutations in peripheral white blood cells from patients with malignant melanoma treated with dacarbazine and hydroxyurea

    NARCIS (Netherlands)

    Philip, P.A.; Souliotis, V.L.; Harris, A.L.; Salisbury, A.; Tates, A.D.; Mitchell, K.; Delft, J.H.M. van; Ganesan, T.S.; Kyrtopoulos, S.A.

    1996-01-01

    Dacarbazine (DTIC) is a DNA-methylating drug used in the treatment of malignant melanoma. Among the DNA dducts induced by DTIC are N7-methylguanine (N7-meG) and O6-methylguamne (O6-meG). The latter adduct, in particular, may be important in the mutagenic as well as the cytotoxic activity of DTIC.

  10. Bacillus subtilis guanine deaminase is encoded by the yknA gene and is induced during growth with purines as the nitrogen source

    DEFF Research Database (Denmark)

    Nygaard, P.; Bested, S. M.; Andersen, K. A. K.

    2000-01-01

    amino acid polypeptide and was preceded by a promoter sequence that is recognized by the A form of RNA polymerase. High levels of GDEase were found in cells grown with purines and intermediary compounds of the purine catabolic pathway as nitrogen sources. Allantoic acid, most likely, is a low molecular...

  11. Theoretical study on the structure, stability, and electronic properties of the guanine-Zn-cytosine base pair in M-DNA

    Czech Academy of Sciences Publication Activity Database

    Fuentes-Cabrera, M.; Sumpter, B.G.; Šponer, Judit E.; Šponer, Jiří; Petit, L.; Wells, J.C.

    2007-01-01

    Roč. 111, č. 4 (2007), s. 870-879 ISSN 1520-6106 R&D Projects: GA AV ČR(CZ) 1QS500040581; GA ČR(CZ) GA203/05/0388; GA ČR(CZ) GA203/05/0009; GA MŠk(CZ) LC512 Institutional research plan: CEZ:AV0Z50040702; CEZ:AV0Z40550506 Keywords : metalated DNA * ab initio * electronic properties of DNA Subject RIV: BO - Biophysics Impact factor: 4.086, year: 2007

  12. Adenine versus Guanine Quartets in Aqueous Solution. Dispersion-Corrected DFT Study on the Differences in π-Stacking and Hydrogen-Bonding Behavior

    NARCIS (Netherlands)

    Fonseca Guerra, C.; van der Wijst, T.; Poater, J.; Swart, M.; Bickelhaupt, F.M.

    2010-01-01

    We have investigated the performance of the dispersion-corrected density functionals (BLYP-D, BP86-D and PBE-D) and the widely used B3LYP functional for describing the hydrogen bonds and the stacking interactions in DNA base dimers. For the gas-phase situation, the bonding energies have been

  13. Influence of Hydration on Proton Transfer in the Guanine-Cytosine Radical Cation (G•+-C) Base Pair: A Density Functional Theory Study

    Science.gov (United States)

    Kumar, Anil; Sevilla, Michael D.

    2009-01-01

    On one-electron oxidation all molecules including DNA bases become more acidic in nature. For the GC base pair experiments suggest that a facile proton transfer takes place in the G•+-C base pair from N1 of G•+ to N3 of cytosine. This intra-base pair proton transfer reaction has been extensively considered using theoretical methods for the gas phase and it is predicted that the proton transfer is slightly unfavorable in disagreement with experiment. In the present study, we consider the effect of the first hydration layer on the proton transfer reaction in G•+-C by the use of density functional theory (DFT), B3LYP/6-31+G** calculations of the G•+-C base pair in the presence of 6 and 11 water molecules. Under the influence of hydration of 11 waters, a facile proton transfer from N1 of G•+ to N3 of C is predicted. The zero point energy (ZPE) corrected forward and backward energy barriers, for the proton transfer from N1 of G•+ to N3 of C, was found to be 1.4 and 2.6 kcal/mol, respectively. The proton transferred G•-(H+)C + 11H2O was found to be 1.2 kcal/mol more stable than G•+-C + 11H2O in agreement with experiment. The present calculation demonstrates that the inclusion of the first hydration shell around G•+-C base pair has an important effect on the internal proton transfer energetics. PMID:19485319

  14. Thermodynamic and spectroscopic investigations of TMPyP4 association with guanine- and cytosine-rich DNA and RNA repeats of C9orf72.

    Science.gov (United States)

    Alniss, Hasan; Zamiri, Bita; Khalaj, Melisa; Pearson, Christopher E; Macgregor, Robert B

    2018-01-22

    An expansion of the hexanucleotide repeat (GGGGCC)n·(GGCCCC)n in the C9orf72 promoter has been shown to be the cause of Amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD). The C9orf72 repeat can form four-stranded structures; the cationic porphyrin (TMPyP4) binds and distorts these structures. Isothermal titration calorimetry (ITC), and circular dichroism (CD) were used to study the binding of TMPyP4 to the C-rich and G-rich DNA and RNA oligos containing the hexanucleotide repeat at pH 7.5 and 0.1 M K + . The CD spectra of G-rich DNA and RNA TMPyP4 complexes showed features of antiparallel and parallel G-quadruplexes, respectively. The shoulder at 260 nm in the CD spectrum becomes more intense upon formation of complexes between TMPyP4 and the C-rich DNA. The peak at 290 nm becomes more intense in the c-rich RNA molecules, suggesting induction of an i-motif structure. The ITC data showed that TMPyP4 binds at two independent sites for all DNA and RNA molecules. For DNA, the data are consistent with TMPyP4 stacking on the terminal tetrads and intercalation. For RNA, the thermodynamics of the two binding modes are consistent with groove binding and intercalation. In both cases, intercalation is the weaker binding mode. These findings are considered with respect to the structural differences of the folded DNA and RNA molecules and the energetics of the processes that drive site-specific recognition by TMPyP4; these data will be helpful in efforts to optimize the specificity and affinity of the binding of porphyrin-like molecules. Copyright © 2018 Elsevier Inc. All rights reserved.

  15. Genetic deficiency of the α subunit of the guanine nucleotide-binding protein G/sub s/ as the molecular basis for Albright hereditary osteodystrophy

    International Nuclear Information System (INIS)

    Levine, M.A.; Ahn, T.G.; Klupt, S.F.; Kaufman, K.D.; Smallwood, P.M.; Bourne, H.R.; Sullivan, K.A.; Van Dop, C.

    1988-01-01

    Patients who have pseudohypoparathyroidism type I associated with Albright hereditary osteodystrophy commonly have a genetic deficiency of the α subunit of the G protein that stimulated adenylyl cyclase αG/sub s/. To discover the molecular mechanism that causes αG/sub s/ deficiency in these patients, the authors examined eight kindreds with one or more members affected with Albright hereditary osteodystrophy or pseudohypoparathyroidism and αG/sub s/ deficiency. In these families, αG/sub s/, deficiency and the Albright hereditary osteodystrophy phenotype were transmitted together in a dominant inheritance pattern. Using a cDNA hybridization probe for αG/sub s/, restriction analysis with several analysis with several endonucleases showed no abnormalities of restriction fragments or gene dosage. RNA blot and dot blot analysis of total RNA from cultured fibroblasts obtained from the patients revealed ∼ 50% reduced mRNA levels for αG/sub s/ in affected members of six of the pedigrees but normal levels in affected members of the two other pedigrees, compared to mRNA levels in fibroblasts from unaffected individuals. By contrast, mRNA levels encoding the α subunit of the G protein that inhibits adenylyl cyclase were not altered. These findings suggest that several molecular mechanisms produce αG/sub s/ deficiency in patients with pseudohypoparathyroidism type Ia and that major gene rearrangements or deletions are not a common cause for αG/sub s/ deficiency in pseudohypoparathyroidism type I

  16. Synthesis of 9-phosphonoalkyl and 9-phosphonoalkoxyalkyl purines: Evaluation of their ability to act as inhibitors of Plasmodium falciparum, Plasmodium vivax and human hypoxanthine-guanine-(xanthine) phosphoribosyltransferases

    Czech Academy of Sciences Publication Activity Database

    Česnek, Michal; Hocková, Dana; Holý, Antonín; Dračínský, Martin; Baszczyňski, Ondřej; de Jersey, J.; Keough, D. T.; Guddat, L. W.

    2012-01-01

    Roč. 20, č. 2 (2012), s. 1076-1089 ISSN 0968-0896 R&D Projects: GA MŠk 1M0508; GA ČR GAP207/11/0108 Institutional research plan: CEZ:AV0Z40550506 Keywords : Plasmodium * malaria * acyclic nucleoside phosphonates Subject RIV: CC - Organic Chemistry Impact factor: 2.903, year: 2012

  17. Theoretical study on the structure, stability, and electronic properties of the guanine-Zn-cytosine base pair in M-DNA

    International Nuclear Information System (INIS)

    Fuentes-Cabrera, Miguel A.; Sumpter, Bobby G.; Sponer, Judit; Sponer, Jiri; Petit, Leon; Wells, Jack C.

    2007-01-01

    M-DNA is a type of metalated DNA that forms at high pH and in the presence of Zn, Ni, and Co, with the metals placed in between each base pair, as in G-Zn-C. Experiments have found that M-DNA could be a promising candidate for a variety of nanotechnological applications, as it is speculated that the metal d-states enhance the conductivity, but controversy still clouds these findings. In this paper, we carry out a comprehensive ab initio study of eight G-Zn-C models in the gas phase to help discern the structure and electronic properties of Zn-DNA. Specifically, we study whether a model prefers to be planar and has electronic properties that correlate with Zn-DNA having a metallic-like conductivity. Out of all the studied models, there is only one which preserves its planarity upon full geometry optimization. Nevertheless, starting from this model, one can deduce a parallel Zn-DNA architecture only. This duplex would contain the imino proton, in contrast to what has been proposed experimentally. Among the nonplanar models, there is one that requires less than 8 kcal/mol to flatten (both in gas and solvent conditions), and we propose that it is a plausible model for building an antiparallel duplex. In this duplex, the imino proton would be replaced by Zn, in accordance with experimental models. Neither planar nor nonplanar models have electronic properties that correlate with Zn-DNA having a metallic-like conductivity due to Zn d-states. To understand whether density functional theory (DFT) can describe appropriately the electronic properties of M-DNAs, we have investigated the electronic properties of G-Co-C base pairs. We have found that when self-interaction corrections (SIC) are not included the HOMO state contains Co d-levels, whereas these levels are moved below the HOMO state when SIC are considered. This result indicates that caution should be exercised when studying the electronic properties of M-DNAs with functionals that do not account for strong electronic correlations

  18. ARF6 Activated by the LHCG Receptor through the Cytohesin Family of Guanine Nucleotide Exchange Factors Mediates the Receptor Internalization and Signaling*

    Science.gov (United States)

    Kanamarlapudi, Venkateswarlu; Thompson, Aiysha; Kelly, Eamonn; López Bernal, Andrés

    2012-01-01

    The luteinizing hormone chorionic gonadotropin receptor (LHCGR) is a Gs-coupled GPCR that is essential for the maturation and function of the ovary and testis. LHCGR is internalized following its activation, which regulates the biological responsiveness of the receptor. Previous studies indicated that ADP-ribosylation factor (ARF)6 and its GTP-exchange factor (GEF) cytohesin 2 regulate LHCGR internalization in follicular membranes. However, the mechanisms by which ARF6 and cytohesin 2 regulate LHCGR internalization remain incompletely understood. Here we investigated the role of the ARF6 signaling pathway in the internalization of heterologously expressed human LHCGR (HLHCGR) in intact cells using a combination of pharmacological inhibitors, siRNA and the expression of mutant proteins. We found that human CG (HCG)-induced HLHCGR internalization, cAMP accumulation and ARF6 activation were inhibited by Gallein (βγ inhibitor), Wortmannin (PI 3-kinase inhibitor), SecinH3 (cytohesin ARF GEF inhibitor), QS11 (an ARF GAP inhibitor), an ARF6 inhibitory peptide and ARF6 siRNA. However, Dynasore (dynamin inhibitor), the dominant negative mutants of NM23-H1 (dynamin activator) and clathrin, and PBP10 (PtdIns 4,5-P2-binding peptide) inhibited agonist-induced HLHCGR and cAMP accumulation but not ARF6 activation. These results indicate that heterotrimeric G-protein, phosphatidylinositol (PI) 3-kinase (PI3K), cytohesin ARF GEF and ARF GAP function upstream of ARF6 whereas dynamin and clathrin act downstream of ARF6 in the regulation of HCG-induced HLHCGR internalization and signaling. In conclusion, we have identified the components and molecular details of the ARF6 signaling pathway required for agonist-induced HLHCGR internalization. PMID:22523074

  19. Developmental changes in the role of a pertussis toxin sensitive guanine nucleotide binding protein in the rat cardiac alpha1-adrenergic system

    International Nuclear Information System (INIS)

    Han, H.M.

    1989-01-01

    During development, the cardiac alpha 1 -adrenergic chronotropic response changes from positive in the neonate to negative in the adult. This thesis examined the possibility of a developmental change in coupling of a PT-sensitive G-protein to the alpha 1 -adrenergic receptor. Radioligand binding experiments performed with the iodinated alpha 1 -selective radioligand [ 125 I]-I-2-[β-(4-hydroxphenyl)ethylaminomethyl]tetralone ([ 125 I]-IBE 2254) demonstrated that the alpha 1 -adrenergic receptor is coupled to a G-protein in both neonatal and adult rat hearts. However, in the neonate the alpha 1 -adrenergic receptor is coupled to a PT-insensitive G-protein, whereas in the adult the alpha 1 -adrenergic receptor is coupled to both a PT-insensitive and a PT-sensitive G-protein. Consistent with the results from binding experiments, PT did not have any effect on the alpha 1 -mediated positive chronotropic response in the neonate, whereas in the adult the alpha 1 -mediated negative chronotropic response was completely converted to a positive one after PT-treatment. This thesis also examined the possibility of an alteration in coupling of the alpha 1 -adrenergic receptor to its effector under certain circumstances such as high potassium (K + ) depolarization in nerve-muscle (NM) co-cultures, a system which has been previously shown to be a convenient in vitro model to study the mature inhibitory alpha 1 -response

  20. Serine34 phosphorylation of RHO guanine dissociation inhibitor (RHOGDI{alpha}) links signaling from conventional protein kinase C to RHO GTPase in cell adhesion

    DEFF Research Database (Denmark)

    Dovas, Athanassios; Choi, Youngsil; Yoneda, Atsuko

    2010-01-01

    . Phosphospecific antibodies reveal endogenous phosphorylation in several cell types that is sensitive to adhesion events triggered, for example, by hepatocyte growth factor. Phosphorylation is also sensitive to PKC inhibition. Together with FRET microscopy sensing GTP-RhoA levels, the data reveal a common pathway...

  1. The effect of novel [3-fluoro-(2-phosphonoethoxy)propyl]purines on the inhibition of Plasmodium falciparum, Plasmodium vivax and human hypoxanthine-guanine-(xanthine) phosphoribosyltransferases

    Czech Academy of Sciences Publication Activity Database

    Baszczyňski, Ondřej; Hocková, Dana; Janeba, Zlatko; Holý, Antonín; Jansa, Petr; Dračínský, Martin; Keough, D. T.; Guddat, L. W.

    2013-01-01

    Roč. 67, Sep (2013), s. 81-89 ISSN 0223-5234 R&D Projects: GA MV VG20102015046; GA ČR GAP207/11/0108 Grant - others:National Health and Madical Research Council(AU) 1030353; National Health and Medical Research Council(AU) 569703 Institutional support: RVO:61388963 Keywords : Malaria * nucleoside phosphonates * fluorine * purines * FPMP Subject RIV: FR - Pharmacology ; Medidal Chemistry Impact factor: 3.432, year: 2013

  2. Stimulation through the T cell receptor induces Cbl association with Crk proteins and the guanine nucleotide exchange protein C3G

    NARCIS (Netherlands)

    Reedquist, K. A.; Fukazawa, T.; Panchamoorthy, G.; Langdon, W. Y.; Shoelson, S. E.; Druker, B. J.; Band, H.

    1996-01-01

    We and others have recently identified Cbl, the protein product of the c-cbl protooncogene, as an early tyrosine kinase substrate upon T cell activation and have shown that Cbl forms in vivo complexes with Src family tyrosine kinases, Grb2 adaptor protein, and the p85 subunit of PI-3 kinase. Here we

  3. WNT5A inhibits human dental papilla cell proliferation and migration

    International Nuclear Information System (INIS)

    Peng, L.; Ye, L.; Dong, G.; Ren, L.B.; Wang, C.L.; Xu, P.; Zhou, X.D.

    2009-01-01

    WNT proteins are a large family of cysteine-rich secreted molecules that are linked to both canonical and non-canonical signal pathways, and have been implicated in oncogenesis and tissue development. Canonical WNT proteins have been proven to play critical roles in tooth development, while little is known about the role of non-canonical WNT proteins such as WNT5A. In this study, WNT5A was localized to human dental papilla tissue and human dental papilla cells (HDPCs) cultured in vitro, using immunochemistry and RT-PCR. Recombinant adenovirus encoding full-length Wnt5a cDNA was constructed to investigate the biological role of WNT5A on HDPCs. The BrdU incorporation assay, the MTT assay and flow cytometric analysis showed that over-expression of Wnt5a strongly inhibited the proliferation of HDPCs in vitro. Wound healing and transwell migration assays indicated that over-expression of WNT5A reduced migration of HDPCs. In conclusion, our results showed that WNT5A negatively regulates both proliferation and migration of HDPCs, suggesting its important role in odontogenesis via controlling the HDPCs.

  4. Middle East Respiratory Coronavirus Accessory Protein 4a Inhibits PKR-Mediated Antiviral Stress Responses

    NARCIS (Netherlands)

    Rabouw, Huib H; Langereis, Martijn A; Knaap, Robert C M; Dalebout, Tim J; Canton, Javier; Sola, Isabel; Enjuanes, Luis; Bredenbeek, Peter J; Kikkert, Marjolein; de Groot, Raoul J; van Kuppeveld, Frank J M

    2016-01-01

    Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory infections that can be life-threatening. To establish an infection and spread, MERS-CoV, like most other viruses, must navigate through an intricate network of antiviral host responses. Besides the well-known type I

  5. Cerebral monoamine oxidase A inhibition in tobacco smokers confirmed with PET and [11C]Befloxatone

    International Nuclear Information System (INIS)

    Leroy, C.; Bragulat, V.; Penttila, J.; Artiges, E.; Martinot, J.L.; Trichard, Ch.; Leroy, C.; Bragulat, V.; Penttila, J.; Artiges, E.; Martinot, J.L.; Trichard, Ch.; Leroy, C.; Bragulat, V.; Penttila, J.; Artiges, E.; Martinot, J.L.; Trichard, Ch.; Berlin, I.; Gregoire, M.C.; Bottlaender, M.; Roumenov, D.; Dolle, F.; Bourgeois, S.; Artiges, E.; Trichard, Ch.

    2009-01-01

    The inhibition of cerebral monoamine oxidases (MAOs) by cigarette smoke components could participate to the tobacco addiction. However, the actual extent of this inhibition in vivo in smokers is still poorly known. We investigated cerebral MAO-A availability in 7 tobacco-dependent subjects and 6 healthy nonsmokers, using positron emission tomography (PET) and the MAO-A selective radioligand [ 11 C]befloxatone. In comparison to nonsmokers, smokers showed a significant overall reduction of [ 11 C]befloxatone binding potential (BP) in cortical areas (average reduction, -60%) and a similar trend in caudate and thalamus (-40%). Our findings confirm a widespread inhibition of cerebral MAO-A in smokers. This mechanism may contribute to tobacco addiction and for a possible mood-modulating effect of tobacco. (authors)

  6. Oxamate, but Not Selective Targeting of LDH-A, Inhibits Medulloblastoma Cell Glycolysis, Growth and Motility

    Directory of Open Access Journals (Sweden)

    Cara J. Valvona

    2018-03-01

    Full Text Available Medulloblastoma is the most common malignant paediatric brain tumour and current therapies often leave patients with severe neurological disabilities. Four major molecular groups of medulloblastoma have been identified (Wnt, Shh, Group 3 and Group 4, which include additional, recently defined subgroups with different prognosis and genetic characteristics. Lactate dehydrogenase A (LDHA is a key enzyme in the aerobic glycolysis pathway, an abnormal metabolic pathway commonly observed in cancers, associated with tumour progression and metastasis. Studies indicate MBs have a glycolytic phenotype; however, LDHA has not yet been explored as a therapeutic target for medulloblastoma. LDHA expression was examined in medulloblastoma subgroups and cell lines. The effects of LDHA inhibition by oxamate or LDHA siRNA on medulloblastoma cell line metabolism, migration and proliferation were examined. LDHA was significantly overexpressed in Group 3 and Wnt MBs compared to non-neoplastic cerebellum. Furthermore, we found that oxamate significantly attenuated glycolysis, proliferation and motility in medulloblastoma cell lines, but LDHA siRNA did not. We established that aerobic glycolysis is a potential therapeutic target for medulloblastoma, but broader LDH inhibition (LDHA, B, and C may be more appropriate than LDHA inhibition alone.

  7. Helicobacter pylori CagA Inhibits PAR1-MARK Family Kinases by Mimicking Host Substrates

    Energy Technology Data Exchange (ETDEWEB)

    Nesic, D.; Miller, M; Quinkert, Z; Stein, M; Chait, B; Stebbins, C

    2010-01-01

    The CagA protein of Helicobacter pylori interacts with numerous cellular factors and is associated with increased virulence and risk of gastric carcinoma. We present here the cocrystal structure of a subdomain of CagA with the human kinase PAR1b/MARK2, revealing that a CagA peptide mimics substrates of this kinase family, resembling eukaryotic protein kinase inhibitors. Mutagenesis of conserved residues central to this interaction renders CagA inactive as an inhibitor of MARK2.

  8. Withaferin A Inhibits STAT3 and Induces Tumor Cell Death in Neuroblastoma and Multiple Myeloma

    Directory of Open Access Journals (Sweden)

    Lisette P. Yco

    2014-01-01

    Full Text Available Signal transducer and activator of transcription 3 (STAT3 is an oncogenic transcription factor that has been implicated in many human cancers and has emerged as an ideal target for cancer therapy. Withaferin A (WFA is a natural product with promising antiproliferative properties through its association with a number of molecular targets including STAT3. However, the effect of WFA in pediatric neuroblastoma (NB and its interaction with STAT3 have not been reported. In this study, we found that WFA effectively induces dose-dependent cell death in high-risk and drug-resistant NB as well as multiple myeloma (MM tumor cells, prevented interleukin-6 (IL-6–mediated and persistently activated STAT3 phosphorylation at Y705, and blocked the transcriptional activity of STAT3. We further provide computational models that show that WFA binds STAT3 near the Y705 phosphotyrosine residue of the STAT3 Src homology 2 (SH2 domain, suggesting that WFA prevents STAT3 dimer formation similar to BP-1-102, a well-established STAT3 inhibitor. Our findings propose that the antitumor activity of WFA is mediated at least in part through inhibition of STAT3 and provide a rationale for further drug development and clinical use in NB and MM.

  9. Surfactant protein A (SP-A) inhibits agglomeration and macrophage uptake of toxic amine modified nanoparticles.

    Science.gov (United States)

    McKenzie, Zofi; Kendall, Michaela; Mackay, Rose-Marie; Whitwell, Harry; Elgy, Christine; Ding, Ping; Mahajan, Sumeet; Morgan, Cliff; Griffiths, Mark; Clark, Howard; Madsen, Jens

    2015-01-01

    The lung provides the main route for nanomaterial exposure. Surfactant protein A (SP-A) is an important respiratory innate immune molecule with the ability to bind or opsonise pathogens to enhance phagocytic removal from the airways. We hypothesised that SP-A, like surfactant protein D, may interact with inhaled nanoparticulates, and that this interaction will be affected by nanoparticle (NP) surface characteristics. In this study, we characterise the interaction of SP-A with unmodified (U-PS) and amine-modified (A-PS) polystyrene particles of varying size and zeta potential using dynamic light scatter analysis. SP-A associated with both 100 nm U-PS and A-PS in a calcium-independent manner. SP-A induced significant calcium-dependent agglomeration of 100 nm U-PS NPs but resulted in calcium-independent inhibition of A-PS self agglomeration. SP-A enhanced uptake of 100 nm U-PS into macrophage-like RAW264.7 cells in a dose-dependent manner but in contrast inhibited A-PS uptake. Reduced association of A-PS particles in RAW264.7 cells following pre-incubation of SP-A was also observed with coherent anti-Stokes Raman spectroscopy. Consistent with these findings, alveolar macrophages (AMs) from SP-A(-/-) mice were more efficient at uptake of 100 nm A-PS compared with wild type C57Bl/6 macrophages. No difference in uptake was observed with 500 nm U-PS or A-PS particles. Pre-incubation with SP-A resulted in a significant decrease in uptake of 100 nm A-PS in macrophages isolated from both groups of mice. In contrast, increased uptake by AMs of U-PS was observed after pre-incubation with SP-A. Thus we have demonstrated that SP-A promotes uptake of non-toxic U-PS particles but inhibits the clearance of potentially toxic A-PS particles by blocking uptake into macrophages.

  10. Secondary metabolite from Nostoc XPORK14A inhibits photosynthesis and growth of Synechocystis PCC 6803.

    Science.gov (United States)

    Shunmugam, Sumathy; Jokela, Jouni; Wahlsten, Matti; Battchikova, Natalia; Ateeq ur Rehman; Vass, Imre; Karonen, Maarit; Sinkkonen, Jari; Permi, Perttu; Sivonen, Kaarina; Aro, Eva-Mari; Allahverdiyeva, Yagut

    2014-06-01

    Screening of 55 different cyanobacterial strains revealed that an extract from Nostoc XPORK14A drastically modifies the amplitude and kinetics of chlorophyll a fluorescence induction of Synechocystis PCC6803 cells.After 2 d exposure to the Nostoc XPORK14A extract, Synechocystis PCC 6803 cells displayed reduced net photosynthetic activity and significantly modified electron transport properties of photosystem II under both light and dark conditions. However, the maximum oxidizable amount of P700 was not strongly affected. The extract also induced strong oxidative stress in Synechocystis PCC 6803 cells in both light and darkness. We identified the secondary metabolite of Nostoc XPORK14A causing these pronounced effects on Synechocystis cells. Mass spectrometry and nuclear magnetic resonance analyses revealed that this compound, designated as M22, has a non-peptide structure. We propose that M22 possesses a dualaction mechanism: firstly, by photogeneration of reactive oxygen species in the presence of light, which in turn affects the photosynthetic machinery of Synechocystis PCC 6803; and secondly, by altering the in vivo redox status of cells, possibly through inhibition of protein kinases.

  11. BolA inhibits cell elongation and regulates MreB expression levels.

    Science.gov (United States)

    Freire, Patrick; Moreira, Ricardo Neves; Arraiano, Cecília Maria

    2009-02-06

    The morphogene bolA is a general stress response gene in Escherichia coli that induces a round morphology when overexpressed. Results presented in this report show that increased BolA levels can inhibit cell elongation mechanisms. MreB polymerization is crucial for the bacterial cell cytoskeleton, and this protein is essential for the maintenance of a cellular rod shape. In this report, we demonstrate that bolA overexpression affects the architecture of MreB filaments. An increase in BolA leads to a significant reduction in MreB protein levels and mreB transcripts. BolA affects the mreBCD operon in vivo at the level of transcription. Furthermore, our results show that BolA is a new transcriptional repressor of MreB. The alterations in cell morphology induced by bolA seem to be mediated by a complex pathway that integrates PBP5, PBP6, MreB, and probably other regulators of cell morphology/elongation.

  12. Salient aspects of PBP2A-inhibition; A QSAR Study.

    Science.gov (United States)

    Ogunleye, Adewale J; Eniafe, Gabriel O; Inyang, Olumide K; Adewumi, Benjamin; Omotuyi, Olaposi I

    2018-05-15

    Backgound: Inhibition of penicillin binding protein 2A (PBP2A) represents a sound drug design strategy in combatting Methicillin resistant Staphylococcus aureus (MRSA). Considering the urgent need for effective antimicrobials in combatting MRSA infections, we have developed a statistically robust ensemble of molecular descriptors (1, 2, & 3-D) from compounds targeting PBP2A in vivo. 37 (training set: 26, test set: 11) PBP2A-inhibitors were submitted for descriptor generation after which an unsupervised, non-exhaustive genetic algorithm (GA) was deployed for fishing out the best descriptor subset. Assignment of descriptors to a regression model was accomplished with the Partial Least Square (PLS) algorithm. At the end, an ensemble of 30 descriptors accurately predicted the ligand bioactivity, IC50 (R = 0.9996, R2 = 0.9992, R2a = 0.9949, SEE =, 0.2297 Q2LOO = 0.9741). Inferentially, we noticed that the overall efficacy of this model greatly depends on atomic polarizability and negative charge (electron) density. Besides the formula derived, the high dimensional model also offers critical insights into salient cheminformatics parameter to note during hit-to-lead PBP2A-antagonist optimization. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  13. MicroRNA-125a Inhibits Autophagy Activation and Antimicrobial Responses during Mycobacterial Infection.

    Science.gov (United States)

    Kim, Jin Kyung; Yuk, Jae-Min; Kim, Soo Yeon; Kim, Tae Sung; Jin, Hyo Sun; Yang, Chul-Su; Jo, Eun-Kyeong

    2015-06-01

    MicroRNAs (miRNAs) are small noncoding nucleotides that play critical roles in the regulation of diverse biological functions, including the response of host immune cells. Autophagy plays a key role in activating the antimicrobial host defense against Mycobacterium tuberculosis. Although the pathways associated with autophagy must be tightly regulated at a posttranscriptional level, the contribution of miRNAs and whether they specifically influence the activation of macrophage autophagy during M. tuberculosis infection are largely unknown. In this study, we demonstrate that M. tuberculosis infection of macrophages leads to increased expression of miRNA-125a-3p (miR-125a), which targets UV radiation resistance-associated gene (UVRAG), to inhibit autophagy activation and antimicrobial responses to M. tuberculosis. Forced expression of miR-125a significantly blocked M. tuberculosis-induced activation of autophagy and phagosomal maturation in macrophages, and inhibitors of miR-125a counteracted these effects. Both TLR2 and MyD88 were required for biogenesis of miR-125a during M. tuberculosis infection. Notably, activation of the AMP-activated protein kinase significantly inhibited the expression of miR-125a in M. tuberculosis-infected macrophages. Moreover, either overexpression of miR-125a or silencing of UVRAG significantly attenuated the antimicrobial effects of macrophages against M. tuberculosis. Taken together, these data indicate that miR-125a regulates the innate host defense by inhibiting the activation of autophagy and antimicrobial effects against M. tuberculosis through targeting UVRAG. Copyright © 2015 by The American Association of Immunologists, Inc.

  14. miR-135a Inhibits the Invasion of Cancer Cells via Suppression of ERRα.

    Directory of Open Access Journals (Sweden)

    Violaine Tribollet

    Full Text Available MicroRNA-135a (miR-135a down-modulates parameters of cancer progression and its expression is decreased in metastatic breast cancers (as compared to non-metastatic tumors as well as in prostate tumors relative to normal tissue. These expression and activity patterns are opposite to those of the Estrogen-Related Receptor α (ERRα, an orphan member of the nuclear receptor family. Indeed high expression of ERRα correlates with poor prognosis in breast and prostate cancers, and the receptor promotes various traits of cancer aggressiveness including cell invasion. Here we show that miR-135a down-regulates the expression of ERRα through specific sequences of its 3'UTR. As a consequence miR-135a also reduces the expression of downstream targets of ERRα. miR-135a also decreases cell invasive potential in an ERRα-dependent manner. Our results suggest that the decreased expression of miR-135a in metastatic tumors leads to elevated ERRα expression, resulting in increased cell invasion capacities.

  15. miR-135a Inhibits the Invasion of Cancer Cells via Suppression of ERRα.

    Science.gov (United States)

    Tribollet, Violaine; Barenton, Bruno; Kroiss, Auriane; Vincent, Séverine; Zhang, Ling; Forcet, Christelle; Cerutti, Catherine; Périan, Séverine; Allioli, Nathalie; Samarut, Jacques; Vanacker, Jean-Marc

    2016-01-01

    MicroRNA-135a (miR-135a) down-modulates parameters of cancer progression and its expression is decreased in metastatic breast cancers (as compared to non-metastatic tumors) as well as in prostate tumors relative to normal tissue. These expression and activity patterns are opposite to those of the Estrogen-Related Receptor α (ERRα), an orphan member of the nuclear receptor family. Indeed high expression of ERRα correlates with poor prognosis in breast and prostate cancers, and the receptor promotes various traits of cancer aggressiveness including cell invasion. Here we show that miR-135a down-regulates the expression of ERRα through specific sequences of its 3'UTR. As a consequence miR-135a also reduces the expression of downstream targets of ERRα. miR-135a also decreases cell invasive potential in an ERRα-dependent manner. Our results suggest that the decreased expression of miR-135a in metastatic tumors leads to elevated ERRα expression, resulting in increased cell invasion capacities.

  16. Marine Longilenes, Oxasqualenoids with Ser-Thr Protein Phosphatase 2A Inhibition Activity

    Directory of Open Access Journals (Sweden)

    Francisco Cen-Pacheco

    2018-04-01

    Full Text Available The red seaweed Laurencia viridis is a rich source of oxygenated secondary metabolites that were derived from squalene. We report here the structures of three novel compounds, (+-longilene peroxide (1, longilene (2, and (+-prelongilene (3 that were isolated from this alga, in addition to other substances, 4 and 5, resulting from their acid-mediated degradation. The effect of compounds 1 and 3 against Ser-Thr protein phosphatase type 2A (PP2A was evaluated, showing that (+-longilene peroxide (1 inhibited PP2A (IC50 11.3 μM. In order to explain the interaction between PP2A and compounds 1 and 3, molecular docking simulations onto the PP2A enzyme-binding region were used.

  17. Glaucocalyxin A inhibits platelet activation and thrombus formation preferentially via GPVI signaling pathway.

    Directory of Open Access Journals (Sweden)

    Wei Li

    Full Text Available Platelets play a pivotal role in atherothrombosis and the antiplatelet agents have been proved to be useful in preventing onset of acute clinical events including myocardial infarction and stroke. Increasing number of natural compounds has been identified to be potential antiplatelet agents. Here we report the antiplatelet effect of glaucocalyxin A (GLA, an ent-diterpenoid that we isolated and purified from the aerial parts of Rabdosia japonica (Burm. f. var. glaucocalyx (Maxim. Hara, and investigate the molecular mechanisms by which GLA inhibits platelet activation and thrombus formation. The effect of GLA on platelet activation was measured using platelets freshly isolated from peripheral blood of healthy donors. Results showed that pretreatment of human platelets with lower concentrations of GLA (0.01 μg/ml, 0.1 μg/ml significantly inhibited platelet aggregation induced by collagen (P<0.001 and CRP (P<0.01, a synthetic GPVI ligand, but not by ADP and U46619. Accordingly, GLA inhibited collagen-stimulated tyrosine phosphorylation of Syk, LAT, and phospholipase Cγ2, the signaling events in collagen receptor GPⅥ pathway. GLA also inhibited platelet p-selectin secretion and integrin activation by convulxin, a GPVI selective ligand. Additionally, GLA was found to inhibit low-dose thrombin-induced platelet activation. Using a flow chamber device, GLA was found to attenuate platelet adhesion on collagen surfaces in high shear condition. In vivo studies showed that GLA administration increased the time for complete occlusion upon vascular injury in mice, but did not extend tail-bleeding time when mice were administered with relatively lower doses of GLA. Therefore, the present results provide the molecular basis for the inhibition effect of GLA on platelet activation and its in vivo effect on thrombus formation, suggesting that GLA could potentially be developed as an antiplatelet and antithrombotic agent.

  18. Adenylate Cyclase Toxin Subverts Phagocyte Function by RhoA Inhibition and Unproductive Ruffling

    Czech Academy of Sciences Publication Activity Database

    Kamanová, Jana; Kofroňová, Olga; Mašín, Jiří; Genth, H.; Vojtová, Jana; Linhartová, Irena; Benada, Oldřich; Just, I.; Šebo, Peter

    2008-01-01

    Roč. 181, č. 8 (2008), s. 5587-5597 ISSN 0022-1767 R&D Projects: GA MŠk 1M0506; GA MŠk 2B06161; GA ČR GA310/08/0447 Grant - others:XE(XE) LSHB-CT-2003-503582 Institutional research plan: CEZ:AV0Z50200510 Keywords : bordetella * adenylate cyclase toxin * rhoa Subject RIV: EC - Immunology Impact factor: 6.000, year: 2008

  19. Activin A inhibits BMP-signaling by binding ACVR2A and ACVR2B

    DEFF Research Database (Denmark)

    Olsen, Oddrun Elise; Wader, Karin Fahl; Hella, Hanne

    2015-01-01

    BACKGROUND: Activins are members of the TGF-β family of ligands that have multiple biological functions in embryonic stem cells as well as in differentiated tissue. Serum levels of activin A were found to be elevated in pathological conditions such as cachexia, osteoporosis and cancer. Signaling...

  20. Phosphodiesterase 10A inhibition attenuates sleep deprivation-induced deficits in long-term fear memory.

    Science.gov (United States)

    Guo, Lengqiu; Guo, Zhuangli; Luo, Xiaoqing; Liang, Rui; Yang, Shui; Ren, Haigang; Wang, Guanghui; Zhen, Xuechu

    2016-12-02

    Sleep, particularly rapid eye movement (REM) sleep, is implicated in the consolidation of emotional memories. In the present study, we investigated the protective effects of a phosphodiesterase 10A (PDE10A) inhibitor MP-10 on deficits in long-term fear memory induced by REM sleep deprivation (REM-SD). REM-SD caused deficits in long-term fear memory, however, MP-10 administration ameliorated the deleterious effects of REM-SD on long term fear memory. Brain-derived neurotropic factor (BDNF) and phosphorylated cAMP response element-binding protein (pCREB) were altered in specific brain regions associated with learning and memory in REM-SD rats. Accordingly, REM-SD caused a significant decrease of pCREB in hippocampus and striatum and a significant decrease of BDNF in the hippocampus, striatum and amygdala, however, MP-10 reversed the effects of REM-SD in a dose-dependent manner. Our findings suggest that REM-SD disrupts the consolidation of long-term fear memory and that administration of MP-10 protects the REM-SD-induced deficits in fear memory, which may be due to the MP-10-induced expression of BDNF in the hippocampus, striatum and amygdala, and phosphorylation of CREB in the hippocampus and striatum. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  1. Trichostatin A inhibits beta-casein expression in mammary epithelial cells

    International Nuclear Information System (INIS)

    Pujuguet, Philippe; Radisky, Derek; Levy, Dinah; Lacza, Charlemagne; Bissell, Mina J.

    2002-01-01

    Many aspects of cellular behavior are affected by information derived from association of the extracellular matrix (ECM) and with cell membrane receptors. When cultured in the presence of laminin-containing ECM and prolactin (Prl), normal mammary epithelial cells express the milk protein beta-casein. Previously, we defined the minimal ECM- and Prl-responsive enhancer element BCE-1 from the upstream region of the beta-casein gene. We also found that BCE-1 was only active when stably integrated into chromatin, and that trichostatin A (TSA), a reagent that leads to alterations in chromatin structure, was able to activate the integrated enhancer element. We now show that endogenous b-casein gene, which is controlled by a genetic assembly that is highly similar to that of BCE-1 and which is also activated by incubation in ECM and Prl, is instead inhibited by TSA. We provide evidence that the differing response of b-casein and BCE-1 to TSA is neither due to an unusual effect of TSA on mammary epithelial cells, nor to secondary consequences from the expression of a separate gene, nor to a particular property of the BCE-1 construct. As a component of this investigation, we also showed that ECM could mediate rapid histone deacetylation in mammary epithelial cells. These results are discussed in combination with previous work showing that TSA mediates the differentiation of many types of cancer cells but inhibits differentiation of some nonmalignant cell types

  2. MicroRNA-133a Inhibits Osteosarcoma Cells Proliferation and Invasion via Targeting IGF-1R

    Directory of Open Access Journals (Sweden)

    Guangnan Chen

    2016-02-01

    Full Text Available Background/Aims: MicroRNAs (miRNAs are a class of small noncoding RNAs that regulate gene expression by repressing translation or cleaving RNA transcripts in a sequence-specific manner. Downregulated microRNAs and their roles in cancer development have attracted much attention. A growing body of evidence showed that microRNA-133a (miR-133a has inhibitory effects on cell proliferation, migration, invasion, and metastasis of osteosarcoma. Methods: MiR-133a expression in human osteosarcoma cell lines and human normal osteoblastic cell line hFOB was investigated by real-time PCR (RT-PCR. The role of miR-133a in human osteosarcoma growth and invasion was assessed in cell lines in vitro and in vivo. Then, luciferase reporter assay validated IGF-1R as a downstream and functional target of miR-133a, and functional studies revealed that the anti-tumor effect of miR-133a was probably due to targeting and repressing of IGF-1R expression. Results: MiR-133a was lower expressed in human osteosarcoma cell lines than human normal osteoblastic cell line hFOB and its effect on inhibiting proliferation, invasion and metastasis is mediated by its direct interaction with the IGF-1R. Furthermore, the tumour-suppressive function of miR-133a probably contributed to inhibiting the activation AKT and ERK signaling pathway. Conclusion: MiR-133a suppresses osteosarcoma progression and metastasis by targeting IGF-1R in human osteosarcoma cells, providing a novel candidate prognostic factor and a potential anti-metastasis therapeutic target in osteosarcoma.

  3. TMEM16A inhibition impedes capacitation and acquisition of hyperactivated motility in guinea pig sperm.

    Science.gov (United States)

    Cordero-Martínez, Joaquín; Reyes-Miguel, Tania; Rodríguez-Páez, Lorena; Garduño-Siciliano, Leticia; Maldonado-García, Deneb; Roa-Espitia, Ana L; Hernández-González, Enrique O

    2018-07-01

    Ca 2+ -activated Cl - channels (CaCCs) are anionic channels that regulate many important physiological functions associated with chloride and calcium flux in some somatic cells. The molecular identity of CaCCs was revealed to be TMEM16A and TMEM16B (also known as Anoctamin or ANO1 and ANO2, respectively) in all eukaryotes. A recent study suggests the presence of TMEM16A in human sperm and a relationship with the rhZP-induced acrosome reaction. However, to the best of our knowledge, little is known about the role of TMEM16A in other spermatic processes such as capacitation or motility. In this study, we evaluated the effects of two TMEM16A antagonists on capacitation, acrosome reaction, and motility in guinea pig sperm; these antagonists were T16Ainh-A01, belonging to a second generation of potent antagonists of TMEM16A, and niflumic acid (NFA), a well-known antagonist of TMEM16A (CaCCs). First of all, we confirmed that the absence of Cl - in the capacitation medium changes motility parameters, capacitation, and the progesterone-induced acrosome reaction. Using a specific antibody, TMEM16A was found as a protein band of ∼120 kDa, which localization was in the apical crest of the acrosome and the middle piece of the flagellum. Inhibition of TMEM16A by T16Ainh-A01 affected sperm physiology by reducing capacitation, blocking the progesterone-induced acrosome reaction under optimal capacitation conditions, inhibiting progressive motility, and the acquisition of hyperactivated motility, diminishing [Ca 2+ ]i, and increasing [Cl - ]i. These changes in sperm kinematic parameters provide new evidence of the important role played by TMEM16A in the production of sperm capable of fertilizing oocytes. © 2018 Wiley Periodicals, Inc.

  4. Protein Kinase-A Inhibition Is Sufficient to Support Human Neural Stem Cells Self-Renewal.

    Science.gov (United States)

    Georges, Pauline; Boissart, Claire; Poulet, Aurélie; Peschanski, Marc; Benchoua, Alexandra

    2015-12-01

    Human pluripotent stem cell-derived neural stem cells offer unprecedented opportunities for producing specific types of neurons for several biomedical applications. However, to achieve it, protocols of production and amplification of human neural stem cells need to be standardized, cost effective, and safe. This means that small molecules should progressively replace the use of media containing cocktails of protein-based growth factors. Here we have conducted a phenotypical screening to identify pathways involved in the regulation of hNSC self-renewal. We analyzed 80 small molecules acting as kinase inhibitors and identified compounds of the 5-isoquinolinesulfonamide family, described as protein kinase A (PKA) and protein kinase G inhibitors, as candidates to support hNSC self-renewal. Investigating the mode of action of these compounds, we found that modulation of PKA activity was central in controlling the choice between self-renewal or terminal neuronal differentiation of hNSC. We finally demonstrated that the pharmacological inhibition of PKA using the small molecule HA1004 was sufficient to support the full derivation, propagation, and long-term maintenance of stable hNSC in absence of any other extrinsic signals. Our results indicated that tuning of PKA activity is a core mechanism regulating hNSC self-renewal and differentiation and delineate the minimal culture media requirement to maintain undifferentiated hNSC in vitro. © 2015 AlphaMed Press.

  5. [Tricostantin A inhibits self-renewal of breast cancer stem cells in vitro].

    Science.gov (United States)

    Peng, Li; Li, Fu-Xi; Shao, Wen-Feng; Xiong, Jing-Bo

    2013-10-01

    To investigate the effect of tricostantin A (TSA) on self-renewal of breast cancer stem cells and explore the mechanisms. Breast cancer cell lines MDA-MB-468, MDA-MB-231, MCF-7 and SKBR3 were cultured in suspension and treated with different concentrations of TSA for 7 days, using 0.1% DMSO as the control. Secondary mammosphere formation efficiency and percentage of CD44(+)/CD24(-) sub-population in the primary mammospheres were used to evaluate the effects of TSA on self-renewal of breast cancer stem cells. The breast cancer stem cell surface marker CD44(+)/CD24(-) and the percentage of apoptosis in the primary mammospheres were assayed using flow cytometry. The mRNA expressions of Nanog, Sox2 and Oct4 in the primary mammospheres were assayed with quantitative PCR. TSA at both 100 and 500 nmol/L, but not at 10 nmol/L, partially inhibited the self-renewal of breast cancer stem cells from the 4 cell lines. TSA at 500 nmol/L induced cell apoptosis in the primary mammospheres. TSA down-regulated the mRNA expression of Nanog and Sox2 in the primary mammospheres. TSA can partially inhibit the self-renewal of breast cancer stem cells through a mechanism involving the down-regulation of Nanog and Sox2 expression, indicating the value of combined treatments with low-dose TSA and other anticancer drugs to achieve maximum inhibition of breast cancer stem cell self-renewal. The core transcriptional factor of embryonic stem cells Nanog and Sox2 can be potential targets of anticancer therapy.

  6. Huperzine A inhibits CCL2 production in experimental autoimmune encephalomyelitis mice and in cultured astrocyte.

    Science.gov (United States)

    Tian, G X; Zhu, X Q; Chen, Y; Wu, G C; Wang, J

    2013-01-01

    The active role of chemokines and inflammatory cytokines in the central nervous system (CNS) during the pathogenesis of experimental autoimmune encephalomyelitis (EAE) has been clearly established. Recent studies from our laboratory reported that Huperzine A (HupA) can attenuate the disease process in EAE by the inhibition of inflammation, demyelination, and axonal injury in the spinal cord as well as encephalomyelitic T-cell proliferation. In this study, the effects of low dose HupA on CCL2, TNF-alpha, IL-6, and IL-1beta expression were evaluated in EAE. The effect of HupA on lipopolysachharide (LPS)-induced inflammatory molecule secretion was investigated in cultured-astrocytes in vitro. In MOG35-55-induced EAE mice, intraperitoneal injections of HupA (0.1 mg/kg•d−1) significantly suppressed the expression of CCL2, IL-6, TNF-alpha, and IL-1beta in the spinal cord. HupA also repressed LPS-induced CCL2 production, but with little influence on pro-inflammatory cytokines in primary cultured astrocytes. The inhibition effect of HupA on CCL2 is PPARgamma-dependent and nicotine receptor-independent. Conditioned culture media from HupA-treated astrocyte decreased PBMC migration in vitro. Collectively, these results suggest that HupA can ameliorate EAE by inhibiting CCL2 production in astrocyte, which may consequently decrease inflammatory cell infiltration in the spinal cord. HupA may have a potential therapeutic value for the treatment of MS and other neuroinflammatory diseases.

  7. Ochratoxin A Inhibits Mouse Embryonic Development by Activating a Mitochondrion-Dependent Apoptotic Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Yan-Der Hsuuw

    2013-01-01

    Full Text Available Ochratoxin A (OTA, a mycotoxin found in many foods worldwide, causes nephrotoxicity, hepatotoxicity, and immunotoxicity, both in vitro and in vivo. In the present study, we explored the cytotoxic effects exerted by OTA on the blastocyst stage of mouse embryos, on subsequent embryonic attachment, on outgrowth in vitro, and following in vivo implantation via embryo transfer. Mouse blastocysts were incubated with or without OTA (1, 5, or 10 μM for 24 h. Cell proliferation and growth were investigated using dual differential staining; apoptosis was measured using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL assay; and embryo implantation and post-implantation development were assessed by examination of in vitro growth and the outcome of in vivo embryo transfer, respectively. Blastocysts treated with 10 μM OTA displayed a significantly increased level of apoptosis and a reduction in total cell number. Interestingly, we observed no marked difference in implantation success rate between OTA-pretreated and control blastocysts either during in vitro embryonic development (following implantation in a fibronectin-coated culture dish or after in vivo embryo transfer. However, in vitro treatment with 10 μM OTA was associated with increased resorption of post-implantation embryos by the mouse uterus, and decreased fetal weight upon embryo transfer. Our results collectively indicate that in vitro exposure to OTA triggers apoptosis and retards early post-implantation development after transfer of embryos to host mice. In addition, OTA induces apoptosis-mediated injury of mouse blastocysts, via reactive oxygen species (ROS generation, and promotes mitochondrion-dependent apoptotic signaling processes that impair subsequent embryonic development.

  8. The therapeutic effect of Chlorogenic acid against Staphylococcus aureus infection through Sortase A inhibition

    Directory of Open Access Journals (Sweden)

    Lin eWang

    2015-10-01

    Full Text Available The emergence and wide spread of multi-drug resistant Staphylococcus aureus (S. aureus requires the development of new therapeutic agents with alternative modes of action. Anti-virulence strategies are hoped to meet that need. Sortase A (SrtA has attracted great interest as a potential drug target to treat infections caused by S. aureus, as many of the surface proteins displayed by SrtA function as virulence factors by mediating bacterial adhesion to specific organ tissues, invasion of host cells, and evasion of the host-immune responses. It has been suggested that inhibitors of SrtA might be promising candidates for the treatment and/or prevention of S. aureus infections. In this study, we report that Chlorogenic acid (CHA, a natural compound that lacks significant anti–S. aureus activity, inhibit the activity of SrtA in vitro (IC50=33.86±5.55μg/ml and the binding of S. aureus to fibrinogen (Fg. Using molecular dynamics simulations and mutagenesis assays, we further demonstrate that CHA binds to the binding sites of C184 and G192 in the SrtA. In vivo studies demonstrated that CHA prevent mice from S. aureus-induced renal abscess, resulting in a significant survival advantage. These findings indicate that CHA is a promising therapeutic compound against SrtA during S. aureus infections.

  9. Transient HIF2A inhibition promotes satellite cell proliferation and muscle regeneration.

    Science.gov (United States)

    Xie, Liwei; Yin, Amelia; Nichenko, Anna S; Beedle, Aaron M; Call, Jarrod A; Yin, Hang

    2018-03-13

    The remarkable regeneration capability of skeletal muscle depends on coordinated proliferation and differentiation of satellite cells. The self-renewal of satellite cells is critical for long-term maintenance of muscle regeneration potential. Hypoxia profoundly affects the proliferation, differentiation, and self-renewal of cultured myoblasts. However, the physiological relevance of hypoxia and hypoxia signaling in satellite cells in vivo remains largely unknown. Here, we report that satellite cells are in an intrinsic hypoxic state in vivo and express hypoxia-inducible factor 2A (HIF2A). HIF2A promotes the stemness and long-term homeostatic maintenance of satellite cells by maintaining the quiescence, increasing the self-renewal and blocking the myogenic differentiation of satellite cells. HIF2A stabilization in satellite cells cultured under normoxia augmented their engraftment potential in regenerative muscle. Reversely, HIF2A ablation led to the depletion of satellite cells and the consequent regenerative failure in the long-term. In contrast, transient pharmacological inhibition of HIF2A accelerated muscle regeneration by increasing satellite cell proliferation and differentiation. Mechanistically, HIF2A induces the quiescence/self-renewal of satellite cells by binding the promoter of Spry1 gene and activating Spry1 expression. These findings suggest that HIF2A is a pivotal mediator of hypoxia signaling in satellite cells and may be therapeutically targeted to improve muscle regeneration.

  10. MAO-A inhibition profiles of some benzophenone glucosides from Gentiana verna subsp. pontica

    DEFF Research Database (Denmark)

    Kaya, Duygu; Jäger, Anna; Yalçin, Funda N

    2014-01-01

    Gentiana verna L. subsp. pontica (Soltok.) Hayek, G. pyrenaica L., and G. verna L. subsp. balcanica Pritchard from Turkey were tested for their MAO-A inhibitory effects. A photometric peroxidase linked MAO-A bioassay performed on the H20 extracts prepared from the methanolic extracts of the title...

  11. Phosphorylation of InhA inhibits mycolic acid biosynthesis and growth of Mycobacterium tuberculosis

    Energy Technology Data Exchange (ETDEWEB)

    Molle, Virginie; Gulten, Gulcin; Vilchèze, Catherine; Veyron-Churlet, Romain; Zanella-Cléon, Isabelle; Sacchettini, James C.; Jacobs, Jr, William R.; Kremer, Laurent (CNRS-UMR); (Einstein); (TAM)

    2011-08-24

    The remarkable survival ability of Mycobacterium tuberculosis in infected hosts is related to the presence of cell wall-associated mycolic acids. Despite their importance, the mechanisms that modulate expression of these lipids in response to environmental changes are unknown. Here we demonstrate that the enoyl-ACP reductase activity of InhA, an essential enzyme of the mycolic acid biosynthetic pathway and the primary target of the anti-tubercular drug isoniazid, is controlled via phosphorylation. Thr-266 is the unique kinase phosphoacceptor, both in vitro and in vivo. The physiological relevance of Thr-266 phosphorylation was demonstrated using inhA phosphoablative (T266A) or phosphomimetic (T266D/E) mutants. Enoyl reductase activity was severely impaired in the mimetic mutants in vitro, as a consequence of a reduced binding affinity to NADH. Importantly, introduction of inhA{_}T266D/E failed to complement growth and mycolic acid defects of an inhA-thermosensitive Mycobacterium smegmatis strain, in a similar manner to what is observed following isoniazid treatment. This study suggests that phosphorylation of InhA may represent an unusual mechanism that allows M. tuberculosis to regulate its mycolic acid content, thus offering a new approach to future anti-tuberculosis drug development.

  12. Phosphorylation of InhA inhibits mycolic acid biosynthesis and growth of Mycobacterium tuberculosis.

    Science.gov (United States)

    Molle, Virginie; Gulten, Gulcin; Vilchèze, Catherine; Veyron-Churlet, Romain; Zanella-Cléon, Isabelle; Sacchettini, James C; Jacobs, William R; Kremer, Laurent

    2010-12-01

    The remarkable survival ability of Mycobacterium tuberculosis in infected hosts is related to the presence of cell wall-associated mycolic acids. Despite their importance, the mechanisms that modulate expression of these lipids in response to environmental changes are unknown. Here we demonstrate that the enoyl-ACP reductase activity of InhA, an essential enzyme of the mycolic acid biosynthetic pathway and the primary target of the anti-tubercular drug isoniazid, is controlled via phosphorylation. Thr-266 is the unique kinase phosphoacceptor, both in vitro and in vivo. The physiological relevance of Thr-266 phosphorylation was demonstrated using inhA phosphoablative (T266A) or phosphomimetic (T266D/E) mutants. Enoyl reductase activity was severely impaired in the mimetic mutants in vitro, as a consequence of a reduced binding affinity to NADH. Importantly, introduction of inhA_T266D/E failed to complement growth and mycolic acid defects of an inhA-thermosensitive Mycobacterium smegmatis strain, in a similar manner to what is observed following isoniazid treatment. This study suggests that phosphorylation of InhA may represent an unusual mechanism that allows M. tuberculosis to regulate its mycolic acid content, thus offering a new approach to future anti-tuberculosis drug development. © 2010 Blackwell Publishing Ltd.

  13. Semaphorin 5A inhibits synaptogenesis in early postnatal- and adult-born hippocampal dentate granule cells.

    Science.gov (United States)

    Duan, Yuntao; Wang, Shih-Hsiu; Song, Juan; Mironova, Yevgeniya; Ming, Guo-li; Kolodkin, Alex L; Giger, Roman J

    2014-10-14

    Human SEMAPHORIN 5A (SEMA5A) is an autism susceptibility gene; however, its function in brain development is unknown. In this study, we show that mouse Sema5A negatively regulates synaptogenesis in early, developmentally born, hippocampal dentate granule cells (GCs). Sema5A is strongly expressed by GCs and regulates dendritic spine density in a cell-autonomous manner. In the adult mouse brain, newly born Sema5A-/- GCs show an increase in dendritic spine density and increased AMPA-type synaptic responses. Sema5A signals through PlexinA2 co-expressed by GCs, and the PlexinA2-RasGAP activity is necessary to suppress spinogenesis. Like Sema5A-/- mutants, PlexinA2-/- mice show an increase in GC glutamatergic synapses, and we show that Sema5A and PlexinA2 genetically interact with respect to GC spine phenotypes. Sema5A-/- mice display deficits in social interaction, a hallmark of autism-spectrum-disorders. These experiments identify novel intra-dendritic Sema5A/PlexinA2 interactions that inhibit excitatory synapse formation in developmentally born and adult-born GCs, and they provide support for SEMA5A contributions to autism-spectrum-disorders.

  14. Semaphorin 5A inhibits synaptogenesis in early postnatal- and adult-born hippocampal dentate granule cells

    OpenAIRE

    Duan, Yuntao; Wang, Shih-Hsiu; Song, Juan; Mironova, Yevgeniya; Ming, Guo-li; Kolodkin, Alex L; Giger, Roman J

    2014-01-01

    eLife digest Neurons communicate with one another at specialized junctions called synapses. There are two types of synapses, called excitatory synapses and inhibitory synapses, and the density and strength of both are tightly regulated because small deviations from the normal density and/or strength may lead to illness. For example, an excess of excitatory synapses has been observed in patients who have autism spectrum disorders and exhibit difficulties in social interaction. The gene that co...

  15. SNARE-mediated trafficking of α5β1 integrin is required for spreading in CHO cells

    International Nuclear Information System (INIS)

    Skalski, Michael; Coppolino, Marc G.

    2005-01-01

    In this study, the role of SNARE-mediated membrane traffic in regulating integrin localization was examined and the requirement for SNARE function in cellular spreading was quantitatively assessed. Membrane traffic was inhibited with the VAMP-specific catalytic light chain from tetanus toxin (TeTx-LC), a dominant-negative form (E329Q) of N-ethylmaleimide-sensitive fusion protein (NSF), and brefeldin A (BfA). Inhibition of membrane traffic with either E329Q-NSF or TeTx-LC, but not BfA, significantly inhibited spreading of CHO cells on fibronectin. Spreading was rescued in TeTx-LC-expressing cells by co-transfection with a TeTx-resistant cellubrevin/VAMP3. E329Q-NSF, a general inhibitor of SNARE function, was a more potent inhibitor of cell spreading than TeTx-LC, suggesting that tetanus toxin-insensitive SNAREs contribute to adhesion. It was found that E329Q-NSF prevented trafficking of α 5 β 1 integrins from a central Rab11-containing compartment to sites of protrusion during cell adhesion, while TeTx-LC delayed this trafficking. These results are consistent with a model of cellular adhesion that implicates SNARE function as an important component of integrin trafficking during the process of cell spreading

  16. Factor VII-Induced MicroRNA-135a Inhibits Autophagy and Is Associated with Poor Prognosis in Hepatocellular Carcinoma

    Directory of Open Access Journals (Sweden)

    Kuang-Tzu Huang

    2017-12-01

    Here, we identified miR-135a as a highly upregulated miRNA in HCC in response to TF/FVII/PAR2 activation. Analyzing 103 HCC patient specimens, we confirmed that miR-135a was frequently elevated in HCC tissues with higher FVII expression compared to adjacent non-cancerous counterparts. Increased miR-135a levels in HCC were also associated with tumor staging, recurrence, microvascular invasion, and decreased disease-free survival. We subsequently identified Atg14, a key component that regulates the formation of autophagosome as a direct target of miR-135a. Ectopic expression of miR-135a suppressed Atg14 levels and inhibited the autophagic processes. Our results indicate strong positive correlations between miR-135a levels and malignant behaviors in HCC patients and also suggest novel functions of miR-135a in regulation of autophagy, which could be useful as a potential target for prognostic and therapeutic uses.

  17. Cerebral monoamine oxidase A inhibition in tobacco smokers confirmed with PET and [{sup 11}C]Befloxatone

    Energy Technology Data Exchange (ETDEWEB)

    Leroy, C.; Bragulat, V.; Penttila, J.; Artiges, E.; Martinot, J.L.; Trichard, Ch. [INSERM U797, Research Unit ' Neuroimaging and Psychiatry' , Orsay (France); Leroy, C.; Bragulat, V.; Penttila, J.; Artiges, E.; Martinot, J.L.; Trichard, Ch. [CEA, ' Neuroimaging and Psychiatry, U797 Unit, Hospital Department Frederic Joliot and Neurospin (France); Leroy, C.; Bragulat, V.; Penttila, J.; Artiges, E.; Martinot, J.L.; Trichard, Ch. [Paris sud University - Paris Descartes University, UMR U797 (France); Berlin, I. [Service de Pharmacologie, Hopital Pitie-Salpetriere - Universite Paris6 - INSERM U677, Paris (France); Gregoire, M.C.; Bottlaender, M.; Roumenov, D.; Dolle, F.; Bourgeois, S. [CEA, DSV, I2BM, Service Hospitalier Frederic Joliot, Orsay (France); Artiges, E.; Trichard, Ch. [Psychiatry Department, Orsay Hospital, Orsay (France)

    2009-07-01

    The inhibition of cerebral monoamine oxidases (MAOs) by cigarette smoke components could participate to the tobacco addiction. However, the actual extent of this inhibition in vivo in smokers is still poorly known. We investigated cerebral MAO-A availability in 7 tobacco-dependent subjects and 6 healthy nonsmokers, using positron emission tomography (PET) and the MAO-A selective radioligand [{sup 11}C]befloxatone. In comparison to nonsmokers, smokers showed a significant overall reduction of [{sup 11}C]befloxatone binding potential (BP) in cortical areas (average reduction, -60%) and a similar trend in caudate and thalamus (-40%). Our findings confirm a widespread inhibition of cerebral MAO-A in smokers. This mechanism may contribute to tobacco addiction and for a possible mood-modulating effect of tobacco. (authors)

  18. The acute-phase response and serum amyloid A inhibit the inflammatory response to Acinetobacter baumannii Pneumonia

    NARCIS (Netherlands)

    Renckens, Rosemarijn; Roelofs, Joris J. T. H.; Knapp, Sylvia; de Vos, Alex F.; Florquin, Sandrine; van der Poll, Tom

    2006-01-01

    BACKGROUND: Acinetobacter baumannii is an emerging pathogen in nosocomial pneumonia. Trauma and postsurgical patients display a profound acute-phase protein response and are susceptible to pneumonia. METHODS: To study the way in which the acute-phase response induced by sterile tissue injury

  19. Human Immunodeficiency Virus Tat-Activated Expression of Poliovirus Protein 2A Inhibits mRNA Translation

    Science.gov (United States)

    Sun, Xiao-Hong; Baltimore, David

    1989-04-01

    To study the effect of poliovirus protein 2A on cellular RNA translation, the tat control system of human immunodeficiency virus (HIV) was used. Protein 2A was expressed from a plasmid construct (pHIV/2A) incorporating the HIV long terminal repeat. Protein synthesis was measured by using chloramphenicol acetyltransferase as a reporter gene driven by the Rous sarcoma virus long terminal repeat. When HIV/2A was contransfected with the reporter, addition of a tat-producing plasmid caused at least a 50-fold drop in chloramphenicol acetyltransferase synthesis. A HeLa cell line carrying HIV/2A was established. In it, tat expression caused more than a 10-fold drop in chloramphenicol acetyltransferase synthesis from the reporter plasmid. Furthermore, 2A induction by tat caused cleavage of the cellular translation factor P220, a part of eukaryotic translation initiation factor 4F. Thus protein 2A can, by itself, carry out the inhibition of cellular protein synthesis characteristic of a poliovirus infection. Also, the HIV tat activation provides a very effective method to control gene expression in mammalian cells.

  20. CYP1A inhibition in fish gill filaments: A novel assay applied on pharmaceuticals and other chemicals

    Energy Technology Data Exchange (ETDEWEB)

    Beijer, Kristina; Abrahamson, Alexandra; Brunstroem, Bjoern [Department of Environmental Toxicology, Uppsala University, Norbyvaegen 18A, SE-752 36 Uppsala (Sweden); Brandt, Ingvar, E-mail: ingvar.brandt@ebc.uu.se [Department of Environmental Toxicology, Uppsala University, Norbyvaegen 18A, SE-752 36 Uppsala (Sweden)

    2010-01-31

    The gill filament 7-ethoxyresorufin O-deethylase (EROD) assay was originally developed as a biomarker for cytochrome P4501A (CYP1A) induction by Ah-receptor agonists in water. In this study, the assay was adapted to measure inhibition of CYP1A activity in fish gill filaments ex vivo. The experiments were carried out using gill arch filaments from {beta}-naphthoflavone ({beta}NF)-exposed three-spined stickleback (Gasterosteus aculeatus). Candidate CYP1A inhibitors were added to the assay buffer. Nine selected pharmaceuticals and five known or suspected CYP1A-modulating chemicals were examined with regard to their ability to reduce EROD activity in gill filaments. Ellipticine, a well characterized CYP1A inhibitor, was the most effective inhibitor of the compounds tested. At a concentration in the assay buffer of 1 {mu}M the antifungal azoles ketoconazole, miconazole and bitertanol, and the plant flavonoid acacetin reduced gill EROD activity by more than 50%, implying IC50 values below 1 {mu}M. These compounds have previously been shown to inhibit EROD activity in liver microsomes from fish and mammals at similar concentrations. The proton pump inhibitor omeprazole reduced the gill EROD activity by 39% at 10 {mu}M. It is concluded that the modified gill filament EROD assay is useful to screen for waterborne pollutants that inhibit catalytic CYP1A activity in fish gills.

  1. CYP1A inhibition in fish gill filaments: A novel assay applied on pharmaceuticals and other chemicals

    International Nuclear Information System (INIS)

    Beijer, Kristina; Abrahamson, Alexandra; Brunstroem, Bjoern; Brandt, Ingvar

    2010-01-01

    The gill filament 7-ethoxyresorufin O-deethylase (EROD) assay was originally developed as a biomarker for cytochrome P4501A (CYP1A) induction by Ah-receptor agonists in water. In this study, the assay was adapted to measure inhibition of CYP1A activity in fish gill filaments ex vivo. The experiments were carried out using gill arch filaments from β-naphthoflavone (βNF)-exposed three-spined stickleback (Gasterosteus aculeatus). Candidate CYP1A inhibitors were added to the assay buffer. Nine selected pharmaceuticals and five known or suspected CYP1A-modulating chemicals were examined with regard to their ability to reduce EROD activity in gill filaments. Ellipticine, a well characterized CYP1A inhibitor, was the most effective inhibitor of the compounds tested. At a concentration in the assay buffer of 1 μM the antifungal azoles ketoconazole, miconazole and bitertanol, and the plant flavonoid acacetin reduced gill EROD activity by more than 50%, implying IC50 values below 1 μM. These compounds have previously been shown to inhibit EROD activity in liver microsomes from fish and mammals at similar concentrations. The proton pump inhibitor omeprazole reduced the gill EROD activity by 39% at 10 μM. It is concluded that the modified gill filament EROD assay is useful to screen for waterborne pollutants that inhibit catalytic CYP1A activity in fish gills.

  2. Human milk containing specific secretory IgA inhibits binding of Giardia lamblia to nylon and glass surfaces.

    Science.gov (United States)

    Samra, H K; Ganguly, N K; Mahajan, R C

    1991-06-01

    The effects of human milk, containing specific secretory IgA, on the adherence of Giardia lamblia trophozoites in the presence and in the absence of intestinal mucus in vitro were studied. It was found that the trophozoites treated with breast milk, containing specific secretory IgA to G. lamblia, showed a significant decrease (p less than 0.01) in adherence to nylon fibre columns and glass surfaces than did trophozoites treated with milk containing no SIgA antibodies. The adherence to glass surfaces was significantly more (p less than 0.01) in the presence of intestinal mucus than when the mucus was absent. Milk that did not contain specific secretory SIgA to G. lamblia did not decrease the adherence to glass surfaces either in the presence or in the absence of mucus. The fluorescence study revealed the binding of specific secretory IgA on the trophozoite surface. The results suggest that binding of SIgA antibodies in milk to G. lamblia trophozoites inhibits parasite adherence, thus protecting against this infection in breast-fed babies.

  3. Nonselective matrix metalloproteinase but not tumor necrosis factor-a inhibition effectively preserves the early critical colon anastomotic integrity

    DEFF Research Database (Denmark)

    Ågren, Magnus S.; Andersen, Thomas L.; Andersen, Line

    2011-01-01

    Increased matrix metalloproteinase (MMP) activity has been implicated in the pathogenesis of colorectal anastomotic leakage. Tumor necrosis factor-a (TNF-a) induces MMPs and may influence anastomosis repair....

  4. Forsythoside A Inhibits BVDV Replication via TRAF2-Dependent CD28-4-1BB Signaling in Bovine PBMCs.

    Directory of Open Access Journals (Sweden)

    Quan-Jiang Song

    Full Text Available Bovine viral diarrhea virus (BVDV, the causative agent of bovine viral diarrhea/mucosal disease (BVD/MD, is an important pathogen of cattle and other wild animals throughout the world. BVDV infection typically leads to an impaired immune response in cattle. In the present study, we investigated the effect of Forsythoside A (FTA on BVDV infection of bovine peripheral blood mononuclear cells (PBMCs. We found that Forsythoside A could not only promote proliferation of PBMCs and T cells activation but also inhibit the replication of BVDV as well as apoptosis induced by BVDV. FTA treatment could counteract the BVDV-induced overproduction of IFN-γ to maintain the immune homeostasis in bovine PBMCs. At same time, FTA can enhance the secretion of IL-2. What's more, BVDV promotes the expression of CD28, 4-1BB and TRAF-2, which can be modulated by FTA. Our data suggest that FTA protects PBMCs from BVDV infection possibly via TRAF2-dependent CD28-4-1BB signaling, which may activate PBMCs in response to BVDV infection. Therefore, this aids in the development of an effective adjuvant for vaccines against BVDV and other specific FTA-based therapies for preventing BVDV infection.

  5. Botulinum Toxin Type A Inhibits α-Smooth Muscle Actin and Myosin II Expression in Fibroblasts Derived From Scar Contracture.

    Science.gov (United States)

    Chen, Minliang; Yan, Tongtong; Ma, Kui; Lai, Linying; Liu, Chang; Liang, Liming; Fu, Xiaobing

    2016-09-01

    Scar contracture (SC) is one of the most common complications resulting from major burn injuries. Numerous treatments are currently available but they do not always yield excellent therapeutic results. Recent reports suggest that botulinum toxin type A (BTXA) is effective at reducing SC clinically, but the molecular mechanism for this action is unknown. α-Smooth muscle actin (α-SMA) and myosin II are the main components of stress fibers, which are the contractile structures of fibroblasts. The effects of BTXA on α-SMA and myosin II in SC are still unknown. This study aimed to explore the effect of BTXA on α-SMA and myosin II expression in fibroblasts derived from SC and to elucidate its actual mechanism further. Fibroblasts were isolated from tissue specimens of SC. Fibroblasts were cultured in Dulbecco modified Eagle medium with different concentrations of BTXA and their proliferation was analyzed through the tetrazolium-based colorimetric method at 1, 4, and 7 days. Proteins of α-SMA and myosin II were checked using Western blot in fibroblasts treated with different concentrations of BTXA at 1, 4, and 7 days. Fibroblasts without BTXA treatment had a higher proliferation than that in other groups, which indicated that the proliferation of fibroblasts was significantly inhibited by BTXA (P < 0.05). Proteins of α-SMA and myosin II between fibroblasts with BTXA and fibroblasts without BTXA are statistically significant (P < 0.05). These results suggest that BTXA effectively inhibited the growth of fibroblasts derived from SC and reduced the expression of α-SMA and myosin II, which provided theoretical support for the application of BTXA to control SC.

  6. Forsythoside A Inhibits BVDV Replication via TRAF2-Dependent CD28–4-1BB Signaling in Bovine PBMCs

    Science.gov (United States)

    Song, Quan-Jiang; Weng, Xiao-Gang; Cai, Dong-Jie; Zhang, Wang; Wang, Jiu-Feng

    2016-01-01

    Bovine viral diarrhea virus (BVDV), the causative agent of bovine viral diarrhea/mucosal disease (BVD/MD), is an important pathogen of cattle and other wild animals throughout the world. BVDV infection typically leads to an impaired immune response in cattle. In the present study, we investigated the effect of Forsythoside A (FTA) on BVDV infection of bovine peripheral blood mononuclear cells (PBMCs). We found that Forsythoside A could not only promote proliferation of PBMCs and T cells activation but also inhibit the replication of BVDV as well as apoptosis induced by BVDV. FTA treatment could counteract the BVDV-induced overproduction of IFN-γ to maintain the immune homeostasis in bovine PBMCs. At same time, FTA can enhance the secretion of IL-2. What’s more, BVDV promotes the expression of CD28, 4-1BB and TRAF-2, which can be modulated by FTA. Our data suggest that FTA protects PBMCs from BVDV infection possibly via TRAF2-dependent CD28–4-1BB signaling, which may activate PBMCs in response to BVDV infection. Therefore, this aids in the development of an effective adjuvant for vaccines against BVDV and other specific FTA-based therapies for preventing BVDV infection. PMID:27617959

  7. MS2 VLP-based delivery of microRNA-146a inhibits autoantibody production in lupus-prone mice

    Directory of Open Access Journals (Sweden)

    Pan Y

    2012-12-01

    Full Text Available Yang Pan,1,2 Tingting Jia,1,2 Yuan Zhang,1,2 Kuo Zhang,1 Rui Zhang,1 Jinming Li,1 Lunan Wang11National Center for Clinical Laboratories, Beijing Hospital of the Ministry of Health, Beijing, People’s Republic of China; 2Graduate School, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, People’s Republic of ChinaBackground: Systemic lupus erythematosus (SLE is a chronic autoimmune disease characterized by the presence of pathogenic autoantibodies. Recent studies suggest that microRNAs (miRNAs play an essential role in immunoregulation and may be involved in the pathogenesis of SLE. Therefore, it was of interest to investigate the potential therapeutic application of miRNAs in SLE, a concept that has not been thoroughly investigated thus far. Virus-like particles (VLPs are a type of recombinant nanoparticle enveloped by certain proteins derived from the outer coat of a virus. Herein, we describe a novel miRNA-delivery approach via bacteriophage MS2 VLPs and investigate the therapeutic effects of miR-146a, a well-studied and SLE-related miRNA, in BXSB lupus-prone mice.Methods: VLPs containing miR-146a, and the control VLPs, were prepared using an Escherichia coli expression system and then administered to lupus-prone mice over a 12-day period. We performed an enzyme-linked immunosorbent assay to evaluate the anti-dsDNA antibody, autoantibody to nuclear antigen (ANA, total IgG and total IgM levels in serum. The expression of miR-146a was analyzed by qRT-PCR. SLE-related cytokines as well as some toll-like receptor signaling pathway molecules were also measured.Results: Treatment with MS2-miR146a VLP showed profound effects on lupus-prone BXSB mice, including an increased level of mature miR-146a, which led to a significant reduction in the expression of autoantibodies and total IgG. Remarkably, these mice also exhibited reduced levels of proinflammatorycytokines, including IFN-Interferon-α (IFN-α, Interleukin-1β (Il-1β and Interleukin-6 (Il-6. Moreover, we showed that the toll-like receptor pathway was involved in this regulation.Conclusion: Restoring the loss of miR-146a was effective in eliminating the production of autoantibodies and ameliorating SLE progression in lupus-prone mice. Thus, the induction of dysregulated miRNAs by an MS2 VLP-based delivery system may lead to novel therapies.Keywords: systemic lupus erythematosus, anti-dsDNA antibody, autoantibody to nuclear antigen, Toll-like receptor, BXSB mice, gene therapy.

  8. E2F1 interactions with hHR23A inhibit its degradation and promote DNA repair.

    Science.gov (United States)

    Singh, Randeep K; Dagnino, Lina

    2016-05-03

    Nucleotide excision repair (NER) is a major mechanism for removal of DNA lesions induced by exposure to UV radiation in the epidermis. Recognition of damaged DNA sites is the initial step in their repair, and requires multiprotein complexes that contain XPC and hHR23 proteins, or their orthologues. A variety of transcription factors are also involved in NER, including E2F1. In epidermal keratinocytes, UV exposure induces E2F1 phosphorylation, which allows it to recruit various NER factors to sites of DNA damage. However, the relationship between E2F1 and hHR23 proteins vis-à-vis NER has remained unexplored. We now show that E2F1 and hHR23 proteins can interact, and this interaction stabilizes E2F1, inhibiting its proteasomal degradation. Reciprocally, E2F1 regulates hHR23A subcellular localization, recruiting it to sites of DNA photodamage. As a result, E2F1 and hHR23A enhance DNA repair following exposure to UV radiation, contributing to genomic stability in the epidermis.

  9. Rab3A Inhibition of Ca2+ -Dependent Dopamine Release From PC12 Cells Involves Interaction With Synaptotagmin I.

    Science.gov (United States)

    Dai, Zhipan; Tang, Xia; Chen, Jia; Tang, Xiaochao; Wang, Xianchun

    2017-11-01

    Rab3 and synaptotagmin have been suggested to play important roles in the regulation of neurotransmitter release and, however, the molecular mechanism has not been completely clear. Here, we studied the effects of Rab3A and synaptotagmin I (Syt I) on dopamine release using PC12 cells as a model system. Rab3A was demonstrated to have effects on both Ca 2+ -independent and Ca 2+ -dependent dopamine releases from the PC12 cells. Application of Rab3A (up to 2500 nM) gradually decreased the amount of Ca 2+ -dependently released dopamine, indicating that Rab3A is a negative modulator that was further supported by the increase in dopamine release caused by Rab3A knockdown. Syt I knockdown weakened the Ca 2+ -dependent dopamine release, suggesting that Syt I plays a positive regulatory role in the cellular process. Treatment of the Syt I-knocked down PC12 cells with Rab3A further decreased Ca 2+ -dependent dopamine release and, however, the decrease magnitude was significantly reduced compared with that before Syt I knockdown, thus for the first time demonstrating that the inhibitory effect of Rab3A on Ca 2+ -dependent dopamine release involves the interaction with Syt I. This work has shed new light on the molecular mechanism for Rab3 and synaptotamin regulation of neurotransmitter release. J. Cell. Biochem. 118: 3696-3705, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  10. MAGE-A inhibits apoptosis in proliferating myeloma cells through repression of Bax and maintenance of survivin.

    Science.gov (United States)

    Nardiello, Tricia; Jungbluth, Achim A; Mei, Anna; Diliberto, Maurizio; Huang, Xiangao; Dabrowski, Ania; Andrade, Valéria C C; Wasserstrum, Rebecca; Ely, Scott; Niesvizky, Ruben; Pearse, Roger; Coleman, Morton; Jayabalan, David S; Bhardwaj, Nina; Old, Lloyd J; Chen-Kiang, Selina; Cho, Hearn Jay

    2011-07-01

    The type I Melanoma Antigen GEnes (MAGEs) are commonly expressed in cancers, fueling speculation that they may be therapeutic targets with oncogenic potential. They form complexes with RING domain proteins that have E3 ubiquitin ligase activity and promote p53 degradation. MAGE-A3 was detected in tumor specimens from patients with multiple myeloma and its expression correlated with higher frequencies of Ki-67(+) malignant cells. In this report, we examine the mechanistic role of MAGE-A in promoting survival of proliferating multiple myeloma cells. The impact of MAGE-A3 expression on survival and proliferation in vivo was examined by immunohistochemical analysis in an independent set of tumor specimens segregated into two groups: newly diagnosed, untreated patients and patients who had relapsed after chemotherapy. The mechanisms of MAGE-A3 activity were investigated in vitro by silencing its expression by short hairpin RNA interference in myeloma cell lines and primary cells and assessing the resultant effects on proliferation and apoptosis. MAGE-A3 was detected in a significantly higher percentage of relapsed patients compared with newly diagnosed, establishing a novel correlation with progression of disease. Silencing of MAGE-A showed that it was dispensable for cell cycling, but was required for survival of proliferating myeloma cells. Loss of MAGE-A led to apoptosis mediated by p53-dependent activation of proapoptotic Bax expression and by reduction of survivin expression through both p53-dependent and -independent mechanisms. These data support a role for MAGE-A in the pathogenesis and progression of multiple myeloma by inhibiting apoptosis in proliferating myeloma cells through two novel mechanisms.

  11. [Lentivirus-mediated shRNA silencing of LAMP2A inhibits the proliferation of multiple myeloma cells].

    Science.gov (United States)

    Li, Lixuan; Li, Jia

    2015-05-01

    To study the effects of lentivirus-mediated short hairpin RNA (shRNA) silencing of lysosome-associated membrane protein type 2A (LAMP2A) expression on the proliferation of multiple myeloma cells. The constructed shRNA lentiviral vector was applied to infect human multiple myeloma cell line MM.1S, and stable expression cell line was obtained by puromycin screening. Western blotting was used to verify the inhibitory effect on LAMP2A protein expression. MTT assay was conducted to detect the effect of knocked-down LAMP2A on MM.1S cell proliferation, and the anti-tumor potency of suberoylanilide hydroxamic acid (SAHA) against the obtained MM.1S LAMP2A(shRNA) stable cell line. Lactate assay was performed to observe the impact of low LAMP2A expression on cell glycolysis. The stable cell line with low LAMP2A expression were obtained with the constructed human LAMP2A-shRNA lentiviral vector. Down-regulation of LAMP2A expression significantly inhibited MM.1S cell proliferation and enhanced the anti-tumor activity of SAHA. Interestingly, decreased LAMP2A expression also inhibited MM.1S cell lactic acid secretion. Down-regulation of LAMP2A expression could inhibit cell proliferation in multiple myeloma cells.

  12. Channeling through Two Stacked Guanine Quartets of One and Two Alkali Cations in the Li+, Na+, K+, and Rb+ Series. Assessment of the Accuracy of the SIBFA Anisotropic Polarizable Molecular Mechanics Potential

    Czech Academy of Sciences Publication Activity Database

    Gresh, N.; Naseem-Khan, S.; Lagardere, L.; Piquemal, J.P.; Šponer, Judit E.; Šponer, Jiří

    2017-01-01

    Roč. 121, č. 16 (2017), s. 3997-4014 ISSN 1520-6106 R&D Projects: GA ČR(CZ) GA16-13721S; GA MŠk(CZ) LQ1601 Institutional support: RVO:68081707 Keywords : intermolecular interaction energies * quantum-chemical computations * classical drude oscillator * kit promoter quadruplex Subject RIV: BO - Biophysics OBOR OECD: Physical chemistry Impact factor: 3.177, year: 2016

  13. A novel Golgi retention signal RPWS for tumor suppressor UBIAD1.

    Directory of Open Access Journals (Sweden)

    Xian Wang

    Full Text Available UBIAD1 plays critical roles in physiology including vitamin K and CoQ10 biosynthesis as well as pathophysiology including dyslipimedia-induced SCD (Schnyder's corneal dystrophy, Parkinson's disease, cardiovascular disease and bladder carcinoma. Since the subcellular localization of UBIAD1 varies in different cell types, characterization of the exact subcellular localization of UBIAD1 in specific human disease is vital for understanding its molecular mechanism. As UBIAD1 suppresses bladder carcinoma, we studied its subcellular localization in human bladder carcinoma cell line T24. Since fluorescent images of UBIAD1-EGFP in T24, human prostate cancer cell line PC-3, human embryonic kidney cell line HEK293 and human hepatocyte cell line L02 are similar, these four cell lines were used for present study. Using a combination of fluorescent microscopy and immunohistochemistry, it was found that UBIAD1 localized on the Golgi and endoplasmic reticulum (ER, but not on the plasma membrane, of T24 and HEK293 cells. Using scanning electron microscopy and western blot analysis, we found that UBIAD1 is enriched in the Golgi fraction extracted from the L02 cells, verifying the Golgi localization of UBAID1. Site-directed mutagenesis showed that the RPWS motif, which forms an Arginine finger on the UBIAD1 N terminus, serves as the Golgi retention signal. With both cycloheximide and brefeldin A inhibition assays, it was shown that UBIAD1 may be transported from the endoplasmic reticulum (ER to the Golgi by a COPII-mediated mechanism. Based upon flow cytometry analysis, it is shown that mutation of the RPWS motif reduced the UBIAD1-induced apoptosis of T24 cells, indicating that the proper Golgi localization of UBIAD1 influences its tumor suppressant activity. This study paves the way for further understanding the molecular mechanism of UBIAD1 in human diseases.

  14. Cytotoxic T cell recognition of an endogenous class I HLA peptide presented by a class II HLA molecule.

    Science.gov (United States)

    Chen, B P; Madrigal, A; Parham, P

    1990-09-01

    Human leukocytes were stimulated in vitro with peptides corresponding in sequence to the highly variable helix of the alpha 1 domain of various HLA-B and -C molecules. A CD4+ CD8- cytotoxic T cell line, CTL-AV, that is specific for the HLA-B7 peptide presented by HLA-DR11.1 was obtained. The HLA-DR11.2 molecule, which only differs at three residues from HLA-DR11.1, did not present the HLA-B7 peptide to CTL-AV. Peptides from the alpha 1 domain helix of other HLA-A and HLA-B molecules, but not HLA-C molecules, competed with the HLA-B7 peptide for binding to HLA-DR11.1. A cell line (WT50) that coexpresses HLA-B7 and HLA-DR11.1 was killed by CTL-AV in the absence of any added HLA-B7 peptide. The processing and presentation of HLA-B7 in these cells appears to be through the endogenous, and not the exogenous, pathway of antigen presentation. Thus, Brefeldin A inhibits presentation and chloroquine does not. Furthermore, introduction of purified HLA-B7 molecules into HLA-DR11.1+, HLA-B7- cells by cytoplasmic loading via osmotic lysis of pinosomes, but not by simple incubation, rendered them susceptible to CTL-AV killing. These results provide an example of class II major histocompatibility complex (MHC) presentation of a constitutively synthesized self protein that uses the endogenous pathway of antigen presentation. They also emphasize the capacity for presentation of MHC peptides by MHC molecules.

  15. GLUT4 in cultured skeletal myotubes is segregated from the transferrin receptor and stored in vesicles associated with TGN

    DEFF Research Database (Denmark)

    Ralston, E; Ploug, Thorkil

    1996-01-01

    of the constitutive endosomal-lysosomal pathway. To address this question, we have investigated the localization of the endogenous GLUT4 in non-stimulated skeletal myotubes from the cell line C2, by immunofluorescence and immunoelectron microscopy. We have used a panel of antibodies to markers of the Golgi complex...... and in vesicles just beyond, i.e. in the structures that constitute the trans-Golgi network (TGN). In myotubes treated with brefeldin A, the immunofluorescence pattern of GLUT4 is modified, but it differs from both Golgi complex markers and TGN38. Instead, it resembles the pattern of the transferrin receptor...... to the GLUT4-containing tubulo-vesicular elements. In brefeldin A-treated cells, a network of tubules of approximately 70 nm diameter, studded with varicosities, stains for both GLUT4 and transferrin receptor, suggesting that brefeldin A has caused fusion of the transferrin receptor and GLUT4-containing...

  16. Reciprocal regulation of ARPP-16 by PKA and MAST3 kinases provides a cAMP-regulated switch in protein phosphatase 2A inhibition

    Science.gov (United States)

    Musante, Veronica; Li, Lu; Kanyo, Jean; Lam, Tukiet T; Colangelo, Christopher M; Cheng, Shuk Kei; Brody, A Harrison; Greengard, Paul; Le Novère, Nicolas; Nairn, Angus C

    2017-01-01

    ARPP-16, ARPP-19, and ENSA are inhibitors of protein phosphatase PP2A. ARPP-19 and ENSA phosphorylated by Greatwall kinase inhibit PP2A during mitosis. ARPP-16 is expressed in striatal neurons where basal phosphorylation by MAST3 kinase inhibits PP2A and regulates key components of striatal signaling. The ARPP-16/19 proteins were discovered as substrates for PKA, but the function of PKA phosphorylation is unknown. We find that phosphorylation by PKA or MAST3 mutually suppresses the ability of the other kinase to act on ARPP-16. Phosphorylation by PKA also acts to prevent inhibition of PP2A by ARPP-16 phosphorylated by MAST3. Moreover, PKA phosphorylates MAST3 at multiple sites resulting in its inhibition. Mathematical modeling highlights the role of these three regulatory interactions to create a switch-like response to cAMP. Together, the results suggest a complex antagonistic interplay between the control of ARPP-16 by MAST3 and PKA that creates a mechanism whereby cAMP mediates PP2A disinhibition. DOI: http://dx.doi.org/10.7554/eLife.24998.001 PMID:28613156

  17. Activation of p53 pathway by Nutlin-3a inhibits the expression of the therapeutic target alpha 5 integrin in colon cancer cells

    Czech Academy of Sciences Publication Activity Database

    Janoušková, Hana; Ray, A.M.; Noulet, F.; Lelong-Rebel, I.; Choulier, L.; Schaffner, F.; Lehmann, M.; Martin, S.; Teisinger, Jan; Dontenwill, M.

    2013-01-01

    Roč. 336, č. 2 (2013), s. 307-318 ISSN 0304-3835 Institutional support: RVO:67985823 Keywords : colon cancer * integrin alpha 5 beta 1 * p53 * Nutlin-3a Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.016, year: 2013

  18. Decreased miR-106a inhibits glioma cell glucose uptake and proliferation by targeting SLC2A3 in GBM.

    Science.gov (United States)

    Dai, Dong-Wei; Lu, Qiong; Wang, Lai-Xing; Zhao, Wen-Yuan; Cao, Yi-Qun; Li, Ya-Nan; Han, Guo-Sheng; Liu, Jian-Min; Yue, Zhi-Jian

    2013-10-14

    MiR-106a is frequently down-regulated in various types of human cancer. However the underlying mechanism of miR-106a involved in glioma remains elusive. The association of miR-106a with glioma grade and patient survival was analyzed. The biological function and target of miR-106a were determined by bioinformatic analysis and cell experiments (Western blot, luciferase reporter, cell cycle, ntracellular ATP production and glucose uptake assay). Finally, rescue expression of its target SLC2A3 was used to test the role of SLC2A3 in miR-106a-mediated cell glycolysis and proliferation. Here we showed that miR-106a was a tumor suppressor miRNA was involved in GBM cell glucose uptake and proliferation. Decreased miR-106a in GBM tissues and conferred a poor survival of GBM patients. SLC2A3 was identified as a core target of miR-106a in GBM cells. Inhibition of SLC2A3 by miR-106a attenuated cell proliferation and inhibited glucose uptake. In addition, for each biological process we identified ontology-associated transcripts that significantly correlated with SLC2A3 expression. Finally, the expression of SLC2A3 largely abrogated miR-106a-mediated cell proliferation and glucose uptake in GBM cells. Taken together, miR-106a and SLC2A3 could be potential therapeutic approaches for GBM.

  19. MicroRNAs miR-17 and miR-20a inhibit T cell activation genes and are under-expressed in MS whole blood.

    Directory of Open Access Journals (Sweden)

    Mathew B Cox

    Full Text Available It is well established that Multiple Sclerosis (MS is an immune mediated disease. Little is known about what drives the differential control of the immune system in MS patients compared to unaffected individuals. MicroRNAs (miRNAs are small non-coding nucleic acids that are involved in the control of gene expression. Their potential role in T cell activation and neurodegenerative disease has recently been recognised and they are therefore excellent candidates for further studies in MS. We investigated the transcriptome of currently known miRNAs using miRNA microarray analysis in peripheral blood samples of 59 treatment naïve MS patients and 37 controls. Of these 59, 18 had a primary progressive, 17 a secondary progressive and 24 a relapsing remitting disease course. In all MS subtypes miR-17 and miR-20a were significantly under-expressed in MS, confirmed by RT-PCR. We demonstrate that these miRNAs modulate T cell activation genes in a knock-in and knock-down T cell model. The same T cell activation genes are also up-regulated in MS whole blood mRNA, suggesting these miRNAs or their analogues may provide useful targets for new therapeutic approaches.

  20. miR-644a Inhibits Cellular Proliferation and Invasion via Suppression of CtBP1 in Gastric Cancer Cells.

    Science.gov (United States)

    Li, Yingchao; Yan, Xiaoni; Ren, Li; Li, Yang

    2018-01-19

    Epithelial-mesenchymal transition (EMT) is one of the most important mechanisms in the metastasis of various cancers, including gastric cancer (GC). In this study, we explored the putative significance of miR-644a and its role in EMT-mediated metastasis of GC. We first detected the expression of miR-644a in a cohort of 107 GC tissues using quantitative RT-PCR. The expression of miR-644a was suppressed in GC tissues and was associated with a later clinical stage and tumor metastasis. Restoring the expression of miR-644a could significantly suppress the migration and invasion of HGC-27 and SGC-7901 cells, which might be correlated to its suppressive effect on the EMT process. We also found that carboxyl-terminal-binding protein 1 (CtBP1) was a putative target gene of miR-644a in GC and might be involved in the suppressive effect. Collectively, through targeting CtBP1-mediated suppression of the EMT process, miR-644a might suppress the tumor metastasis of GC cells.

  1. Natriuretic peptide receptor A inhibition suppresses gastric cancer development through reactive oxygen species-mediated G2/M cell cycle arrest and cell death.

    Science.gov (United States)

    Li, Zheng; Wang, Ji-Wei; Wang, Wei-Zhi; Zhi, Xiao-Fei; Zhang, Qun; Li, Bo-Wen; Wang, Lin-Jun; Xie, Kun-Ling; Tao, Jin-Qiu; Tang, Jie; Wei, Song; Zhu, Yi; Xu, Hao; Zhang, Dian-Cai; Yang, Li; Xu, Ze-Kuan

    2016-10-01

    Natriuretic peptide receptor A (NPRA), the major receptor for atrial natriuretic peptide (ANP), has been implicated in tumorigenesis; however, the role of ANP-NPRA signaling in the development of gastric cancer remains unclear. Immunohistochemical analyses indicated that NPRA expression was positively associated with gastric tumor size and cancer stage. NPRA inhibition by shRNA induced G2/M cell cycle arrest, cell death, and autophagy in gastric cancer cells, due to accumulation of reactive oxygen species (ROS). Either genetic or pharmacologic inhibition of autophagy led to caspase-dependent cell death. Therefore, autophagy induced by NPRA silencing may represent a cytoprotective mechanism. ROS accumulation activated c-Jun N-terminal kinase (JNK) and AMP-activated protein kinase (AMPK). ROS-mediated activation of JNK inhibited cell proliferation by disturbing cell cycle and decreased cell viability. In addition, AMPK activation promoted autophagy in NPRA-downregulated cancer cells. Overall, our results indicate that the inhibition of NPRA suppresses gastric cancer development and targeting NPRA may represent a promising strategy for the treatment of gastric cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Myrigalone A Inhibits Lepidium sativum Seed Germination by Interference with Gibberellin Metabolism and Apoplastic Superoxide Production Required for Embryo Extension Growth and Endosperm Rupture

    Czech Academy of Sciences Publication Activity Database

    Oracz, K.; Voegele, A.; Tarkowská, Danuše; Jacquemoud, D.; Turečková, Veronika; Urbanová, Terezie; Strnad, Miroslav; Sliwinska, E.; Leubner-Metzger, G.

    2012-01-01

    Roč. 53, č. 1 (2012), s. 81-95 ISSN 0032-0781 R&D Projects: GA AV ČR KAN200380801; GA MŠk ED0007/01/01; GA ČR GD522/08/H003 Keywords : Embryo cell extension growth * Endoreduplication * Endosperm rupture * Gibberellin metabolism * Lepidium sativum * Myrica gale * Phytotoxicity * Reactive oxygen species Subject RIV: EF - Botanics Impact factor: 4.134, year: 2012

  3. miR‑30a inhibits epithelial‑mesenchymal transition and metastasis in triple‑negative breast cancer by targeting ROR1.

    Science.gov (United States)

    Wang, Xin; Qiu, Huisi; Tang, Ruiming; Song, Huisheng; Pan, Huilin; Feng, Zhengfu; Chen, Longhua

    2018-04-18

    Triple‑negative breast cancer (TNBC) is a highly aggressive breast cancer subtype that lacks effective targeted therapies. In the present study, we revealed that the expression of miR‑30a was significantly decreased in TNBC, and TNBC patients with low expression of miR‑30a were associated with high histological grade and more lymph node metastasis. Moreover, we found that miR‑30a suppressed TNBC cell epithelial‑mesenchymal transition (EMT), as demonstrated by the overexpression of miR‑30a which increased the expression of epithelial marker E‑cadherin but decreased the expression of mesenchymal markers N‑cadherin and vimentin. Furthermore, we demonstrated that overexpression of miR‑30a significantly suppressed TNBC cell invasion and migration, as well as inhibited tumor growth and metastasis in vivo. More importantly, RTK‑like orphan receptor 1 (ROR1) was predicted as the direct target of miR‑30a, which was subsequently confirmed by luciferase assays. Forced expression of miR‑30a in TNBC cells decreased ROR1 expression, whereas the overexpression of ROR1 reversed the suppressive effects of miR‑30a in TNBC cell migration and invasion. Collectively, this study indicated that miR‑30a functions as a tumor‑metastasis suppressor miRNA in TNBC by directly targeting ROR1 and that miR‑30a may serve as a novel therapeutic target for TNBC.

  4. Reciprocal regulation of ARPP-16 by PKA and MAST3 kinases provides a cAMP-regulated switch in protein phosphatase 2A inhibition.

    Science.gov (United States)

    Musante, Veronica; Li, Lu; Kanyo, Jean; Lam, Tukiet T; Colangelo, Christopher M; Cheng, Shuk Kei; Brody, A Harrison; Greengard, Paul; Le Novère, Nicolas; Nairn, Angus C

    2017-06-14

    ARPP-16, ARPP-19, and ENSA are inhibitors of protein phosphatase PP2A. ARPP-19 and ENSA phosphorylated by Greatwall kinase inhibit PP2A during mitosis. ARPP-16 is expressed in striatal neurons where basal phosphorylation by MAST3 kinase inhibits PP2A and regulates key components of striatal signaling. The ARPP-16/19 proteins were discovered as substrates for PKA, but the function of PKA phosphorylation is unknown. We find that phosphorylation by PKA or MAST3 mutually suppresses the ability of the other kinase to act on ARPP-16. Phosphorylation by PKA also acts to prevent inhibition of PP2A by ARPP-16 phosphorylated by MAST3. Moreover, PKA phosphorylates MAST3 at multiple sites resulting in its inhibition. Mathematical modeling highlights the role of these three regulatory interactions to create a switch-like response to cAMP. Together, the results suggest a complex antagonistic interplay between the control of ARPP-16 by MAST3 and PKA that creates a mechanism whereby cAMP mediates PP2A disinhibition.

  5. Activin A Inhibits MPTP and LPS-Induced Increases in Inflammatory Cell Populations and Loss of Dopamine Neurons in the Mouse Midbrain In Vivo.

    Science.gov (United States)

    Stayte, Sandy; Rentsch, Peggy; Tröscher, Anna R; Bamberger, Maximilian; Li, Kong M; Vissel, Bryce

    2017-01-01

    Parkinson's disease is a chronic neurodegenerative disease characterized by a significant loss of dopaminergic neurons within the substantia nigra pars compacta region and a subsequent loss of dopamine within the striatum. A promising avenue of research has been the administration of growth factors to promote the survival of remaining midbrain neurons, although the mechanism by which they provide neuroprotection is not understood. Activin A, a member of the transforming growth factor β superfamily, has been shown to be a potent anti-inflammatory following acute brain injury and has been demonstrated to play a role in the neuroprotection of midbrain neurons against MPP+-induced degeneration in vitro. We hypothesized that activin A may offer similar anti-inflammatory and neuroprotective effects in in vivo mouse models of Parkinson's disease. We found that activin A significantly attenuated the inflammatory response induced by both MPTP and intranigral administration of lipopolysaccharide in C57BL/6 mice. We found that administration of activin A promoted survival of dopaminergic and total neuron populations in the pars compacta region both 8 days and 8 weeks after MPTP-induced degeneration. Surprisingly, no corresponding protection of striatal dopamine levels was found. Furthermore, activin A failed to protect against loss of striatal dopamine transporter expression in the striatum, suggesting the neuroprotective action of activin A may be localized to the substantia nigra. Together, these results provide the first evidence that activin A exerts potent neuroprotection and anti-inflammatory effects in the MPTP and lipopolysaccharide mouse models of Parkinson's disease.

  6. Flow-mediated lipoprotein retention; Pregnancy-associated plasma protein-A inhibition in atherosclerosis and its release during myocardial ischemia

    DEFF Research Database (Denmark)

    Steffensen, Lasse Bach

    , which states that retention of lipoproteins is the key initiating event of atherosclerosis development. Pregnancy-associated plasma protein-A (PAPP-A) regulates the insulin-like growth factor (IGF) system by proteolytic cleavage of a subset of IGF-binding proteins. Studies in mice have established PAPP...

  7. Ochratoxin A inhibits the production of tissue factor and plasminogen activator inhibitor-2 by human blood mononuclear cells: Another potential mechanism of immune-suppression

    International Nuclear Information System (INIS)

    Rossiello, Maria R.; Rotunno, Crescenzia; Coluccia, Addolorata; Carratu, Maria R.; Di Santo, Angelomaria; Evangelista, Virgilio; Semeraro, Nicola; Colucci, Mario

    2008-01-01

    The mycotoxin ochratoxin A (OTA), an ubiquitous contaminant of food products endowed with a wide spectrum of toxicity, affects several functions of mononuclear leukocytes. Monocytes/macrophages play a major role in fibrin accumulation associated with immune-inflammatory processes through the production of tissue factor (TF) and plasminogen activator inhibitor 2 (PAI-2). We studied the effect of OTA on TF and PAI-2 production by human blood mononuclear cells (MNC). The cells were incubated for 3 or 18 h at 37 deg. C with non toxic OTA concentrations in the absence and in the presence of lipopolysaccharide (LPS) or other inflammatory agents. TF activity was measured by a one-stage clotting test. Antigen assays were performed by specific ELISAs in cell extracts or conditioned media and specific mRNAs were assessed by RT-PCR. OTA had no direct effect on TF and PAI-2 production by MNC. However, OTA caused a dose-dependent reduction in LPS-induced TF (activity, antigen and mRNA) and PAI-2 (antigen and mRNA) production with > 85% inhibition at 1 μg/ml. Similar results were obtained when monocyte-enriched preparations were used instead of MNC. TF production was also impaired by OTA (1 μg/ml) when MNC were stimulated with phorbol myristate acetate (98% inhibition), IL-1β (83%) or TNF-α (62%). The inhibition of TF and PAI-2 induction might represent a hitherto unrecognized mechanism whereby OTA exerts immunosuppressant activity

  8. miR-34a Inhibits Proliferation and Invasion of Bladder Cancer Cells by Targeting Orphan Nuclear Receptor HNF4G

    Directory of Open Access Journals (Sweden)

    Huaibin Sun

    2015-01-01

    Full Text Available miR-34a is a member of the miR-34 family and acts as a tumor suppressor in bladder cancer. This study explored the regulative role of miR-34a on an orphan nuclear receptor HNF4G, which has a well-confirmed role in bladder tumor growth and invasion. qRT-PCR analysis was applied to measure miR-34a expression in two tumorigenic bladder cancer cell lines 5637 and T24 and one normal human urothelial cell line SV-HUC-1. Luciferase assay was performed to verify the putative binding between miR-34a and HNF4G. The influence of miR-34a-HNF4G axis on cell viability, colony formation, and invasion was assessed with loss- and gain-of-function analysis. This study observed that the miR-34a expressions in 5637 and T24 cells were significantly lower than in SV-HUC-1, while the muscle invasive cell sublines 5637-M and T24-M had even lower miR-34a expression than in the nonmuscle invasive sublines. HNF4G has a 3′-UTR binding site with miR-34a and is a direct downstream target of miR-34a. miR-34a can directly downregulate the expression of HNF4G and thus inhibit tumor cell viability, colony formation, and invasion. Therefore, miR-34a-HNF4G axis is an important pathway modulating cell viability, proliferation, and invasion of bladder cancer cells.

  9. p16(INK4A) inhibits the pro-metastatic potentials of osteosarcoma cells through targeting the ERK pathway and TGF-β1.

    Science.gov (United States)

    Silva, Gabriela; Aboussekhra, Abdelilah

    2016-05-01

    Extracellular signal-regulated kinase (ERK) is a downstream component of the evolutionarily conserved mitogen-activated protein kinase-signaling pathway, which controls the expression of a plethora of genes implicated in various physiological processes. This pathway is often hyper-activated by mutations or abnormal extracellular signaling in different types of human cancer, including the most common primary malignant bone tumor osteosarcomas. p16(INK4A) is an important tumor suppressor gene frequently lost in osteosarcomas, and is associated with the progression of these malignancies. We have shown, here, that the ERK1/2 protein kinase is also activated by p16(INK4A) down-regulation in osteosarcoma cells and normal human as well as mouse cells. This inhibitory effect is associated with the suppression of the upstream kinase MEK1/2, and is mediated via the repression of miR-21-5p and the consequent up-regulation of the MEK/ERK antagonist SPRY2 in osteosarcoma cells. Furthermore, we have shown that p16(INK4) inhibits the migration/invasion abilities of these cells through miR-21-5p-dependent inhibition of ERK1/2. In addition, we present clear evidence that p16(INK4) represses the paracrine pro-migratory effect of osteosarcoma cells on stromal fibroblasts through the inhibition of the TGF-β1 expression/secretion. This effect is also ERK1/2-dependent, indicating that in addition to their cell-autonomous actions, p16(INK4) and ERK1/2 have also non-cell-autonomous cancer-related functions. Together, these results indicate that the tumor suppressor p16(INK4) protein represses the carcinogenic process of osteosarcoma cells not only as a cell cycle regulator, but also as a negative regulator of pro-carcinogenic/-metastatic pathways. This indicates that targeting the ERK pathway is of utmost therapeutic value. © 2015 Wiley Periodicals, Inc.

  10. 31P NMR saturation-transfer measurements in Saccharomyces cerevisiae: characterization of phosphate exchange reactions by iodoacetate and antimycin A inhibition

    International Nuclear Information System (INIS)

    Campbell-Burk, S.L.; Jones, K.A.; Shulman, R.G.

    1987-01-01

    31 P nuclear magnetic resonance (NMR) saturation-transfer (ST) techniques have been used to measure steady-state flows through phosphate-adenosine 5'-triphosphate (ATP) exchange reactions in glucose-grown derepressed yeast. The results have revealed that the reactions catalyzed by glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase (GAPDH/PGK) and by the mitochondrial ATPase contribute to the observed ST. Contributions from these reactions were evaluated by performing ST studies under various metabolic conditions in the presence and absence of either iodoacetate, a specific inhibitor of GAPDH, or the respiratory chain inhibitor antimycin A. Intracellular phosphate (P/sub i/) longitudinal relaxation times were determined by performing inversion recovery experiments during steady-state ATP/sub λ/ saturation and were used in combination with ST data to determine P/sub i/ consumption rates. 13 C NMR and O 2 electrode measurements were also conducted to monitor changes in rates of glucose consumption and O 2 consumption, respectively, under the various metabolic conditions examined. The results suggest that GAPDH/PGK-catalyzed P/sub i/-ATP exchange is responsible for antimycin-resistant saturation transfer observed in anaerobic and aerobic glucose-fed yeast. Kinetics through GAPDH/PGK were found to depend on metabolic conditions. The coupled system appears to operate in a unidirectional manner during anaerobic glucose metabolism and bidirectionally when the cells are respiring on exogenously supplied ethanol. Additionally, mitochondrial ATPase activity appears to be responsible for the transfer observed in iodoacetate-treated aerobic cells supplied with either glucose or ethanol, with synthesis of ATP occurring unidirectionally

  11. Mouse mannose-binding lectin-A and ficolin-A inhibit lipopolysaccharide-mediated pro-inflammatory responses on mast cells

    DEFF Research Database (Denmark)

    Ma, Ying Jie; Kang, Hee Jung; Kim, Ji Yeon

    2013-01-01

    It is unknown how soluble pattern-recognition receptors in blood, such as mannose-binding lectin (MBL) and ficolins, modulate mast cell-mediated inflammatory responses. We investigate how mouse MBL-A or ficolin-A regulate mouse bone marrow-derived mast cells (mBMMCs)-derived inflammatory response...... cytokine production by LPS-mediated TLR4 in mBMMCs appears to be down-regulated, indicating that mouse MBL and ficolin may have an inhibitory function toward mouse TLR4-mediated excessive inflammation on the mast cells.......It is unknown how soluble pattern-recognition receptors in blood, such as mannose-binding lectin (MBL) and ficolins, modulate mast cell-mediated inflammatory responses. We investigate how mouse MBL-A or ficolin-A regulate mouse bone marrow-derived mast cells (mBMMCs)-derived inflammatory response...

  12. Protein kinase A inhibition modulates the intracellular routing of gene delivery vehicles in HeLa cells, leading to productive transfection

    NARCIS (Netherlands)

    Rehman, Zia Ur; Hoekstra, Dick; Zuhorn, Inge S.

    2011-01-01

    Cellular entry of nanoparticles for drug- and gene delivery relies on various endocytic pathways, including clathrin-and caveolae-mediated endocytosis. To improve delivery, i.e., the therapeutic and/or cell biological impact, current efforts are aimed at avoiding processing of the carriers along the

  13. Hydroxysafflor Yellow A Inhibits LPS-Induced NLRP3 Inflammasome Activation via Binding to Xanthine Oxidase in Mouse RAW264.7 Macrophages

    Directory of Open Access Journals (Sweden)

    Xiaolong Xu

    2016-01-01

    Full Text Available Hydroxysafflor yellow A (HSYA is an effective therapeutic agent for inflammatory diseases and autoimmune disorders; however, its regulatory effect on NLRP3 inflammasome activation in macrophages has not been investigated. In this study, we predicted the potential interaction between HSYA and xanthine oxidase (XO via PharmMapper inverse docking and confirmed the binding inhibition via inhibitory test (IC50 = 40.04 μM. Computation docking illustrated that, in this HSYA-XO complex, HSYA was surrounded by Leu 648, Leu 712, His 875, Leu 873, Ser 876, Glu 879, Phe 649, and Asn 650 with a binding energy of −5.77 kcal/M and formed hydrogen bonds with the hydroxyl groups of HSYA at Glu 879, Asn 650, and His 875. We then found that HSYA significantly decreased the activity of XO in RAW264.7 macrophages and suppressed LPS-induced ROS generation. Moreover, we proved that HSYA markedly inhibited LPS-induced cleaved caspase-1 activation via suppressing the sensitization of NLRP3 inflammasome and prevented the mature IL-1β formation from pro-IL-1β form. These findings suggest that XO may be a potential target of HSYA via direct binding inhibition and the combination of HSYA-XO suppresses LPS-induced ROS generation, contributing to the depression of NLRP3 inflammasome and inhibition of IL-1β secretion in macrophages.

  14. Antisense locked nucleic acids targeting agrA inhibit quorum sensing and pathogenesis of community-associated methicillin-resistant Staphylococcus aureus.

    Science.gov (United States)

    Da, F; Yao, L; Su, Z; Hou, Z; Li, Z; Xue, X; Meng, J; Luo, X

    2017-01-01

    Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is commonly associated with nonnosocomial skin and soft tissue infections due to its virulence, which is mainly controlled by the accessory gene regulator (agr) quorum sensing (QS) system. In this study (KFF) 3 K peptide-conjugated locked nucleic acids (PLNAs) targeting agrA mRNA were developed to inhibit agr activity and arrest the pathogenicity of CA-MRSA. Two PLNAs were designed, and synthesized, after predicting the secondary structure of agrA mRNA. The influence on bacterial growth was tested using a growth curve assay. RT-qPCR, haemolysis assay, lactate dehydrogenase release assay and chemotaxis assay were used to evaluate the effects of the PLNAs on inhibiting agr QS. A mouse skin infection model was employed to test the protective effect of the PLNAs in vivo. None of the PLNAs were found to be bacteriostatic or bactericidal in vitro. However, one PLNA, PLNA34, showed strong ability to suppress expression of agrA and the effector molecule RNAIII in USA300 LAC strain. Furthermore, PLNA34 inhibited the expression of virulence genes that are upregulated by agr, including hla, psmα, psmβ and pvl. The haemolytic activity of the supernatants from PLNA34-treated bacteria was also dramatically reduced, as well as the capacity to lyse and recruit neutrophils. Moreover, PLNA34 showed high levels of protection in the CA-MRSA mouse skin infection model. The anti-agrA PLNA34 can effectively inhibit the agr QS and suppress CA-MRSA pathogenicity. agrA is a promising target for the development of antisense oligonucleotides to block agr QS. Journal of Applied Microbiology © 2016 The Society for Applied Microbiology.

  15. Site-specific modification of the lactose operator with acetylaminofluorene

    Energy Technology Data Exchange (ETDEWEB)

    Stoerhrer, G; Osband, J A; Alvarado-Urbina, G

    1983-01-01

    The authors have synthesized the tetradecamer GAGCXGATAACAAG containing a part of the sequence of the lactose operator. A guanine base in the sequence is replaced by the adduct of the carcinogen 2-acetylaminofluorene with guanine. Under the standard conditions of de-protection, the fluorene moiety is lost, leaving behind a guanine oxidation product. New conditions of de-protection have been developed which allow the isolation of an oligonucleotide containing the adduct of 2-aminofluorene with guanine. The presence of the amino-fluorene adduct greatly increases retention on reverse phase chromatography and produces a unique pattern of sequencing bands. 10 references, 6 figures.

  16. Extent of intramolecular pi stacks in aqueous solution in mixed-ligand copper(II) complexes formed by heteroaromatic amines and the anticancer and antivirally active 9[2-(phosphonomethoxy)ethyl]guanine (PMEG). A comparison with related acyclic nucleotide analogues

    Czech Academy of Sciences Publication Activity Database

    Blindauer, C. A.; Sigel, A.; Operschall, B. P.; Griesser, R.; Holý, Antonín; Sigel, H.

    2016-01-01

    Roč. 103, Jan 8 (2016), s. 248-260 ISSN 0277-5387 Institutional support: RVO:61388963 Keywords : anticancer activity * antivirals * aromatic-ring stacking * isomeric equilibria * nucleotide analogues Subject RIV: CC - Organic Chemistry Impact factor: 1.926, year: 2016

  17. Human cystatin C forms an inactive dimer during intracellular trafficking in transfected CHO cells

    DEFF Research Database (Denmark)

    Merz, G S; Benedikz, Eirikur; Schwenk, V

    1997-01-01

    To define the cellular processing of human cystatin C as well as to lay the groundwork for investigating its contribution to lcelandic Hereditary Cerebral Hemorrhage with Amyloidosis (HCHWA-I), we have characterized the trafficking, secretion, and extracellular fate of human cystatin C...... that the cystatin C dimer, formed during intracellular trafficking, is converted to monomer at or before secretion. Cells in which exit from the endoplasmic reticulum (ER) was blocked with brefeldin A contained the 33 kDa species, indicating that cystatin C dimerization occurs in the ER. After removal of brefeldin......, presumably as a consequence of the low pH of late endosome/lysosomes. As a dimer, cystatin C would be prevented from inhibiting the lysosomal cysteine proteases. These results reveal a novel mechanism, transient dimerization, by which cystatin C is inactivated during the early part of its trafficking through...

  18. Ultra-sensitive detection of leukemia by graphene

    Science.gov (United States)

    Akhavan, Omid; Ghaderi, Elham; Hashemi, Ehsan; Rahighi, Reza

    2014-11-01

    Graphene oxide nanoplatelets (GONPs) with extremely sharp edges (lateral dimensions ~20-200 nm and thicknesses leukemia cells. The blood serums containing the extracted guanine were used in differential pulse voltammetry (DPV) with reduced graphene oxide nanowall (rGONW) electrodes to develop fast and ultra-sensitive electrochemical detection of leukemia cells at leukemia fractions (LFs) of ~10-11 (as the lower detection limit). The stability of the DPV signals obtained by oxidation of the extracted guanine on the rGONWs was studied after 20 cycles. Without the guanine extraction, the DPV peaks relating to guanine oxidation of normal and abnormal cells overlapped at LFs diagnosis.Graphene oxide nanoplatelets (GONPs) with extremely sharp edges (lateral dimensions ~20-200 nm and thicknesses leukemia cells. The blood serums containing the extracted guanine were used in differential pulse voltammetry (DPV) with reduced graphene oxide nanowall (rGONW) electrodes to develop fast and ultra-sensitive electrochemical detection of leukemia cells at leukemia fractions (LFs) of ~10-11 (as the lower detection limit). The stability of the DPV signals obtained by oxidation of the extracted guanine on the rGONWs was studied after 20 cycles. Without the guanine extraction, the DPV peaks relating to guanine oxidation of normal and abnormal cells overlapped at LFs diagnosis. Electronic supplementary information (ESI) available. See DOI: 10.1039/C4NR04589K

  19. MicroRNA-99a inhibits insulin-induced proliferation, migration, dedifferentiation, and rapamycin resistance of vascular smooth muscle cells by inhibiting insulin-like growth factor-1 receptor and mammalian target of rapamycin

    International Nuclear Information System (INIS)

    Zhang, Zi-wei; Guo, Rui-wei; Lv, Jin-lin; Wang, Xian-mei; Ye, Jin-shan; Lu, Ni-hong; Liang, Xing; Yang, Li-xia

    2017-01-01

    Patients with type 2 diabetes mellitus (T2DM) are characterized by insulin resistance and are subsequently at high risk for atherosclerosis. Hyperinsulinemia has been associated with proliferation, migration, and dedifferentiation of vascular smooth muscle cells (VSMCs) during the pathogenesis of atherosclerosis. Moreover, insulin-like growth factor-1 receptor (IGF-1R) and mammalian target of rapamycin (mTOR) have been demonstrated to be the underlying signaling pathways. Recently, microRNA-99a (miR-99a) has been suggested to regulate the phenotypic changes of VSMCs in cancer cells. However, whether it is involved in insulin-induced changes of VSCMs has not been determined. In this study, we found that insulin induced proliferation, migration, and dedifferentiation of mouse VSMCs in a dose-dependent manner. Furthermore, the stimulating effects of high-dose insulin on proliferation, migration, and dedifferentiation of mouse VSMCs were found to be associated with the attenuation of the inhibitory effects of miR-99a on IGF-1R and mTOR signaling activities. Finally, we found that the inducing effect of high-dose insulin on proliferation, migration, and dedifferentiation of VSMCs was partially inhibited by an active mimic of miR-99a. Taken together, these results suggest that miR-99a plays a key regulatory role in the pathogenesis of insulin-induced proliferation, migration, and phenotype conversion of VSMCs at least partly via inhibition of IGF-1R and mTOR signaling. Our results provide evidence that miR-99a may be a novel target for the treatment of hyperinsulinemia-induced atherosclerosis. - Highlights: • Suggesting a new mechanism of insulin-triggered VSMC functions. • Providing a new therapeutic strategies that target atherosclerosis in T2DM patients. • Providing a new strategies that target in-stent restenosis in T2DM patients.

  20. Brugia malayi Antigen (BmA Inhibits HIV-1 Trans-Infection but Neither BmA nor ES-62 Alter HIV-1 Infectivity of DC Induced CD4+ Th-Cells.

    Directory of Open Access Journals (Sweden)

    Emily E I M Mouser

    Full Text Available One of the hallmarks of HIV-1 disease is the association of heightened CD4+ T-cell activation with HIV-1 replication. Parasitic helminths including filarial nematodes have evolved numerous and complex mechanisms to skew, dampen and evade human immune responses suggesting that HIV-1 infection may be modulated in co-infected individuals. Here we studied the effects of two filarial nematode products, adult worm antigen from Brugia malayi (BmA and excretory-secretory product 62 (ES-62 from Acanthocheilonema viteae on HIV-1 infection in vitro. Neither BmA nor ES-62 influenced HIV-1 replication in CD4+ enriched T-cells, with either a CCR5- or CXCR4-using virus. BmA, but not ES-62, had the capacity to bind the C-type lectin dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN thereby inhibiting HIV-1 trans-infection of CD4+ enriched T-cells. As for their effect on DCs, neither BmA nor ES-62 could enhance or inhibit DC maturation as determined by CD83, CD86 and HLA-DR expression, or the production of IL-6, IL-10, IL-12 and TNF-α. As expected, due to the unaltered DC phenotype, no differences were found in CD4+ T helper (Th cell phenotypes induced by DCs treated with either BmA or ES-62. Moreover, the HIV-1 susceptibility of the Th-cell populations induced by BmA or ES-62 exposed DCs was unaffected for both CCR5- and CXCR4-using HIV-1 viruses. In conclusion, although BmA has the potential capacity to interfere with HIV-1 transmission or initial viral dissemination through preventing the virus from interacting with DCs, no differences in the Th-cell polarizing capacity of DCs exposed to BmA or ES-62 were observed. Neither antigenic source demonstrated beneficial or detrimental effects on the HIV-1 susceptibility of CD4+ Th-cells induced by exposed DCs.

  1. microRNA-146a inhibits G protein-coupled receptor-mediated activation of NF-κB by targeting CARD10 and COPS8 in gastric cancer

    DEFF Research Database (Denmark)

    Crone, Stephanie Geisler; Jacobsen, Anders; Federspiel, Birgitte

    2012-01-01

    Gastric cancer is the second most common cause of cancer-related death in the world. Inflammatory signals originating from gastric cancer cells are important for recruiting inflammatory cells and regulation of metastasis of gastric cancer. Several microRNAs (miRNA) have been shown to be involved...... in development and progression of gastric cancer. miRNA-146a (miR-146a) is a modulator of inflammatory signals, but little is known about its importance in gastric cancer. We therefore wanted to identify targets of miR-146a in gastric cancer and examine its biological roles....

  2. 7-Alkylguanine adduct levels in urine, lungs and liver of mice exposed to styrene by inhalation

    International Nuclear Information System (INIS)

    Vodicka, Pavel Erik; Linhart, Igor; Novak, Jan; Koskinen, Mikko; Vodickova, Ludmila; Hemminki, Kari

    2006-01-01

    This study describes urinary excretion of two nucleobase adducts derived from styrene 7,8-oxide (SO), i.e., 7-(2-hydroxy-1-phenylethyl)guanine (N7αG) and 7-(2-hydroxy-2-phenylethyl)guanine (N7βG), as well as a formation of N7-SO-guanine adducts in lungs and liver of two month old male NMRI mice exposed to styrene by inhalation in a 3-week subacute study. Strikingly higher excretion of both isomeric nucleobase adducts in the first day of exposure was recorded, while the daily excretion of nucleobase adducts in following time intervals reached the steady-state level at 4.32 + 1.14 and 6.91 + 1.17 pmol/animal for lower and higher styrene exposure, respectively. β-SO-guanine DNA adducts in lungs increased with exposure in a linear way (F = 13.7 for linearity and 0.17 for non-linearity, respectively), reaching at the 21st day the level of 23.0 adducts/10 8 normal nucleotides, i.e., 0.74 fmol/μg DNA of 7-alkylguanine DNA adducts for the concentration of 1500 mg/m 3 , while no 7-SO-guanine DNA adducts were detected in the liver after 21 days of inhalation exposure to both of styrene concentrations. A comparison of 7-alkylguanines excreted in urine with 7-SO-guanines in lungs (after correction for depurination and for missing α-isomers) revealed that persisting 7-SO-guanine DNA adducts in lungs account for about 0.5% of the total alkylation at N7 of guanine. The total styrene-specific 7-guanine alkylation accounts for about 1.0 x 10 -5 % of the total styrene uptake, while N1-adenine alkylation contributes to this percentage only negligibly

  3. Catabolite repression of enzyme synthesis does not prevent sporulation.

    OpenAIRE

    Lopez, J M; Uratani-Wong, B; Freese, E

    1980-01-01

    In the presence of excess glucose, a decrease of guanine nucleotides in Bacillus subtilis initiated sporulation but did not prevent catabolite repression of three enzymes. Therefore, the ultimate mechanism(s) repressing enzyme synthesis differs from that suppressing sporulation.

  4. Indicator Based and Indicator - Free Electrochemical DNA Biosensors

    National Research Council Canada - National Science Library

    Kerman, Kagan

    2001-01-01

    The utility and advantages of an indicator free and MB based sequence specific DNA hybridization biosensor based on guanine and adenine oxidation signals and MB reduction signals have been demonstrated...

  5. ORF Alignment: NT_033779 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available rEMBL::g2674107:GUANINE NUCLEOTIDE-EXCHANGE ... PROTEIN. organism:BOS TAURUS (BOVINE). dbxref:GenBank...; ... AF023451; g2674107; -.'', species:''BOS TAURUS ... Length = 185 ... Query: 586 METGIELFNRKP

  6. Mild Lesch-Nyhan Disease in a Boy with a Null Mutation inHPRT1

    DEFF Research Database (Denmark)

    Bayat, Allan; Christensen, Mette; Wibrand, Flemming

    2015-01-01

    Hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency results in a continuous spectrum of clinical phenotypes though all include overproduction of uric acid with hyperuricaemia, urate nephrolithiasis and gout. HPRT1 mutations that result in very low or no HPRT enzyme activities...

  7. 75 FR 24711 - Food and Drug Administration Modernization Act of 1997: Modifications to the List of Recognized...

    Science.gov (United States)

    2010-05-05

    ... Guanine Relevant guidance Phosphoribosyl Transferase Gene Mutation Assay 2-137 ASTM E1263-97 (Reapproved... Antifungal Disk Diffusion Susceptibility Testing of Yeasts 7-37 NCCLS I/LA06-A Detection and Quantitation of...

  8. Low Melatonin Production During Adulthood - Phase 2: Association with Levels of Hydroxyl Radical Scavenging and DNA Damage

    National Research Council Canada - National Science Library

    Sobel, Eugene L; Davanipour, Zoreh; Poulsen, Henrik

    2004-01-01

    The primary purpose of the proposed study is to develop cross-sectional evidence concerning whether or not lower melatonin production levels are associated with increased oxidative DNA guanine damage...

  9. Genetics Home Reference: Lesch-Nyhan syndrome

    Science.gov (United States)

    ... Potier MC, Dauphinot L, Shirley TL, Torero-Ibad R, Fuchs J, Jinnah HA. Hypoxanthine-guanine phosphoribosyl transferase regulates early developmental programming of dopamine neurons: implications for Lesch-Nyhan disease ...

  10. Presidential Green Chemistry Challenge: 2000 Greener Synthetic Pathways Award

    Science.gov (United States)

    Presidential Green Chemistry Challenge 2000 award winner, Roche Colorado, developed a greener synthesis for gancyclovir (Cytovene, a potent antiviral drug) that uses a second-generation Guanine Triester (GTE) process.

  11. Chlorophyll and its degradation products in the two-spotted spider mite, Tetranychus urticae: observations using epifluorescence and confocal laser scanning microscopy.

    Science.gov (United States)

    Occhipinti, Andrea; Maffei, Massimo E

    2013-10-01

    Chlorophyll and chlorophyll degradation products were observed in the two-spotted spider mite (Tetranychus urticae) using epifluorescence microscopy (EFM) and confocal laser scanning microscopy (CLSM). A clear red fluorescence (EFM) and a fluorescence induced by a laser wavelength of 650 nm (CLSM) were observed. In the lateral caeca, in the ventriculus and in the excretory organ, a bright light blue fluorescence was observed in close association with chlorophyll by using EFM. The same material can be localized with CLSM by using a laser with a wavelength of 488 nm. By comparison with synthetic guanine, this bright fluorescence is supposed to be guanine. The presence of guanine fluorescence in the mite pellets confirms this hypothesis. A possible mechanism for guanine formation is discussed.

  12. Toxicity, Mutagenicity, and Mutational Spectra of Vinyl Chloride, 2- Chloroethylene Oxide, and Chloracetaldehyde in a Human Lymphoblastoid Line Expressing Cytochrome P450IIE1.

    Science.gov (United States)

    1992-01-01

    concluded that CEO was the alkylating agent involved in conversion of adenosine to l,N 6 -ethenoadenosine. 1,N6 - Ethenoadenosine was not produced by CEO...guanines as nearest neighbors upon the alkylation of a guanine residue in DNA. N-methyl-N- nitrosourea (MNU) was reacted with a synthetic polynucleotide...the alkylating agent MNNG or the intercalating agent ICR-191. In the study they determined that mutants comprising at least one percent of the total

  13. Identifying the impact of G-quadruplexes on Affymetrix 3' arrays using cloud computing.

    Science.gov (United States)

    Memon, Farhat N; Owen, Anne M; Sanchez-Graillet, Olivia; Upton, Graham J G; Harrison, Andrew P

    2010-01-15

    A tetramer quadruplex structure is formed by four parallel strands of DNA/ RNA containing runs of guanine. These quadruplexes are able to form because guanine can Hoogsteen hydrogen bond to other guanines, and a tetrad of guanines can form a stable arrangement. Recently we have discovered that probes on Affymetrix GeneChips that contain runs of guanine do not measure gene expression reliably. We associate this finding with the likelihood that quadruplexes are forming on the surface of GeneChips. In order to cope with the rapidly expanding size of GeneChip array datasets in the public domain, we are exploring the use of cloud computing to replicate our experiments on 3' arrays to look at the effect of the location of G-spots (runs of guanines). Cloud computing is a recently introduced high-performance solution that takes advantage of the computational infrastructure of large organisations such as Amazon and Google. We expect that cloud computing will become widely adopted because it enables bioinformaticians to avoid capital expenditure on expensive computing resources and to only pay a cloud computing provider for what is used. Moreover, as well as financial efficiency, cloud computing is an ecologically-friendly technology, it enables efficient data-sharing and we expect it to be faster for development purposes. Here we propose the advantageous use of cloud computing to perform a large data-mining analysis of public domain 3' arrays.

  14. Broadband and polarization reflectors in the lookdown, Selene vomer.

    Science.gov (United States)

    Zhao, Shulei; Brady, Parrish Clawson; Gao, Meng; Etheredge, Robert Ian; Kattawar, George W; Cummings, Molly E

    2015-03-06

    Predator evasion in the open ocean is difficult because there are no objects to hide behind. The silvery surface of fish plays an important role in open water camouflage. Various models have been proposed to account for the broadband reflectance by the fish skin that involve one-dimensional variations in the arrangement of guanine crystal reflectors, yet the three-dimensional organization of these guanine platelets have not been well characterized. Here, we report the three-dimensional organization and the optical properties of integumentary guanine platelets in a silvery marine fish, the lookdown (Selene vomer). Our structural analysis and computational modelling show that stacks of guanine platelets with random yaw angles in the fish skin produce broadband reflectance via colour mixing. Optical axes of the guanine platelets and the collagen layer are aligned closely and provide bulk birefringence properties that influence the polarization reflectance by the skin. These data demonstrate how the lookdown preserves or alters polarization states at different incident polarization angles. These optical properties resulted from the organization of these guanine platelets and the collagen layer may have implications for open ocean camouflage in varying light fields. © 2015 The Author(s) Published by the Royal Society. All rights reserved.

  15. Inhibitors of plant hormone transport

    Czech Academy of Sciences Publication Activity Database

    Klíma, Petr; Laňková, Martina; Zažímalová, Eva

    2016-01-01

    Roč. 253, č. 6 (2016), s. 1391-1404 ISSN 0033-183X R&D Projects: GA MŠk(CZ) LD15088 Institutional support: RVO:61389030 Keywords : polar auxin transport * acid-binding protein * gnom arf-gef * equilibrative nucleoside transporter * efflux carrier polarity * plasma-membrane-protein * cultured tobacco cells * arabidopsis-thaliana * gravitropic response * brefeldin-a * Plant hormones * Transport * Inhibitors * Auxin * Cytokinins * Strigolactones * Abscisic acid * Cell biology Subject RIV: ED - Physiology Impact factor: 2.870, year: 2016

  16. The Apical Actin Fringe Contributes to Localized Cell Wall Deposition and Polarized Growth in the Lily Pollen Tube1[W][OPEN

    Science.gov (United States)

    Rounds, Caleb M.; Hepler, Peter K.; Winship, Lawrence J.

    2014-01-01

    In lily (Lilium formosanum) pollen tubes, pectin, a major component of the cell wall, is delivered through regulated exocytosis. The targeted transport and secretion of the pectin-containing vesicles may be controlled by the cortical actin fringe at the pollen tube apex. Here, we address the role of the actin fringe using three different inhibitors of growth: brefeldin A, latrunculin B, and potassium cyanide. Brefeldin A blocks membrane trafficking and inhibits exocytosis in pollen tubes; it also leads to the degradation of the actin fringe and the formation of an aggregate of filamentous actin at the base of the clear zone. Latrunculin B, which depolymerizes filamentous actin, markedly slows growth but allows focused pectin deposition to continue. Of note, the locus of deposition shifts frequently and correlates with changes in the direction of growth. Finally, potassium cyanide, an electron transport chain inhibitor, briefly stops growth while causing the actin fringe to completely disappear. Pectin deposition continues but lacks focus, instead being delivered in a wide arc across the pollen tube tip. These data support a model in which the actin fringe contributes to the focused secretion of pectin to the apical cell wall and, thus, to the polarized growth of the pollen tube. PMID:25037212

  17. The apical actin fringe contributes to localized cell wall deposition and polarized growth in the lily pollen tube.

    Science.gov (United States)

    Rounds, Caleb M; Hepler, Peter K; Winship, Lawrence J

    2014-09-01

    In lily (Lilium formosanum) pollen tubes, pectin, a major component of the cell wall, is delivered through regulated exocytosis. The targeted transport and secretion of the pectin-containing vesicles may be controlled by the cortical actin fringe at the pollen tube apex. Here, we address the role of the actin fringe using three different inhibitors of growth: brefeldin A, latrunculin B, and potassium cyanide. Brefeldin A blocks membrane trafficking and inhibits exocytosis in pollen tubes; it also leads to the degradation of the actin fringe and the formation of an aggregate of filamentous actin at the base of the clear zone. Latrunculin B, which depolymerizes filamentous actin, markedly slows growth but allows focused pectin deposition to continue. Of note, the locus of deposition shifts frequently and correlates with changes in the direction of growth. Finally, potassium cyanide, an electron transport chain inhibitor, briefly stops growth while causing the actin fringe to completely disappear. Pectin deposition continues but lacks focus, instead being delivered in a wide arc across the pollen tube tip. These data support a model in which the actin fringe contributes to the focused secretion of pectin to the apical cell wall and, thus, to the polarized growth of the pollen tube. © 2014 American Society of Plant Biologists. All Rights Reserved.

  18. Products obtained after in vitro reaction of 7,12-dimethylbenz[alpha]anthracene 5,6-oxide with nucleic acids.

    Science.gov (United States)

    Blobstein, S H; Weinstein, I B; Grunberger, D; Weisgras, J; Harvey, R G

    1975-07-29

    Several lines of evidence suggest that oxide derivatives of carcinogenic polycyclic hydrocarbons are the reactive intermediates for in vivo binding to cellular nucleic acids. In the present study the covalent binding of 7,12-dimethylbenz[alpha]anthracene 5,6-oxide to synthetic homopolymers and nucleic acids in aqueous-acetone solutions has been investigated. Poly(G) was found to be the most reactive nucleic acid and underwent approximately 7-10% modification. Alkaline hydrolysis of the poly(G)-dimethylbenzathracene conjugate yielded chromatographically distinct polycyclic hydrocarbon-modified nucleotides which were further characterized by spectral analyses and enzymatic and chemical degradation. When the oxide was allowed to react with GMP or dGMP, at least two products were obtained in about 1% yield. Acid hydrolysis of the dGMP-dimethylbenzanthracene conjugates liberated the corresponding guanine-dimethylbenzathracene products. Mass spectral analysis of the modified bases provided direct evidence that we had obtained covalent binding of the poly-cyclic hydrocarbon to guanine. The mass spectral cleavage pattern suggest that one of these products is a hydroxydihydro derivative of dimethylbenzanthracene bound to guanine and the other is a dimethylbenzanthracene-guanine conjugate. Additional structural aspects of these guanine derivatives are discussed.

  19. Designing a New Class of Bases for Nucleic Acid Quadruplexes and Quadruplex-Active Ligands.

    Science.gov (United States)

    Bazzi, Sophia; Novotný, Jan; Yurenko, Yevgen P; Marek, Radek

    2015-06-22

    A new class of quadruplex nucleobases, derived from 3-deazaguanine, has been designed for various applications as smart quadruplex ligands as well as quadruplex-based aptamers, receptors, and sensors. An efficient strategy for modifying the guanine quadruplex core has been developed and tested by using quantum chemistry methods. Several potential guanine derivatives modified at the 3- or 8-position or both are analyzed, and the results compared to reference systems containing natural guanine. Analysis of the formation energies (BLYP-D3(BJ)/def2-TZVPP level of theory, in combination with the COSMO model for water) in model systems consisting of two and three stacked tetrads with Na(+) /K(+) ion(s) inside the internal channel indicates that the formation of structures with 3-halo-3-deazaguanine bases leads to a substantial gain in energy, as compared to the corresponding reference guanine complexes. The results cast light on changes in the noncovalent interactions (hydrogen bonding, stacking, and ion coordination) in a quadruplex stem upon modification of the guanine core. In particular, the enhanced stability of the modified quadruplexes was shown to originate mainly from increased π-π stacking. Our study suggests the 3-halo-3-deazaguanine skeleton as a potential building unit for quadruplex systems and smart G-quadruplex ligands. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. High-throughput screening assay of hepatitis C virus helicase inhibitors using fluorescence-quenching phenomenon

    International Nuclear Information System (INIS)

    Tani, Hidenori; Akimitsu, Nobuyoshi; Fujita, Osamu; Matsuda, Yasuyoshi; Miyata, Ryo; Tsuneda, Satoshi; Igarashi, Masayuki; Sekiguchi, Yuji; Noda, Naohiro

    2009-01-01

    We have developed a novel high-throughput screening assay of hepatitis C virus (HCV) nonstructural protein 3 (NS3) helicase inhibitors using the fluorescence-quenching phenomenon via photoinduced electron transfer between fluorescent dyes and guanine bases. We prepared double-stranded DNA (dsDNA) with a 5'-fluorescent-dye (BODIPY FL)-labeled strand hybridized with a complementary strand, the 3'-end of which has guanine bases. When dsDNA is unwound by helicase, the dye emits fluorescence owing to its release from the guanine bases. Our results demonstrate that this assay is suitable for quantitative assay of HCV NS3 helicase activity and useful for high-throughput screening for inhibitors. Furthermore, we applied this assay to the screening for NS3 helicase inhibitors from cell extracts of microorganisms, and found several cell extracts containing potential inhibitors.

  1. Site-specifically modified oligodeoxyribonucleotides as templates for Escherichia coli DNA polymerase I

    International Nuclear Information System (INIS)

    O'Connor, D.; Stoehrer, G.

    1985-01-01

    Oligodeoxyribonucleotides with site-specific modifications have been used as substrates for Escherichia coli DNA polymerase I holoenzyme and Klenow fragment. Modifications included the bulky guanine-8-aminofluorene adduct and a guanine oxidation product resembling the product of photosensitized DNA oxidation. By a combination of primers and nick-mers, conditions of single-strand-directed DNA synthesis and nick-translation could be created. The results show that the polymerase can bypass both types of lesions. Bypass occurs on a single-stranded template but is facilitated on a nicked, double-stranded template. Only purines, with guanine more favored than adenine, are incorporated across both lesions. The results indicate that site-specifically modified oligonucleotides can be sensitive probes for the action of polymerases on damaged templates. They also suggest a function for polymerase I, in its nick-translation capacity, during DNA repair and mutagenesis

  2. DNA lability induced by nimustine and ramustine in rat glioma cells.

    Science.gov (United States)

    Mineura, K; Fushimi, S; Itoh, Y; Kowada, M

    1988-01-01

    The DNA labile sites induced by two nitrosoureas, nimustine (ACNU) and ramustine (MCNU) synthesised in Japan, have been examined in highly reiterated DNA sequences of rat glioma cells. Reiterated fragments of 167 and 203 base pairs (bp), obtained after Hind III and Hae III restriction endonuclease digestion of rat glioma cells DNA, were used as target DNA sequences to determine the labile sites. In vitro reaction with ACNU and MCNU resulted in scission products corresponding to the locations of guanine. Subsequent piperidine hydrolysis produced more frequent breaks of the phosphodiester bonds at guanine positions, thus forming alkali-labile sites. Images PMID:3236017

  3. Simultaneous quantification of purine and pyrimidine bases, nucleosides and their degradation products in bovine blood plasma by high performance liquid chromatography tandem mass spectrometry

    DEFF Research Database (Denmark)

    Nielsen, Charlotte Stentoft; Vestergaard, Mogens; Løvendahl, Peter

    2014-01-01

    describes the development and validation of a sensitive and specific method for simultaneous determination of 20 purines (adenine, guanine, guanosine, inosine, 2′-deoxyguanosine, 2′-deoxyinosine, xanthine, hypoxanthine), pyrimidines (cytosine, thymine, uracil, cytidine, uridine, thymidine, 2′-deoxyuridine...... investigated. It was confirmed that using a log-calibration model rather than a linear calibration model resulted in lower CV% and a lack of fit test demonstrated a satisfying linear regression. The method covers concentration ranges for each metabolite according to that in actual samples, e.g. guanine: 0...... veins and, with a few exceptions, also for other species such as chicken, pig, mink, human and rat....

  4. GMP reverses the facilitatory effect of glutamate on inhibitory avoidance task in rats.

    Science.gov (United States)

    Rubin, M A; Jurach, A; da Costa Júnior, E M; Lima, T T; Jiménez-Bernal, R E; Begnini, J; Souza, D O; de Mello, C F

    1996-09-02

    Previous studies have demonstrated that post-training intrahippocampal glutamate administration improves inhibitory avoidance task performance in rats. Antagonism of the agonist actions of glutamate by guanine nucleotides has been shown at the molecular and behavioural level. In the present investigation we demonstrate that intrahippocampal co-administration of GMP (guanosine 5'-monophosphate) reverses the facilitatory effect of glutamate on the inhibitory avoidance learning paradigm and inhibits [3H]glutamate binding in hippocampal synaptic plasma membranes. These results suggest that guanine nucleotides may modulate glutamate actions.

  5. Novel radiosynthesis of PET HSV-tk gene reporter probes [18F]FHPG and [18F]FHBG employing dual Sep-Pak SPE techniques.

    Science.gov (United States)

    Wang, Ji-Quan; Zheng, Qi-Huang; Fei, Xiangshu; Mock, Bruce H; Hutchins, Gary D

    2003-11-17

    Positron emission tomography (PET) herpes simplex virus thymidine kinase (HSV-tk) gene reporter probes 9-[(3-[(18)F]fluoro-1-hydroxy-2-propoxy)methyl]guanine ([(18)F]FHPG) and 9-(4-[(18)F]fluoro-3-hydroxymethylbutyl)guanine ([(18)F]FHBG) were prepared by nucleophilic substitution of the appropriate tosylated precursors with [(18)F]KF/Kryptofix 2.2.2 followed by a quick deprotection reaction and purification with a simplified dual Silica Sep-Pak solid-phase extraction (SPE) method in 15-30% radiochemical yield.

  6. Herpes simplex virus 1 induces de novo phospholipid synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Sutter, Esther [Electron Microscopy, Institute of Veterinary Anatomy, University of Zuerich (Switzerland); Oliveira, Anna Paula de; Tobler, Kurt [Electron microscopy, Institute of Virology, University of Zuerich (Switzerland); Schraner, Elisabeth M. [Electron Microscopy, Institute of Veterinary Anatomy, University of Zuerich (Switzerland); Sonda, Sabrina [Institute of Parasitology, University of Zuerich (Switzerland); Kaech, Andres [Center for Microscopy and Image Analysis, University of Zuerich (Switzerland); Lucas, Miriam S. [Electron Microscopy ETH Zuerich (EMEZ), Swiss Federal Institute of Technology, Zuerich (Switzerland); Ackermann, Mathias [Electron microscopy, Institute of Virology, University of Zuerich (Switzerland); Wild, Peter, E-mail: pewild@access.uzh.ch [Electron Microscopy, Institute of Veterinary Anatomy, University of Zuerich (Switzerland)

    2012-08-01

    Herpes simplex virus type 1 capsids bud at nuclear membranes and Golgi membranes acquiring an envelope composed of phospholipids. Hence, we measured incorporation of phospholipid precursors into these membranes, and quantified changes in size of cellular compartments by morphometric analysis. Incorporation of [{sup 3}H]-choline into both nuclear and cytoplasmic membranes was significantly enhanced upon infection. [{sup 3}H]-choline was also part of isolated virions even grown in the presence of brefeldin A. Nuclei expanded early in infection. The Golgi complex and vacuoles increased substantially whereas the endoplasmic reticulum enlarged only temporarily. The data suggest that HSV-1 stimulates phospholipid synthesis, and that de novo synthesized phospholipids are inserted into nuclear and cytoplasmic membranes to i) maintain membrane integrity in the course of nuclear and cellular expansion, ii) to supply membrane constituents for envelopment of capsids by budding at nuclear membranes and Golgi membranes, and iii) to provide membranes for formation of transport vacuoles.

  7. ER-mediated stress induces mitochondrial-dependent caspases activation in NT2 neuron-like cells.

    Science.gov (United States)

    Arduino, Daniela M; Esteves, A Raquel; Domingues, A Filipa; Pereira, Claudia M F; Cardoso, Sandra M; Oliveira, Catarina R

    2009-11-30

    Recent studies have revealed that endoplasmic reticulum (ER) disturbance is involved in the pathophysiology of neurodegenerative disorders, contributing to the activation of the ER stress-mediated apoptotic pathway. Therefore, we investigated here the molecular mechanisms underlying the ER-mitochondria axis, focusing on calcium as a potential mediator of cell death signals. Using NT2 cells treated with brefeldin A or tunicamycin, we observed that ER stress induces changes in the mitochondrial function, impairing mitochondrial membrane potential and distressing mitochondrial respiratory chain complex Moreover, stress stimuli at ER level evoked calcium fluxes between ER and mitochondria. Under these conditions, ER stress activated the unfolded protein response by an overexpression of GRP78, and also caspase-4 and-2, both involved upstream of caspase-9. Our findings show that ER and mitochondria interconnection plays a prominent role in the induction of neuronal cell death under particular stress circumstances.

  8. Arabidopsis thaliana Somatic Embryogenesis Receptor Kinase 1 protein is present in sporophytic and gametophytic cells and undergoes endocytosis

    DEFF Research Database (Denmark)

    Kwaaitaal, Mark Adrianus Cornelis J; de Vries, S C; Russinova, E

    2005-01-01

    Arabidopsis thaliana plants expressing AtSERK1 fused to yellow-fluorescent protein were generated. Fluorescence was detected predominantly at the cell periphery, most likely the plasma membrane, of cells in ovules, embryo sacs, anthers, and embryos and in seedlings. The AtSERK1 protein was detected...... in diverse cell types including the epidermis and the vascular bundles. In some cells, fluorescent receptors were seen in small vesicle-like compartments. After application of the fungal toxin Brefeldin A, the fluorescent receptors were rapidly internalized in the root meristem and root vascular tissue. We...... conclude that the AtSERK1 receptor functions in a common signalling pathway employed in both sporophytic and gametophytic cells....

  9. Fate of cerium dioxide nanoparticles in endothelial cells: exocytosis

    Energy Technology Data Exchange (ETDEWEB)

    Strobel, Claudia, E-mail: Claudia.Strobel@med.uni-jena.de [Jena University Hospital – Friedrich Schiller University Jena, Department of Experimental Radiology, Institute of Diagnostic and Interventional Radiology (Germany); Oehring, Hartmut [Jena University Hospital – Friedrich Schiller University Jena, Institute of Anatomy II (Germany); Herrmann, Rudolf [University of Augsburg, Department of Physics (Germany); Förster, Martin [Jena University Hospital – Friedrich Schiller University Jena, Department of Internal Medicine I, Division of Pulmonary Medicine and Allergy/Immunology (Germany); Reller, Armin [University of Augsburg, Department of Physics (Germany); Hilger, Ingrid, E-mail: ingrid.hilger@med.uni-jena.de [Jena University Hospital – Friedrich Schiller University Jena, Department of Experimental Radiology, Institute of Diagnostic and Interventional Radiology (Germany)

    2015-05-15

    Although cytotoxicity and endocytosis of nanoparticles have been the subject of numerous studies, investigations regarding exocytosis as an important mechanism to reduce intracellular nanoparticle accumulation are rather rare and there is a distinct lack of knowledge. The current study investigated the behavior of human microvascular endothelial cells to exocytose cerium dioxide (CeO{sub 2}) nanoparticles (18.8 nm) by utilization of specific inhibitors [brefeldin A; nocodazole; methyl-β-cyclodextrin (MβcD)] and different analytical methods (flow cytometry, transmission electron microscopy, inductively coupled plasma mass spectrometry). Overall, it was found that endothelial cells were able to release CeO{sub 2} nanoparticles via exocytosis after the migration of nanoparticle containing endosomes toward the plasma membrane. The exocytosis process occurred mainly by fusion of vesicular membranes with plasma membrane resulting in the discharge of vesicular content to extracellular environment. Nevertheless, it seems to be likely that nanoparticles present in the cytosol could leave the cells in a direct manner. MβcD treatment led to the strongest inhibition of the nanoparticle exocytosis indicating a significant role of the plasma membrane cholesterol content in the exocytosis process. Brefeldin A (inhibitor of Golgi-to-cell-surface-transport) caused a higher inhibitory effect on exocytosis than nocodazole (inhibitor of microtubules). Thus, the transfer from distal Golgi compartments to the cell surface influenced the exocytosis process of the CeO{sub 2} nanoparticles more than the microtubule-associated transport. In conclusion, endothelial cells, which came in contact with nanoparticles, e.g., after intravenously applied nano-based drugs, can regulate their intracellular nanoparticle amount, which is necessary to avoid adverse nanoparticle effects on cells.

  10. The management of house dust mite allergies

    NARCIS (Netherlands)

    Bronswijk, van J.E.M.H.; Schober, G.; Kniest, F.M.

    1990-01-01

    A safe and practical home sanitation procedure for the removal of house dust mites (Dermatophagoides pteronyssinus) and their allergens is described. The severity of mite infestation was assessed with the use of the Acarex test, which measures the concentration of guanine in house dust, and all

  11. Reversal of diet-induced obesity and insulin resistance by inducible genetic ablation of GRK2

    NARCIS (Netherlands)

    Vila-Bedmar, Rocio; Cruces-Sande, Marta; Lucas, Elisa; Willemen, Hanneke L D M; Heijnen, Cobi J; Kavelaars, Annemieke; Mayor, Federico; Murga, Cristina

    2015-01-01

    Insulin resistance is a common feature of obesity and predisposes individuals to various prevalent pathological conditions. G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor kinase 2 (GRK2) integrates several signal transduction pathways and is emerging as a

  12. Development of a Novel Fluorescence Assay Based on the Use of the Thrombin-Binding Aptamer for the Detection of O6-Alkylguanine-DNA Alkyltransferase Activity

    Directory of Open Access Journals (Sweden)

    Maria Tintoré

    2010-01-01

    Full Text Available Human O6-alkylguanine-DNA alkyltransferase (hAGT is a DNA repair protein that reverses the effects of alkylating agents by removing DNA adducts from the O6 position of guanine. Here, we developed a real-time fluorescence hAGT activity assay that is based on the detection of conformational changes of the thrombin-binding aptamer (TBA. The quadruplex structure of TBA is disrupted when a central guanine is replaced by an O6-methyl-guanine. The sequence also contains a fluorophore (fluorescein and a quencher (dabsyl attached to the opposite ends. In the unfolded structure, the fluorophore and the quencher are separated. When hAGT removes the methyl group from the central guanine of TBA, it folds back immediately into its quadruplex structure. Consequently, the fluorophore and the quencher come into close proximity, thereby resulting in decreased fluorescence intensity. Here, we developed a new method to quantify the hAGT without using radioactivity. This new fluorescence resonance energy transfer assay has been designed to detect the conformational change of TBA that is induced by the removal of the O6-methyl group.

  13. Digital PCR quantification of MGMT methylation refines prediction of clinical benefit from alkylating agents in glioblastoma and metastatic colorectal cancer

    NARCIS (Netherlands)

    Barault, L.; Amatu, A.; Bleeker, F. E.; Moutinho, C.; Falcomatà, C.; Fiano, V.; Cassingena, A.; Siravegna, G.; Milione, M.; Cassoni, P.; de Braud, F.; Rudà, R.; Soffietti, R.; Venesio, T.; Bardelli, A.; Wesseling, P.; de Witt Hamer, P.; Pietrantonio, F.; Siena, S.; Esteller, M.; Sartore-Bianchi, A.; Di Nicolantonio, F.

    2015-01-01

    O(6)-methyl-guanine-methyl-transferase (MGMT) silencing by promoter methylation may identify cancer patients responding to the alkylating agents dacarbazine or temozolomide. We evaluated the prognostic and predictive value of MGMT methylation testing both in tumor and cell-free circulating DNA

  14. Digital PCR quantification of MGMT methylation refines prediction of clinical benefit from alkylating agents in glioblastoma and metastatic colorectal cancer

    NARCIS (Netherlands)

    Barault, L.; Amatu, A.; Bleeker, F. E.; Moutinho, C.; Falcomatà, C.; Fiano, V.; Cassingena, A.; Siravegna, G.; Milione, M.; Cassoni, P.; de Braud, F.; Rudà, R.; Soffietti, R.; Venesio, T.; Bardelli, A.; Wesseling, P.; de Witt Hamer, P.; Pietrantonio, F.; Siena, S.; Esteller, M.; Sartore-Bianchi, A.; di Nicolantonio, Federica

    2015-01-01

    Background: O6-methyl-guanine-methyl-transferase (MGMT) silencing by promoter methylation may identify cancer patients responding to the alkylating agents dacarbazine or temozolomide. Patients and methods: We evaluated the prognostic and predictive value of MGMT methylation testing both in tumor and

  15. M3 muscarinic receptor interaction with phospholipase C beta3 determines its signaling efficiency

    NARCIS (Netherlands)

    Kan, W.; Adjobo-Hermans, M.J.; Burroughs, M.; Faibis, G.; Malik, S.; Tall, G.G.; Smrcka, A.V.

    2014-01-01

    Phospholipase Cbeta (PLCbeta) enzymes are activated by G protein-coupled receptors through receptor-catalyzed guanine nucleotide exchange on Galphabetagamma heterotrimers containing Gq family G proteins. Here we report evidence for a direct interaction between M3 muscarinic receptor (M3R) and

  16. Structure-function relationship of a plant NCS1 member - Homology modeling and mutagenesis identified residues critical for substrate specificity of PLUTO, a nucleobase transporter from arabidopsis

    KAUST Repository

    Witz, Sandra

    2014-03-12

    Plastidic uracil salvage is essential for plant growth and development. So far, PLUTO, the plastidic nucleobase transporter from Arabidopsis thaliana is the only known uracil importer at the inner plastidic membrane which represents the permeability barrier of this organelle. We present the first homology model of PLUTO, the sole plant NCS1 member from Arabidopsis based on the crystal structure of the benzyl hydantoin transporter MHP1 from Microbacterium liquefaciens and validated by molecular dynamics simulations. Polar side chains of residues Glu-227 and backbones of Val-145, Gly-147 and Thr-425 are proposed to form the binding site for the three PLUTO substrates uracil, adenine and guanine. Mutational analysis and competition studies identified Glu-227 as an important residue for uracil and to a lesser extent for guanine transport. A differential response in substrate transport was apparent with PLUTO double mutants E227Q G147Q and E227Q T425A, both of which most strongly affected adenine transport, and in V145A G147Q, which markedly affected guanine transport. These differences could be explained by docking studies, showing that uracil and guanine exhibit a similar binding mode whereas adenine binds deep into the catalytic pocket of PLUTO. Furthermore, competition studies confirmed these results. The present study defines the molecular determinants for PLUTO substrate binding and demonstrates key differences in structure-function relations between PLUTO and other NCS1 family members. 2014 Witz et al.

  17. CHAPTER 1

    African Journals Online (AJOL)

    Dr Olaleye

    form of natural DNA. .... either covalent or non-covalent bonding. In a covalent binding the reactive part of the compounds interacts with a nitrogen base of DNA such as guanine N7 e.g., ... electrostatic and groove binding of cationic metal.

  18. Cyclic AMP signalling in Dictyostelium : G-proteins activate separate Ras pathways using specific RasGEFs

    NARCIS (Netherlands)

    Kae, Helmut; Kortholt, Arjan; Rehmann, Holger; Insall, RobertH.; Van Haastert, Peter J. M.; Spiegelman, George B.; Weeks, Gerald

    In general, mammalian Ras guanine nucleotide exchange factors (RasGEFs) show little substrate specificity, although they are often thought to regulate specific pathways. Here, we provide in vitro and in vivo evidence that two RasGEFs can each act on specific Ras proteins. During Dictyostelium

  19. High-Resolution Scanning Tunneling Microscopy Studies of Molecular Nanostructures on Surfaces

    DEFF Research Database (Denmark)

    Song, Xin

    . First, to study the role of hydrogen bonding in self-assembly, we investigate the monomolecular self-assembled system of pyrene-4,5,9,10-tetrone and phenanthrene- 9,10-dione molecules on Au(111) and HOPG surface respectively and the binary molecular self-assembled system of stearic acid and guanine...

  20. Purine Bases in Blood Plasma of Patients with Chronic Pulmonary Diseases

    Directory of Open Access Journals (Sweden)

    Larissa E. Muravluyova

    2012-09-01

    Full Text Available The article is focused on the study of purine bases and intermediates of purine catabolism in plasma of patients with chronic obstructive bronchitis and idiopathic interstitial pneumonia. Decrease of adenine and hypoxantine in plasma of patients with idiopathic interstitial pneumonia was registered. Increase of guanine in plasma of patients with chronic obstructive pulmonary disease was established.

  1. Dicty_cDB: AFI568 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available nine... 72 8e-12 EU170368_1( EU170368 |pid:none) Dipolydora quadrilobata guanine nu... 72 8e-12 AM888287_1( AM888287 |pid:none) Sorda...ria macrospora partial gsa3 g... 72 1e-11 AF452097_1( AF

  2. Large normal-range TBP and ATXN7 CAG repeat lengths are associated with increased lifetime risk of depression

    DEFF Research Database (Denmark)

    Gardiner, S. L.; van Belzen, M. J.; Boogaard, M. W.

    2017-01-01

    Depression is one of the most prevalent and debilitating psychiatric disorders worldwide. Recently, we showed that both relatively short and relatively long cytosine–adenine–guanine (CAG) repeats in the huntingtin gene (HTT) are associated with an increased risk of lifetime depression. However, t...

  3. Sequence Classification: 893846 [

    Lifescience Database Archive (English)

    Full Text Available ds zinc, found both on membranes and in the cytosol; guanine nucleotide dissociation stimulator; Dss4p || http://www.ncbi.nlm.nih.gov/protein/6325274 ... ...tide release factor functioning in the post-Golgi secretory pathway, required for ER-to-Golgi transport, bin

  4. Sequence Classification: 785961 [

    Lifescience Database Archive (English)

    Full Text Available hways (157.5 kD) (aex-3) || http://www.ncbi.nlm.nih.gov/protein/25147747 ... ...ulsion defective AEX-3, Rab3 Guanine nucleotide Exchange Factor, regulator of presynaptic activity, regulates the rab-3 and cab-1 pat

  5. The focal adhesion-associated proteins DOCK5 and GIT2 comprise a rheostat in control of epithelial invasion

    DEFF Research Database (Denmark)

    Frank, Scott R; Köllmann, C P; van Lidth de Jeude, J F

    2017-01-01

    DOCK proteins are guanine nucleotide exchange factors for Rac and Cdc42 GTPases. DOCK1 is the founding member of the family and acts downstream of integrins via the canonical Crk-p130Cas complex to activate Rac GTPases in numerous contexts. In contrast, DOCK5, which possesses the greatest similar......:10.1038/onc.2016.345....

  6. Isolation of deletion alleles by G4 DNA-induced mutagenesis

    NARCIS (Netherlands)

    Pontier, Daphne B; Kruisselbrink, Evelien; Guryev, Victor; Tijsterman, Marcel

    Metazoan genomes contain thousands of sequence tracts that match the guanine-quadruplex (G4) DNA signature G(3)N(x)G(3)N(x)G(3)N(x)G(3), a motif that is intrinsically mutagenic, probably because it can form secondary structures during DNA replication. Here we show how and to what extent this feature

  7. Oxidative stress and dopamine deficiency in a genetic mouse model of Lesch-Nyhan disease.

    NARCIS (Netherlands)

    Visser, J.E.; Smith, D.W.; Moy, S.S.; Breese, G.R.; Friedmann, T.; Rothstein, J.D.; Jinnah, H.A.

    2002-01-01

    Lesch-Nyhan disease, a neurogenetic disorder caused by congenital deficiency of the purine salvage enzyme hypoxanthine guanine phosphoribosyl transferase, is associated with a prominent loss of striatal dopamine. The current studies address the hypothesis that oxidant stress causes damage or

  8. Signaling efficiency of Galphaq through its effectors p63RhoGEF and GEFT depends on their subcellular location

    NARCIS (Netherlands)

    Goedhart, J.; Unen, J. van; Adjobo-Hermans, M.J.W.; Gadella, T.W.

    2013-01-01

    The p63RhoGEF and GEFT proteins are encoded by the same gene and both members of the Dbl family of guanine nucleotide exchange factors. These proteins can be activated by the heterotrimeric G-protein subunit Galphaq. We show that p63RhoGEF is located at the plasma membrane, whereas GEFT is confined

  9. Signaling efficiency of Gαq through its effectors p63RhoGEF and GEFT depends on their subcellular location.

    NARCIS (Netherlands)

    Goedhart, J.; van Unen, J.; Adjobo-Hermans, M.J.W.; Gadella (jr.), T.W.J.

    2013-01-01

    The p63RhoGEF and GEFT proteins are encoded by the same gene and both members of the Dbl family of guanine nucleotide exchange factors. These proteins can be activated by the heterotrimeric G-protein subunit Galphaq. We show that p63RhoGEF is located at the plasma membrane, whereas GEFT is confined

  10. Monitoring of herpes simplex virus thymidine kinase enzyme activity using positron emission tomography

    NARCIS (Netherlands)

    Hospers, GAP; Calogero, Anna; van Waarde, A; Doze, P; Vaalburg, W; Mulder, NH; de Vries, EFJ

    2000-01-01

    9-[(1-[F-18]Fluoro-3-hydroxy-2-propoxy)methyl]guanine ([F-18]FHPG) wasevaluated as a tracer for noninvasive positron emission tomography (PET) imaging of herpes simplex virus type 1 thymidine kinase (HSV-tk) gene expression. C6 rat glioma cells with and without the HSV-tk gene were incubated with

  11. [C-11]FMAU and [F-18]FHPG as PET tracers for herpes simplex virus thymidine kinase enzyme activity and human cytomegalovirus infections

    NARCIS (Netherlands)

    de Vries, EFJ; van Waarde, A; Harmsen, MC; Mulder, NH; Vaalburg, W; Hospers, GAP

    [C-11]-2'-Fluoro-5-methyl-1-beta-D-arabinofuranosyluracil ([C-11]FMAU) and [F-18]-9-[(3-fluoro-1-hydroxy-2-propoxy)methyl]guanine ([F-18]FHPG), radiolabeled representatives of two classes of antiviral agents, were evaluated as tracers for measuring herpes simplex virus thymidine kinase (HSV-tk)

  12. Response to Comment on "Positive Selection of Tyrosine Loss in Metazoan Evolution"

    DEFF Research Database (Denmark)

    Tan, Chris Soon Heng; Schoof, Erwin; Creixell, Pau

    2011-01-01

    Su et al. claim guanine-cytosine (GC) content variation can largely explain the observed tyrosine frequency variation, independent of adaptive evolution of cell-signaling complexity. We found that GC content variation, in the absence of selection for amino acid changes, can only maximally account...

  13. Comparison on the interaction of Al3+/nano-Al13 with calf thymus DNA /salmon sperm DNA

    Science.gov (United States)

    Ma, Fei; Ma, Yue; Du, Changwen; Yang, Xiaodi; Shen, Renfang

    2015-11-01

    The conformation change, binding mode and binding site between Al3+/nano-Al13 and calf thymus DNA/salmon sperm DNA were investigated by UV-vis absorption, FTIR spectra, Raman spectroscopy and CD spectra, as well as melting curves measurement. The UV-vis spectra and circular dichroism spectra results suggested that the phosphate group structure was changed when Al3+ interacted with DNA, while the double-helix was distorted when nano-Al13 interacted with DNA. The FTIR and Raman spectroscopy revealed that the binding sites were Al3+ … PO2, Al3+ … N7/guanine PO2 … Al13 … N7-C8/guanine with calf thymus DNA, and Al3+ … N3-O2/cytosine, Al3+ … N7-C8/guanine, PO2 … Al13 … N7-C8/guanine, PO2 … Al13 … N1/adenine with salmon sperm DNA, respectively. The electrostatic binding was existed between Al3+ and DNA, and the electrostatic binding and complexing were found between nano-Al13 and DNA.

  14. Unusual hydrogen bonding patterns in AF [aminofluorene] and AAF [acetylaminofluorene] modified DNA

    International Nuclear Information System (INIS)

    Broyde, S.; Hingerty, B.E.; Shapiro, R.; Norman, D.; Oak Ridge National Lab., TN; New York Univ., NY; Columbia Univ., New York, NY

    1989-01-01

    New structures are presented for AF and AAF modified DNAs that place the carcinogen in the minor groove of a B-DNA helix. These structures employ non-Watson-Crick base pairing schemes with syn guanine at the modification site. 32 refs., 9 figs

  15. The Juxtamembrane and carboxy-terminal domains of Arabidopsis PRK2 are critical for ROP-induced growth in pollen tubes

    Science.gov (United States)

    Polarized growth of pollen tubes is a critical step for successful reproduction in angiosperms and is controlled by ROP GTPases. Spatiotemporal activation of ROP (Rho GTPases of plants) necessitates a complex and sophisticated regulatory system, in which guanine nucleotide exchange factors (RopGEFs)...

  16. Hydrogen Bonding in DNA Base Pairs: Reconciliation of Theory and Experiment

    NARCIS (Netherlands)

    Fonseca Guerra, C.; Bickelhaupt, F.M.; Snijders, J.G.; Baerends, E.J.

    2000-01-01

    Up till now, there has been a significant disagreement between theory and experiment regarding hydrogen bond lengths in Watson - Crick base pairs. To investigate the possible sources of this discrepancy, we have studied numerous model systems for adenine - thymine (AT) and guanine - cytosine (GC)

  17. Spectroscopic insights into quadruplexes of five-repeat telomere DNA sequences upon G-block damage

    Czech Academy of Sciences Publication Activity Database

    Dvořáková, Zuzana; Vorlíčková, Michaela; Renčiuk, Daniel

    2017-01-01

    Roč. 1861, č. 11 (2017), s. 2750-2757 ISSN 0304-4165 R&D Projects: GA ČR(CZ) GJ17-19170Y Institutional support: RVO:68081707 Keywords : k+ solution * guanine quadruplexes Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 4.702, year: 2016

  18. GPCR engineering yields high-resolution structural insights into beta2-adrenergic receptor function

    DEFF Research Database (Denmark)

    Rosenbaum, Daniel M; Cherezov, Vadim; Hanson, Michael A

    2007-01-01

    The beta2-adrenergic receptor (beta2AR) is a well-studied prototype for heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) that respond to diffusible hormones and neurotransmitters. To overcome the structural flexibility of the beta2AR and to facilitate its cr...

  19. High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor

    DEFF Research Database (Denmark)

    Cherezov, Vadim; Rosenbaum, Daniel M; Hanson, Michael A

    2007-01-01

    Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors constitute the largest family of eukaryotic signal transduction proteins that communicate across the membrane. We report the crystal structure of a human beta2-adrenergic receptor-T4 lysozyme fusion protein bound to t...

  20. Mechanism Underlying the Nucleobase-Distinguishing Ability of Benzopyridopyrimidine (BPP).

    Science.gov (United States)

    Kochman, Michał A; Bil, Andrzej; Miller, R J Dwayne

    2017-11-02

    Benzopyridopyrimidine (BPP) is a fluorescent nucleobase analogue capable of forming base pairs with adenine (A) and guanine (G) at different sites. When incorporated into oligodeoxynucleotides, it is capable of differentiating between the two purine nucleobases by virtue of the fact that its fluorescence is largely quenched when it is base-paired to guanine, whereas base-pairing to adenine causes only a slight reduction of the fluorescence quantum yield. In the present article, the photophysics of BPP is investigated through computer simulations. BPP is found to be a good charge acceptor, as demonstrated by its positive and appreciably large electron affinity. The selective quenching process is attributed to charge transfer (CT) from the purine nucleobase, which is predicted to be efficient in the BPP-G base pair, but essentially inoperative in the BPP-A base pair. The CT process owes its high selectivity to a combination of two factors: the ionization potential of guanine is lower than that of adenine, and less obviously, the site occupied by guanine enables a greater stabilization of the CT state through electrostatic interactions than the one occupied by adenine. The case of BPP illustrates that molecular recognition via hydrogen bonding can enhance the selectivity of photoinduced CT processes.

  1. Inhibition of the Escherichia coli 6-oxopurine phosphoribosyltransferases by nucleoside phosphonates: potential for new antibacterial agents

    Czech Academy of Sciences Publication Activity Database

    Keough, D. T.; Hocková, Dana; Rejman, Dominik; Špaček, Petr; Vrbková, Silvie; Krečmerová, Marcela; Eng, W. S.; Jans, H.; West, N. P.; Naesens, L. M. J.; de Jersey, J.; Guddat, L. W.

    2013-01-01

    Roč. 56, č. 17 (2013), s. 6967-6984 ISSN 0022-2623 R&D Projects: GA ČR GAP207/11/0108 Institutional support: RVO:61388963 Keywords : nucleoside phosphonates * antibacterial agents * hypoxanthine-guanine phosphoribosyltransferase * state analog inhibitor * antimalarial chemotherapy Subject RIV: CC - Organic Chemistry Impact factor: 5.480, year: 2013

  2. Progressive osseous heteroplasia (POH): an Egyptian patient | El ...

    African Journals Online (AJOL)

    Progressive osseous heteroplasia is a rare genetic disorder characterized by cutaneous ossification during infancy and progressive ossification of subcutaneous and deep connective tissue including muscle and fascia during childhood. It is at the severe end of a spectrum of Guanine Nucleotide-binding protein, ...

  3. The Rac1 hypervariable region in targeting and signaling: a tail of many stories

    NARCIS (Netherlands)

    Lam, B. Daniel; Hordijk, Peter L.

    2013-01-01

    Cellular signaling by small GTPases is critically dependent on proper spatio-temporal orchestration of activation and output. In addition to their core G (guanine nucleotide binding)-domain, small GTPases comprise a hypervariable region (HVR) and a lipid anchor that are generally accepted to control

  4. O-6-benzylguanine potentiates BCNU but not busulfan toxicity in hematopoietic stem cells

    NARCIS (Netherlands)

    Westerhof, GR; Down, JD; Blokland, [No Value; Wood, M; Boudewijn, A; Watson, AJ; McGown, AT; Ploemacher, RE; Margison, GP

    Objective. Busulfan (BU) is often used in conditioning regimens prior to bone marrow transplantation, but its mechanism of action remains to be resolved. We have examined the possibility that BU may exert part of its toxic effects via DNA alkylation at the Oh position of guanine as this might

  5. Site-directed mutagenesis of Arg58 and Asp86 of elongation factor Tu from Escherichia coli: effects on the GTPase reaction and aminoacyl-tRNA binding

    DEFF Research Database (Denmark)

    Knudsen, Charlotte Rohde; Clark, Brian F. C.

    1996-01-01

    Elongation factor Tu from Escherichia coli was mutated separately at positions Asp86 and Arg58, in order to shed light both on the GTPase mechanism of elongation factor Tu and on the binding of aminoacyl-tRNA. In addition, the binding of guanine nucleotides was investigated by determination...

  6. Loss of GPR3 reduces the amyloid plaque burden and improves memory in Alzheimer's disease mouse models

    NARCIS (Netherlands)

    Huang, Yunhong; Skwarek-Maruszewska, Aneta; Horré, Katrien; Vandewyer, Elke; Wolfs, Leen; Snellinx, An; Saito, Takashi; Radaelli, Enrico; Corthout, Nikky; Colombelli, Julien; Lo, Adrian C; Van Aerschot, Leen; Callaerts-Vegh, Zsuzsanna; Trabzuni, Daniah; Bossers, Koen; Verhaagen, Joost; Ryten, Mina; Munck, Sebastian; D'Hooge, Rudi; Swaab, Dick F; Hardy, John; Saido, Takaomi C; De Strooper, Bart; Thathiah, Amantha

    2015-01-01

    The orphan G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR) GPR3 regulates activity of the γ-secretase complex in the absence of an effect on Notch proteolysis, providing a potential therapeutic target for Alzheimer's disease (AD). However, given the vast

  7. Effect of Chimaerins, Novel Receptors for Phorbol Esters, on Breast Cancer Cell Proliferation and Cell Cycle Progression

    Science.gov (United States)

    2006-07-01

    Microbiology . All Rights Reserved. Essential Role for Rac in Heregulin 1 Mitogenic Signaling: a Mechanism That Involves Epidermal Growth Factor Receptor... Prescott , A. Gray, G. S. Kular, H. Stewart, and C. P. Downes. 2004. Inositol phospholipids regulate the guanine-nucle- otide-exchange factor Tiam1

  8. Epac : effectors and biological functions

    NARCIS (Netherlands)

    Roscioni, Sara S.; Elzinga, Carolina R. S.; Schmidt, Martina

    Epac1 (also known as cAMP-GEF-I) and Epac2 (also known as cAMP-GEF-II) are cyclic AMP-activated guanine nucleotide exchange factors for Ras-like GTPases. Since their discovery about 10 years ago, it is now accepted that Epac proteins are novel cAMP sensors that regulate several pivotal cellular

  9. Attenuated Variants of Lesch-Nyhan Disease

    Science.gov (United States)

    Jinnah, H. A.; Ceballos-Picot, Irene; Torres, Rosa J.; Visser, Jasper E.; Schretlen, David J.; Verdu, Alfonso; Larovere, Laura E.; Chen, Chung-Jen; Cossu, Antonello; Wu, Chien-Hui; Sampat, Radhika; Chang, Shun-Jen; de Kremer, Raquel Dodelson; Nyhan, William; Harris, James C.; Reich, Stephen G.; Puig, Juan G.

    2010-01-01

    Lesch-Nyhan disease is a neurogenetic disorder caused by deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase. The classic form of the disease is described by a characteristic syndrome that includes overproduction of uric acid, severe generalized dystonia, cognitive disability and self-injurious behaviour. In addition to the…

  10. Synthesis and spectroscopy of clay intercalated Cu(II) bio-monomer complexes: coordination of Cu(II) with purines and nucleotides

    NARCIS (Netherlands)

    Weckhuysen, B.M.; Leeman, H.; Schoonheydt, R.A.

    1999-01-01

    The spectroscopic properties of Cu(bio-monomer)nm+ complexes [BM=bio-monomer (purine, adenine, guanine, hypoxanthine, 5-ADP and 5-GMP)] in saponite clays have been investigated by diffuse reflectance spectroscopy (DRS) in the UV-Vis-NIR region and electron paramagnetic resonance (EPR) at X-band.

  11. GenBank blastx search result: AK240924 [KOME

    Lifescience Database Archive (English)

    Full Text Available 1810012H11Rik and D130071N09), the Rpl26 gene for ribosomal protein L26, gene Oppo1-pending... (oppo 1), gene RAN guanine nucleotide release factor (Rangnrf-pending) and three CpG islands, complete sequence. ROD 7e-20 1 ...

  12. GenBank blastx search result: AK108325 [KOME

    Lifescience Database Archive (English)

    Full Text Available H11Rik and D130071N09), the Rpl26 gene for ribosomal protein L26, gene Oppo1-pending... (oppo 1), gene RAN guanine nucleotide release factor (Rangnrf-pending) and three CpG islands, complete sequence.|ROD ROD 5e-34 +3 ...

  13. GenBank blastx search result: AK058868 [KOME

    Lifescience Database Archive (English)

    Full Text Available H11Rik and D130071N09), the Rpl26 gene for ribosomal protein L26, gene Oppo1-pending... (oppo 1), gene RAN guanine nucleotide release factor (Rangnrf-pending) and three CpG islands, complete sequence.|ROD ROD 2e-18 +2 ...

  14. GenBank blastx search result: AK058511 [KOME

    Lifescience Database Archive (English)

    Full Text Available H11Rik and D130071N09), the Rpl26 gene for ribosomal protein L26, gene Oppo1-pending... (oppo 1), gene RAN guanine nucleotide release factor (Rangnrf-pending) and three CpG islands, complete sequence.|ROD ROD 3e-17 +2 ...

  15. GenBank blastx search result: AK104090 [KOME

    Lifescience Database Archive (English)

    Full Text Available H11Rik and D130071N09), the Rpl26 gene for ribosomal protein L26, gene Oppo1-pending... (oppo 1), gene RAN guanine nucleotide release factor (Rangnrf-pending) and three CpG islands, complete sequence.|ROD ROD 6e-16 +2 ...

  16. GenBank blastx search result: AK062017 [KOME

    Lifescience Database Archive (English)

    Full Text Available H11Rik and D130071N09), the Rpl26 gene for ribosomal protein L26, gene Oppo1-pending... (oppo 1), gene RAN guanine nucleotide release factor (Rangnrf-pending) and three CpG islands, complete sequence.|ROD ROD 1e-10 +1 ...

  17. GenBank blastx search result: AK105075 [KOME

    Lifescience Database Archive (English)

    Full Text Available H11Rik and D130071N09), the Rpl26 gene for ribosomal protein L26, gene Oppo1-pending... (oppo 1), gene RAN guanine nucleotide release factor (Rangnrf-pending) and three CpG islands, complete sequence.|ROD ROD 3e-38 +3 ...

  18. GenBank blastx search result: AK058591 [KOME

    Lifescience Database Archive (English)

    Full Text Available H11Rik and D130071N09), the Rpl26 gene for ribosomal protein L26, gene Oppo1-pending... (oppo 1), gene RAN guanine nucleotide release factor (Rangnrf-pending) and three CpG islands, complete sequence.|ROD ROD 2e-67 +2 ...

  19. GenBank blastx search result: AK061954 [KOME

    Lifescience Database Archive (English)

    Full Text Available H11Rik and D130071N09), the Rpl26 gene for ribosomal protein L26, gene Oppo1-pending... (oppo 1), gene RAN guanine nucleotide release factor (Rangnrf-pending) and three CpG islands, complete sequence.|ROD ROD 3e-12 +1 ...

  20. GenBank blastx search result: AK109916 [KOME

    Lifescience Database Archive (English)

    Full Text Available H11Rik and D130071N09), the Rpl26 gene for ribosomal protein L26, gene Oppo1-pending... (oppo 1), gene RAN guanine nucleotide release factor (Rangnrf-pending) and three CpG islands, complete sequence.|ROD ROD 2e-34 +3 ...

  1. GenBank blastx search result: AK059612 [KOME

    Lifescience Database Archive (English)

    Full Text Available H11Rik and D130071N09), the Rpl26 gene for ribosomal protein L26, gene Oppo1-pending... (oppo 1), gene RAN guanine nucleotide release factor (Rangnrf-pending) and three CpG islands, complete sequence.|ROD ROD 4e-18 +2 ...

  2. GenBank blastx search result: AK103990 [KOME

    Lifescience Database Archive (English)

    Full Text Available H11Rik and D130071N09), the Rpl26 gene for ribosomal protein L26, gene Oppo1-pending... (oppo 1), gene RAN guanine nucleotide release factor (Rangnrf-pending) and three CpG islands, complete sequence.|ROD ROD 5e-11 +3 ...

  3. Nye molekylaere markører ved de kroniske myeloproliferative sygdomme

    DEFF Research Database (Denmark)

    Larsen, Thomas Stauffer; Pallisgaard, Niels; Christensen, Jacob Haaber

    2006-01-01

    The Philadelphia-negative chronic myeloproliferative disorders feature autonomous myeloid hyperproliferation and hypersensitivity to a number of growth factors, which most recently have been shown to be explained by a guanine-to-thymidine mutation in the Janus tyrosine kinase (JAK2) gene, implica...

  4. Formation of covalent complexes between human O sup 6 -alkylguanine-DNA alkyltransferase and BCNU-treated defined length synthetic oligodeoxynucleotides

    Energy Technology Data Exchange (ETDEWEB)

    Brent, T.P.; Remack, J.S. (St. Jude Children' s Research Hospital, Memphis, TN (USA))

    1988-07-25

    Repair of chloroethylnitrosourea (CENU)-induced precursors of DNA interstrand cross-links by O{sup 6}-alkylguanine-DNA alkyltransferase (GAT or GATase) appears to be a factor in tumor resistance to therapy with this class of antineoplastic drugs. Since human GAT is highly specific for O{sup 6}-guanine, yet the probably cross-link structure is N{prime}-Guanine N{sup 3}cytosine ethane, rearrangement of the initial O{sup 6}-guanine adduct via O{sup 6},N{sup 1}ethanoguanine has been proposed. The authors suggested that GAT reaction with this intermediate would produce DNA covalently linked to protein through an ethane link from N{sup 1}-guanine to the alkylacceptor site on GAT. In preliminary studies they demonstrated a covalent complex between GAT and carmustine (BCNU)-treated DNA by a precipitation assay method. They have now developed a method for isolating the reaction product of BCNU-treated synthetic 14-mer ({sup 32}P)-labeled oligodeoxynucleotide and GAT using polyacrylamide gel electrophoresis. This approach can be used to characterize the adducts induced by CENUs that lead to complex formation with GAT.

  5. Kinetics of hydrogen-deuterium exchange in guanosine 5'-monophosphate and guanosine 3':5'-monophosphate determined by laser-Raman spectroscopy.

    Science.gov (United States)

    Lane, M J; Thomas, G J

    1979-09-04

    Pseudo-first-order rate constants governing the deuterium exchange of 8-CH groups in guanosine 5'-monophosphate (5'-rGMP) and guanosine 3':5'-monophosphate (cGMP) were determined as a function of temperature in the range 30-80 degrees C by means of laser-Raman spectroscopy. For each guanine nucleotide the logarithm of the rate constant exhibits a strictly linear dependence on reciprocal temperature: i.e., k psi = Ae-Ea/RT with A = 8.84 X 10(14) h-1 and Ea = 24.6 kcal/mol for 5'-rGMP and A = 3.33 X 10(13) h-1 and Ea = 22.2 kcal/mol for cGMP. Exchange of the 8-CH groups in guanine nucleotides is generally 2-3 times more rapid than in adenine nucleotides [cf. g. j. thomas, Jr., & J. Livramento (1975) Biochemistry 14, 5210-5218]. As in the case of adenine nucleotides, cyclic and 5' nucleotides of guanine exchange at markedly different rates at lower temperatures, with exchange in the cyclic nucleotide being the more facile. Each of the guanine nucleotides was prepared in four different isotopic modifications for Raman spectral analysis. The Raman frequency shifts resulting from the various isotopic substitutions have been tabulated, and assignments have been given for most of the observed vibrational frequencies.

  6. MECP2 Is a Frequently Amplified Oncogene with a Novel Epigenetic Mechanism That Mimics the Role of Activated RAS in Malignancy

    DEFF Research Database (Denmark)

    Neupane, Manish; Clark, Allison P.; Landini, Serena

    2016-01-01

    An unbiased genome-scale screen for unmutated genes that drive cancer growth when overexpressed identified methyl cytosine-guanine dinucleotide (CpG) binding protein 2 (MECP2) as a novel oncogene. MECP2 resides in a region of the X-chromosome that is significantly amplified across 18% of cancers,...

  7. Novel guanidine-based inhibitors of inosine monophosphate dehydrogenase.

    Science.gov (United States)

    Iwanowicz, Edwin J; Watterson, Scott H; Liu, Chunjian; Gu, Henry H; Mitt, Toomas; Leftheris, Katerina; Barrish, Joel C; Fleener, Catherine A; Rouleau, Katherine; Sherbina, N Z; Hollenbaugh, Diane L

    2002-10-21

    A series of novel guanidine-based small molecule inhibitors of inosine monophosphate dehydrogenase (IMPDH) was explored. IMPDH catalyzes the rate determining step in guanine nucleotide biosynthesis and is a target for anticancer, immunosuppressive and antiviral therapy. The synthesis and the structure-activity relationships (SARs), derived from in vitro studies, for this new series of inhibitors is given.

  8. 3-cyanoindole-based inhibitors of inosine monophosphate dehydrogenase: synthesis and initial structure-activity relationships.

    Science.gov (United States)

    Dhar, T G Murali; Shen, Zhongqi; Gu, Henry H; Chen, Ping; Norris, Derek; Watterson, Scott H; Ballentine, Shelley K; Fleener, Catherine A; Rouleau, Katherine A; Barrish, Joel C; Townsend, Robert; Hollenbaugh, Diane L; Iwanowicz, Edwin J

    2003-10-20

    A series of novel small molecule inhibitors of inosine monophosphate dehydrogenase (IMPDH), based upon a 3-cyanoindole core, were explored. IMPDH catalyzes the rate determining step in guanine nucleotide biosynthesis and is a target for anticancer, immunosuppressive and antiviral therapy. The synthesis and the structure-activity relationships (SAR), derived from in vitro studies, for this new series of inhibitors is given.

  9. The Rac-RhoGDI complex and the structural basis for the regulation of Rho proteins by RhoGDI

    DEFF Research Database (Denmark)

    Scheffzek, K; Stephan, I; Jensen, Ole Nørregaard

    2000-01-01

    Rho family-specific guanine nucleotide dissociation inhibitors (RhoGDIs) decrease the rate of nucleotide dissociation and release Rho proteins such as RhoA, Rac and Cdc42 from membranes, forming tight complexes that shuttle between cytosol and membrane compartments. We have solved the crystal...

  10. 6-Oxopurine Phosphoribosyltransferase: A Target for the Development of Antimalarial Drugs

    Czech Academy of Sciences Publication Activity Database

    de Jersey, J.; Holý, Antonín; Hocková, Dana; Naesens, L.; Keough, D. T.; Guddat, L. W.

    2011-01-01

    Roč. 11, č. 16 (2011), s. 2085-2102 ISSN 1568-0266 R&D Projects: GA MŠk 1M0508 Institutional research plan: CEZ:AV0Z40550506 Keywords : Malaria * acyclic nucleoside phosphonates * hypoxanthine * guanine phosphoribosyl transferase Subject RIV: CC - Organic Chemistry Impact factor: 4.174, year: 2011

  11. Positive Relationship between Abdominal Coloration and Dermal Melanin Density in Phrynosomatid Lizards

    Science.gov (United States)

    Vanessa S. Quinn; Diana K. Hews

    2003-01-01

    Phrynosomatid lizards show considerable variation among species in the occurrence of a secondary sexual trait, blue abdominal coloration. The production of blue skin may be controlled by at least two cellular components, melanin in melanophores, and guanine in iridophores. To examine the hypothesis that a mechanism producing variation in abdominal coloration is...

  12. Characterization of genetic miscoding lesions caused by postmortem damage

    DEFF Research Database (Denmark)

    Gilbert, M Thomas P; Hansen, Anders J; Willerslev, Eske

    2002-01-01

    The spectrum of postmortem damage in mitochondrial DNA was analyzed in a large data set of cloned sequences from ancient human specimens. The most common forms of damage observed are two complementary groups of transitions, termed "type 1" (adenine-->guanine/thymine-->cytosine) and "type 2...

  13. Huntingtin gene repeat size variations affect risk of lifetime depression

    DEFF Research Database (Denmark)

    Gardiner, Sarah L.; van Belzen, Martine J.; Boogaard, Merel W.

    2017-01-01

    Huntington disease (HD) is a severe neuropsychiatric disorder caused by a cytosine-adenine-guanine (CAG) repeat expansion in the HTT gene. Although HD is frequently complicated by depression, it is still unknown to what extent common HTT CAG repeat size variations in the normal range could affect...

  14. Detection and interpretation of 8-oxodG and 8-oxoGua in urine, plasma and cerebrospinal fluid

    DEFF Research Database (Denmark)

    Poulsen, Henrik E; Nadal, Laia Llovera; Broedbaek, Kasper

    2014-01-01

    DNA and RNA oxidations have been linked to diseases such as cancer, arteriosclerosis, neurodegeneration and diabetes. The prototype base modification studied is the 8-hydroxylation of guanine. DNA integrity is maintained by elaborate repair systems and RNA integrity is less studied but relies mai...

  15. Voltammetry and Molecular Assembly of G-quadruplex DNAzyme on Single-crystal Au(111)-electrode Surfaces – Hemin as an Electrochemical Intercalator

    DEFF Research Database (Denmark)

    Zhang, Ling; Ulstrup, Jens; Zhang, Jingdong

    2016-01-01

    DNA quadruplexes (qs’s) are a class of “non-canonical” oligonucleotides (OGNs) composed of stacked guanine (G) quartets generally stabilized by monovalent cations. Metal porphyrins selectively bind to G-qs complexes to form DNAzyme, which can exhibit peroxidase and other catalytic activity simila...

  16. LRRK2 kinase activity is dependent on LRRK2 GTP binding capacity but independent of LRRK2 GTP binding.

    Directory of Open Access Journals (Sweden)

    Jean-Marc Taymans

    Full Text Available Leucine rich repeat kinase 2 (LRRK2 is a Parkinson's disease (PD gene that encodes a large multidomain protein including both a GTPase and a kinase domain. GTPases often regulate kinases within signal transduction cascades, where GTPases act as molecular switches cycling between a GTP bound "on" state and a GDP bound "off" state. It has been proposed that LRRK2 kinase activity may be increased upon GTP binding at the LRRK2 Ras of complex proteins (ROC GTPase domain. Here we extensively test this hypothesis by measuring LRRK2 phosphorylation activity under influence of GDP, GTP or non-hydrolyzable GTP analogues GTPγS or GMPPCP. We show that autophosphorylation and lrrktide phosphorylation activity of recombinant LRRK2 protein is unaltered by guanine nucleotides, when co-incubated with LRRK2 during phosphorylation reactions. Also phosphorylation activity of LRRK2 is unchanged when the LRRK2 guanine nucleotide binding pocket is previously saturated with various nucleotides, in contrast to the greatly reduced activity measured for the guanine nucleotide binding site mutant T1348N. Interestingly, when nucleotides were incubated with cell lysates prior to purification of LRRK2, kinase activity was slightly enhanced by GTPγS or GMPPCP compared to GDP, pointing to an upstream guanine nucleotide binding protein that may activate LRRK2 in a GTP-dependent manner. Using metabolic labeling, we also found that cellular phosphorylation of LRRK2 was not significantly modulated by nucleotides, although labeling is significantly reduced by guanine nucleotide binding site mutants. We conclude that while kinase activity of LRRK2 requires an intact ROC-GTPase domain, it is independent of GDP or GTP binding to ROC.

  17. Fluorescent quenching-based quantitative detection of specific DNA/RNA using a BODIPY® FL-labeled probe or primer

    Science.gov (United States)

    Kurata, Shinya; Kanagawa, Takahiro; Yamada, Kazutaka; Torimura, Masaki; Yokomaku, Toyokazu; Kamagata, Yoichi; Kurane, Ryuichiro

    2001-01-01

    We have developed a simple method for the quantitative detection of specific DNA or RNA molecules based on the finding that BODIPY® FL fluorescence was quenched by its interaction with a uniquely positioned guanine. This approach makes use of an oligonucleotide probe or primer containing a BODIPY® FL-modified cytosine at its 5′-end. When such a probe was hybridized with a target DNA, its fluorescence was quenched by the guanine in the target, complementary to the modified cytosine, and the quench rate was proportional to the amount of target DNA. This widely applicable technique will be used directly with larger samples or in conjunction with the polymerase chain reaction to quantify small DNA samples. PMID:11239011

  18. Investigating the Fundamentals of Molecular Depth Profiling Using Strong-field Photoionization of Sputtered Neutrals

    Science.gov (United States)

    Willingham, D.; Brenes, D. A.; Winograd, N.; Wucher, A.

    2010-01-01

    Molecular depth profiles of model organic thin films were performed using a 40 keV C60+ cluster ion source in concert with TOF-SIMS. Strong-field photoionization of intact neutral molecules sputtered by 40 keV C60+ primary ions was used to analyze changes in the chemical environment of the guanine thin films as a function of ion fluence. Direct comparison of the secondary ion and neutral components of the molecular depth profiles yields valuable information about chemical damage accumulation as well as changes in the molecular ionization probability. An analytical protocol based on the erosion dynamics model is developed and evaluated using guanine and trehalose molecular secondary ion signals with and without comparable laser photoionization data. PMID:26269660

  19. AMPD2 regulates GTP synthesis and is mutated in a potentially treatable neurodegenerative brainstem disorder.

    Science.gov (United States)

    Akizu, Naiara; Cantagrel, Vincent; Schroth, Jana; Cai, Na; Vaux, Keith; McCloskey, Douglas; Naviaux, Robert K; Van Vleet, Jeremy; Fenstermaker, Ali G; Silhavy, Jennifer L; Scheliga, Judith S; Toyama, Keiko; Morisaki, Hiroko; Sonmez, Fatma M; Celep, Figen; Oraby, Azza; Zaki, Maha S; Al-Baradie, Raidah; Faqeih, Eissa A; Saleh, Mohammed A M; Spencer, Emily; Rosti, Rasim Ozgur; Scott, Eric; Nickerson, Elizabeth; Gabriel, Stacey; Morisaki, Takayuki; Holmes, Edward W; Gleeson, Joseph G

    2013-08-01

    Purine biosynthesis and metabolism, conserved in all living organisms, is essential for cellular energy homeostasis and nucleic acid synthesis. The de novo synthesis of purine precursors is under tight negative feedback regulation mediated by adenosine and guanine nucleotides. We describe a distinct early-onset neurodegenerative condition resulting from mutations in the adenosine monophosphate deaminase 2 gene (AMPD2). Patients have characteristic brain imaging features of pontocerebellar hypoplasia (PCH) due to loss of brainstem and cerebellar parenchyma. We found that AMPD2 plays an evolutionary conserved role in the maintenance of cellular guanine nucleotide pools by regulating the feedback inhibition of adenosine derivatives on de novo purine synthesis. AMPD2 deficiency results in defective GTP-dependent initiation of protein translation, which can be rescued by administration of purine precursors. These data suggest AMPD2-related PCH as a potentially treatable early-onset neurodegenerative disease. Copyright © 2013 Elsevier Inc. All rights reserved.

  20. AMPD2 Regulates GTP Synthesis and is Mutated in a Potentially-Treatable Neurodegenerative Brainstem Disorder

    Science.gov (United States)

    Akizu, Naiara; Cantagrel, Vincent; Schroth, Jana; Cai, Na; Vaux, Keith; McCloskey, Douglas; Naviaux, Robert K.; Vleet, Jeremy Van; Fenstermaker, Ali G.; Silhavy, Jennifer L.; Scheliga, Judith S.; Toyama, Keiko; Morisaki, Hiroko; Sonmez, Fatma Mujgan; Celep, Figen; Oraby, Azza; Zaki, Maha S.; Al-Baradie, Raidah; Faqeih, Eissa; Saleh, Mohammad; Spencer, Emily; Rosti, Rasim Ozgur; Scott, Eric; Nickerson, Elizabeth; Gabriel, Stacey; Morisaki, Takayuki; Holmes, Edward W.; Gleeson, Joseph G.

    2013-01-01

    Purine biosynthesis and metabolism, conserved in all living organisms, is essential for cellular energy homeostasis and nucleic acids synthesis. The de novo synthesis of purine precursors is under tight negative feedback regulation mediated by adenosine and guanine nucleotides. We describe a new distinct early-onset neurodegenerative condition resulting from mutations in the adenosine monophosphate deaminase 2 gene (AMPD2). Patients have characteristic brain imaging features of pontocerebellar hypoplasia (PCH), due to loss of brainstem and cerebellar parenchyma. We found that AMPD2 plays an evolutionary conserved role in the maintenance of cellular guanine nucleotide pools by regulating the feedback inhibition of adenosine derivatives on de novo purine synthesis. AMPD2 deficiency results in defective GTP-dependent initiation of protein translation, which can be rescued by administration of purine precursors. These data suggest AMPD2-related PCH as a new, potentially treatable early-onset neurodegenerative disease. PMID:23911318

  1. Rapid and reproducible radiosynthesis of [{sup 18}F] FHBG

    Energy Technology Data Exchange (ETDEWEB)

    Ponde, Datta E.; Dence, Carmen S.; Schuster, Daniel P.; Welch, Michael J. E-mail: Welchm@wustl.edu

    2004-01-01

    9-(4-[{sup 18}F] Fluoro-3-hydroxymethylbutyl) guanine ([{sup 18}F] FHBG), an imaging agent for gene therapy using PET, was prepared in a one-pot, two-step synthesis. Microwave (MW) mediated nucleophilic fluorination of N{sup 2}, monomethoxytrityl-9-[4-(tosyl)-3-monomethoxytrityl-methylbutyl] guanine using no-carrier-added [{sup 18}F] fluoride, followed by deprotection with hydrochloric acid and HPLC purification, gave [{sup 18}F] FHBG. The radiochemical yield (decay corrected) was 12{+-}5% (n = 35), the synthesis time was 55-60 min, and the radiochemical purity was >99%. The compound was used for lung imaging and was injected into Sprague-Dawley rats previously infected with the herpes simplex virus type 1 thymidine kinase (HSV1-tk) reporter gene. MicroPET imaging showed accumulation confined to the lungs.

  2. Repair of DNA treated with γ-irradiation and chemical carcinogens. Comprehensive report of entire period of ERDA support from June 1, 1975--January 15, 1978

    International Nuclear Information System (INIS)

    Goldthwait, D.A.

    1978-01-01

    A partially purified enzyme fraction isolated from E. coli showed an N-glycosidase activity as well as a phosphodiesterase activity on DNA treated with methylnitrosourea, and with 7-bromomethylbenz(a)anthracene and a phosphodiesterase activity against γ-irradiated DNA. Both 0-6 methyl guanine and 3-methyladenine were released from DNA treated with MNU; the adenine and guanine derivatives from the DNA treated with 7-bromomethyl-12-methylbenz(a)anthracene were also liberated. Progress is also reported on studies on Endonucleases II and VI and Exonuclease III of E. coli; methods for assay and for synthesis of substrates; attempts at purification of repair enzymes from mammalian tissues; and β-propiolactone reactions with deoxynucleosides and with DNA

  3. Ionic liquid-functionalized graphene as modifier for electrochemical and electrocatalytic improvement: comparison of different carbon electrodes.

    Science.gov (United States)

    Du, Meng; Yang, Tao; Ma, Suyan; Zhao, Changzhi; Jiao, Kui

    2011-04-01

    Electrochemical activities of typically electrochemical targets at three kinds of modified carbon electrodes, i.e. carbon ionic liquid electrode (CILE), graphene/carbon paste electrode (CPE), and ionic liquid-functionalized graphene (IL-graphene)/CPE, were compared in detail. The redox processes of the probes at IL-graphene/CPE were faster than those at CILE and graphene/CPE from cyclic voltammetry. An electrochemical method for the simultaneous determination of guanine and adenine was described with detection limits of 6.5×10(-8) mol L(-1) (guanine) and 3.2×10(-8) mol L(-1) (adenine). Single A→G mutation of sequence-specific DNA could be discriminated by the IL-graphene/CPE. Copyright © 2011 Elsevier B.V. All rights reserved.

  4. Microfabricated electrochemical sensor for the detection of radiation-induced DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    Wang, J.; Rivas, G.; Ozsoz, M.; Grant, D.H.; Cai, X.; Parrado, C. [New Mexico State Univ., Las Cruces, NM (United States)

    1997-04-01

    An electrochemical biosensor protocol for the detection of radiation-induced DNA damage is described. The procedure employs a dsDNA-coated screen-printed electrode and relies on changes in the guanine-DNA oxidation signal upon exposure to ultraviolet radiation. The decreased signal is ascribed primarily to conformational changes in the DNA and to the photoconversion of the guanine-DNA moiety to a nonelectroactive monomeric base product. Factors influencing the response of these microfabricated DNA sensors, such as irradiation time, wavelength, and distance, are explored, and future prospects are discussed. Similar results are given for the use of bare strip electrodes in connection with irradiated DNA solutions. 8 refs., 4 figs.

  5. Current approaches to improve the anticancer chemotherapy with alkylating agents: state of the problem in world and Ukraine.

    Directory of Open Access Journals (Sweden)

    Iatsyshyna A. P.

    2012-01-01

    Full Text Available Alkylating agents are frequently used in many established anticancer chemotherapies. They alkylate the genomic DNA at various sites. Alkylation of the guanine at the O6-position is cytotoxic, it has the strongest mutagenic potential, as well as can cause the tumor development. Alkyl groups at the O6-position of guanine are removed by the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT. The effectiveness of alkylating chemotherapy is limited by MGMT in cancer cells and adverse toxic side effects in normal cells. Different approaches consisting in the modulation of the MGMT expression and activity are under development now to improve the cancer chemotherapy. They include two main directions, in particular, the increase in chemosensitivity of cancer cells to alkylating drugs and the protection of normal cells from the toxic side effects of chemotherapy. This review is focused on current attempts to improve the alkylating chemotherapy of malignant tumours worldwide and state of the issue in Ukraine

  6. DNA minor groove alkylating agents.

    Science.gov (United States)

    Denny, W A

    2001-04-01

    Recent work on a number of different classes of anticancer agents that alkylate DNA in the minor groove is reviewed. There has been much work with nitrogen mustards, where attachment of the mustard unit to carrier molecules can change the normal patterns of both regio- and sequence-selectivity, from reaction primarily at most guanine N7 sites in the major groove to a few adenine N3 sites at the 3'-end of poly(A/T) sequences in the minor groove. Carrier molecules discussed for mustards are intercalators, polypyrroles, polyimidazoles, bis(benzimidazoles), polybenzamides and anilinoquinolinium salts. In contrast, similar targeting of pyrrolizidine alkylators by a variety of carriers has little effect of their patterns of alkylation (at the 2-amino group of guanine). Recent work on the pyrrolobenzodiazepine and cyclopropaindolone classes of natural product minor groove binders is also reviewed.

  7. Mutation of the conserved Gly94 and Gly126 in elongation factor Tu from Escherichia coli. Elucidation of their structural and functional roles

    DEFF Research Database (Denmark)

    Knudsen, Charlotte Rohde; Kjaersgård, I V; Wiborg, O

    1995-01-01

    All guanine-nucleotide-binding proteins cycle between an inactive, GDP-bound and an active, GTP-bound conformation whereby they function as molecular switches. Elongation factor Tu from Escherichia coli is used as a model for defining residues important in the switch mechanism. Gly94 and Gly126...... were separately mutated to alanine residues to study their role in the switch mechanism. The mutant proteins are denoted [G94A]EF-Tu and [G126A]EF-Tu, respectively. Both mutations affect the affinities for guanine nucleotides considerably, resulting in a decrease in the characteristic preference...... for GDP over GTP. Furthermore the [G94A]EF-Tu mutant possesses an increased GTPase activity. The aminoacyl-tRNA affinity is much reduced for [G94A]EF-Tu, as reflected in an increase of the dissociation rate constant for the ternary complex by a factor of 40. Surprisingly, however, both mutants...

  8. Kennedy's disease and partial androgen insensitivity syndrome. Report of 4 cases and literature review.

    Science.gov (United States)

    Valera Yepes, Rocío; Virgili Casas, Maria; Povedano Panades, Monica; Guerrero Gual, Mireia; Villabona Artero, Carles

    2015-05-01

    Kennedy's disease, also known as bulbospinal muscular atrophy, is a rare, X-linked recessive neurodegenerative disorder affecting adult males. It is caused by expansion of an unstable cytosine-adenine-guanine tandem-repeat in exon 1 of the androgen-receptor gene on chromosome Xq11-12, and is characterized by spinal motor neuron progressive degeneration. Endocrinologically, these patients often have the features of hypogonadism associated to the androgen insensitivity syndrome, particularly its partial forms. We report 4 cases with the typical neurological presentation, consisting of slowly progressing generalized muscle weakness with atrophy and bulbar muscle involvement; these patients also had several endocrine manifestations; the most common non-neurological manifestation was gynecomastia. In all cases reported, molecular analysis showed an abnormal cytosine-adenine-guanine triplet repeat expansion in the androgen receptor gene. Copyright © 2014 SEEN. Published by Elsevier España, S.L.U. All rights reserved.

  9. Biochemical techniques for the characterization of G-quadruplex structures: EMSA, DMS footprinting, and DNA polymerase stop assay.

    Science.gov (United States)

    Sun, Daekyu; Hurley, Laurence H

    2010-01-01

    The proximal promoter region of many human growth-related genes contains a polypurine/polypyrimidine tract that serves as multiple binding sites for Sp1 or other transcription factors. These tracts often contain a guanine-rich sequence consisting of four runs of three or more contiguous guanines separated by one or more bases, corresponding to a general motif known for the formation of an intramolecular G-quadruplex. Recent results provide strong evidence that specific G-quadruplex structures form naturally within these polypurine/polypyrimidine tracts in many human promoter regions, raising the possibility that the transcriptional control of these genes can be modulated by G-quadruplex-interactive agents. In this chapter, we describe three general biochemical methodologies, electrophoretic mobility shift assay (EMSA), dimethylsulfate (DMS) footprinting, and the DNA polymerase stop assay, which can be useful for initial characterization of G-quadruplex structures formed by G-rich sequences.

  10. Biological (molecular and cellular) markers of toxicity

    International Nuclear Information System (INIS)

    Shugart, L.R.; D'Surney, S.J.; Gettys-Hull, C.; Greeley, M.S. Jr.

    1991-01-01

    Several molecular and cellular markers of genotoxicity were adapted for measurement in the Medaka (Oryzias latipes), and were used to describe the effects of treatment of the organism with diethylnitrosamine (DEN). NO 6 -ethyl guanine adducts were detected, and a slight statistically significant, increase in DNA strand breaks was observed. These results are consistent with the hypothesis that prolonged exposure to high levels of DEN induced alkyltransferase activity which enzymatically removes any O 6 -ethyl guanine adducts but does not result in strand breaks or hypomethylation of the DNA such as might be expected from excision repair of chemically modified DNA. Following a five week continuous DEN exposure with 100 percent renewal of DEN-water every third day, the F values (DNA double strandedness) increased considerably and to similar extent in fish exposed to 25, 50, and 100 ppM DEN. This has been observed also in medaka exposed to BaP

  11. Structural and Functional Studies on Nucleotide Excision Repair From Recognition to Incision.

    Energy Technology Data Exchange (ETDEWEB)

    Caroline Kisker

    2001-01-01

    Maintenance of the correct genetic information is crucial for all living organisms because mutations are the primary cause of hereditary diseases, as well as cancer and may also be involved in aging. The importance of genomic integrity is underscored by the fact that 80 to 90% of all human cancers are ultimately due to DNA damage. Among the different repair mechanisms that have evolved to protect the genome, nucleotide excision repair (NER) is a universal pathway found in all organisms. NER removes a wide variety of bulky DNA adducts including the carcinogenic cyclobutane pyrimidine dimers induced by UV radiation, benzo(a)pyrene-guanine adducts caused by smoking and the guanine-cisplatin adducts induced by chemotherapy. The importance of this repair mechanism is reflected by three severe inherited diseases in humans, which are due to defects in NER: xeroderma pigmentosum, Cockayne's syndrome and trichothiodystrophy.

  12. The deoxyribonucleic acid of Micrococcus radiodurans

    Science.gov (United States)

    Schein, Arnold H.

    1966-01-01

    The DNA of Micrococcus radiodurans was prepared by three methods. Although the recovery of DNA varied considerably, the percentage molar base ratios of the DNA from the three preparations were essentially the same: guanine, 33±2; adenine, 18±1; cytosine, 33±2; thymine, 17±1. Base compositions calculated from Tm values and from density in caesium chloride gradients also yielded guanine+cytosine contents of 66 and 68% of total bases respectively. No unusual bases were observed. The S20,w values were characteristic of high-molecular-weight DNA. Electron microscopy showed the purified DNA in long strands; occasionally these were coiled. Images(a)(b)(c)(d)(e)Fig. 1. PMID:16742439

  13. Inverting the G-Tetrad Polarity of a G-Quadruplex by Using Xanthine and 8-Oxoguanine.

    Science.gov (United States)

    Cheong, Vee Vee; Lech, Christopher Jacques; Heddi, Brahim; Phan, Anh Tuân

    2016-01-04

    G-quadruplexes are four-stranded nucleic acid structures that are built from consecutively stacked guanine tetrad (G-tetrad) assemblies. The simultaneous incorporation of two guanine base lesions, xanthine (X) and 8-oxoguanine (O), within a single G-tetrad of a G-quadruplex was recently shown to lead to the formation of a stable G⋅G⋅X⋅O tetrad. Herein, a judicious introduction of X and O into a human telomeric G-quadruplex-forming sequence is shown to reverse the hydrogen-bond polarity of the modified G-tetrad while preserving the original folding topology. The control exerted over G-tetrad polarity by joint X⋅O modification will be valuable for the design and programming of G-quadruplex structures and their properties. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Proceedings of the Annual Conference on Environmental Toxicology (5th) Held at Fairborn, OH on 24-26 September 1974

    Science.gov (United States)

    1974-12-01

    naphthylamine, benzidine and derivatives, 4-biphenylamine, 4-nitrobiphenyl, auramine and magenta) Alkylating Agents (chlornaphthazlne, mustard gas...recently developed carborane-silicone columns (Willeboordse et al., 1968; Yancey and Lynn, 1974). Alkylating Agents Several alkylating agents have emerged...Magee, "Nitrosamine-Induced Carcinogenesis. The Alkylation of N-7 of Guanine of Nucleic Acids of the Rat by Diethylnitrosamine N-Ethyl-N- Nitrosourea

  15. [The biochemical mechanisms of the action of N-alkyl-N-nitrosoureas. The possible reasons for drug resistance to these compounds].

    Science.gov (United States)

    Syrkin, A B; Gorbacheva, L B

    1996-01-01

    N-alkyl-N-nitrosoureas exhibit a wide spectrum of antitumor activity. They react as alkylating agents at nucleophilic sites in purine and pyrimidine moieties of DNA. The predominant site of this alkylation is N7 of guanine, which is followed by the site N3 of adenine and 06 of guanine. The formation and persistence of 0(6)-alkylguanine (0(6)-AG) may be of primary importance in cytotoxicity of the nitrosoureas. 0(6)-AG adducts of DNA of the tumor cells are repaired by protein 0(6)-alkylguanine-DNA transferase (0(6)-AGT) which transfers the alkyl group to internal cysteine residue being the acceptor protein for the alkyl group in an irreversible transfer reaction. 0(6)-AGT can protect the tumor cells against 0(6)-AG adducts by the way of inhibiting the formation of the DNA interstrand cross-links 0(6)-AGT plays an important role in the drug resistance because it repairs the DNA alkyl adducts at the 0(6) position of guanine. The 0(6)-AGT activity inversely correlates with the cytotoxic effect of the nitrosoureas. The agents like 0(6)-methylguanosine, 0(6)-methyl-2'-deoxyguanosine, and some 0(6)-benzylated guanine derivatives are effective inactivators of 0(6)-AGT, and thus can be used to enhance the cytotoxicity of N-nitrosoureas. The activation of 0(6)-AGT and other repairing enzymes such as alpha and beta DNA-polymerases as well as an increase in the level of reduced glutathione may be used in developing the resistance to the nitrosoureas.

  16. Comparative Biochemistry and Metabolism. Part 1. Carcinogenesis

    Science.gov (United States)

    1982-08-01

    1968), Nitrosamine-induced carcino- genesis. The alkylation of nucleic acids of the rat by N-methvl- N- nitrosourea , dimethylnitrosamine...inorganic reducing agent , hydrazine, is toxic and weakly carcinogenic. In earlier studies it was found that oral administration of a toxic dose of...metabolically activated to a methylatinj agent . Liver DNA from mice and hamsters contained considerably more 7-methyl- guanine and 0 6-methylguanine

  17. Vav1: Friend and Foe of Cancer.

    Science.gov (United States)

    Guo, Fukun; Zheng, Yi

    2017-12-01

    A recent study shows that the protumorigenic guanine nucleotide exchange factor (GEF) Vav1 functions as a tumor suppressor in T cell acute lymphoblastic leukemia (T-ALL) through its ability to complex with the Cbl-b ubiquitin ligase and the intracellular domain of Notch1 (ICN1) and to promote ICN1 degradation. Vav1can act as a double-edged sword in tumorigenesis. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. The invisible hand: regulation of RHO GTPases by RHOGDIs

    OpenAIRE

    Garcia-Mata, Rafael; Boulter, Etienne; Burridge, Keith

    2011-01-01

    The 'invisible hand' is a term originally coined by Adam Smith in the Theory of Moral Sentiments to describe the forces of self-interest, competition, and supply and demand that regulate the resources in society. This metaphor continues to be used by economists to describe the self-regulating nature of a market economy. The same metaphor can be used to describe the RHO-specific guanine nucleotide dissociation inhibitor (RHOGDI) family, which operates in the background, as an invisible hand, u...

  19. Benzo(a)pyrene diol epoxides as intermediates in nucleic acid binding in vitro and in vivo.

    Science.gov (United States)

    Weinstein, I B; Jeffrey, A M; Jennette, K W; Blobstein, S H; Harvey, R G; Harris, C; Autrup, H; Kasai, H; Nakanishi, K

    1976-08-13

    Evidence has been obtained that a specific isomer of a diol epoxide derivative of benzo(a)pyrene, (+/-)-7 beta,8alpha-dihydroxy-9alpha, 10alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene, is an intermediate in the binding of benzo(a)pyrene to RNA in cultured bovine bronchial mucosa. An adduct is formed between position 10 of this derivative and the 2-amino group of guanine.

  20. Structure of 2,4-Diaminopyrimidine-Theobromine Alternate Base Pairs

    Czech Academy of Sciences Publication Activity Database

    Gengeliczki, Z.; Callahan, M. P.; Kabeláč, Martin; Rijs, A. M.; de Vries, M. S.

    2011-01-01

    Roč. 115, č. 41 (2011), s. 11423-11427 ISSN 1089-5639 R&D Projects: GA MŠk LC512; GA AV ČR IAA400550808 Institutional research plan: CEZ:AV0Z40550506 Keywords : ab-initio calculations * double resonance spectroscopy * basis-set * guanine * cytosine Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 2.946, year: 2011