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Sample records for alpha ligands activate

  1. Catalposide is a natural agonistic ligand of peroxisome proliferator-activated receptor-{alpha}

    SciTech Connect

    Lee, Ji Hae; Jun, Hee-jin; Hoang, Minh-Hien; Jia, Yaoyao; Han, Xiang Hua; Lee, Dong-Ho; Lee, Hak-Ju; Hwang, Bang Yeon; Lee, Sung-Joon

    2012-06-15

    Highlights: Black-Right-Pointing-Pointer Catalposide is a novel ligand for PPAR{alpha}. Black-Right-Pointing-Pointer Cell stimulated with catalposide improved fatty acid uptake, regulated target genes in fatty acid {beta}-oxidation and synthesis. Black-Right-Pointing-Pointer Catalposdie reduces hepatic triacylglycerides. Black-Right-Pointing-Pointer Theses demonstrate catalposide could ameliorate hyperlipidemia and hepatic steatosis. -- Abstract: Peroxisome proliferator-activated receptor-alpha (PPAR{alpha}) is a nuclear receptor that regulates the expression of genes related to cellular lipid uptake and oxidation. Thus, PPAR{alpha} agonists may be important in the treatment of hypertriglyceridemia and hepatic steatosis. In this study, we demonstrated that catalposide is a novel natural PPAR{alpha} agonist, identified from reporter gene assay-based activity screening with approximately 900 natural plant and seaweed extracts. Results of time-resolved fluorescence resonance energy transfer analyses suggested that the compound interacted directly with the ligand-binding domain of PPAR{alpha}. Cultured hepatocytes stimulated with catalposide exhibited significantly reduced cellular triglyceride concentrations, by 21%, while cellular uptake of fatty acids was increased, by 70% (P < 0.05). Quantitative PCR analysis revealed that the increase in cellular fatty acid uptake was due to upregulation of fatty acid transporter protein-4 (+19% vs. the control) in cells stimulated with catalposide. Additionally, expression of genes related to fatty acid oxidation and high-density lipoprotein metabolism were upregulated, while that of genes related to fatty acid synthesis were suppressed. In conclusion, catalposide is hypolipidemic by activation of PPAR{alpha} via a ligand-mediated mechanism that modulates the expression of in lipid metabolism genes in hepatocytes.

  2. Implications of a peroxisome proliferator-activated receptor alpha (PPARα) ligand clofibrate in breast cancer

    PubMed Central

    Goswami, Sudeshna

    2016-01-01

    Inflammatory and invasive breast cancers are aggressive and require better understanding for the development of new treatments and more accurate prognosis. Here, we detected high expression of PPARα in human primary inflammatory (SUM149PT) and highly invasive (SUM1315MO2) breast cancer cells, and tissue sections of human breast cancer. PPARα ligands are clinically used to treat dyslipidemia. Among lipid lowering drugs clofibrate, fenofibrate and WY14643, clofibrate showed high chemo-sensitivity towards breast cancer cells. Clofibrate treatment significantly induced PPARα DNA binding activity, and remarkably reduced cyclooxygenase-2/PGE2 and 5-lipoxygenase/LTB4 inflammatory pathways. Clofibrate treatment reduced the proliferation of breast cancer cells probably by inhibiting NF-κB and ERK1/2 activation, reducing cyclinD1, cyclinA, cyclinE, and inducing pro-apoptotic P21 levels. Surprisingly, the expression of lipogenic pathway genes including SREBP-1c (sterol regulatory element-binding protein-1c), HMG-CoA synthase, SPTLC1 (serine palmitoyltransferase long-chain), and Acyl-CoA oxidase (ACO) decreased with a concurrent increase in fatty acid oxidation genes such as CPT-1a (carnitine palmitoyltransferase 1a) and SREBP-2 (Sterol regulatory element-binding protein-2). Clofibrate treatment induced secretion of free fatty acids and effectively decreased the level of phosphorylated active form of fatty acid synthase (FASN), an enzyme catalyzing de novo synthesis of fatty acids. High level of coactivators steroid receptor coactivator-1 (SRC-1) and histone acetylase CBP-300 (CREB binding protein-300) were observed in the nuclear complexes of clofibrate treated breast cancer cells. These findings implicate that stimulating PPARα by safe, well-tolerated, and clinically approved clofibrate may provide a safer and more effective strategy to target the signaling, lipogenic, and inflammatory pathways in aggressive forms of breast cancer. PMID:26621841

  3. Ligand-induced interaction between. alpha. - and. beta. -type platelet-derived growth factor (PDGF) receptors: Role of receptor heterodimers in kinase activation

    SciTech Connect

    Kanakaraj, P.; Raj, S.; Bishayee, S. ); Khan, S.A. )

    1991-02-19

    Two types of PDGF receptors have been cloned and sequenced. Both receptors are transmembrane glycoproteins with a ligand-stimulatable tyrosine kinase site. The authors have shown earlier that ligand-induced activation of the {beta}-type PDGF receptor is due to the conversion of the monomeric form of the receptor to the dimeric form. In the present studies, they have established the ligand-binding specificity of two receptor types and extended it further to investigate the ligand-induced association state of the {alpha}-receptor and the role of {alpha}-receptor in the activation of {beta}-receptor. These studies were conducted with cells that express one or the other type of PDGF receptor as well as with cells that express both types of receptors. Moreover, ligand-binding characteristics of the receptor were confirmed by immunoprecipitation of the receptor-{sup 125}I-PDGF covalent complex with type-specific anti-PDGF receptor antibodies. These studies revealed that all three isoforms of PDGF bind to {alpha}-receptor, and such binding leads to dimerization as well as activation of the receptor. In contrast, {beta}-receptor can be activated only by PDGF BB and not by PDGF AB or PDGF AA. However, by using antipeptide antibodies that are specific for {alpha}- or {beta}-type PDGF receptor, they demonstrated that in the presence of {alpha}-receptor, {beta}-receptor kinase can be activated by PDGF AB. They present here direct evidence that strongly suggests that such PDGF AB induced activation of {beta}-receptor is due to the formation of a noncovalently linked {alpha}-{beta} receptor heterodimer.

  4. Antioxidant alpha-lipoic acid inhibits osteoclast differentiation by reducing nuclear factor-kappaB DNA binding and prevents in vivo bone resorption induced by receptor activator of nuclear factor-kappaB ligand and tumor necrosis factor-alpha.

    PubMed

    Kim, Hyon Jong; Chang, Eun-Ju; Kim, Hyun-Man; Lee, Seung Bok; Kim, Hyun-Duck; Su Kim, Ghi; Kim, Hong-Hee

    2006-05-01

    The relationship between oxidative stress and bone mineral density or osteoporosis has recently been reported. As bone loss occurring in osteoporosis and inflammatory diseases is primarily due to increases in osteoclast number, reactive oxygen species (ROS) may be relevant to osteoclast differentiation, which requires receptor activator of nuclear factor-kappaB ligand (RANKL). Tumor necrosis factor-alpha (TNF-alpha) frequently present in inflammatory conditions has a profound synergy with RANKL in osteoclastogenesis. In this study, we investigated the effects of alpha-lipoic acid (alpha-LA), a strong antioxidant clinically used for some time, on osteoclast differentiation and bone resorption. At concentrations showing no growth inhibition, alpha-LA potently suppressed osteoclastogenesis from bone marrow-derived precursor cells driven either by a high-dose RANKL alone or by a low-dose RANKL plus TNF-alpha (RANKL/TNF-alpha). alpha-LA abolished ROS elevation by RANKL or RANKL/TNF-alpha and inhibited NF-kappaB activation in osteoclast precursor cells. Specifically, alpha-LA reduced DNA binding of NF-kappaB but did not inhibit IKK activation. Furthermore, alpha-LA greatly suppressed in vivo bone loss induced by RANKL or TNF-alpha in a calvarial remodeling model. Therefore, our data provide evidence that ROS plays an important role in osteoclast differentiation through NF-kappaB regulation and the antioxidant alpha-lipoic acid has a therapeutic potential for bone erosive diseases.

  5. Critical role of charged residues in helix 7 of the ligand binding domain in Hepatocyte Nuclear Factor 4alpha dimerisation and transcriptional activity.

    PubMed

    Eeckhoute, Jérôme; Oxombre, Bénédicte; Formstecher, Pierre; Lefebvre, Philippe; Laine, Bernard

    2003-11-15

    Hepatocyte Nuclear Factor 4alpha (HNF4alpha, NR2A1) is central to hepatocyte and pancreatic beta-cell functions. Along with retinoid X receptor alpha (RXRalpha), HNF4alpha belongs to the nuclear receptor subfamily 2 (NR2), characterised by a conserved arginyl residue and a glutamate residue insert in helix 7 (H7) of the ligand binding domain (LBD). Crystallographic studies indicate that R348 and E352 residues in RXRalpha H7 are involved in charge-driven interactions that improve dimerisation. Consistent with these findings, we showed that removing the charge of the corresponding residues in HNF4alpha H7, R258 and E262, impaired dimerisation in solution. Moreover, our results provide a new concept according to which helices of the HNF4alpha LBD dimerisation interface contribute differently to dimerisation required for DNA binding; unlike H9 and H10, H7 is not involved in DNA binding. Substitutions of E262 decreased the repression of HNF4alpha transcriptional activity by a dominant-negative HNF4alpha mutant, highlighting the importance of this residue for dimerisation in the cell context. The E262 insert is crucial for HNF4alpha function since its deletion abolished HNF4alpha transcriptional activity and coactivator recruitment. The glutamate residue insert and the conserved arginyl residue in H7 most probably represent a signature of the NR2 subfamily of nuclear receptors.

  6. Bioisosteric phentolamine analogs as selective human alpha(2)- versus alpha(1)-adrenoceptor ligands.

    PubMed

    Bavadekar, Supriya A; Hong, Seoung-Soo; Lee, Sang-Ii; Miller, Duane D; Feller, Dennis R

    2008-08-20

    Phentolamine is known to act as a competitive, non-subtype-selective alpha-adrenoceptor antagonist. In an attempt to improve alpha(2)- versus alpha(1)-adrenoceptor selectivity and alpha(2)-adrenoceptor subtype-selectivity, two new chemical series of bioisosteric phentolamine analogs were prepared and evaluated. These compounds were evaluated for binding affinities on alpha(1)- (alpha(1A)-, alpha(1B)-, alpha(1D)-) and alpha(2)- (alpha(2A)-, alpha(2B)-, alpha(2C)-) adrenoceptor subtypes that had been stably expressed in human embryonic kidney and Chinese hamster ovary cell lines, respectively. Methylation of the phenolic hydroxy group and replacement of the 4-methyl group of phentolamine with varying lipophilic substituents yielded bioisosteric analogs selective for the alpha(2)- versus alpha(1)-adrenoceptors. Within the alpha(2)-adrenoceptors, these analogs bound with higher affinity at the alpha(2A)- and alpha(2C)-subtypes as compared to the alpha(2B)-subtype. In particular, the t-butyl analog was found to be the most selective, its binding at the alpha(2C)-adrenoceptor (Ki=3.6 nM) being 37- to 173-fold higher than that at the alpha(1)-adrenoceptors, and around 2- and 19-fold higher than at the alpha(2A)- and alpha(2B)-adrenoceptors, respectively. Data from luciferase reporter gene assays confirmed the functional antagonist activities of selected compounds from the bioisosteric series on human alpha(1A)- and alpha(2C)-adrenoceptors. Thus, the results with these bioisosteric analogs of phentolamine provide a lead to the rational design of potent and selective alpha(2)-adrenoceptor ligands that may be useful in improving the therapeutic profile of this drug class for human disorders.

  7. The alpha 4 integrin chain is a ligand for alpha 4 beta 7 and alpha 4 beta 1

    PubMed Central

    1995-01-01

    The heterodimeric alpha 4 integrins alpha 4 beta 7 lymphocyte Peyer's patch adhesion molecule ([LPAM]-1) and alpha 4 beta 1 (very late antigen-4) are cell surface adhesion molecules involved in lymphocyte trafficking and lymphocyte-cell and matrix interactions. Known cellular ligands include vascular cell adhesion molecule (VCAM)-1, which binds to alpha 4 beta 1 and alpha 4 beta 7, and the mucosal addressin cell adhesion molecule (MAdCAM)-1, which binds to alpha 4 beta 7. Here we show that the alpha 4 chain of these integrins can itself serve as a ligand. The alpha 4 chain, immunoaffinity purified and immobilized on glass slides, binds thymocytes and T lymphocytes. Binding exhibits divalent cation requirements and temperature sensitivity which are characteristic of integrin-mediated interactions, and is specifically inhibited by anti-alpha 4 integrin antibodies, which exert their effect at the cell surface. Cells expressing exclusively alpha 4 beta 7 (TK-1) or alpha 4 beta 1 (L1-2) both bound avidly, whereas alpha 4-negative cells did not. A soluble 34-kD alpha 4 chain fragment retained binding activity, and it inhibited lymphocyte adhesion to alpha 4 ligands. It has been shown that alpha 4 integrin binding to fibronectin involves an leucine-aspartic acid-valine (LDV) motif in the HepII/IIICS region of fibronectin (CS-1 peptide), and homologous sequences are important in binding to VCAM-1 and MAdCAM-1. Three conserved LDV motifs occur in the extracellular sequence of alpha 4. A synthetic LDV-containing alpha 4- derived oligopeptide supports alpha 4-integrin-dependent lymphocyte adhesion and blocks binding to the 34-kD alpha 4 chain fragment. Our results suggest that alpha 4 beta 7 and alpha 4 beta 1 integrins may be able to bind to the alpha 4 subunit on adjacent cells, providing a novel mechanism for alpha 4 integrin-mediated and activation-regulated lymphocyte interactions during immune responses. PMID:7629498

  8. Interpreting EEG alpha activity.

    PubMed

    Bazanova, O M; Vernon, D

    2014-07-01

    Exploring EEG alpha oscillations has generated considerable interest, in particular with regards to the role they play in cognitive, psychomotor, psycho-emotional and physiological aspects of human life. However, there is no clearly agreed upon definition of what constitutes 'alpha activity' or which of the many indices should be used to characterize it. To address these issues this review attempts to delineate EEG alpha-activity, its physical, molecular and morphological nature, and examine the following indices: (1) the individual alpha peak frequency; (2) activation magnitude, as measured by alpha amplitude suppression across the individual alpha bandwidth in response to eyes opening, and (3) alpha "auto-rhythmicity" indices: which include intra-spindle amplitude variability, spindle length and steepness. Throughout, the article offers a number of suggestions regarding the mechanism(s) of alpha activity related to inter and intra-individual variability. In addition, it provides some insights into the various psychophysiological indices of alpha activity and highlights their role in optimal functioning and behavior.

  9. Ligand specificities of recombinant retinoic acid receptors RAR alpha and RAR beta.

    PubMed Central

    Crettaz, M; Baron, A; Siegenthaler, G; Hunziker, W

    1990-01-01

    Binding of retinoic acid (RA) to specific RA receptors alpha and beta (RAR alpha and RAR beta) was studied. Receptors were obtained in two ways: (1) full-length receptors were produced by transient expression of the respective human cDNAs in COS 1 cells; and (2) the ligand-binding domains of RAR alpha and RAR beta were produced in Escherichia coli. RA binding to the wild-type and truncated forms of the receptor was identical for both RAR alpha and RAR beta, indicating that the ligand-binding domains have retained the binding characteristics of the intact receptors. Furthermore, RA bound with the same affinity to both RAR alpha and RAR beta. Only retinoid analogues with an acidic end-group were able to actively bind to both receptors. On measuring the binding of various retinoids, we have found that the properties of the ligand-binding sites of RAR alpha and RAR beta were rather similar. Two retinoid analogues were capable of binding preferentially to either RAR alpha or RAR beta, suggesting that it may be possible to synthesize specific ligands for RAR alpha and RAR beta. PMID:2176462

  10. Recombinant human endostatin inhibits TNF-alpha-induced receptor activator of NF-κB ligand expression in fibroblast-like synoviocytes in mice with adjuvant arthritis.

    PubMed

    Gao, Qiu-Fang; Zhang, Xiu-Hong; Yuan, Feng-Lai; Zhao, Ming-Dong; Li, Xia

    2016-12-01

    Bone loss is a critical pathology responsible for the functional disability in patients with rheumatoid arthritis (RA). It is well known that receptor activator of nuclear factor kappa-B (NF-κB) ligand (RANKL) plays a crucial role in bone loss in RA. The purpose of this study was to determine whether recombinant human endostatin (rh-endostatin) mediates bone erosion in RA by regulation of RANKL expression in an experimental model of RA, consisting of mice with adjuvant-induced arthritis (AA). Cultured AA fibroblast-like synoviocytes (FLSs) obtained from these mice were induced by tumor necrosis factor-α (TNF-α) combined with or without rh-endostatin. The levels of RANKL and osteoprotegerin (OPG) mRNA, soluble and membrane-bound proteins were assessed by real-time PCR, ELISA, and Western blotting. Western blotting and the luciferase reporter assay were used to study related signaling pathways. Rh-endostatin inhibited RANKL mRNA expression, soluble and membrane-bound protein expression in AA FLSs but not in CD4+ T cells. However, OPG expression and secretion was not affected by rh-endostatin in AA FLSs. Molecular analysis demonstrated that rh-endostatin significantly inhibited TNF-α-induced MAPK and AP-1 signaling pathways. Moreover, rh-endostatin attenuated TNF-α-induced NF-κB signaling by suppressing the phosphorylation level of inhibitor kappaBα (IκBα) and nuclear translocation of NF-κB p65 in FLSs from mice with AA. These results provide the first evidence that rh-endostatin inhibits TNF-α-induced RANKL expression in AA FLSs.

  11. PANP is a novel O-glycosylated PILR{alpha} ligand expressed in neural tissues

    SciTech Connect

    Kogure, Amane; Shiratori, Ikuo; Wang, Jing; Lanier, Lewis L.; Arase, Hisashi

    2011-02-18

    Research highlights: {yields} A Novel molecule, PANP, was identified to be a PILR{alpha} ligand. {yields} Sialylated O-glycan structures on PANP were required for PILR{alpha} recognition. {yields} Transcription of PANP was mainly observed in neural tissues. {yields} PANP seems to be involved in immune regulation as a ligand for PILR{alpha}. -- Abstract: PILR{alpha} is an immune inhibitory receptor possessing an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain enabling it to deliver inhibitory signals. Binding of PILR{alpha} to its ligand CD99 is involved in immune regulation; however, whether there are other PILR{alpha} ligands in addition to CD99 is not known. Here, we report that a novel molecule, PILR-associating neural protein (PANP), acts as an additional ligand for PILR{alpha}. Transcription of PANP was mainly observed in neural tissues. PILR{alpha}-Ig fusion protein bound cells transfected with PANP and the transfectants stimulated PILR{alpha} reporter cells. Specific O-glycan structures on PANP were found to be required for PILR recognition of this ligand. These results suggest that PANP is involved in immune regulation as a ligand of the PILR{alpha}.

  12. Novel ligands for the opioid receptors: synthesis and structure-activity relationships among 5'-aryl and 5'-heteroaryl 17-cyclopropylmethyl-4,5 alpha-epoxypyrido[2',3':6,7]morphinans.

    PubMed

    Ananthan, Subramaniam; Khare, Naveen K; Saini, Surendra K; Davis, Peg; Dersch, Christina M; Porreca, Frank; Rothman, Richard B

    2003-09-01

    A series of pyridomorphinans possessing an aryl (10a-s) or heteroaryl (11a-h) substituent at the 5'-position of the pyridine ring of 17-cyclopropylmethyl-4,5 alpha-epoxypyrido[2',3':6,7]morphinan was synthesized and evaluated for binding and functional activity at the opioid delta, mu, and kappa receptors. All of these pyridomorphinans bound with higher affinity at the delta site than at mu or kappa sites. The binding data on isomeric compounds revealed that there exists greater bulk tolerance for substituents placed at the o-position of the phenyl ring than at m- or p-positions. Among the ligands examined, the 2-chlorophenyl (10l), 2-nitrophenyl (10n), 2-pyridyl (11a), and 4-quinolinyl (11g) compounds bound to the delta receptor with subnanomolar affinity. Compound 10c with the p-tolyl substituent displayed the highest mu/delta selectivity (ratio=42) whereas compound 10l with the 2-chlorophenyl substituent displayed the highest kappa/delta selectivity (ratio=23). At 10 microM concentration, the in vitro functional activity determined using [(35)S]GTP-gamma-S binding assays showed that all of the compounds were antagonists devoid of any significant agonist activity at the delta, mu, and kappa receptors. Antagonist potency determinations of three selected ligands revealed that the p-tolyl compound 10c is a potent delta selective antagonist. In the [(35)S]GTP-gamma-S assays this compound had a functional antagonist K(i) value of 0.2, 4.52, and 7.62 nM at the delta, mu, and kappa receptors, respectively. In the smooth muscle assays 10c displayed delta antagonist potency with a K(e) value of 0.88 nM. As an antagonist, it was 70-fold more potent at the delta receptors in the MVD than at the mu receptors in the GPI. The in vitro delta antagonist profile of this pyridomorphinan 10c resembles that of the widely used delta selective antagonist ligand naltrindole.

  13. Ligand binding affinities of arctigenin and its demethylated metabolites to estrogen receptor alpha.

    PubMed

    Jin, Jong-Sik; Lee, Jong-Hyun; Hattori, Masao

    2013-01-16

    Phytoestrogens are defined as plant-derived compounds with estrogen-like activities according to their chemical structures and activities. Plant lignans are generally categorized as phytoestrogens. It was reported that (-)-arctigenin, the aglycone of arctiin, was demethylated to (-)-dihydroxyenterolactone (DHENL) by Eubacterium (E.) sp. ARC-2. Through stepwise demethylation, E. sp. ARC-2 produced six intermediates, three mono-desmethylarctigenins and three di-desmethylarctigenins. In the present study, ligand binding affinities of (-)-arctigenin and its seven metabolites, including DHENL, were investigated for an estrogen receptor alpha, and found that demethylated metabolites had stronger binding affinities than (-)-arctigenin using a ligand binding screen assay method. The IC(50) value of (2R,3R)-2-(4-hydroxy-3-methoxybenzyl)-3-(3,4-dihydroxybenzyl)-butyrolactone was 7.9 × 10⁻⁴ M.

  14. Brucella abortus Invasion of Osteocytes Modulates Connexin 43 and Integrin Expression and Induces Osteoclastogenesis via Receptor Activator of NF-κB Ligand and Tumor Necrosis Factor Alpha Secretion

    PubMed Central

    Pesce Viglietti, Ayelén Ivana; Arriola Benitez, Paula Constanza; Gentilini, María Virginia; Velásquez, Lis Noelia; Fossati, Carlos Alberto; Giambartolomei, Guillermo Hernán

    2015-01-01

    Osteoarticular brucellosis is the most common localization of human active disease. Osteocytes are the most abundant cells of bone. They secrete factors that regulate the differentiation of both osteoblasts and osteoclasts during bone remodeling. The aim of this study is to determine if Brucella abortus infection modifies osteocyte function. Our results indicate that B. abortus infection induced matrix metalloproteinase 2 (MMP-2), receptor activator for NF-κB ligand (RANKL), proinflammatory cytokines, and keratinocyte chemoattractant (KC) secretion by osteocytes. In addition, supernatants from B. abortus-infected osteocytes induced bone marrow-derived monocytes (BMM) to undergo osteoclastogenesis. Using neutralizing antibodies against tumor necrosis factor alpha (TNF-α) or osteoprotegerin (OPG), RANKL's decoy receptor, we determined that TNF-α and RANKL are involved in osteoclastogenesis induced by supernatants from B. abortus-infected osteocytes. Connexin 43 (Cx43) and the integrins E11/gp38, integrin-α, integrin-β, and CD44 are involved in cell-cell interactions necessary for osteocyte survival. B. abortus infection inhibited the expression of Cx43 but did not modify the expression of integrins. Yet the expression of both Cx43 and integrins was inhibited by supernatants from B. abortus-infected macrophages. B. abortus infection was not capable of inducing osteocyte apoptosis. However, supernatants from B. abortus-infected macrophages induced osteocyte apoptosis in a dose-dependent manner. Taken together, our results indicate that B. abortus infection could alter osteocyte function, contributing to bone damage. PMID:26459511

  15. Artificial ligand binding within the HIF2alpha PAS-B domain of the HIF2 transcription factor.

    PubMed

    Scheuermann, Thomas H; Tomchick, Diana R; Machius, Mischa; Guo, Yan; Bruick, Richard K; Gardner, Kevin H

    2009-01-13

    The hypoxia-inducible factor (HIF) basic helix-loop-helix Per-aryl hydrocarbon receptor nuclear translocator (ARNT)-Sim (bHLH-PAS) transcription factors are master regulators of the conserved molecular mechanism by which metazoans sense and respond to reductions in local oxygen concentrations. In humans, HIF is critically important for the sustained growth and metastasis of solid tumors. Here, we describe crystal structures of the heterodimer formed by the C-terminal PAS domains from the HIF2alpha and ARNT subunits of the HIF2 transcription factor, both in the absence and presence of an artificial ligand. Unexpectedly, the HIF2alpha PAS-B domain contains a large internal cavity that accommodates ligands identified from a small-molecule screen. Binding one of these ligands to HIF2alpha PAS-B modulates the affinity of the HIF2alpha:ARNT PAS-B heterodimer in vitro. Given the essential role of PAS domains in forming active HIF heterodimers, these results suggest a presently uncharacterized ligand-mediated mechanism for regulating HIF2 activity in endogenous and clinical settings.

  16. Artificial ligand binding within the HIF2[alpha] PAS-B domain of the HIF2 transcription factor

    SciTech Connect

    Scheuermann, Thomas H.; Tomchick, Diana R.; Machius, Mischa; Guo, Yan; Bruick, Richard K.; Gardner, Kevin H.

    2009-05-12

    The hypoxia-inducible factor (HIF) basic helix-loop-helix Per-aryl hydrocarbon receptor nuclear translocator (ARNT)-Sim (bHLH-PAS) transcription factors are master regulators of the conserved molecular mechanism by which metazoans sense and respond to reductions in local oxygen concentrations. In humans, HIF is critically important for the sustained growth and metastasis of solid tumors. Here, we describe crystal structures of the heterodimer formed by the C-terminal PAS domains from the HIF2{alpha} and ARNT subunits of the HIF2 transcription factor, both in the absence and presence of an artificial ligand. Unexpectedly, the HIF2{alpha} PAS-B domain contains a large internal cavity that accommodates ligands identified from a small-molecule screen. Binding one of these ligands to HIF2{alpha} PAS-B modulates the affinity of the HIF2{alpha}:ARNT PAS-B heterodimer in vitro. Given the essential role of PAS domains in forming active HIF heterodimers, these results suggest a presently uncharacterized ligand-mediated mechanism for regulating HIF2 activity in endogenous and clinical settings.

  17. AFM imaging of ligand binding to platelet integrin alphaIIbbeta3 receptors reconstituted into planar lipid bilayers.

    PubMed

    Hussain, Mohammad A; Agnihotri, Aashiish; Siedlecki, Christopher A

    2005-07-19

    The platelet integrin alphaIIbbeta3 plays a key role in platelet adhesion, activation, and aggregation at the subendothelium and at protein-coated synthetic biomaterials. In this study, interactions between alphaIIbbeta3 and both protein and peptide ligands for the receptor were imaged under physiological conditions by high-resolution atomic force microscopy (AFM). To directly image the ligand-receptor interactions, alphaIIbbeta3 receptors were reconstituted into a supported lipid bilayer formed on a mica surface in the AFM fluid cell assembly and subsequently activated with Mn2+. Fibrinogen, the natural protein ligand for the integrin, as well as a nanogold-labeled peptide ligand (an RGD-containing heptamer) were infused into the AFM fluid cell, incubated with the reconstituted and activated receptors, and imaged under buffer. Height images illustrating topographical features showed the integrin reconstituted in the bilayer. Fibrinogen molecules binding to the receptors were easily observed in the height images, with fibrinogen showing its characteristic trinodular structure and occasionally bridging integrin receptors. Fibrinogen was observed to bind to integrins at the D-domain consistent with the location of the gamma-chain dodecapeptide, while fibrinogen bridging integrins bound to receptors on opposite sides of the protein consistent with a 2-fold axis of symmetry. Peptide ligands were not visible in height images; however, phase images that map the mechanical properties detected the nanogold labels and demonstrated the presence of peptide ligands bound to the receptors. The results demonstrate the ability of this high-resolution microscopy technique to directly visualize single ligand/receptor interactions in a dynamic and physiologically relevant environment, and establish a framework for future fundamental studies of single protein/receptor interactions during normal pathological processes as well as biomaterial surface-induced thrombosis.

  18. Electronic spectra and photophysics of platinum(II) complexes with alpha-diimine ligands - Solid-state effects. I - Monomers and ligand pi dimers

    NASA Technical Reports Server (NTRS)

    Miskowski, Vincent M.; Houlding, Virginia H.

    1989-01-01

    Two types of emission behavior for Pt(II) complexes containing alpha-diimine ligands have been observed in dilute solution. If the complex also has weak field ligands such as chloride, ligand field (d-d) excited states become the lowest energy excited states. If only strong field ligands are present, a diimine 3(pi-pi/asterisk/) state becomes the lowest. In none of the cases studied did metal-to-ligand charge transfer excited state lie lowest.

  19. Labeled ALPHA4BETA2 ligands and methods therefor

    DOEpatents

    Mukherjee, Jogeshwar; Pichika, Ramaiah; Potkin, Steven; Leslie, Frances; Chattopadhyay, Sankha

    2013-02-19

    Contemplated compositions and methods are employed to bind in vitro and in vivo to an .alpha.4.beta.2 nicotinic acetylcholine receptor in a highly selective manner. Where such compounds are labeled, compositions and methods employing such compounds can be used for PET and SPECT analysis. Alternatively, and/or additionally contemplated compounds can be used as antagonists, partial agonists or agonists in the treatment of diseases or conditions associated with .alpha.4.beta..beta.2 dysfunction.

  20. Principles of Ligand Binding within a Completely Buried Cavity in HIF2[alpha] PAS-B

    SciTech Connect

    Key, Jason; Scheuermann, Thomas H.; Anderson, Peter C.; Daggett, Valerie; Gardner, Kevin H.

    2010-04-19

    Hypoxia-inducible factors (HIFs) are heterodimeric transcription factors responsible for the metazoan hypoxia response and promote tumor growth, metastasis, and resistance to cancer treatment. The C-terminal Per-ARNT-Sim (PAS) domain of HIF2{alpha} (HIF2{alpha} PAS-B) contains a preformed solvent-inaccessible cavity that binds artificial ligands that allosterically perturb the formation of the HIF heterodimer. To better understand how small molecules bind within this domain, we examined the structures and equilibrium and transition-state thermodynamics of HIF2{alpha} PAS-B with several artificial ligands using isothermal titration calorimetry, NMR exchange spectroscopy, and X-ray crystallography. Rapid association rates reveal that ligand binding is not dependent upon a slow conformational change in the protein to permit ligand access, despite the closed conformation observed in the NMR and crystal structures. Compensating enthalpic and entropic contributions to the thermodynamic barrier for ligand binding suggest a binding-competent transition state characterized by increased structural disorder. Finally, molecular dynamics simulations reveal conversion between open and closed conformations of the protein and pathways of ligand entry into the binding pocket.

  1. Noncontiguous domains of the alpha-factor receptor of yeasts confer ligand specificity.

    PubMed

    Sen, M; Marsh, L

    1994-01-14

    The Saccharomyces cerevisiae alpha-factor receptor has a 3400-fold higher affinity for the S. cerevisiae alpha-factor peptide (c-alpha-f) than for the Saccharomyces kluyveri alpha-factor peptide (k-alpha-f) as determined by competition for [3H] c-alpha-f binding. The S. kluyveri alpha-factor receptor has an approximately 2-fold higher affinity for k-alpha-f than for c-alpha-f. The S. kluyveri receptor gene (k-STE2) is incompletely regulated by S. cerevisiae mating type and poorly expressed on the surface of an S. cerevisiae mating type a strain. A chimeric receptor (c/k1) with amino acid residues 1-45 derived from S. cerevisiae and amino acid residues 46-427 from S. kluyveri exhibits the binding specificity of the S. kluyveri receptor. However, chimeric receptors containing residues 1-168 (c/k2) or 1-250 (c/k3) from S. cerevisiae and the remainder from the S. kluyveri receptor exhibit specificities similar to one another, but intermediate between the parent S. cerevisiae and S. kluyveri receptors. The relative ability of c-alpha-f and k-alpha-f to induce growth arrest in strains expressing chimeric receptors parallels relative affinity. Thus, two noncontiguous domains that include putative extracellular loops 1 and 3 and associated transmembrane segments, but exclude the extracellular NH2 terminus and loop 2, appear to contribute to alpha-factor receptor ligand specificity. COOH-terminal regions of the S. kluyveri receptor appear to confer a desensitization defect when expressed in S. cerevisiae. The S. cerevisiae receptor truncated at residue 296 retains ligand specificity for growth arrest.

  2. Phytol directly activates peroxisome proliferator-activated receptor {alpha} (PPAR{alpha}) and regulates gene expression involved in lipid metabolism in PPAR{alpha}-expressing HepG2 hepatocytes

    SciTech Connect

    Goto, Tsuyoshi; Takahashi, Nobuyuki; Kato, Sota; Egawa, Kahori; Ebisu, Shogo; Moriyama, Tatsuya; Fushiki, Tohru; Kawada, Teruo . E-mail: fat@kais.kyoto-u.ac.jp

    2005-11-18

    The peroxisome proliferator-activated receptor (PPAR) is one of the indispensable transcription factors for regulating lipid metabolism in various tissues. In our screening for natural compounds that activate PPAR using luciferase assays, a branched-carbon-chain alcohol (a component of chlorophylls), phytol, has been identified as a PPAR{alpha}-specific activator. Phytol induced the increase in PPAR{alpha}-dependent luciferase activity and the degree of in vitro binding of a coactivator, SRC-1, to GST-PPAR{alpha}. Moreover, the addition of phytol upregulated the expression of PPAR{alpha}-target genes at both mRNA and protein levels in PPAR{alpha}-expressing HepG2 hepatocytes. These findings indicate that phytol is functional as a PPAR{alpha} ligand and that it stimulates the expression of PPAR{alpha}-target genes in intact cells. Because PPAR{alpha} activation enhances circulating lipid clearance, phytol may be important in managing abnormalities in lipid metabolism.

  3. Peroxisome proliferator-activated receptor alpha (PPARalpha) agonists down-regulate alpha2-macroglobulin expression by a PPARalpha-dependent mechanism.

    EPA Science Inventory

    Peroxisome proliferator-activated receptor alpha (PPARα) regulates transcription of genes involved both in lipid and glucose metabolism as well as inflammation. Fibrates are PPARα ligands used to normalize lipid and glucose parameters and exert anti-inflammatory effects. Fibrates...

  4. Epiligrin, a component of epithelial basement membranes, is an adhesive ligand for alpha 3 beta 1 positive T lymphocytes

    PubMed Central

    1993-01-01

    The cutaneous T cell lymphomas (CTCL), typified by mycosis fungoides, and several chronic T cell mediated dermatoses are characterized by the migration of T lymphocytes into the epidermis (epidermotropism). Alternatively, other types of cutaneous inflammation (malignant cutaneous B cell lymphoma, CBCL, or lymphocytoma cutis, non-malignant T or B cell type) do not show evidence of epidermotropism. This suggests that certain T lymphocyte subpopulations are able to interact with and penetrate the epidermal basement membrane. We show here that T lymphocytes derived from patients with CTCL (HUT 78 or HUT 102 cells), adhere to the detergent-insoluble extracellular matrix prepared from cultured basal keratinocytes (HFK ECM). HUT cell adhesion to HFK ECM was inhibitable with monoclonal antibodies (mAbs) directed to the alpha 3 (P1B5) or beta 1 (P4C10) integrin receptors, and could be up- regulated by an activating anti-beta 1 mAb (P4G11). An inhibitory mAb, P3H9-2, raised against keratinocytes identified epiligrin as the ligand for alpha 3 beta 1 positive T cells in HFK ECM. Interestingly, two lymphocyte populations could be clearly distinguished relative to expression of alpha 3 beta 1 by flow cytometry analysis. Lymphokine activated killer cells, alloreactive cytotoxic T cells and T cells derived from patients with CTCL expressed high levels of alpha 3 beta 1 (alpha 3 beta 1high). Non-adherent peripheral blood mononuclear cells, acute T or B lymphocytic leukemias, or non-cutaneous T or B lymphocyte cell lines expressed low levels of alpha 3 beta 1 (alpha 3 beta 1low). Resting PBL or alpha 3 beta 1low T or B cell lines did not adhere to HFK ECM or purified epiligrin. However, adhesion to epiligrin could be up-regulated by mAbs which activate the beta 1 subunit indicating that alpha 3 beta 1 activity is a function of expression and affinity. In skin derived from patients with graft-vs.-host (GVH) disease, experimentally induced delayed hypersensitivity reactions, and CTCL

  5. Fatty Acid Amide Hydrolase (FAAH) Inhibition Enhances Memory Acquisition through Activation of PPAR-alpha Nuclear Receptors

    ERIC Educational Resources Information Center

    Mazzola, Carmen; Medalie, Julie; Scherma, Maria; Panlilio, Leigh V.; Solinas, Marcello; Tanda, Gianluigi; Drago, Filippo; Cadet, Jean Lud; Goldberg, Steven R.; Yasar, Sevil

    2009-01-01

    Inhibitors of fatty acid amide hydrolase (FAAH) increase endogenous levels of anandamide (a cannabinoid CB[subscript 1]-receptor ligand) and oleoylethanolamide and palmitoylethanolamide (OEA and PEA, ligands for alpha-type peroxisome proliferator-activated nuclear receptors, PPAR-alpha) when and where they are naturally released in the brain.…

  6. 3-Methylcholanthrene and other aryl hydrocarbon receptor agonists directly activate estrogen receptor alpha.

    PubMed

    Abdelrahim, Maen; Ariazi, Eric; Kim, Kyounghyun; Khan, Shaheen; Barhoumi, Rola; Burghardt, Robert; Liu, Shengxi; Hill, Denise; Finnell, Richard; Wlodarczyk, Bogdan; Jordan, V Craig; Safe, Stephen

    2006-02-15

    3-Methylcholanthrene (3MC) is an aryl hydrocarbon receptor (AhR) agonist, and it has been reported that 3MC induces estrogenic activity through AhR-estrogen receptor alpha (ER alpha) interactions. In this study, we used 3MC and 3,3',4,4',5-pentachlorobiphenyl (PCB) as prototypical AhR ligands, and both compounds activated estrogen-responsive reporter genes/gene products (cathepsin D) in MCF-7 breast cancer cells. The estrogenic responses induced by these AhR ligands were inhibited by the antiestrogen ICI 182780 and by the transfection of a small inhibitory RNA for ER alpha but were not affected by the small inhibitory RNA for AhR. These results suggest that 3MC and PCB directly activate ER alpha, and this was confirmed in a competitive ER alpha binding assay and in a fluorescence resonance energy transfer experiment in which PCB and 3MC induced CFP-ER alpha/YFP-ER alpha interactions. In a chromatin immunoprecipitation assay, PCB and 3MC enhanced ER alpha (but not AhR) association with the estrogen-responsive region of the pS2 gene promoter. Moreover, in AhR knockout mice, 3MC increased uterine weights and induced expression of cyclin D1 mRNA levels. These results show that PCB and 3MC directly activate ER alpha-dependent transactivation and extend the number of ligands that activate both AhR and ER alpha.

  7. A photoregulated ligand for the nuclear import receptor karyopherin alpha.

    PubMed

    Park, S B; Standaert, R F

    2001-12-01

    The ability to orchestrate the transport of proteins between nucleus and cytoplasm provides cells with a powerful regulatory mechanism. Selective translocation between these compartments is often used to propagate cellular signals, and it is an intimate part of the processes that control cell division, viral replication, and other cellular events. Therefore, precise experimental control over protein localization, through the agency of light, would provide a powerful tool for the study and manipulation of these events. To this end, a prototype photoregulated nuclear localization signal (NLS) was derived from a native NLS. A library of 30 mutants of the bipartite NLS from Xenopus laevis nucleoplasmin containing a novel, photoisomerizable amino acid was prepared by parallel, solid-phase synthesis and screened in vitro for binding to the nuclear import receptor karyopherin alpha, which mediates the nuclear import of cellular proteins. A single peptide was identified in which the cis and trans photoisomers bind the receptor differentially. The strategy used to obtain this peptide is systematic and empirical; therefore, it is potentially applicable to any peptide-receptor system.

  8. Correlation between chemical structure, receptor binding, and biological activity of some novel, highly active, 16 alpha, 17 alpha-acetal-substituted glucocorticoids.

    PubMed

    Dahlberg, E; Thalén, A; Brattsand, R; Gustafsson, J A; Johansson, U; Roempke, K; Saartok, T

    1984-01-01

    The affinity for the glucocorticoid receptor in rat skeletal muscle of some glucocorticoids with a new type of 16 alpha, 17 alpha-acetal substituent has been estimated and correlated to the glucocorticoid activities in three in vivo systems in rats. Budesonide (an approximately 1:1 mixture of the C(22) epimers of 11 beta, 21-dihydroxy-16 alpha, 17 alpha-[(22R,S)-propylmethylenedioxy]-pregna-1,4-diene-3,20-dione) and the isolated (22R)- and (22S)-epimers bound to the same binding site as the potent glucocorticoids dexamethasone (DEX) or triamcinolone 16 alpha, 17 alpha-acetonide (TA), but with even higher affinity than DEX or TA, despite the lack of a 9 alpha-fluoro atom in budesonide and its epimers. The (22R)-epimer was twice as active as the (22S)-epimer, 4 times more active than TA, and 14 times more active than DEX. The introduction of a 9 alpha-fluoro atom slightly decreased the binding affinity of the (22R)-epimer of budesonide, in contrast to the positive effect of 9 alpha-fluorination of, e.g., 16 alpha, 17 alpha-acetonides. The negative influence of 9 alpha-fluorination of the (22R)-epimer was partially reversed in the 6 alpha, 9 alpha-difluorinated (22R)-epimer. Nevertheless, the fluorinated compounds were more active than DEX and TA (8 and 11 times more active than DEX, and 2 and 3 times more active than TA, in case of the 9 alpha-fluoro- and 6 alpha, 9 alpha-difluoro-derivatives of the (22R)-epimer, respectively). Budesonide is metabolized mainly to 16 alpha-hydroxyprednisolone (11 beta, 16 alpha, 17 alpha, 21-tetrahydroxy-pregna-1,4-diene-3,20-dione) and 6 beta-hydroxy-budesonide. Both metabolites were very weak competitors for the ligand-binding sites on the receptor (3% and 6% of the affinity of DEX, respectively). The affinity for the receptor in vitro was closely correlated to the topical glucocorticoid activity in vivo for the 12 steroids compared (r = 0.98; R = 0.98), which supports the contention that in vitro tests for receptor affinity are

  9. The co-crystal structure of unliganded bovine alpha-thrombin and prethrombin-2: movement of the Tyr-Pro-Pro-Trp segment and active site residues upon ligand binding.

    PubMed Central

    Malkowski, M. G.; Martin, P. D.; Guzik, J. C.; Edwards, B. F.

    1997-01-01

    Unliganded bovine alpha-thrombin and prethrombin-2 have been co-crystallized, in space group P21212, using either ammonium sulfate or polyethylene glycol 2000 (PEG2K), and their structures determined at 2.2 A and 2.3 A, respectively. Initial phases were determined by molecular replacement and refined using XPLOR to final R factors of 0.187 (Rfree = 0.255) and 0.190 (Rfree = 0.282) for the salt and PEG2K models, respectively. The apo-enzyme form of bovine alpha-thrombin shows dramatic shifts in placement for the Tyr-Pro-Pro-Trp segment, for Glu-192, and for the catalytic residues His-57 and Ser-195, when compared to 4 thrombin complexes representing different states of catalysis, namely (1) the Michaelis complex (residues 7-19 of fibrinogen A alpha with a non-cleavable scissile bond), (2) enzyme-inhibitor complex (D-Phe-Pro-Arg chloromethylketone), (3) enzyme product complex (residues 7-16 of fibrinopeptide A), and (4) the exosite complex (residues 53-64 of hirudin). The structures of bovine and human prethrombin-2 are generally similar to one another (RMS deviation of 0.68 A) but differ significantly in the Arg-15/Ile-16 cleavage region and in the three activation domains, which are disordered in bovine prethrombin-2, analogous to that seen for trypsinogen. PMID:9232645

  10. Cis-interactions between Notch and its ligands block ligand-independent Notch activity

    PubMed Central

    Palmer, William Hunt; Jia, Dongyu; Deng, Wu-Min

    2014-01-01

    The Notch pathway is integrated into numerous developmental processes and therefore is fine-tuned on many levels, including receptor production, endocytosis, and degradation. Notch is further characterized by a twofold relationship with its Delta-Serrate (DSL) ligands, as ligands from opposing cells (trans-ligands) activate Notch, whereas ligands expressed in the same cell (cis-ligands) inhibit signaling. We show that cells without both cis- and trans-ligands can mediate Notch-dependent developmental events during Drosophila oogenesis, indicating ligand-independent Notch activity occurs when the receptor is free of cis- and trans-ligands. Furthermore, cis-ligands can reduce Notch activity in endogenous and genetically induced situations of elevated trans-ligand-independent Notch signaling. We conclude that cis-expressed ligands exert their repressive effect on Notch signaling in cases of trans-ligand-independent activation, and propose a new function of cis-inhibition which buffers cells against accidental Notch activity. DOI: http://dx.doi.org/10.7554/eLife.04415.001 PMID:25486593

  11. Activation of human alpha1 and alpha2 homomeric glycine receptors by taurine and GABA.

    PubMed

    De Saint Jan, D; David-Watine, B; Korn, H; Bregestovski, P

    2001-09-15

    1. Two ligand binding alpha subunits, alpha1 and alpha2, of the human (H) glycine receptor (GlyR) are involved at inhibitory synapses in the adult and neonatal spinal cord, respectively. The ability of homomeric alphaH1 and alphaH2 GlyRs to be activated by glycine, taurine and GABA was studied in Xenopus oocytes or in the human embryonic kidney HEK-293 cell line. 2. In outside-out patches from HEK cells, glycine, taurine and GABA activated both GlyRs with the same main unitary conductance, i.e. 85 +/- 3 pS (n = 6) for alphaH1, and 95 +/- 5 pS (n = 4) for alphaH2. 3. The sensitivity of both alphaH1 and alphaH2 GlyRs to glycine was highly variable. In Xenopus oocytes the EC50 for glycine (EC50gly) was between 25 and 280 microM for alphaH1 (n = 44) and between 46 and 541 microM for alphaH2 (n = 52). For both receptors, the highest EC50gly values were found on cells with low maximal glycine responses. 4. The actions of taurine and GABA were dependent on the EC50gly: (i) their EC50 values were linearly correlated to EC50gly, with EC50tau approximately 10 EC50gly and EC50GABA approximately 500-800 EC50gly; (ii) they could act either as full or weak agonists depending on the EC50gly. 5. The Hill coefficient (n(H)) of glycine remained stable regardless of the EC50gly whereas n(H) for taurine decreased with increasing EC50tau. 6. The degree of desensitization, evaluated by fast application of saturating concentrations of agonist on outside-out patches from Xenopus oocytes, was similar for glycine and taurine on both GlyRs and did not exceed 50 %. 7. Our data concerning the variations of EC50gly and the subsequent behaviour of taurine and GABA could be qualitatively described by the simple del Castillo-Katz scheme, assuming that the agonist gating constant varies whereas the binding constants are stable. However, the stability of the Hill coefficient for glycine was not explained by this model, suggesting that other mechanisms are involved in the modulation of EC50.

  12. A 3D structure model of integrin alpha 4 beta 1 complex: I. Construction of a homology model of beta 1 and ligand binding analysis.

    PubMed Central

    You, Tony J; Maxwell, David S; Kogan, Timothy P; Chen, Qi; Li, Jian; Kassir, Jamal; Holland, George W; Dixon, Richard A F

    2002-01-01

    It is well established that integrin alpha 4 beta 1 binds to the vascular cell adhesion molecule (VCAM) and fibronectin and plays an important role in signal transduction. Blocking the binding of VCAM to alpha 4 beta 1 is thought to be a way of controlling a number of disease processes. To better understand how various inhibitors might block the interaction of VCAM and fibronectin with alpha 4 beta 1, we began constructing a structure model for the integrin alpha 4 beta 1 complex. As the first step, we have built a homology model of the beta 1 subunit based on the I domain of the integrin CD11B subunit. The model, including a bound Mg(2+) ion, was optimized through a specially designed relaxation scheme involving restrained minimization and dynamics steps. The native ligand VCAM and two highly active small molecules (TBC772 and TBC3486) shown to inhibit binding of CS-1 and VCAM to alpha 4 beta 1 were docked into the active site of the refined model. Results from the binding analysis fit well with a pharmacophore model that was independently derived from active analog studies. A critical examination of residues in the binding site and analysis of docked ligands that are both potent and selective led to the proposal of a mechanism for beta 1/beta 7 ligand binding selectivity. PMID:11751331

  13. LAR, liprin alpha and the regulation of active zone morphogenesis.

    PubMed

    Stryker, Emily; Johnson, Karl G

    2007-11-01

    Active zones are protein-rich regions of neurons that act as sites of synaptic vesicle fusion and neurotransmitter release at the pre-synaptic terminus. Although the discovery that the receptor protein tyrosine phosphatase LAR and its cytoplasmic binding partner liprin alpha are essential for proper active zone formation is nearly a decade old, the underlying mechanisms are still poorly understood. Recent studies have identified a number of binding partners for both LAR and liprin alpha, several of which play key roles in active zone assembly. These include nidogen, dallylike and syndecan--extracellular ligands for LAR that regulate synapse morphogenesis. In addition, liprin-alpha-interacting proteins such as ERC2, RIM and the MALS/Veli-Cask-Mint1 complex cooperate to form a dense molecular scaffold at the active zone that is crucial for proper synaptic function. These studies allow us to propose testable models of LAR and liprin alpha function, and provide insights into the fundamental molecular mechanisms of synapse formation and stabilization.

  14. The thrombin receptor extracellular domain contains sites crucial for peptide ligand-induced activation.

    PubMed Central

    Bahou, W F; Coller, B S; Potter, C L; Norton, K J; Kutok, J L; Goligorsky, M S

    1993-01-01

    A thrombin receptor (TR) demonstrating a unique activation mechanism has recently been isolated from a megakaryocytic (Dami) cell line. To further study determinants of peptide ligand-mediated activation phenomenon, we have isolated, cloned, and stably expressed the identical receptor from a human umbilical vein endothelial cell (HUVEC) library. Chinese hamster ovary (CHO) cells expressing a functional TR (CHO-TR), platelets, and HUVECs were then used to specifically characterize alpha-thrombin- and peptide ligand-induced activation responses using two different antibodies: anti-TR34-52 directed against a 20-amino acid peptide spanning the thrombin cleavage site, and anti-TR1-160 generated against the NH2-terminal 160 amino acids of the TR expressed as a chimeric protein in Escherichia coli. Activation-dependent responses to both alpha-thrombin (10 nM) and peptide ligand (20 microM) were studied using fura 2-loaded cells and microspectrofluorimetry. Whereas preincubation of CHO-TR with anti-TR34-52 abolished only alpha-thrombin-induced [Ca2+]i transients, preincubation with anti-TR1-160 abrogated both alpha-thrombin- and peptide ligand-induced responses. This latter inhibitory effect was dose dependent and similar for both agonists, with an EC50 of approximately 90 micrograms/ml. Anti-TR1-160 similarly abolished peptide ligand-induced [Ca2+]i transients in platelets and HUVECs, whereas qualitatively different responses characterized by delayed but sustained elevations in [Ca2+]i transients were evident using alpha-thrombin. Platelet aggregation to low concentrations of both ligands was nearly abolished by anti-TR1-160, although some shape change remained; anti-TR34-52 only inhibited alpha-thrombin-induced aggregation. These data establish that a critical recognition sequence for peptide ligand-mediated receptor activation is contained on the NH2-terminal portion of the receptor, upstream from the first transmembrane domain. Furthermore, alpha

  15. PPAR{alpha} gene expression is up-regulated by LXR and PXR activators in the small intestine

    SciTech Connect

    Inoue, Jun; Satoh, Shin-ichi; Kita, Mariko; Nakahara, Mayuko; Hachimura, Satoshi; Miyata, Masaaki; Nishimaki-Mogami, Tomoko; Sato, Ryuichiro

    2008-07-11

    LXR, PXR, and PPAR{alpha} are members of a nuclear receptor family which regulate the expression of genes involved in lipid metabolism. Here, we show the administration of T0901317 stimulates PPAR{alpha} gene expression in the small intestine but not in the liver of both normal and FXR-null mice. The administration of LXR specific ligand GW3965, or PXR specific ligand PCN has the same effect, indicating that ligand-dependent activation of LXR and PXR, but not FXR, is responsible for the increased gene expression of PPAR{alpha} in the mouse small intestine.

  16. Competitive antagonism between the nicotinic allosteric potentiating ligand galantamine and kynurenic acid at alpha7* nicotinic receptors.

    PubMed

    Lopes, Cristiane; Pereira, Edna F R; Wu, Hui-Qiu; Purushottamachar, Puranik; Njar, Vincent; Schwarcz, Robert; Albuquerque, Edson X

    2007-07-01

    Galantamine, a drug used to treat Alzheimer's disease, is a nicotinic allosteric potentiating ligand, and kynurenic acid (KYNA), a neuroactive metabolite of the kynurenine pathway, is an endogenous noncompetitive inhibitor of alpha7* nicotinic receptors (nAChRs) [the asterisk next to the nAChR subunit is intended to indicate that the exact subunit composition of the receptor is not known (Pharmacol Rev 51:397-401, 1999)]. Here, possible interactions between KYNA and galantamine at alpha7* nAChRs were examined in vitro and in vivo. In the presence of tetrodotoxin (TTX), approximately 85% of cultured hippocampal neurons responded to choline (0.3-30 mM) with alpha7* nAChR-subserved whole-cell (type IA) currents. In the absence of TTX and in the presence of glutamate receptor antagonists, choline triggered inhibitory postsynaptic currents (IPSCs) by activating alpha7* nAChRs on GABAergic neurons synapsing onto the neurons under study. Galantamine (1-10 microM) potentiated, whereas KYNA (10 nM-1 mM) inhibited, choline-triggered responses. Galantamine (1 microM), applied before KYNA, shifted to the right the concentration-response relationship for KYNA to inhibit type IA currents, increasing the IC(50) of KYNA from 13.9 +/- 8.3 to 271 +/- 131 microM. Galantamine, applied before or after KYNA, antagonized inhibition of choline-triggered IPSCs by KYNA. Local infusion of KYNA (100 nM) in the rat striatum reduced extracellular dopamine levels in vivo. This effect resulted from alpha7* nAChR inhibition and was blocked by coapplied galantamine (1-5 microM). It is concluded that galantamine competitively antagonizes the actions of KYNA on alpha7* nAChRs. Reducing alpha7* nAChR inhibition by endogenous KYNA may be an important determinant of the effectiveness of galantamine in neurological and psychiatric disorders associated with decreased alpha7* nAChR activity in the brain.

  17. Validated ligand mapping of ACE active site

    NASA Astrophysics Data System (ADS)

    Kuster, Daniel J.; Marshall, Garland R.

    2005-08-01

    Crystal structures of angiotensin-converting enzyme (ACE) complexed with three inhibitors (lisinopril, captopril, enalapril) provided experimental data for testing the validity of a prior active site model predicting the bound conformation of the inhibitors. The ACE active site model - predicted over 18 years ago using a series of potent ACE inhibitors of diverse chemical structure - was recreated using published data and commercial software. Comparison between the predicted structures of the three inhibitors bound to the active site of ACE and those determined experimentally yielded root mean square deviation (RMSD) values of 0.43-0.81 Å, among the distances defining the active site map. The bound conformations of the chemically relevant atoms were accurately deduced from the geometry of ligands, applying the assumption that the geometry of the active site groups responsible for binding and catalysis of amide hydrolysis was constrained. The mapping of bound inhibitors at the ACE active site was validated for known experimental compounds, so that the constrained conformational search methodology may be applied with confidence when no experimentally determined structure of the enzyme yet exists, but potent, diverse inhibitors are available.

  18. Structural Basis for Iloprost as a Dual Peroxisome Proliferator-activated Receptor [alpha/delta] Agonist

    SciTech Connect

    Jin, Lihua; Lin, Shengchen; Rong, Hui; Zheng, Songyang; Jin, Shikan; Wang, Rui; Li, Yong

    2012-03-15

    Iloprost is a prostacyclin analog that has been used to treat many vascular conditions. Peroxisome proliferator-activated receptors (PPARs) are ligand-regulated transcription factors with various important biological effects such as metabolic and cardiovascular physiology. Here, we report the crystal structures of the PPAR{alpha} ligand-binding domain and PPAR{delta} ligand-binding domain bound to iloprost, thus providing unambiguous evidence for the direct interaction between iloprost and PPARs and a structural basis for the recognition of PPAR{alpha}/{delta} by this prostacyclin analog. In addition to conserved contacts for all PPAR{alpha} ligands, iloprost also initiates several specific interactions with PPARs using its unique structural groups. Structural and functional studies of receptor-ligand interactions reveal strong functional correlations of the iloprost-PPAR{alpha}/{delta} interactions as well as the molecular basis of PPAR subtype selectivity toward iloprost ligand. As such, the structural mechanism may provide a more rational template for designing novel compounds targeting PPARs with more favorable pharmacologic impact based on existing iloprost drugs.

  19. Ectodomain cleavage of the EGF ligands HB-EGF, neuregulin1-beta, and TGF-alpha is specifically triggered by different stimuli and involves different PKC isoenzymes.

    PubMed

    Herrlich, Andreas; Klinman, Eva; Fu, Jonathan; Sadegh, Cameron; Lodish, Harvey

    2008-12-01

    Metalloproteinase cleavage of transmembrane proteins (ectodomain cleavage), including the epidermal growth factor (EGF) ligands heparin-binding EGF-like growth factor (HB-EGF), neuregulin (NRG), and transforming growth factor-alpha (TGF-alpha), is important in many cellular signaling pathways and is disregulated in many diseases. It is largely unknown how physiological stimuli of ectodomain cleavage--hypertonic stress, phorbol ester, or activation of G-protein-coupled receptors [e.g., by lysophosphatidic acid (LPA)]--are molecularly connected to metalloproteinase activation. To study this question, we developed a fluorescence-activated cell sorting (FACS)- based assay that measures cleavage of EGF ligands in single living cells. EGF ligands expressed in mouse lung epithelial cells are differentially and specifically cleaved depending on the stimulus. Inhibition of protein kinase C (PKC) isoenzymes or metalloproteinase inhibition by batimastat (BB94) showed that different regulatory signals are used by different stimuli and EGF substrates, suggesting differential effects that act on the substrate, the metalloproteinase, or both. For example, hypertonic stress led to strong cleavage of HB-EGF and NRG but only moderate cleavage of TGF-alpha. HB-EGF, NRG, and TGF-alpha cleavage was not dependent on PKC, and only HB-EGF and NRG cleavage were inhibited by BB94. In contrast, phorbol 12-myristate-13-acetate (TPA) -induced cleavage of HB-EGF, NRG, and TGF-alpha was dependent on PKC and sensitive to BB94 inhibition. LPA led to significant cleavage of only NRG and TGF-alpha and was inhibited by BB94; only LPA-induced NRG cleavage required PKC. Surprisingly, specific inhibition of atypical PKCs zeta and iota [not activated by diacylglycerol (DAG) and calcium] significantly enhanced TPA-induced NRG cleavage. Employed in a high-throughput cloning strategy, our cleavage assay should allow the identification of candidate proteins involved in signal transduction of different

  20. Crystal structure of the extracellular segment of integrin {alpha}V{beta}3 in complex with an Arg-Gly-Asp ligand.

    SciTech Connect

    Xiong, J.-P.; Stehle, T.; Zhang, R.; Joachimiak, A.; Goodman, S.; Arnaout, M. A.; Biosciences Division; Massachusetts General Hospital; Harvard Medical School

    2002-04-05

    The structural basis for the divalent cation-dependent binding of heterodimeric alpha beta integrins to their ligands, which contain the prototypical Arg-Gly-Asp sequence, is unknown. Interaction with ligands triggers tertiary and quaternary structural rearrangements in integrins that are needed for cell signaling. Here we report the crystal structure of the extracellular segment of integrin alpha Vbeta 3 in complex with a cyclic peptide presenting the Arg-Gly-Asp sequence. The ligand binds at the major interface between the alpha V and beta 3 subunits and makes extensive contacts with both. Both tertiary and quaternary changes are observed in the presence of ligand. The tertiary rearrangements take place in beta A, the ligand-binding domain of beta 3; in the complex, beta A acquires two cations, one of which contacts the ligand Asp directly and the other stabilizes the ligand-binding surface. Ligand binding induces small changes in the orientation of alpha V relative to beta 3.

  1. Automated docking of alpha-(1-->4)- and alpha-(1-->6)-linked glucosyl trisaccharides and maltopentaose into the soybean beta-amylase active site.

    PubMed

    Rockey, W M; Laederach, A; Reilly, P J

    2000-08-01

    The Lamarckian genetic algorithm of AutoDock 3.0 was used to dock alpha-maltotriose, methyl alpha-panoside, methyl alpha-isopanoside, methyl alpha-isomaltotrioside, methyl alpha-(6(1)-alpha-glucopyranosyl)-maltoside, and alpha-maltopentaose into the closed and, except for alpha-maltopentaose, into the open conformation of the soybean beta-amylase active site. In the closed conformation, the hinged flap at the mouth of the active site closes over the substrate. The nonreducing end of alpha-maltotriose docks preferentially to subsites -2 or +1, the latter yielding nonproductive binding. Some ligands dock into less optimal conformations with the nonreducing end at subsite -1. The reducing-end glucosyl residue of nonproductively-bound alpha-maltotriose is close to residue Gln194, which likely contributes to binding to subsite +3. In the open conformation, the substrate hydrogen-bonds with several residues of the open flap. When the flap closes, the substrate productively docks if the nonreducing end is near subsites -2 or -1. Trisaccharides with alpha-(1-->6) bonds do not successfully dock except for methyl alpha-isopanoside, whose first and second glucosyl rings dock exceptionally well into subsites -2 and -1. The alpha-(1-->6) bond between the second and third glucosyl units causes the latter to be improperly positioned into subsite +1; the fact that isopanose is not a substrate of beta-amylase indicates that binding to this subsite is critical for hydrolysis.

  2. Pre-clinical validation of a novel alpha-7 nicotinic receptor radiotracer, [(3)H]AZ11637326: target localization, biodistribution and ligand occupancy in the rat brain.

    PubMed

    Maier, Donna L; Hill, Geraldine; Ding, Min; Tuke, David; Einstein, Emily; Gurley, David; Gordon, John C; Bock, Mary J; Smith, Jeff S; Bialecki, Russell; Eisman, Mark; Elmore, Charles S; Werkheiser, Jennifer L

    2011-01-01

    The alpha-7 neuronal nicotinic receptor is a novel pharmacological target for psychiatric and cognitive disorders. Selective radiotracer tools for pre-clinical receptor occupancy can facilitate the interpretation of the biological actions of small molecules at a target receptor. We discovered a high affinity nicotinic alpha-7 subtype-selective ligand, AZ11637326, with physical-chemical and pharmacokinetic properties suitable for an in vivo radioligand tool. [(3)H]AZ11637326 synthesis by tritiodehalogenation of the corresponding tribromide precursor yielded a high specific activity radiotracer with high affinity alpha-7 receptor binding in the rat hippocampus determined by autoradiography (Kd = 0.2 nM). When [(3)H]AZ11637326 was administered to rats by intravenous bolus, rapid uptake was measured in the brain followed by a 3-4 fold greater specific binding in regions containing the alpha-7 receptor (frontal cortex, hippocampus, hypothalamus and midbrain) when compared to non-target regions (striatum and cerebellum). Systemic administration of the high affinity alpha-7 receptor antagonist, methyllycaconitine (MLA), or pretreatment with alpha-7 selective agonists (AR-R17779, PyrQTC, DBCO-4-POM, and DBCO-3-POM) significantly blocked the alpha-7 specific binding of [(3)H]AZ11637326 in the rat brain. The rank order of ligand ED(50) values for in vivo alpha-7 receptor occupancy in rat hippocampus was: DBCO-4-POM > DBCO-3-POM ∼ MLA > PyrQTC > AR-R17779. The occupancy affinity shift was consistent with in vitro binding affinity in autoradiography. Our studies established the optimal conditions for [(3)H]AZ11637326 in vivo specific binding in the rat brain and support the use of [(3)H]AZ11637326 as a pre-clinical tool for assessment of novel alpha-7 compounds in drug discovery.

  3. Activating Cell Death Ligand Signaling Through Proteasome Inhibition

    DTIC Science & Technology

    2009-05-01

    Activating Cell Death Ligand Signaling Through Proteasome Inhibition PRINCIPAL INVESTIGATOR: Steven R Schwarze...SUBTITLE Activating Cell Death Ligand Signaling Through 5a. CONTRACT NUMBER Proteasome Inhibition 5b. GRANT NUMBER W81XWH-08-1-0392 5c...proteasome inhibition can act as an anti-neoplastic agent in vivo by sensitizing cancer cells to cell death ligands in the tumor microenvironment

  4. Ligand interaction of human alpha 2-macroglobulin-alpha 2-macroglobulin receptor studied by partitioning in aqueous two-phase systems.

    PubMed

    Birkenmeier, G; Kunath, M

    1996-05-17

    Alpha 2-macroglobulin (alpha 2-M) is a major proteinase inhibitor in human blood and tissue. Besides its antiproteolytic potential, alpha 2-M was found to modulate antigen- and mitogen-driven immune responses and cell growth by binding and transporting distinct cytokines, growth factors and hormones. The inhibitor is cleared from circulation by binding to a multifunctional cellular receptor present on different cell types. Alpha 2-M, as well as its receptor, are capable of binding a variety of ligands. In the present study we have applied aqueous two-phase systems to analyze the interaction of IL-1 beta and alpha 2-M receptor to different forms of alpha 2-M. The partition of IL-1 beta was changed by addition of transformed alpha 2-M to the two-phase systems rather than by the native inhibitor. The interaction between IL-1 beta and alpha 2-M was enhanced by divalent cations. In addition, the complex formation between 125I-labelled receptor and alpha 2-M could clearly be demonstrated by partitioning. In the presence of divalent cations, transformed alpha 2-M, in contrast to the native inhibitor, effectively changed the partition of the receptor. However, the observed alteration of the partition coefficient was found to be less compared with the values obtained by partitioning of the receptor in the presence of whole plasma containing the inhibitor in equivalent concentrations. The results indicate that other components of the plasma exist which competitively bind to the receptor but independent of Ca2+-ions.

  5. Peroxisome proliferator-activated receptor alpha target genes.

    PubMed

    Rakhshandehroo, Maryam; Knoch, Bianca; Müller, Michael; Kersten, Sander

    2010-01-01

    The peroxisome proliferator-activated receptor alpha (PPARα) is a ligand-activated transcription factor involved in the regulation of a variety of processes, ranging from inflammation and immunity to nutrient metabolism and energy homeostasis. PPARα serves as a molecular target for hypolipidemic fibrates drugs which bind the receptor with high affinity. Furthermore, PPARα binds and is activated by numerous fatty acids and fatty acid-derived compounds. PPARα governs biological processes by altering the expression of a large number of target genes. Accordingly, the specific role of PPARα is directly related to the biological function of its target genes. Here, we present an overview of the involvement of PPARα in lipid metabolism and other pathways through a detailed analysis of the different known or putative PPARα target genes. The emphasis is on gene regulation by PPARα in liver although many of the results likely apply to other organs and tissues as well.

  6. Peroxisome Proliferator-Activated Receptor Alpha Target Genes

    PubMed Central

    Rakhshandehroo, Maryam; Knoch, Bianca; Müller, Michael; Kersten, Sander

    2010-01-01

    The peroxisome proliferator-activated receptor alpha (PPARα) is a ligand-activated transcription factor involved in the regulation of a variety of processes, ranging from inflammation and immunity to nutrient metabolism and energy homeostasis. PPARα serves as a molecular target for hypolipidemic fibrates drugs which bind the receptor with high affinity. Furthermore, PPARα binds and is activated by numerous fatty acids and fatty acid-derived compounds. PPARα governs biological processes by altering the expression of a large number of target genes. Accordingly, the specific role of PPARα is directly related to the biological function of its target genes. Here, we present an overview of the involvement of PPARα in lipid metabolism and other pathways through a detailed analysis of the different known or putative PPARα target genes. The emphasis is on gene regulation by PPARα in liver although many of the results likely apply to other organs and tissues as well. PMID:20936127

  7. Disulfide Bond Requirements for Active Wnt Ligands*

    PubMed Central

    MacDonald, Bryan T.; Hien, Annie; Zhang, Xinjun; Iranloye, Oladoyin; Virshup, David M.; Waterman, Marian L.; He, Xi

    2014-01-01

    Secreted Wnt lipoproteins are cysteine-rich and lipid-modified morphogens that bind to the Frizzled (FZD) receptor and LDL receptor-related protein 6 (LRP6). Wnt engages FZD through protruding thumb and index finger domains, which are each assembled from paired β strands secured by disulfide bonds and grasp two sides of the FZD ectodomain. The importance of Wnt disulfide bonds has been assumed but uncharacterized. We systematically analyzed cysteines and associated disulfide bonds in the prototypic Wnt3a. Our data show that mutation of any individual cysteine of Wnt3a results in covalent Wnt oligomers through ectopic intermolecular disulfide bond formation and diminishes/abolishes Wnt signaling. Although individual cysteine mutations in the amino part of the saposin-like domain and in the base of the index finger are better tolerated and permit residual Wnt3a secretion/activity, those in the amino terminus, the thumb, and at the tip of the index finger are incompatible with secretion and/or activity. A few select double cysteine mutants based on the disulfide bond pattern restore Wnt secretion/activity. Further, a double cysteine mutation at the index finger tip results in a Wnt3a with normal secretion but minimal FZD binding and dominant negative properties. Our results experimentally validate predictions from the Wnt crystal structure and highlight critical but different roles of the saposin-like and cytokine-like domains, including the thumb and the index finger in Wnt folding/secretion and FZD binding. Finally, we modified existing expression vectors for 19 epitope-tagged human WNT proteins by removal of a tag-supplied ectopic cysteine, thereby generating tagged WNT ligands active in canonical and non-canonical signaling. PMID:24841207

  8. SUMOylation of ROR{alpha} potentiates transcriptional activation function

    SciTech Connect

    Hwang, Eun Ju; Lee, Ji Min; Jeong, Jiyeong; Park, Joo Hyeon; Yang, Young; Lim, Jong-Seok; Kim, Jung Hwa; Baek, Sung Hee; Kim, Keun Il

    2009-01-16

    SUMOylation regulates a variety of cellular processes, including control of transcriptional activities of nuclear receptors. Here, we present SUMOylation of orphan nuclear receptor, ROR{alpha} by both SUMO-1 and SUMO-2. SUMOylation of ROR{alpha} occurred on the 240th lysine residue at the hinge region of human protein. PIAS family members, PIASx{alpha}, PIAS3, and PIASy, increased SUMOylation of ROR{alpha}, whereas SENP2 specifically removed SUMO from ROR{alpha}. SUMOylation-defective mutant form of ROR{alpha} exhibited decreased transcriptional activity on ROR{alpha}-responsive promoters indicating that SUMOylation may positively regulate transcriptional function of ROR{alpha}.

  9. Alpha Radiolysis of Nuclear Solvent Extraction Ligands Used for An(III) and Ln(III) Separations

    SciTech Connect

    Mezyk, Stephen P.; Mincher, Bruce J.; Nilsson, Mikael

    2016-08-01

    This document is the final report for the Nuclear Energy Universities Program (NEUP) grant 10-910 (DE-AC07-05ID14517) “Alpha Radiolysis of Nuclear Solvent Extraction Ligands used for An(III) and Ln(III) Separations”. The goal of this work was to obtain a quantitative understanding of the impacts of both low Linear Energy Transfer (LET, gamma-rays) and high LET (alpha particles) radiation chemistry occurring in future large-scale separations processes. This quantitative understanding of the major radiation effects on diluents and ligands is essential for optimal process implementation, and could result in significant cost savings in the future.

  10. The stromal cell-derived factor-1alpha dependent migration of human cord blood CD34 haematopoietic stem and progenitor cells switches from protein kinase C (PKC)-alpha dependence to PKC-alpha independence upon prolonged culture in the presence of Flt3-ligand and interleukin-6.

    PubMed

    Kasenda, Benjamin; Kassmer, Susannah H; Niggemann, Bernd; Schiermeier, Sven; Hatzmann, Wolfgang; Zänker, Kurt S; Dittmar, Thomas

    2008-09-01

    Addition of the inflammatory cytokine interleukin (IL)-6 to the culture medium of human cord blood haematopoietic stem and progenitor cells (HSPCs) has been shown to lead to an altered stromal cell-derived factor-1alpha-dependent migratory phenotype. This study investigated whether this effect was attributed to a differential engagement of protein kinase C (PKC) isotypes. The migratory activity of both Flt3-ligand and Flt3-ligand/IL-6 cultured cord blood HSPCs was PKC-alpha dependent on day 1, but PKC-alpha independent after 5 d of cultivation. PKC-alpha expression was not down-regulated in cells cultured for 5 d indicating a switch of signalling molecules directing cell migration.

  11. Lucid dreaming and alpha activity: a preliminary report.

    PubMed

    Ogilvie, R D; Hunt, H T; Tyson, P D; Lucescu, M L; Jeakins, D B

    1982-12-01

    10 good dream recallers spent 2 nights in the sleep lab during which they were awakened 4 times per night from REM sleep, twice during their highest alpha activity in REM, and twice during low REM alpha. 5 were given alpha feedback training prior to sleep onset. Arousals from high alpha REM sleep yielded significantly higher lucidity ratings. Alpha feedback had no effect upon lucidity or REM alpha levels. Similarities between lucid dreams and meditative phenomena are discussed.

  12. Fine mapping of inhibitory anti-alpha5 monoclonal antibody epitopes that differentially affect integrin-ligand binding.

    PubMed Central

    Burrows, L; Clark, K; Mould, A P; Humphries, M J

    1999-01-01

    The high-affinity interaction of integrin alpha5beta1 with the central cell-binding domain of fibronectin requires both the Arg-Gly-Asp (RGD) sequence (in the tenth type III repeat) and a second site Pro-His-Ser-Arg-Asn (PHSRN) in the adjacent ninth type III repeat, which synergizes with RGD. Arg-Arg-Glu-Thr-Ala-Trp-Ala (RRETAWA) is a novel peptidic ligand for alpha5beta1, identified by phage display, which blocks alpha5beta1-mediated cell adhesion to fibronectin. A key question is the location of the binding sites for these ligand sequences within the integrin. In this study we have identified residues that form part of the epitopes of three inhibitory anti-alpha5 monoclonal antibodies (mAbs): 16, P1D6 and SNAKA52. These mAbs have distinct functional properties. mAb 16 blocks the recognition of RGD and RRETAWA, whereas P1D6 blocks binding to the synergy sequence. The binding of SNAKA52 is inhibited by anti-beta1 mAbs, indicating that its epitope is close to the interface between the alpha and beta subunits. Residues in human alpha5 were replaced with the corresponding residues in mouse alpha5 by site-directed mutagenesis; wild-type or mutant human alpha5 was expressed on the surface of alpha5-deficient Chinese hamster ovary cells. mAb binding was assessed by flow cytometry and by adhesion to the central cell-binding domain of fibronectin or RRETAWA by cell attachment assay. All three epitopes were located to different putative loops in the N-terminal domain of alpha5. As expected, disruption of these epitopes had no effect on ligand recognition by alpha5beta1. The locations of these epitopes are consistent with the beta-propeller model for integrin alpha-subunit structure and allow us to propose a topological image of the integrin-ligand complex. PMID:10567237

  13. Regulation of ligands for the NKG2D activating receptor

    PubMed Central

    Raulet, David H.; Gasser, Stephan; Gowen, Benjamin G.; Deng, Weiwen; Jung, Heiyoun

    2014-01-01

    NKG2D is an activating receptor expressed by all NK cells and subsets of T cells. It serves as a major recognition receptor for detection and elimination of transformed and infected cells and participates in the genesis of several inflammatory diseases. The ligands for NKG2D are self-proteins that are induced by pathways that are active in certain pathophysiological states. NKG2D ligands are regulated transcriptionally, at the level of mRNA and protein stability, and by cleavage from the cell surface. In some cases, ligand induction can be attributed to pathways that are activated specifically in cancer cells or infected cells. We review the numerous pathways that have been implicated in the regulation of NKG2D ligands, discuss the pathologic states in which those pathways are likely to act, and attempt to synthesize the findings into general schemes of NKG2D ligand regulation in NK cell responses to cancer and infection. PMID:23298206

  14. The role of phosphorylation in activation of the alpha 6A beta 1 laminin receptor.

    PubMed

    Hogervorst, F; Kuikman, I; Noteboom, E; Sonnenberg, A

    1993-09-05

    The phorbol ester phorbol 12-myristate 13-acetate (PMA) induces phosphorylation of serine residues in the cytoplasmic domain of the alpha 6A integrin subunit, as well as activation of the alpha 6A beta 1 laminin receptor. We examined whether phosphorylation correlates with the induction of high affinity binding of laminin by the alpha 6A beta 1 receptor. Two potential phosphorylation sites for protein kinase C, serine 1041 and serine 1048, are present in the cytoplasmic domain of the alpha 6A subunit. We introduced point mutations into the alpha 6A cDNA, replacing either one or both of the serine residues with alanine. Wild-type and mutant alpha 6A cDNAs were transfected into K562 cells. All alpha 6A subunit mutants were expressed at levels similar to those of wild-type alpha 6A and formed heterodimers with endogenous beta 1. Analysis of the phosphorylation state of wild-type and mutant alpha 6A subunits in resting K562 cells and after treatment with PMA showed that serine 1041, but not serine 1048, is the target residue of PMA-induced phosphorylation. Cells expressing alpha 6A mutant subunits or wild-type alpha 6A transfectants all bound laminin in the presence, but not in the absence of PMA; however, the extent of binding differed. Cells transfected with alpha 6A containing the serine to alanine mutation showed a 2-3-fold higher binding to laminin than cells transfected with alpha 6A containing serine 1041. The results indicate that phosphorylation of the alpha 6A cytoplasmic domain is not required for the induction of high affinity of the alpha 6A beta 1 receptor by PMA, and suggest that, in contrast, it may reduce the affinity of this integrin for ligand.

  15. Chaperone-like activities of {alpha}-synuclein: {alpha}-Synuclein assists enzyme activities of esterases

    SciTech Connect

    Ahn, Misun; Kim, SeungBum; Kang, Mira; Ryu, Yeonwoo . E-mail: ywryu@ajou.ac.kr; Doohun Kim, T. . E-mail: doohunkim@ajou.ac.kr

    2006-08-11

    {alpha}-Synuclein, a major constituent of Lewy bodies (LBs), has been implicated to play a critical role in the pathogenesis of Parkinson's disease (PD), although the physiological function of {alpha}-synuclein has not yet been known. Here we have shown that {alpha}-synuclein, which has no well-defined secondary or tertiary structure, can protect the enzyme activity of microbial esterases against stress conditions such as heat, pH, and organic solvents. In particular, the flexibility of {alpha}-synuclein and its C-terminal region seems to be important for complex formation, but the structural integrity of the C-terminal region may not be required for stabilization of enzyme activity. In addition, atomic force microscopy (AFM) and in vivo enzyme assays showed highly specific interactions of esterases with {alpha}-synuclein. Our results indicate that {alpha}-synuclein not only protects the enzyme activity of microbial esterases in vitro, but also can stabilize the active conformation of microbial esterases in vivo.

  16. Regulation of ligands for the activating receptor NKG2D

    PubMed Central

    Mistry, Anita R; O'Callaghan, Chris A

    2007-01-01

    The outcome of an encounter between a cytotoxic cell and a potential target cell depends on the balance of signals from inhibitory and activating receptors. Natural Killer group 2D (NKG2D) has recently emerged as a major activating receptor on T lymphocytes and natural killer cells. In both humans and mice, multiple different genes encode ligands for NKG2D, and these ligands are non-classical major histocompatibility complex class I molecules. The NKG2D–ligand interaction triggers an activating signal in the cell expressing NKG2D and this promotes cytotoxic lysis of the cell expressing the ligand. Most normal tissues do not express ligands for NKG2D, but ligand expression has been documented in tumour and virus-infected cells, leading to lysis of these cells. Tight regulation of ligand expression is important. If there is inappropriate expression in normal tissues, this will favour autoimmune processes, whilst failure to up-regulate the ligands in pathological conditions would favour cancer development or dissemination of intracellular infection. PMID:17614877

  17. Development of a radioiodinated ligand for characterising. cap alpha. /sub 1/-adrenoceptors. [Pentolamine and 2 BETA-(4-hydroxyphenyl)-ethylaminomethyl)-tetralone

    SciTech Connect

    Adams, A.; Jarrott, B.

    1982-03-15

    Two ..cap alpha..-adrenoceptor antagonists, phentolamine and 2-(..beta..-(4-hydroxyphenyl)-ethylaminomethyl)-tetralone (BE 2254) which are phenolic derivatives were radioiodinated after chloramine-T oxidation of Na/sup 125/I and the labelled material isolated by chromatography. /sup 125/I-Phentolamine does not bind selectively to ..cap alpha..-adrenoceptors in guinea pig brain whereas the /sup 125/I-BE 2254 derivative binds rapidly, reversibly and with high affinity to these receptors with a K/sub d/ of 230 pM. At low concentrations of /sup 125/I-BE 2254 (< 100 pM) approx. 90% of the bound radioligand is specifically bound and under these conditions drug displacement studies show that the ligand binds predominantly to the ..cap alpha../sub 1/ subclass of adrenoceptors. Binding measurements to kidney and smooth muscle membrane preparations indicate that /sup 125/I-BE 2254 may also be a useful tool in the study of ..cap alpha..-adrenoceptors in peripheral tissues. The high specific activity of /sup 125/I-BE 2254 permits the use of minimal quantities of membrane material for receptor assay and ligand displacement measurements, e.g. 250 ..mu..g per assay tube, and this provides a significant advantage over the use of existing radioligands such as /sup 3/H-prazosin which requires approx. 40 times as much tissue.

  18. Isolation of a highly specific ligand for the alpha 5 beta 1 integrin from a phage display library

    PubMed Central

    1994-01-01

    Our previous studies showed that the alpha 5 beta 1 integrin selects cysteine pair-containing RGD peptides from a phage display library based on a random hexapeptide. We have therefore searched for more selective peptides for this integrin using a larger phage display library, where heptapeptides are flanked by cysteine residues, thus making the inserts potentially cyclic. Most of the phage sequences that bound to alpha 5 beta 1 (69 of 125) contained the RGD motif. Some of the heptapeptides contained an NGR motif. As the NGR sequence occurs in the cell-binding region of the fibronectin molecule, this sequence could contribute to the specific recognition of fibronectin by alpha 5 beta 1. Selection for high affinity peptides for alpha 5 beta 1 surprisingly yielded a sequence RRETAWA that does not bear obvious resemblance to known integrin ligand sequences. The synthetic cyclic peptide GACRRETAWACGA (*CRRETAWAC*) was a potent inhibitor of alpha 5 beta 1-mediated cell attachment to fibronectin. This peptide is nearly specific for the alpha 5 beta 1 integrin, because much higher concentrations were needed to inhibit the alpha v beta 1 integrin, and there was no effect on alpha v beta 3- and alpha v beta 5-mediated cell attachment to vitronectin. The peptide also did not bind to the alpha IIb beta 3 integrin. *CRRETAWAC* appears to interact with the same or an overlapping binding site in alpha 5 beta 1 as RGD, because cell attachment to *CRRETAWAC* coated on plastic was divalent cation dependent and could be blocked by an RGD-containing peptide. These results reveal a novel binding specificity in the alpha 5 beta 1 integrin. PMID:7507494

  19. Synthesis of four stereoisomers of 1-azabiocyclo[2.2.2]OCT-3-YL-{alpha}-fluoroalkyl-{alpha}-hydroxy-{alpha}-phenylacetate (FQNPe): Potential imaging ligands for the muscarinic-cholinergic receptor (m-AChR) by PET

    SciTech Connect

    Luo, H.; McPherson, D.W.; Knapp, F.F. Jr.

    1996-10-01

    Earlier studies with the racemic 1-azabiocyclo[2.2.2]oct-3-yl {alpha}-fluoroalkyl-{alpha}-hydroxy-{alpha}-phenylacetate (FQNPe) mixture had demonstrated high in vitro binding affinity for the muscarinic-cholinergic receptor (m-AChR). Pre-treatment of rats with this new agent significantly blocked receptor localization of subsequently injected [I-131]-Z-(-,-)-IQNP, which is an established high affinity m-AChR ligand. Syntheses and characterization of the four FQNPe stereoisomers: (-)(-) FQNPe, (-)(+) FQNPe, (+)(-) FQNPe, and (+)(+) FQNPe will be presented. The interesting NMR spectra of the diastereomeric salts formed in the resolution of racemic {alpha}-(1-chloropent-5-yl)-{alpha}-hydroxy {alpha}-phenylacetic acid will also be discussed.

  20. The human alpha 2-macroglobulin receptor: identification of a 420-kD cell surface glycoprotein specific for the activated conformation of alpha 2-macroglobulin

    PubMed Central

    1990-01-01

    Ligand affinity chromatography was used to purify a cell surface alpha 2-macroglobulin (alpha 2M) receptor. Detergent extracts of human placenta were applied to an affinity matrix consisting of alpha 2M, previously reacted with methylamine, coupled to Sepharose. Elution with EDTA specifically released polypeptides with apparent molecular masses of 420 and 39 kD. In some preparations, small amounts of a 90-kD polypeptide were observed. The 420- and 39-kD polypeptides appear specific for the forms of alpha 2M activated by reaction with proteinases or methylamine and do not bind to an affinity matrix consisting of native alpha 2M coupled to Sepharose. Separation of these two polypeptides was accomplished by anion exchange chromatography, and binding activity was exclusively associated with the 420-kD polypeptide. The purified 420-kD protein binds to the conformationally altered forms of alpha 2M that are known to specifically interact with alpha 2M receptors and does not bind to native alpha 2M. Binding of the 420-kD polypeptide to immobilized wheat germ agglutinin indicates that this polypeptide is a glycoprotein. The cell surface localization of the 420-kD glycoprotein was confirmed by affinity chromatography of extracts from surface radioiodinated fibroblasts. These properties suggest that the 420-kD polypeptide is a cell surface receptor for the activated forms of alpha 2M. PMID:1691187

  1. Alexa Fluor 546-ArIB[V11L;V16A] is a potent ligand for selectively labeling alpha 7 nicotinic acetylcholine receptors.

    PubMed

    Hone, Arik J; Whiteaker, Paul; Mohn, Jesse L; Jacob, Michele H; McIntosh, J Michael

    2010-08-01

    The alpha7* (*denotes the possible presence of additional subunits) nicotinic acetylcholine receptor (nAChR) subtype is widely expressed in the vertebrate nervous system and implicated in neuropsychiatric disorders that compromise thought and cognition. In this report, we demonstrate that the recently developed fluorescent ligand Cy3-ArIB[V11L;V16A] labels alpha7 nAChRs in cultured hippocampal neurons. However, photobleaching of this ligand during long image acquisition times prompted us to develop a new derivative. In photostability studies, this new ligand, Alexa Fluor 546-ArIB[V11L;V16A], was significantly more resistant to bleaching than the Cy3 derivative. The classic alpha7 ligand alpha-bungarotoxin binds to alpha1* and alpha9* nAChRs. In contrast, Alexa Fluor 546-ArIB[V11L;V16A] potently (IC(50) 1.8 nM) and selectively blocked alpha7 nAChRs but not alpha1* or alpha9* nAChRs expressed in Xenopus oocytes. Selectivity was further confirmed by competition binding studies of native nAChRs in rat brain membranes. The fluorescence properties of Alexa Fluor 546-ArIB[V11L;V16A] were assessed using human embryonic kidney-293 cells stably transfected with nAChRs; labeling was observed on cells expressing alpha7 but not cells expressing alpha3beta2, alpha3beta4, or alpha4beta2 nAChRs. Further imaging studies demonstrate that Alexa Fluor 546-ArIB[V11L;V16A] labels hippocampal neurons from wild-type mice but not from nAChR alpha7 subunit-null mice. Thus, Alexa Fluor 546-ArIB[V11L;V16A] represents a potent and selective ligand for imaging alpha7 nAChRs.

  2. Miniature Neutron-Alpha Activation Spectrometer

    NASA Astrophysics Data System (ADS)

    Rhodes, Edgar; Holloway, James Paul; He, Zhong; Goldsten, John

    2002-10-01

    We are developing a miniature neutron-alpha activation spectrometer for in-situ analysis of chem-bio samples, including rocks, fines, ices, and drill cores, suitable for a lander or Rover platform for Mars or outer-planet missions. In the neutron-activation mode, penetrating analysis will be performed of the whole sample using a γ spectrometer and in the α-activation mode, the sample surface will be analyzed using Rutherford-backscatter and x-ray spectrometers. Novel in our approach is the development of a switchable radioactive neutron source and a small high-resolution γ detector. The detectors and electronics will benefit from remote unattended operation capabilities resulting from our NEAR XGRS heritage and recent development of a Ge γ detector for MESSENGER. Much of the technology used in this instrument can be adapted to portable or unattended terrestrial applications for detection of explosives, chemical toxins, nuclear weapons, and contraband.

  3. Brassinolide activities of 2alpha,3alpha-diols versus 3alpha,4alpha-diols in the bean second internode bioassay: explanation by molecular modeling methods.

    PubMed

    Sísa, Miroslav; Vilaplana-Polo, Marc; Ballesteros, Carme Brosa; Kohout, Ladislav

    2007-10-01

    In general, the structural requirements postulated for a high brassinolide activity are: 2alpha,3alpha-diol, 6-ketone or better 7-oxalactone in B-ring, A/B trans fused ring junction, a cis C-22,C-23-diol preferentially with RR configurations, and a C-24 methyl or ethyl substituent [Takatsuto S, Yazawa N, Ikekawa N, Takematsu T, Takeuchi Y, Koguchi M. Structure-activity relationship of brassinosteroids. Phytochemistry 1983;22:2437-41; Thompson MJ, Meudt WJ, Mandava NB, Dutky SR, Lusby WR, Spaulding DW. Synthesis of brassinosteroids and relationship of structure to plant growth-promoting effects. Steroids 1982;39:89-105]. We found that the 3alpha,4alpha-diols 4, 6 and 8 are more active than the 2alpha,3alpha-diols 3, 5 and 7 [Sísa M, Budesínský M, Kohout L. Synthesis of 7a-homo and 7a,7b-dihomo-5alpha-cholestane analogues of brassinolide. Collect Czech Chem Commun 2003;68:2171-89]. This fact is in strong contrast with the structure requirements mentioned above. Our hypothesis suggests that the lower activity of 2alpha,3alpha-diols and/or the higher activity of 3alpha,4alpha-diols could be explained by twisting and distortion of the molecule due to the seven- or eight-membered B-ring and also by the position of a carbonyl group relative to the A-ring diol. 3D-SAR computer methodologies as alignments and overlaps of GRID maps and 3D-QSAR analysis GRID-GOLPE (CoMFA-like) were used as an effort to explain the higher bioactivity of 3alpha,4alpha-diols 4, 6 and 8 in comparison with the 2alpha,3alpha-diols 3, 5 and 7 of B-ring enlarged brassinosteroids.

  4. Human neural cell adhesion molecule L1 and rat homologue NILE are ligands for integrin alpha v beta 3

    PubMed Central

    1996-01-01

    Integrin alpha v beta 3 is distinct in its capacity to recognize the sequence Arg-Gly-Asp (RGD) in many extra-cellular matrix (ECM) components. Here, we demonstrate that in addition to the recognition of ECM components, alpha v beta 3 can interact with the neural cell adhesion molecule L1-CAM; a member of the immunoglobulin superfamily (IgSF). M21 melanoma cells displayed significant Ca(++)-dependent adhesion and spreading on immunopurified rat L1 (NILE). This adhesion was found to be dependent on the expression of the alpha v-integrin subunit and could be significantly inhibited by an antibody to the alpha v beta 3 heterodimer. M21 cells also displayed some alpha v beta 3-dependent adhesion and spreading on immunopurified human L1. Ligation between this ligand and alpha v beta 3 was also observed to promote significant haptotactic cell migration. To map the site of alpha v beta 3 ligation we used recombinant L1 fragments comprising the entire extracellular domain of human L1. Significant alpha v beta 3-dependent adhesion and spreading was evident on a L1 fragment containing Ig-like domains 4, 5, and 6. Importantly, mutation of an RGD sequence present in the sixth Ig-like domain of L1 abrogated M21 cell adhesion. We conclude that alpha v beta 3-dependent recognition of human L1 is dependent on ligation of this RGD site. Despite high levels of L1 expression the M21 melanoma cells did not display significant adhesion via a homophilic L1-L1 interaction. These data suggest that M21 melanoma cells recognize and adhere to L1 through a mechanism that is primarily heterophilic and integrin dependent. Finally, we present evidence that melanoma cells can shed and deposit L1 in occluding ECM. In this regard, alpha v beta 3 may recognize L1 in a cell-cell or cell- substrate interaction. PMID:8636223

  5. Ligand activation of peroxisome proliferator-activated receptor-β/δ suppresses liver tumorigenesis in hepatitis B transgenic mice.

    PubMed

    Balandaram, Gayathri; Kramer, Lance R; Kang, Boo-Hyon; Murray, Iain A; Perdew, Gary H; Gonzalez, Frank J; Peters, Jeffrey M

    2016-07-01

    Peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) inhibits steatosis and inflammation, known risk factors for liver cancer. In this study, the effect of ligand activation of PPARβ/δ in modulating liver tumorigenesis in transgenic hepatitis B virus (HBV) mice was examined. Activation of PPARβ/δ in HBV mice reduced steatosis, the average number of liver foci, and tumor multiplicity. Reduced expression of hepatic CYCLIN D1 and c-MYC, tumor necrosis factor alpha (Tnfa) mRNA, serum levels of alanine aminotransaminase, and an increase in apoptotic signaling was also observed following ligand activation of PPARβ/δ in HBV mice compared to controls. Inhibition of Tnfa mRNA expression was not observed in wild-type hepatocytes. Ligand activation of PPARβ/δ inhibited lipopolysaccharide (LPS)-induced mRNA expression of Tnfa in wild-type, but not in Pparβ/δ-null Kupffer cells. Interestingly, LPS-induced expression of Tnfa mRNA was also inhibited in Kupffer cells from a transgenic mouse line that expressed a DNA binding mutant form of PPARβ/δ compared to controls. Combined, these results suggest that ligand activation of PPARβ/δ attenuates hepatic tumorigenesis in HBV transgenic mice by inhibiting steatosis and cell proliferation, enhancing hepatocyte apoptosis, and modulating anti-inflammatory activity in Kupffer cells.

  6. Ligand reorganization and activation energies in nonadiabatic electron transfer reactions

    NASA Astrophysics Data System (ADS)

    Zhu, Jianjun; Wang, Jianji; Stell, George

    2006-10-01

    The activation energy and ligand reorganization energy for nonadiabatic electron transfer reactions in chemical and biological systems are investigated in this paper. The free energy surfaces and the activation energy are derived exactly in the general case in which the ligand vibration frequencies are not equal. The activation energy is derived by free energy minimization at the transition state. Our formulation leads to the Marcus-Hush [J. Chem. Phys. 24, 979 (1956); 98, 7170 (1994); 28, 962 (1958)] results in the equal-frequency limit and also generalizes the Marcus-Sumi [J. Chem. Phys. 84, 4894 (1986)] model in the context of studying the solvent dynamic effect on electron transfer reactions. It is found that when the ligand vibration frequencies are different, the activation energy derived from the Marcus-Hush formula deviates by 5%-10% from the exact value. If the reduced reorganization energy approximation is introduced in the Marcus-Hush formula, the result is almost exact.

  7. Ginsenoside Rf, a component of ginseng, regulates lipoprotein metabolism through peroxisome proliferator-activated receptor {alpha}

    SciTech Connect

    Lee, Hyunghee; Gonzalez, Frank J.; Yoon, Michung . E-mail: yoon60@mokwon.ac.kr

    2006-01-06

    We investigated whether ginseng regulates lipoprotein metabolism by altering peroxisome proliferator-activated receptor {alpha} (PPAR{alpha})-mediated pathways, using a PPAR{alpha}-null mouse model. Administration of ginseng extract, ginsenosides, and ginsenoside Rf (Rf) to wild-type mice not only significantly increased basal levels of hepatic apolipoprotein (apo) A-I and C-III mRNA compared with wild-type controls, but also substantially reversed the reductions in mRNA levels of apo A-I and C-III expected following treatment with the potent PPAR{alpha} ligand Wy14,643. In contrast, no effect was detected in the PPAR{alpha}-null mice. Testing of eight main ginsenosides on PPAR{alpha} reporter gene expression indicated that Rf was responsible for the effects of ginseng on lipoprotein metabolism. Furthermore, the inhibition of PPAR{alpha}-dependent transactivation by Rf seems to occur at the level of DNA binding. These results demonstrate that ginseng component Rf regulates apo A-I and C-III mRNA and the actions of Rf on lipoprotein metabolism are mediated via interactions with PPAR{alpha}.

  8. Thiophene-Core Estrogen Receptor Ligands Having Superagonist Activity

    PubMed Central

    Min, Jian; Wang, Pengcheng; Srinivasan, Sathish; Nwachukwu, Jerome C.; Guo, Pu; Huang, Minjian; Carlson, Kathryn E.; Katzenellenbogen, John A.; Nettles, Kendall W.; Zhou, Hai-Bing

    2013-01-01

    To probe the importance of the heterocyclic core of estrogen receptor (ER) ligands, we prepared a series of thiophene-core ligands by Suzuki cross-coupling of aryl boronic acids with bromo-thiophenes, and we assessed their receptor binding and cell biological activities. The disposition of the phenol substituents on the thiophene core, at alternate or adjacent sites, and the nature of substituents on these phenols all contribute to binding affinity and subtype selectivity. Most of the bis(hydroxyphenyl)-thiophenes were ERβ selective, whereas the tris(hydroxyphenyl)-thiophenes were ERα selective; analogous furan-core compounds generally have lower affinity and less selectivity. Some diarylthiophenes show distinct superagonist activity in reporter gene assays, giving maximal activities 2–3 times that of estradiol, and modeling suggests that these ligands have a different interaction with a hydrogen-bonding residue in helix-11. Ligand-core modification may be a new strategy for developing ER ligands whose selectivity is based on having transcriptional activity greater than that of estradiol. PMID:23586645

  9. A Guided Inquiry Activity for Teaching Ligand Field Theory

    ERIC Educational Resources Information Center

    Johnson, Brian J.; Graham, Kate J.

    2015-01-01

    This paper will describe a guided inquiry activity for teaching ligand field theory. Previous research suggests the guided inquiry approach is highly effective for student learning. This activity familiarizes students with the key concepts of molecular orbital theory applied to coordination complexes. Students will learn to identify factors that…

  10. Ligand binding to thromboxane receptors on human platelets: correlation with biological activity.

    PubMed Central

    Armstrong, R. A.; Jones, R. L.; Wilson, N. H.

    1983-01-01

    The preparation of enantiomerically pure [3H]-15 (S) 9, 11-epoxymethano PGH2 (a thromboxane A2-like agonist) has enabled the binding of ligands to the thromboxane receptor of the human platelet to be studied. The binding of the radio-ligand to washed human platelets has 3 components. One component is not displaceable by 'cold' 9, 11-epoxymethano PGH2 and its concentration-binding plot is roughly linear. The other 2 components are displaceable and saturable, and the larger of the two, which is sensitive to the stereochemistry of the C15 secondary alcohol, appears to represent the thromboxane receptor. About 1700 15(S)9, 11-epoxymethano PGH2 molecules are specifically bound to a single platelet and 50% of this binding is achieved with a concentration of 75 nM. Displacement of [3H]-15(S)9, 11-epoxymethano PGH2 is effected by (a) TXA2 and PGH2 and a number of bicyclic stable analogues (e.g. 9,11-azo PGH2), all of which produce irreversible aggregation of human platelets; (b) analogues of PGF2 alpha with potent thromboxane-like activity (e.g. ICI 79939); (c) compounds with partial agonist activity on the platelet thromboxane system (e.g. CTA2); (d) Thromboxane/endoperoxide analogues which specifically antagonize thromboxane-like actions on the human platelet (e.g. PTA2 and EP 045). Displacement is not achieved with the natural prostaglandins PGE2, PGD2 and PGF2 alpha. Neither the thromboxane-synthetase inhibitor dazoxiben nor R(+)-trimethoquinol have high displacing activity. The correlation of radio-ligand displacement with the biological activity of the competing ligands is discussed in relation to the nature of the thromboxane receptor on the human platelet. PMID:6317122

  11. Protein profiling of mouse livers with peroxisome proliferator-activated receptor alpha activation.

    PubMed

    Chu, Ruiyin; Lim, Hanjo; Brumfield, Laura; Liu, Hong; Herring, Chris; Ulintz, Peter; Reddy, Janardan K; Davison, Matthew

    2004-07-01

    Peroxisome proliferator-activated receptor alpha (PPARalpha) is important in the induction of cell-specific pleiotropic responses, including the development of liver tumors, when it is chronically activated by structurally diverse synthetic ligands such as Wy-14,643 or by unmetabolized endogenous ligands resulting from the disruption of the gene encoding acyl coenzyme A (CoA) oxidase (AOX). Alterations in gene expression patterns in livers with PPARalpha activation were delineated by using a proteomic approach to analyze liver proteins of Wy-14,643-treated and AOX(-/-) mice. We identified 46 differentially expressed proteins in mouse livers with PPARalpha activation. Up-regulated proteins, including acetyl-CoA acetyltransferase, farnesyl pyrophosphate synthase, and carnitine O-octanoyltransferase, are involved in fatty acid metabolism, whereas down-regulated proteins, including ketohexokinase, formiminotransferase-cyclodeaminase, fructose-bisphosphatase aldolase B, sarcosine dehydrogenase, and cysteine sulfinic acid decarboxylase, are involved in carbohydrate and amino acid metabolism. Among stress response and xenobiotic metabolism proteins, selenium-binding protein 2 and catalase showed a dramatic approximately 18-fold decrease in expression and a modest approximately 6-fold increase in expression, respectively. In addition, glycine N-methyltransferase, pyrophosphate phosphohydrolase, and protein phosphatase 1D were down-regulated with PPARalpha activation. These observations establish proteomic profiles reflecting a common and predictable pattern of differential protein expression in livers with PPARalpha activation. We conclude that livers with PPARalpha activation are transcriptionally geared towards fatty acid combustion.

  12. EGF AND TGF-{alpha} motogenic activities are mediated by the EGF receptor via distinct matrix-dependent mechanisms

    SciTech Connect

    Ellis, Ian R.; Schor, Ana M.; Schor, Seth L. . E-mail: s.l.schor@dundee.ac.uk

    2007-02-15

    EGF and TGF-{alpha} induce an equipotent stimulation of fibroblast migration and proliferation. In spite of their homologous structure and ligation by the same receptor (EGFR), we report that their respective motogenic activities are mediated by different signal transduction intermediates, with p70{sup S6K} participating in EGF signalling and phospholipase C{gamma} in TGF-{alpha} signalling. We additionally demonstrate that EGF and TGF-{alpha} motogenic activities may be resolved into two stages: (a) cell 'activation' by a transient exposure to either cytokine, and (b) the subsequent 'manifestation' of an enhanced migratory phenotype in the absence of cytokine. The cell activation and manifestation stages for each cytokine are mediated by distinct matrix-dependent mechanisms: motogenetic activation by EGF requires the concomitant functionality of EGFR and the hyaluronan receptor CD44, whereas activation by TGF-{alpha} requires EGFR and integrin {alpha}v{beta}3. Manifestation of elevated migration no longer requires the continued presence of exogenous cytokine and functional EGFR but does require the above mentioned matrix receptors, as well as their respective ligands, i.e., hyaluronan in the case of EGF, and vitronectin in the case of TGF-{alpha}. In contrast, the mitogenic activities of EGF and TGF-{alpha} are independent of CD44 and {alpha}v{beta}3 functionality. These results demonstrate clear qualitative differences between EGF and TGF-{alpha} pathways and highlight the importance of the extracellular matrix in regulating cytokine bioactivity.

  13. Beta-thiomaltosides as active site probes for alpha-amylase.

    PubMed

    Stankiewicz, P J; Cascio, D; McPherson, A

    1983-12-01

    A series of substituted 1-thio-beta-D-maltopyranosides was synthesized and confirmed by elemental analysis, optical rotation, NMR, and liquid chromatography. These compounds were shown by several biochemical techniques to bind to the active site of alpha-amylase. Steady-state kinetic studies showed the compounds to be competitive inhibitors, with affinities lying within the range of the natural ligands, maltose and maltotriose. Affinity chromatography employing p-aminophenyl-1-thio-beta-D-maltopyranoside linked to Sepharose provides a relatively simple procedure for alpha-amylase purification. The binding of p-bromphenyl-1-thio-beta-D-maltoside was observed in crystals of alpha-amylase using X-ray crystallography, and through the use of difference Fourier analysis its interaction at 5.0-A resolution with the active site of the enzyme has been visualized. The inhibitor binds in a long, deep cleft that divides the two major domains of the enzyme. These studies are believed to provide a first step toward the rational design of ligands for the physiological regulation of starch breakdown and utilization through modulation of alpha-amylase activity.

  14. Immobilized alpha-melanocyte stimulating hormone 10-13 (GKPV) inhibits tumor necrosis factor-alpha stimulated NF-kappaB activity.

    PubMed

    Kelly, J M; Moir, A J G; Carlson, K; Yang, Y; MacNeil, S; Haycock, J W

    2006-02-01

    alpha-MSH is an anti-inflammatory peptide which signals by binding to the melanocortin-1 receptor (MC1R) and elevating cyclic AMP in several different cells and tissues. The carboxyl terminal peptides of alpha-MSH (KPV/GKPV) are the smallest minimal sequences that prevent inflammation, but it is not known if they operate via MC1R or cyclic AMP. The aim of this study was to examine the intracellular signaling potential of the GKPV peptide sequence when immobilized to polystyrene beads via a polyethylene glycol moiety. Beads containing an immobilized GKPV peptide were investigated for their ability to inhibit proinflammatory tumor necrosis factor-alpha (TNF-alpha) stimulated activation of NF-kappaB in HBL cells stably transfected with an NF-kappaB-luciferase reporter construct. Peptide functionalized beads were compared with the ability of soluble peptide alone (alpha-MSH or GKPV) or non-functionalized beads to inhibit TNF-alpha stimulated activation of NF-kappaB. GKPV peptide functionalized beads significantly inhibited NF-kappaB-luciferase activity in comparison to beads containing no peptide moiety in one of two growths conditions investigated. Soluble alpha-MSH and GKPV peptides were also confirmed to inhibit NF-kappaB-luciferase. The present study suggests that the carboxyl terminal MSH peptide acts via a cell receptor-based mechanism and furthermore may support the potential use of such immobilized ligands for anti-inflammatory therapeutic use.

  15. Fas/Fas ligand interactions promote activation-induced cell death of NK T lymphocytes.

    PubMed

    Leite-de-Moraes, M C; Herbelin, A; Gouarin, C; Koezuka, Y; Schneider, E; Dy, M

    2000-10-15

    NKT cells are a versatile population whose immunoregulatory functions are modulated by their microenvironment. We demonstrate herein that in addition to their IFN-gamma production, NKT lymphocytes stimulated with IL-12 plus IL-18 in vitro underwent activation in terms of CD69 expression, blast transformation, and proliferation. Yet they were unable to survive in culture because, once activated, they were rapidly eliminated by apoptosis, even in the presence of their survival factor IL-7. This process was preceded by up-regulation of Fas (CD95) and Fas ligand expression in response to IL-12 plus IL-18 and was blocked by zVAD, a large spectrum caspase inhibitor, as well as by anti-Fas ligand mAb, suggesting the involvement of the Fas pathway. In accordance with this idea, NKT cells from Fas-deficient C57BL/6-lpr/lpr mice did not die in these conditions, although they shared the same features of cell activation as their wild-type counterpart. Activation-induced cell death occurred also after TCR engagement in vivo, since NKT cells became apoptotic after injection of their cognate ligand, alpha-galactosylceramide, in wild-type, but not in Fas-deficient, mice. Taken together, our data provide the first evidence for a new Fas-dependent mechanism allowing the elimination of TCR-dependent or -independent activated NKT cells, which are potentially dangerous to the organism.

  16. Structural and EPR characterisation of single electron and alkyl transfer products from reaction of dimethyl magnesium with bulky alpha-diimine ligands.

    PubMed

    Bailey, Philip J; Coxall, Robert A; Dick, Caroline M; Fabre, Sylvie; Parsons, Simon; Yellowlees, Lesley J

    2005-09-28

    Treatment of dimethylmagnesium with bulky alpha-diimine ligands provides either the biradical methyl-bridged complexes [(alpha-diimine-.)Mg+(mu-CH3)]2 via single electron transfer (SET), or the product of methyl transfer to an imine carbon atom depending upon conditions.

  17. Inhibitory effect on hepatitis B virus in vitro by a peroxisome proliferator-activated receptor-{gamma} ligand, rosiglitazone

    SciTech Connect

    Wakui, Yuta; Inoue, Jun; Ueno, Yoshiyuki; Fukushima, Koji; Kondo, Yasuteru; Kakazu, Eiji; Obara, Noriyuki; Kimura, Osamu; Shimosegawa, Tooru

    2010-05-28

    Although chronic infection of hepatitis B virus (HBV) is currently managed with nucleot(s)ide analogues or interferon-{alpha}, the control of HBV infection still remains a clinical challenge. Peroxisome proliferator-activated receptor (PPAR) is a ligand-activated transcription factor, that plays a role in glucose and lipid metabolism, immune reactions, and inflammation. In this study, the suppressive effect of PPAR ligands on HBV replication was examined in vitro using a PPAR{alpha} ligand, bezafibrate, and a PPAR{gamma} ligand, rosiglitazone. The effects were examined in HepG2 cells transfected with a plasmid containing 1.3-fold HBV genome. Whereas bezafibrate showed no effect against HBV replication, rosiglitazone reduced the amount of HBV DNA, hepatitis B surface antigen, and hepatitis B e antigen in the culture supernatant. Southern blot analysis showed that the replicative intermediates of HBV in the cells were also inhibited. It was confirmed that GW9662, an antagonist of PPAR{gamma}, reduced the suppressive effect of rosiglitazone on HBV. Moreover, rosiglitazone showed a synergistic effect on HBV replication with lamivudine or interferon-{alpha}-2b. In conclusion, this study showed that rosiglitazone inhibited the replication of HBV in vitro, and suggested that the combination therapy of rosiglitazone and nucleot(s)ide analogues or interferon could be a therapeutic option for chronic HBV infection.

  18. Alpha1 and Alpha2 Integrins Mediate Invasive Activity of Mouse Mammary Carcinoma Cells through Regulation of Stromelysin-1 Expression

    SciTech Connect

    Lochter, Andre; Navre, Marc; Werb, Zena; Bissell, Mina J

    1998-06-29

    Tumor cell invasion relies on cell migration and extracellular matrix proteolysis. We investigated the contribution of different integrins to the invasive activity of mouse mammary carcinoma cells. Antibodies against integrin subunits {alpha}6 and {beta}1, but not against {alpha}1 and {alpha}2, inhibited cell locomotion on a reconstituted basement membrane in two-dimensional cell migration assays, whereas antibodies against {beta}1, but not against a6 or {alpha}2, interfered with cell adhesion to basement membrane constituents. Blocking antibodies against {alpha}1 integrins impaired only cell adhesion to type IV collagen. Antibodies against {alpha}1, {alpha}2, {alpha}6, and {beta}1, but not {alpha}5, integrin subunits reduced invasion of a reconstituted basement membrane. Integrins {alpha}1 and {alpha}2, which contributed only marginally to motility and adhesion, regulated proteinase production. Antibodies against {alpha}1 and {alpha}2, but not {alpha}6 and {beta}1, integrin subunits inhibited both transcription and protein expression of the matrix metalloproteinase stromelysin-1. Inhibition of tumor cell invasion by antibodies against {alpha}1 and {alpha}2 was reversed by addition of recombinant stromelysin-1. In contrast, stromelysin-1 could not rescue invasion inhibited by anti-{alpha}6 antibodies. Our data indicate that {alpha}1 and {alpha}2 integrins confer invasive behavior by regulating stromelysin-1 expression, whereas {alpha}6 integrins regulate cell motility. These results provide new insights into the specific functions of integrins during tumor cell invasion.

  19. Crystallographic Analysis of Murine Constitutive Androstane Receptor Ligand-Binding Domain Complexed with 5[alpha]-androst-16-en-3[alpha]-ol

    SciTech Connect

    Vincent, J.; Shan, L.; Fan, M.; Brunzelle, J.S.; Forman, B.M.; Fernandez, E.J.

    2010-03-08

    The constitutive androstane receptor (CAR) is a member of the nuclear receptor superfamily. In contrast to classical nuclear receptors, which possess small-molecule ligand-inducible activity, CAR exhibits constitutive transcriptional activity in the apparent absence of ligand. CAR is among the most important transcription factors; it coordinately regulates the expression of microsomal cytochrome P450 genes and other drug-metabolizing enzymes. The murine CAR ligand-binding domain (LBD) was coexpressed with the steroid receptor coactivator protein (SRC-1) receptor-interacting domain (RID) in Escherichia coli. The mCAR LBD subunit was purified away from SRC-1 by affinity, anion-exchange and size-exclusion chromatography, crystallized with androstenol and the structure of the complex determined by molecular replacement.

  20. Regulation of the human SLC25A20 expression by peroxisome proliferator-activated receptor alpha in human hepatoblastoma cells

    SciTech Connect

    Tachibana, Keisuke; Takeuchi, Kentaro; Inada, Hirohiko; Yamasaki, Daisuke; Ishimoto, Kenji; Tanaka, Toshiya; Hamakubo, Takao; Sakai, Juro; Kodama, Tatsuhiko; Doi, Takefumi

    2009-11-20

    Solute carrier family 25, member 20 (SLC25A20) is a key molecule that transfers acylcarnitine esters in exchange for free carnitine across the mitochondrial membrane in the mitochondrial {beta}-oxidation. The peroxisome proliferator-activated receptor alpha (PPAR{alpha}) is a ligand-activated transcription factor that plays an important role in the regulation of {beta}-oxidation. We previously established tetracycline-regulated human cell line that can be induced to express PPAR{alpha} and found that PPAR{alpha} induces the SLC25A20 expression. In this study, we analyzed the promoter region of the human slc25a20 gene and showed that PPAR{alpha} regulates the expression of human SLC25A20 via the peroxisome proliferator responsive element.

  1. Fast skeletal muscle troponin I is a co-activator of estrogen receptor-related receptor {alpha}

    SciTech Connect

    Li Yuping; Chen Bin; Chen Jian; Lou Guiyu; Chen Shiuan; Zhou Dujin

    2008-05-16

    ERR{alpha} (estrogen receptor-related receptor {alpha}) is a member of the nuclear receptor superfamily. To further our understanding of the detailed molecular mechanism of transcriptional regulation by ERR{alpha}, we searched for ERR{alpha}-interacting proteins using a yeast two-hybrid system by screening a human mammary gland cDNA expression library with the ligand-binding domain (LBD) of ERR{alpha} as the 'bait'. Fast skeletal muscle troponin I (TNNI2), along with several known nuclear receptor co-activators, were isolated. We demonstrated that TNNI2 localizes to the cell nucleus and interacts with ERR{alpha} in co-immunoprecipitation experiments. GST pull-down assays also revealed that TNNI2 interacts directly with ERR{alpha}. Through luciferase reporter gene assays, TNNI2 was found to enhance the transactivity of ERR{alpha}. Combining mutagenesis and yeast two-hybrid assays, we mapped the ERR{alpha}-interacting domain on TNNI2 to a region encompassing amino acids 1-128. These findings reveal a new function for TNNI2 as a co-activator of ERR{alpha}.

  2. Development of Ligand-Transformed Alpha-Fetoprotein for Use Against Breast Cancer in Humans.

    DTIC Science & Technology

    1996-07-01

    Cancer Inst., 64: 1147-1152, 1980. 20. Brock, D. J., and Sutcliffe, R. G. Alpha-fetoprotein in the antenatal diagnosis of anencephaly and spina bifida...G. Alpha-fetoprotein in the antenatal diagnosis of anencephaly and spina bifida. Lancet 1972: 2:197-8. 32. Janerich, D. T., Mayne, S. T., Thompson, W

  3. Signal-transducing mechanisms involved in activation of the platelet collagen receptor integrin alpha(2)beta(1).

    PubMed

    Jung, S M; Moroi, M

    2000-03-17

    Evidence was obtained about the mechanism responsible for platelet integrin alpha(2)beta activation by determining effects of various inhibitors on soluble collagen binding, a parameter to assess integrin alpha(2)beta(1) activation, in stimulated platelets. Agonists that can also activate platelet glycoprotein IIb/IIIa are able to activate integrin alpha(2)beta(1), but those operating via glycoprotein Ib cannot. Activation of alpha(2)beta(1) induced by low thrombin or collagen-related peptide concentrations was almost completely inhibited by apyrase, and the inhibitors wortmannin, 4-amino-5-(chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, bisindolylmaleimide I, and SQ29548 significantly inhibited it. Activation induced by high thrombin or collagen-related peptide concentrations was far less sensitive to these inhibitors. However, only wortmannin markedly inhibited ADP-induced integrin alpha(2)beta(1) activation, and this was not ADP concentration-dependent. These results suggest that at the low agonist concentrations, the released ADP would be a primary inducer of integrin alpha(2)beta(1) activation, while at the high agonist concentrations, there would be several pathways through which integrin alpha(2)beta(1) activation can be induced. Kinetic analyses revealed that ADP-induced platelets had about the same number of binding sites (B(max)) as thrombin-induced platelets, but their affinity (K(d)) for soluble collagen was 3.7-12.7-fold lower, suggesting that activated integrin alpha(2)beta(1) induced by ADP is different from that induced by thrombin. The data are consistent with an activation mechanism involving released ADP and in which there exists two different states of activated integrin alpha(2)beta(1); these activated forms of integrin alpha(2)beta(1) would have different conformations that determine their ligand affinity.

  4. Effect of neohesperidin dihydrochalcone on the activity and stability of alpha-amylase: a comparative study on bacterial, fungal, and mammalian enzymes.

    PubMed

    Kashani-Amin, Elaheh; Ebrahim-Habibi, Azadeh; Larijani, Bagher; Moosavi-Movahedi, Ali Akbar

    2015-10-01

    Neohesperidin dihydrochalcone (NHDC) was recently introduced as an activator of mammalian alpha-amylase. In the current study, the effect of NHDC has been investigated on bacterial and fungal alpha-amylases. Enzyme assays and kinetic analysis demonstrated the capability of NHDC to significantly activate both tested alpha-amylases. The ligand activation pattern was found to be more similar between the fungal and mammalian enzyme in comparison with the bacterial one. Further, thermostability experiments indicated a stability increase in the presence of NHDC for the bacterial enzyme. In silico (docking) test locates a putative binding site for NHDC on alpha-amylase surface in domain B. This domain shows differences in various alpha-amylase types, and the different behavior of the ligand toward the studied enzymes may be attributed to this fact.

  5. Isothermal drop calorimeter provides measurements for alpha active, pyrophoric materials

    NASA Technical Reports Server (NTRS)

    Savage, H.

    1969-01-01

    Isothermal drop calorimeter measures the heat content of intensely alpha active and pyrophoric materials in inert atmospheres. It consists of a furnace, calorimeter, and aluminum isothermal jacket contained within an inert-atmosphere glove box, which permits the use of unencapsulated materials without exposing personnel to alpha contamination.

  6. Inhibition of peroxisome-proliferator-activated receptor (PPAR)alpha by MK886.

    PubMed Central

    Kehrer, J P; Biswal, S S; La, E; Thuillier, P; Datta, K; Fischer, S M; Vanden Heuvel, J P

    2001-01-01

    Although MK886 was originally identified as an inhibitor of 5-lipoxygenase activating protein (FLAP), recent data demonstrate that this activity does not underlie its ability to induce apoptosis [Datta, Biswal and Kehrer (1999) Biochem. J. 340, 371--375]. Since FLAP is a fatty-acid binding protein, it is conceivable that MK886 may affect other such proteins. A family of nuclear receptors that are activated by fatty acids and their metabolites, the peroxisome-proliferator-activated receptors (PPARs), have been implicated in apoptosis and may represent a target for MK886. The ability of MK886 to inhibit PPAR-alpha, -beta and -gamma activity was assessed using reporter assay systems (peroxisome-proliferator response element--luciferase). Using a transient transfection system in monkey kidney fibroblast CV-1 cells, mouse keratinocyte 308 cells and human lung adenocarcinoma A549 cells, 10--20 microM MK886 inhibited Wy14,643 activation of PPAR alpha by approximately 80%. Similar inhibition of PPAR alpha by MK886 was observed with a stable transfection reporter system in CV-1 cells. Only minimal inhibitory effects were seen on PPAR beta and PPAR gamma. MK886 inhibited PPAR alpha by a non-competitive mechanism as shown by its effects on the binding of arachidonic acid to PPAR alpha protein, and a dose-response study using a transient transfection reporter assay in COS-1 cells. An assay assessing PPAR ligand-receptor interactions showed that MK886 prevents the conformational change necessary for active-complex formation. The expression of keratin-1, a protein encoded by a PPAR alpha-responsive gene, was reduced by MK886 in a culture of mouse primary keratinocytes, suggesting that PPAR inhibition has functional consequences in normal cells. Although Jurkat cells express all PPAR isoforms, various PPAR alpha and PPAR gamma agonists were unable to prevent MK886-induced apoptosis. This is consistent with MK886 functioning as a non-competitive inhibitor of PPAR alpha, but may

  7. Supramolecular coordination and antimicrobial activities of constructed mixed ligand complexes

    NASA Astrophysics Data System (ADS)

    El-Sonbati, A. Z.; Diab, M. A.; El-Bindary, A. A.; Abou-Dobara, M. I.; Seyam, H. A.

    2013-03-01

    A novel series of copper(II) and palladium(II) with 4-derivatives benzaldehyde pyrazolone (Ln) were synthesized. The mixed ligand complexes were prepared by using 1,10-phenanthroline (Phen) as second ligand. The structure of these complexes was identified and confirm by elemental analysis, molar conductivity, UV-Vis, IR and 1H NMR spectroscopy and magnetic moment measurements as well as thermal analysis. The ligand behaves as a neutral bidentate ligand through ON donor sites. ESR spectra show the simultaneous presence of a planar trans and a nearly planar cis isomers in the 1:2 ratio for all N,O complexes [Cu(Ln)2]Cl2ṡ2H2O. Schiff bases (Ln) were tested against bacterial species; namely two Gram positive bacteria (Staphylococcus aureus and Bacillus cereus) and two Gram negative bacteria (Escherichia coli and Klebsiella pneumoniae) and fungal species (Aspergillus niger, Fusarium oxysporium, Penicillium italicum and Alternaria alternata). The tested compounds have antibacterial activity against S. aureus, B. cereus and K. pneumoniae.

  8. Identification and synthesis of [1,2,4]triazolo[3,4-a]phthalazine derivatives as high-affinity ligands to the alpha 2 delta-1 subunit of voltage gated calcium channel.

    PubMed

    Lebsack, Alec D; Gunzner, Janet; Wang, Bowei; Pracitto, Richard; Schaffhauser, Hervé; Santini, Angelina; Aiyar, Jayashree; Bezverkov, Robert; Munoz, Benito; Liu, Wensheng; Venkatraman, Shankar

    2004-05-17

    We have identified and synthesized a series of [1,2,4]triazolo[3,4-a]phthalazine derivatives as high-affinity ligands to alpha 2 delta-1 subunit of voltage gated calcium channels. Structure-activity relationship studies directed toward improving the potency and physical properties of 2 lead to the discovery of 20 (IC(50)=15 nM) and (S)-22 (IC(50)=30 nM). A potent and selective radioligand, [(3)H]-(S)-22 was also synthesized to demonstrate that this ligand binds to the same site as gabapentin.

  9. Analysis of ligand binding to the synthetic dodecapeptide 185-196 of the acetylcholine receptor alpha subunit.

    PubMed Central

    Neumann, D; Barchan, D; Fridkin, M; Fuchs, S

    1986-01-01

    A synthetic dodecapeptide corresponding to residues 185-196 of the Torpedo acetylcholine receptor alpha subunit, which contains the adjacent cysteine residues at positions 192 and 193, was recently shown by us to contain the essential elements for alpha-bungarotoxin binding. In the present study, we have used Sepharose-linked peptides for quantitative analysis of the cholinergic binding properties of this and other synthetic peptides. Sepharose-linked peptides corresponding to residues 1-20, 126-143, 143-158, 169-181, 185-196, 193-210, and 394-409 of the alpha subunit of Torpedo acetylcholine receptor, as well as a peptide corresponding to residues 185-196 of the alpha subunit of human acetylcholine receptor, were tested for their toxin-binding capacity. Of these immobilized peptides, only peptide 185-196 of the Torpedo acetylcholine receptor bound toxin significantly, thus verifying that this synthetic peptide contains essential components of the receptor toxin-binding site. Analysis of toxin binding to the peptide yielded a dissociation constant of 3.5 X 10(-5) M. This binding was inhibited by various cholinergic ligands. The inhibition potency obtained was alpha-bungarotoxin greater than Naja naja siamensis toxin greater than d-tubocurarine greater than decamethonium greater than acetylcholine greater than carbamoylcholine. This pharmacological profile resembles that of the nicotinic acetylcholine receptor and therefore suggests that the synthetic dodecapeptide also includes the neurotransmitter binding site. Reduction and carboxymethylation of the cysteine residues on peptide 185-196 inhibit its capacity to bind toxin, demonstrating that an intact disulfide is required for toxin binding. A decrease in toxin binding was also obtained following chemical modification of the tryptophan residue at position 187, thus implying its possible involvement in toxin binding. The failure to detect binding of toxin to the corresponding human sequence 185-196, in which the

  10. Analysis of ligand binding to the synthetic dodecapeptide 185-196 of the acetylcholine receptor alpha subunit.

    PubMed

    Neumann, D; Barchan, D; Fridkin, M; Fuchs, S

    1986-12-01

    A synthetic dodecapeptide corresponding to residues 185-196 of the Torpedo acetylcholine receptor alpha subunit, which contains the adjacent cysteine residues at positions 192 and 193, was recently shown by us to contain the essential elements for alpha-bungarotoxin binding. In the present study, we have used Sepharose-linked peptides for quantitative analysis of the cholinergic binding properties of this and other synthetic peptides. Sepharose-linked peptides corresponding to residues 1-20, 126-143, 143-158, 169-181, 185-196, 193-210, and 394-409 of the alpha subunit of Torpedo acetylcholine receptor, as well as a peptide corresponding to residues 185-196 of the alpha subunit of human acetylcholine receptor, were tested for their toxin-binding capacity. Of these immobilized peptides, only peptide 185-196 of the Torpedo acetylcholine receptor bound toxin significantly, thus verifying that this synthetic peptide contains essential components of the receptor toxin-binding site. Analysis of toxin binding to the peptide yielded a dissociation constant of 3.5 X 10(-5) M. This binding was inhibited by various cholinergic ligands. The inhibition potency obtained was alpha-bungarotoxin greater than Naja naja siamensis toxin greater than d-tubocurarine greater than decamethonium greater than acetylcholine greater than carbamoylcholine. This pharmacological profile resembles that of the nicotinic acetylcholine receptor and therefore suggests that the synthetic dodecapeptide also includes the neurotransmitter binding site. Reduction and carboxymethylation of the cysteine residues on peptide 185-196 inhibit its capacity to bind toxin, demonstrating that an intact disulfide is required for toxin binding. A decrease in toxin binding was also obtained following chemical modification of the tryptophan residue at position 187, thus implying its possible involvement in toxin binding. The failure to detect binding of toxin to the corresponding human sequence 185-196, in which the

  11. CD31/PECAM-1 is a ligand for alpha v beta 3 integrin involved in adhesion of leukocytes to endothelium

    PubMed Central

    1995-01-01

    To protect the body efficiently from infectious organisms, leukocytes circulate as nonadherent cells in the blood and lymph, and migrate as adherent cells into tissues. Circulating leukocytes in the blood have first to adhere to and then to cross the endothelial lining. CD31/PECAM- 1 is an adhesion molecule expressed by vascular endothelial cells, platelets, monocytes, neutrophils, and naive T lymphocytes. It is a transmembrane glycoprotein of the immunoglobulin gene superfamily (IgSF), with six Ig-like homology units mediating leukocyte-endothelial interactions. The adhesive interactions mediated by CD31 are complex and include homophilic (CD31-CD31) or heterophilic (CD31-X) contacts. Soluble, recombinant forms of CD31 allowed us to study the heterophilic interactions in leukocyte adhesion assays. We show that the adhesion molecule alpha v beta 3 integrin is a ligand for CD31. The leukocytes revealed adhesion mediated by the second Ig-like domain of CD31, and this binding was inhibited by alpha v beta 3 integrin-specific antibodies. Moreover alpha v beta 3 was precipitated by recombinant CD31 from cell lysates. These data establish a third IgSF-integrin pair of adhesion molecules, CD31-alpha v beta 3 in addition to VCAM-1, MadCAM-1/alpha 4 integrins, and ICAM/beta 2 integrins, which are major components mediating leukocyte-endothelial adhesion. Identification of a further versatile adhesion pair broadens our current understanding of leukocyte-endothelial interactions and may provide the basis for the treatment of inflammatory disorders and metastasis formation. PMID:7542249

  12. Mechanism of ligand binding to alpha 1-acid glycoprotein (orosomucoid): correlated thermodynamic factors and molecular parameters of polarity.

    PubMed Central

    Urien, S; Giroud, Y; Tsai, R S; Carrupt, P A; Brée, F; Testa, B; Tillement, J P

    1995-01-01

    Eight ligands were used in this study, four basic, three neutral and one acidic. Their binding to serum alpha 1-acid glycoprotein (orosomucoid) was measured at several temperatures, and the data were analysed together by a general model with three unknowns, number of binding sites, delta H0 and delta S0. The partition coefficients of the ligands were measured in octanol/water and heptane/water systems (log Poct. and log Phep.), and their molecular volumes were calculated by molecular modelling techniques. These structural properties allow determination of polarity parameters (delta log Poct.-hep., lambda oct. and lambda hep.) which encode in different proportions the various polar interactions between the solute and the aqueous and organic phases, i.e. hydrogen-bonding capacity and dipolarity/polarizability. This study shows that good correlations exist between delta H0 or delta S0 and polarity parameters, such that the enthalpic contribution to binding increases with increasing polarity of the ligands, mainly hydrogen-bond-donor acidity, whereas their entropic contribution to binding decreases. PMID:7887909

  13. Heterodimeric interaction between retinoid X receptor alpha and orphan nuclear receptor OR1 reveals dimerization-induced activation as a novel mechanism of nuclear receptor activation.

    PubMed Central

    Wiebel, F F; Gustafsson, J A

    1997-01-01

    OR1 is a member of the steroid/thyroid hormone nuclear receptor superfamily which has been described to mediate transcriptional responses to retinoids and oxysterols. On a DR4 response element, an OR1 heterodimer with the nuclear receptor retinoid X receptor alpha (RXR alpha) has been described to convey transcriptional activation in both the absence and presence of the RXR ligand 9-cis retinoic acid, the mechanisms of which have remained unclear. Here, we dissect the effects of RXR alpha and OR1 ligand-binding domain interaction on transcriptional regulation and the role of the respective carboxy-terminal activation domains (AF-2s) in the absence and presence of the RXR ligand, employing chimeras of the nuclear receptors containing the heterologous GAL4 DNA-binding domain as well as natural receptors. The results show that the interaction of the RXR and OR1 ligand-binding domains unleashes a transcription activation potential that is mainly dependent on the AF-2 of OR1, indicating that interaction with RXR activates OR1. This defines dimerization-induced activation as a novel function of heterodimeric interaction and mechanism of receptor activation not previously described for nuclear receptors. Moreover, we present evidence that activation of OR1 occurs by a conformational change induced upon heterodimerization with RXR. PMID:9199332

  14. Increased 5. cap alpha. -reductase activity in idiopathic hirsutism

    SciTech Connect

    Serafini, P.; Lobo, R.A.

    1985-01-01

    In vitro, genital skin 5..cap alpha..-reductase activity (5..cap alpha..-RA) was measured in ten hirsute women with normal androgen levels (idiopathic hirsutism (IH)) and in ten hirsute women with elevated androgen levels (polycystic ovary syndrome (PCO)) in order to determine the influence of secreted androgens on 5..cap alpha..-RA. In vitro 5..cap alpha..-RA was assessed by incubations of skin with /sup 14/C-testosterone (T) for 2 hours, after which steroids were separated and the radioactivity of dihydrotestosterone (DHT) and 5..cap alpha..-androstane 3..cap alpha..-17..beta..-estradiol (3..cap alpha..-diol) in specific eluates were determined. All androgens were normal in IH with the exception of higher levels of 3..cap alpha..-diol glucuronide which were similar to the levels of PCO. The conversion ratio (CR) of T to DHT in IH and PCO were similar, yet significantly greater than the CR of control subjects. The CR of T to 3..cap alpha..-diol in IH and PCO were similar, yet higher than in control subjects. Serum androgens showed no correlation with 5..cap alpha..-RA, while the CR of T to DHT showed a significant positive correlation with the Ferriman and Gallwey score. The increased 5..cap alpha..-RA in IH appears to be independent of serum androgen levels and is, therefore, an inherent abnormality. The term idiopathic is a misnomer, because hirsutism in these patients may be explained on the basis of increased skin 5..cap alpha..-RA.

  15. Predicting Monoamine Oxidase Inhibitory Activity through Ligand-Based Models

    PubMed Central

    Vilar, Santiago; Ferino, Giulio; Quezada, Elias; Santana, Lourdes; Friedman, Carol

    2013-01-01

    The evolution of bio- and cheminformatics associated with the development of specialized software and increasing computer power has produced a great interest in theoretical in silico methods applied in drug rational design. These techniques apply the concept that “similar molecules have similar biological properties” that has been exploited in Medicinal Chemistry for years to design new molecules with desirable pharmacological profiles. Ligand-based methods are not dependent on receptor structural data and take into account two and three-dimensional molecular properties to assess similarity of new compounds in regards to the set of molecules with the biological property under study. Depending on the complexity of the calculation, there are different types of ligand-based methods, such as QSAR (Quantitative Structure-Activity Relationship) with 2D and 3D descriptors, CoMFA (Comparative Molecular Field Analysis) or pharmacophoric approaches. This work provides a description of a series of ligand-based models applied in the prediction of the inhibitory activity of monoamine oxidase (MAO) enzymes. The controlled regulation of the enzymes’ function through the use of MAO inhibitors is used as a treatment in many psychiatric and neurological disorders, such as depression, anxiety, Alzheimer’s and Parkinson’s disease. For this reason, multiple scaffolds, such as substituted coumarins, indolylmethylamine or pyridazine derivatives were synthesized and assayed toward MAO-A and MAO-B inhibition. Our intention is to focus on the description of ligand-based models to provide new insights in the relationship between the MAO inhibitory activity and the molecular structure of the different inhibitors, and further study enzyme selectivity and possible mechanisms of action. PMID:23231398

  16. Hepatic triacylglycerol hydrolysis regulates peroxisome proliferator-activated receptor alpha activity.

    PubMed

    Sapiro, Jessica M; Mashek, Mara T; Greenberg, Andrew S; Mashek, Douglas G

    2009-08-01

    Recent evidence suggests that fatty acids generated from intracellular triacylglycerol (TAG) hydrolysis may have important roles in intracellular signaling. This study was conducted to determine if fatty acids liberated from TAG hydrolysis regulate peroxisome proliferator-activated receptor alpha (PPARalpha). Primary rat hepatocyte cultures were treated with adenoviruses overexpressing adipose differentiation-related protein (ADRP) or adipose triacylglycerol lipase (ATGL) or treated with short interfering RNA (siRNA) targeted against ADRP. Subsequent effects on TAG metabolism and PPARalpha activity and target gene expression were determined. Overexpressing ADRP attenuated TAG hydrolysis, whereas siRNA-mediated knockdown of ADRP or ATGL overexpression resulted in enhanced TAG hydrolysis. Results from PPARalpha reporter activity assays demonstrated that decreasing TAG hydrolysis by ADRP overexpression resulted in a 35-60% reduction in reporter activity under basal conditions or in the presence of fatty acids. As expected, PPARalpha target genes were also decreased in response to ADRP overexpression. However, the PPARalpha ligand, WY-14643, was able to restore PPARalpha activity following ADRP overexpression. Despite its effects on PPARalpha, overexpressing ADRP did not affect PPARgamma activity. Enhancing TAG hydrolysis through ADRP knockdown or ATGL overexpression increased PPARalpha activity. These results indicate that TAG hydrolysis and the consequential release of fatty acids regulate PPARalpha activity.

  17. Structural basis of activation-dependent binding of ligand-mimetic antibody AL-57 to integrin LFA-1

    SciTech Connect

    Zhang, Hongmin; Liu, Jin-huan; Yang, Wei; Springer, Timothy; Shimaoka, Motomu; Wang, Jia-huai

    2010-09-21

    The activity of integrin LFA-1 ({alpha}{sub L}{beta}{sub 2}) to its ligand ICAM-1 is regulated through the conformational changes of its ligand-binding domain, the I domain of {alpha}{sub L} chain, from an inactive, low-affinity closed form (LA), to an intermediate-affinity form (IA), and then finally, to a high-affinity open form (HA). A ligand-mimetic human monoclonal antibody AL-57 (activated LFA-1 clone 57) was identified by phage display to specifically recognize the affinity-upregulated I domain. Here, we describe the crystal structures of the Fab fragment of AL-57 in complex with IA, as well as in its unligated form. We discuss the structural features conferring AL-57's strong selectivity for the high affinity, open conformation of the I domain. The AL-57-binding site overlaps the ICAM-1 binding site on the I domain. Furthermore, an antibody Asp mimics an ICAM Glu by forming a coordination to the metal-ion dependent adhesion site (MIDAS). The structure also reveals better shape complementarity and a more hydrophobic interacting interface in AL-57 binding than in ICAM-1 binding. The results explain AL-57's antagonistic mimicry of LFA-1's natural ligands, the ICAM molecules.

  18. Ligand and Structure-based Methodologies for the Prediction of the Activity of G Protein-Coupled Receptor Ligands

    PubMed Central

    Costanzi, Stefano; Tikhonova, Irina G.; Harden, T. Kendall; Jacobson, Kenneth A.

    2008-01-01

    Summary Accurate in silico models for the quantitative prediction of the activity of G protein-coupled receptor (GPCR) ligands would greatly facilitate the process of drug discovery and development. Several methodologies have been developed based on the properties of the ligands, the direct study of the receptor-ligand interactions, or a combination of both approaches. Ligand-based three-dimensional quantitative structure-activity relationships (3D-QSAR) techniques, not requiring knowledge of the receptor structure, have been historically the first to be applied to the prediction of the activity of GPCR ligands. They are generally endowed with robustness and good ranking ability; however they are highly dependent on training sets. Structure-based techniques generally do not provide the level of accuracy necessary to yield meaningful rankings when applied to GPCR homology models. However, they are essentially independent from training sets and have a sufficient level of accuracy to allow an effective discrimination between binders and nonbinders, thus qualifying as viable lead discovery tools. The combination of ligand and structure-based methodologies in the form of receptor-based 3D-QSAR and ligand and structure-based consensus models results in robust and accurate quantitative predictions. The contribution of the structure-based component to these combined approaches is expected to become more substantial and effective in the future, as more sophisticated scoring functions are developed and more detailed structural information on GPCRs is gathered. PMID:18483766

  19. Ligand and structure-based methodologies for the prediction of the activity of G protein-coupled receptor ligands

    NASA Astrophysics Data System (ADS)

    Costanzi, Stefano; Tikhonova, Irina G.; Harden, T. Kendall; Jacobson, Kenneth A.

    2009-11-01

    Accurate in silico models for the quantitative prediction of the activity of G protein-coupled receptor (GPCR) ligands would greatly facilitate the process of drug discovery and development. Several methodologies have been developed based on the properties of the ligands, the direct study of the receptor-ligand interactions, or a combination of both approaches. Ligand-based three-dimensional quantitative structure-activity relationships (3D-QSAR) techniques, not requiring knowledge of the receptor structure, have been historically the first to be applied to the prediction of the activity of GPCR ligands. They are generally endowed with robustness and good ranking ability; however they are highly dependent on training sets. Structure-based techniques generally do not provide the level of accuracy necessary to yield meaningful rankings when applied to GPCR homology models. However, they are essentially independent from training sets and have a sufficient level of accuracy to allow an effective discrimination between binders and nonbinders, thus qualifying as viable lead discovery tools. The combination of ligand and structure-based methodologies in the form of receptor-based 3D-QSAR and ligand and structure-based consensus models results in robust and accurate quantitative predictions. The contribution of the structure-based component to these combined approaches is expected to become more substantial and effective in the future, as more sophisticated scoring functions are developed and more detailed structural information on GPCRs is gathered.

  20. EGF receptor ligands: recent advances

    PubMed Central

    Singh, Bhuminder; Carpenter, Graham; Coffey, Robert J.

    2016-01-01

    Seven ligands bind to and activate the mammalian epidermal growth factor (EGF) receptor (EGFR/ERBB1/HER1): EGF, transforming growth factor-alpha (TGFA), heparin-binding EGF-like growth factor (HBEGF), betacellulin (BTC), amphiregulin (AREG), epiregulin (EREG), and epigen (EPGN). Of these, EGF, TGFA, HBEGF, and BTC are thought to be high-affinity ligands, whereas AREG, EREG, and EPGN constitute low-affinity ligands. This focused review is meant to highlight recent studies related to actions of the individual EGFR ligands, the interesting biology that has been uncovered, and relevant advances related to ligand interactions with the EGFR. PMID:27635238

  1. Ligand-Gated Ion Channels: Permeation and Activation1

    NASA Astrophysics Data System (ADS)

    Lynch, Joseph W.; Barry, Peter H.

    Ligand-gated ion channels (LGICs) are fast-responding channels in which the receptor, which binds the activating molecule (the ligand), and the ion channel are part of the same nanomolecular protein complex. This chapter will describe the properties and functions of the nicotinic acetylcholine LGIC superfamily, which play a critical role in the fast chemical transmission of electrical signals between nerve cells at synapses and between nerve and muscle cells at endplates. All the processing functions of the brain and the resulting behavioral output depend on chemical transmission across such neuronal interconnections. To describe the properties of the channels of this LGIC superfamily,we will mainly use two examples of this family of channels: the excitatory nicotinic acetylcholine receptor (nAChR) and the inhibitory glycine receptor (GlyR) channels. In the chemical transmission of electrical signals, the arrival of an electrical signal at the synaptic terminal of a nerve causes the release of a chemical signal—a neurotransmitter molecule (the ligand, also referred to as the agonist). The neurotransmitter rapidly diffuses across the very narrow 20-40 nm synaptic gap between the cells and binds to the LGIC receptors in the membrane of the target (postsynaptic) cell and generates a new electrical signal in that cell (e.g., Kandel et al., 2000). How this chemical signal is converted into an electrical one depends on the fundamental properties of LGICs and the ionic composition of the postsynaptic cell and its external solution.

  2. Resveratrol exerts pharmacological preconditioning by activating PGC-1alpha.

    PubMed

    Tan, Lan; Yu, Jin-Tai; Guan, Hua-Shi

    2008-11-01

    Resveratrol (RSV), a polyphenol phytoalexin abundantly found in grape skins and in wines, is currently the focus of intense research as a pharmacological preconditioning agent in kidney, heart, and brain from ischemic injury. However, the exact molecular mechanism of RSV preconditioning remains obscure. The data from current studies indicate that pharmacological preconditioning with RSV were attributed to its role as intracellular antioxidant, anti-inflammatory agent, its ability to induce nitric oxide synthase (NOS) expression, its ability to induce angiogenesis, and its ability to increases sirtuin 1 (SIRT1) activity. Peroxisome proliferators-activated receptor (PPAR) gamma co-activator-1alpha (PGC-1alpha) is a member of a family of transcription coactivators that owns mitochondrial biogenesis, antioxidation, growth factor signaling regulation, and angiogenesis activities. And, almost all the signaling pathways activated by RVS involve in PGC-1alpha activity. Moreover, it has been proofed that RVS could mediate an increase PGC-1alpha activity. These significant conditions support the hypothesis that RSV exerts pharmacological preconditioning by activating PGC-1alpha. Attempts to confirm this hypothesis will provide new directions in the study of pharmaceutical preconditioning and the development of new treatment approaches for reducing the extent of ischemia/reperfusion injury.

  3. Characterization of [35S]-ATP alpha S and [3H]-alpha, beta-MeATP binding sites in rat brain cortical synaptosomes: regulation of ligand binding by divalent cations.

    PubMed

    Schäfer, R; Reiser, G

    1997-07-01

    1. We made a comparative analysis of the binding characteristics of the radioligands [35S]-ATP alpha S and [3H]-alpha, beta-MeATP in order to test whether these ligands can be used to analyse P2-purinoceptors in synaptosomal membranes from rat brain cortex. 2. Synaptosomes possess sites with high affinity for [35S]-ATP alpha S (Kd = 22.2 +/- 9.1 nM, Bmax = 14.8 pmol mg-1 protein). The rank order of the competition potency of the different compounds (ATP alpha S, ATP, ATP gamma S > ADP beta S, 2-MeSATP > deoxyATP, ADP > > UTP, alpha, beta-MeATP, AMP, Reactive Blue-2, suramin, isoPPADS) is consistent with pharmacological properties of P2Y-purinoceptors. 3. Under identical conditions [35S]-ATP alpha S and [3H]-alpha, beta-MeATP bind to different binding sites at synaptosomal membranes from rat brain cortex. The affinity of the [3H]-alpha, beta-MeATP binding sites (Kd = 13.7 +/- 1.8 nM, Bmax = 6.34 +/- 0.28 pmol mg-1 protein) was 38 fold higher than the potency of alpha, beta-MeATP to displace [35S]-ATP alpha S binding (Ki = 0.52 microM). ATP and ADP beta S competed at both binding sites with different affinities, 60 fold and 175 fold, respectively. The other agonists tested (2-MeSATP, UTP, GTP) did not affect specific [35H]-alpha, beta-MeATP binding at concentrations up to 100 microM. The antagonists (suramin, isoPPADS, Evan's Blue) showed completely different affinities for both binding sites. 4. Binding of [35S]-ATP alpha S on synaptosomes was regulated by GTP, which is indicative for G-protein coupled receptors. The Kd value for the high affinity binding site was reduced in the presence of GTP about 5 fold (from 1.8 nM to 8.6 nM). In the presence of Mg2+ the affinity was increased (Kd 1.8 nM versus 22 nM in the absence of Mg2+). 5. The binding of both radioligands was regulated in an opposite manner by physiological concentrations of Ca2+ and Mg2+. Binding of [3H]-alpha, beta-MeATP to synaptosomal membranes was increased 3 fold by raising the Ca2+ concentration

  4. Fibronectin type III5 repeat contains a novel cell adhesion sequence, KLDAPT, which binds activated alpha4beta1 and alpha4beta7 integrins.

    PubMed

    Moyano, J V; Carnemolla, B; Domínguez-Jiménez, C; García-Gila, M; Albar, J P; Sánchez-Aparicio, P; Leprini, A; Querzé, G; Zardi, L; Garcia-Pardo, A

    1997-10-03

    The region of fibronectin encompassing type III repeats 4-6 contains a low affinity heparin binding domain, but its physiological significance is not clear. We have studied whether this domain is able to interact with cells as already shown for other heparin binding domains of fibronectin. A computer search based on homologies with known active sites in fibronectin revealed the sequence KLDAPT located in FN-III5. A synthetic peptide containing this sequence induced lymphoid cell adhesion upon treatment with the activating anti-beta1 monoclonal antibody (mAb) TS2/16 or with Mn2+, indicating that KLDAPT was binding to an integrin. A recombinant fragment containing repeat III5 (FN-III5) also mediated adhesion of TS2/16/Mn2+-treated cells while the FN-III6 fragment did not. Soluble KLDAPT peptide inhibited cell adhesion to FN-III5 as well as to a 38-kDa fibronectin fragment and VCAM-1, two previously known ligands for alpha4beta1 integrin. KLDAPT also competed with the binding of soluble alkaline phosphatase-coupled VCAM-Ig to Mn2+-treated alpha4beta1. Furthermore, mAbs anti-alpha4 and anti-alpha4beta7, but not mAbs to other integrins, inhibited cell adhesion to FN-III5 and KLDAPT. These results therefore establish a cell adhesive function for the FN-III5 repeat and show that KLDAPT is a novel fibronectin ligand for activated alpha4 integrins.

  5. Facile syntheses of thiophene-substituted 1,4-diazabutadiene (alpha-diimine) ligands and their conversion to phosphenium triiodide salts.

    PubMed

    Powell, Adam B; Brown, Jaclyn R; Vasudevan, Kalyan V; Cowley, Alan H

    2009-04-14

    Four novel N-aryl-2-thienyl substituted 1,4-diazabutadiene (alpha-diimine) ligands 5-8 have been prepared by cyanide ion-catalyzed intermolecular coupling of the appropriate aromatic aldimines. A ligand featuring a phenyl spacer moiety between a thiophene carbon atom and each imino nitrogen atom (12) has been prepared by a similar synthetic route. Ligands 5-8 and 12 were characterized on the basis of 1H and 13C NMR, IR and MS-CI spectroscopy. Upon treatment with PI3 in CH2Cl2 solution, ligands 5-8 undergo redox reactions to furnish the triiodide salts of the corresponding phosphenium cations 13-16 which were characterized by 1H, 13C and 31P NMR, and MS-CI spectroscopy. The phosphenium triiodide salt 15, and ligands 5-7 and 12 were also structurally authenticated.

  6. Alpha-mannosidase activity in goats fed with Sida carpinifolia.

    PubMed

    Bedin, Marisete; Moleta Colodel, Edson; Viapiana, Marli; Matte, Ursula; Driemeier, David; Giugliani, Roberto

    2010-03-01

    Human alpha-mannosidosis results from alpha-mannosidase deficiency and progressive accumulation of mannose-rich oligosaccharides in lysosomes. Two days before Saanen goats were fed with Sida carpinifolia, alpha-mannosidase activity in leukocytes was 128+/-28 nmoles4-MU/h/mgprotein (first trial) and 104+/-6 nmoles4-MU/h/mgprotein (second trial). At day 5, after the introduction of S. carpinifolia diet, the alpha-mannosidase activity in leukocytes was significantly increased, both in the first (288+/-13 nmoles4-MU/h/mgprotein) and in the second trial (303+/-45 nmoles4-MU/h/mgprotein), and it returned to normal levels 2 days after the withdrawal of the plant from the diet (114+/-7 nmoles4-MU/h/mgprotein in first trial, and 108+/-25 nmoles4-MU/h/mgprotein in the second one). Plasma alpha-mannosidase activity decreased significantly 4 days after animal exposure to the S. carpinifolia diet (769+/-167 nmoles4-MU/h/ml) and returned to normal values 10 days after the withdrawal of the plant from the diet (1289+/-163 nmoles4-MU/h/ml). Thin-layer chromatography showed an abnormal excretion of oligosaccharides in urine as of day 2 after diet exposure, which persisted until one day after the withdrawal of the plant. Animals presented neurological clinical signs beginning at day 37 (in the first trial) and at day 25 (in the second trial) after being fed with the plant. The results obtained herein suggest that oligosaccharides observed in urine are a result of a decrease in alpha-mannosidase activity in plasma. S. carpinifolia seems to have other compounds that act on alpha-mannosidase enzyme in leukocytes in a competitive manner with swainsonine. The increase in alpha-mannosidase enzyme in leukocytes could be attributed to one of these compounds present in S. carpinifolia.

  7. N-methyl-D-aspartate antagonist activity of alpha- and beta-sulfallorphans.

    PubMed

    Shukla, V K; Lemaire, S

    1997-01-01

    Resolved equatorial (alpha) and axial (beta) forms of S-allylmorphinans, alpha-sulfallorphan and beta-sulfallorphan, were tested for their ability to compete with the binding of phencyclidine and sigma receptor ligands to mouse brain membranes and to antagonize N-methyl-D-aspartate (NMDA)-induced convulsions in mice. alpha- and beta-sulfallorphans displayed distinct binding affinities for phencyclidine and sigma sites, inhibiting the binding of [3H]-(5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten++ +-5, 10-imine ([3H]MK-801) with Ki values of 2.32 and 0.13 microM and that of [3H](+)-pentazocine with Ki values of 1.97 and 1.61 microM, respectively. Intracerebroventricular administration of these compounds in mice caused dose-dependent inhibitions of NMDA-induced convulsions, but did not affect convulsions induced by (R,S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), kainic acid and bicuculline. alpha- and beta-sulfallorphans blocked the convulsive activity of NMDA (1 nmol/mouse; intracerebroventricular) with ED50 values of 0.48 and 0.015 nmol/mouse, as compared with 0.55, 0.039 and 0.013 nmol/mouse for dextrorphan, MK-801 and (+/-)3-(2-carboxypiperazine-4yl)propyl-1-proprionic acid, respectively. The structurally related compound, dextrallorphan, significantly but less potently blocked NMDA-induced convulsions (ED60, 2.68 nmol/mouse). At the protective doses, alpha- and beta-sulfallorphans markedly reduced NMDA- and AMPA-induced mortality without inducing locomotion and falling behavior. These results indicate that alpha- and beta-sulfallorphans are potent and selective NMDA antagonists devoid of motor side effects at protective doses.

  8. Active-site zinc ligands and activated H2O of zinc enzymes.

    PubMed Central

    Vallee, B L; Auld, D S

    1990-01-01

    The x-ray crystallographic structures of 12 zinc enzymes have been chosen as standards of reference to identify the ligands to the catalytic and structural zinc atoms of other members of their respective enzyme families. Universally, H2O is a ligand and critical component of the catalytically active zinc sites. In addition, three protein side chains bind to the catalytic zinc atom, whereas four protein ligands bind to the structural zinc atom. The geometry and coordination number of zinc can vary greatly to accommodate particular ligands. Zinc forms complexes with nitrogen and oxygen just as readily as with sulfur, and this is reflected in catalytic zinc sites having a binding frequency of His much greater than Glu greater than Asp = Cys, three of which bind to the metal atom. The systematic spacing between the ligands is striking. For all catalytic zinc sites except the coenzyme-dependent alcohol dehydrogenase, the first two ligands are separated by a "short-spacer" consisting of 1 to 3 amino acids. These ligands are separated from the third ligand by a "long spacer" of approximately 20 to approximately 120 amino acids. The spacer enables formation of a primary bidentate zinc complex, whereas the long spacer contributes flexibility to the coordination sphere, which can poise the zinc for catalysis as well as bring other catalytic and substrate binding groups into apposition with the active site. The H2O is activated by ionization, polarization, or poised for displacement. Collectively, the data imply that the preferred mechanistic pathway for activating the water--e.g., zinc hydroxide or Lewis acid catalysis--will be determined by the identity of the other three ligands and their spacing. Images PMID:2104979

  9. Tetrahydro-iso-alpha Acids Antagonize Estrogen Receptor Alpha Activity in MCF-7 Breast Cancer Cells.

    PubMed

    Lempereur, Maëlle; Majewska, Claire; Brunquers, Amandine; Wongpramud, Sumalee; Valet, Bénédicte; Janssens, Philippe; Dillemans, Monique; Van Nedervelde, Laurence; Gallo, Dominique

    2016-01-01

    Tetrahydro-iso-alpha acids commonly called THIAA or Tetra are modified hop acids extracted from hop (Humulus lupulus L.) which are frequently used in brewing industry mainly in order to provide beer bitterness and foam stability. Interestingly, molecular structure of tetrahydro-iso-alpha acids is close to a new type of estrogen receptor alpha (ERα) antagonists aimed at disrupting the binding of coactivators containing an LxxLL motif (NR-box). In this work we show that THIAA decreases estradiol-stimulated proliferation of MCF-7 (ERα-positive breast cancer cells). Besides, we show that it inhibits ERα transcriptional activity. Interestingly, this extract fails to compete with estradiol for ERα binding and does not significantly impact the receptor turnover rate in MCF-7 cells, suggesting that it does not act like classical antiestrogens. Hence, we demonstrate that THIAA is able to antagonize ERα estradiol-induced recruitment of the LxxLL binding motif.

  10. Tetrahydro-iso-alpha Acids Antagonize Estrogen Receptor Alpha Activity in MCF-7 Breast Cancer Cells

    PubMed Central

    Lempereur, Maëlle; Majewska, Claire; Brunquers, Amandine; Wongpramud, Sumalee; Valet, Bénédicte; Janssens, Philippe; Dillemans, Monique; Van Nedervelde, Laurence; Gallo, Dominique

    2016-01-01

    Tetrahydro-iso-alpha acids commonly called THIAA or Tetra are modified hop acids extracted from hop (Humulus lupulus L.) which are frequently used in brewing industry mainly in order to provide beer bitterness and foam stability. Interestingly, molecular structure of tetrahydro-iso-alpha acids is close to a new type of estrogen receptor alpha (ERα) antagonists aimed at disrupting the binding of coactivators containing an LxxLL motif (NR-box). In this work we show that THIAA decreases estradiol-stimulated proliferation of MCF-7 (ERα-positive breast cancer cells). Besides, we show that it inhibits ERα transcriptional activity. Interestingly, this extract fails to compete with estradiol for ERα binding and does not significantly impact the receptor turnover rate in MCF-7 cells, suggesting that it does not act like classical antiestrogens. Hence, we demonstrate that THIAA is able to antagonize ERα estradiol-induced recruitment of the LxxLL binding motif. PMID:27190515

  11. Screening of medicinal plants for PPPAR-alpha and PPAR-gamma activation and evaluation of their effects on glucose uptake and 3T3-L1 adipogenesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Medicinal plants are a rich source of ligands for nuclear receptors. The present study was aimed to screen a collection of plant extracts for PPAR-alpha/gamma activating properties and identify the active extract that can stimulate cellular glucose uptake without enhancing the adipogenesis. A report...

  12. Adsorption behavior of alpha -cypermethrin on cork and activated carbon.

    PubMed

    Domingues, Valentina F; Priolo, Giuseppe; Alves, Arminda C; Cabral, Miguel F; Delerue-Matos, Cristina

    2007-08-01

    Studies were undertaken to determine the adsorption behavior of alpha -cypermethrin [R)-alpha -cyano-3-phenoxybenzyl(1S)-cis-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate, and (S)-alpha-cyano-3-phenoxybenzyl (1R)-cis-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate] in solutions on granules of cork and activated carbon (GAC). The adsorption studies were carried out using a batch equilibrium technique. A gas chromatograph with an electron capture detector (GC-ECD) was used to analyze alpha -cypermethrin after solid phase extraction with C18 disks. Physical properties including real density, pore volume, surface area and pore diameter of cork were evaluated by mercury porosimetry. Characterization of cork particles showed variations thereby indicating the highly heterogeneous structure of the material. The average surface area of cork particles was lower than that of GAC. Kinetics adsorption studies allowed the determination of the equilibrium time - 24 hours for both cork (1-2 mm and 3-4 mm) and GAC. For the studied alpha -cypermethrin concentration range, GAC revealed to be a better sorbent. However, adsorption parameters for equilibrium concentrations, obtained through the Langmuir and Freundlich models, showed that granulated cork 1-2 mm have the maximum amount of adsorbed alpha-cypermethrin (q(m)) (303 microg/g); followed by GAC (186 microg/g) and cork 3-4 mm (136 microg/g). The standard deviation (SD) values, demonstrate that Freundlich model better describes the alpha -cypermethrin adsorption phenomena on GAC, while alpha -cypermethrin adsorption on cork (1-2 mm and 3-4 mm) is better described by the Langmuir. In view of the adsorption results obtained in this study it appears that granulated cork may be a better and a cheaper alternative to GAC for removing alpha -cypermethrin from water.

  13. Identifying Activity Cliff Generators of PPAR Ligands Using SAS Maps.

    PubMed

    Méndez-Lucio, Oscar; Pérez-Villanueva, Jaime; Castillo, Rafael; Medina-Franco, José L

    2012-12-01

    Structure-activity relationships (SAR) of compound databases play a key role in hit identification and lead optimization. In particular, activity cliffs, defined as a pair of structurally similar molecules that present large changes in potency, provide valuable SAR information. Herein, we introduce the concept of activity cliff generator, defined as a molecular structure that has a high probability to form activity cliffs with molecules tested in the same biological assay. To illustrate this concept, we discuss a case study where Structure-Activity Similarity maps were used to systematically identify and analyze activity cliff generators present in a dataset of 168 compounds tested against three peroxisome-proliferator-activated receptor (PPAR) subtypes. Single-target and dual-target activity cliff generators for PPARα and δ were identified. In addition, docking calculations of compounds that were classified as cliff generators helped to suggest a hot spot in the target protein responsible of activity cliffs and to analyze its implication in ligand-enzyme interaction.

  14. Tracking variations in the alpha activity in an electroencephalogram

    NASA Technical Reports Server (NTRS)

    Prabhu, K. S.

    1971-01-01

    The problem of tracking Alpha voltage variations in an electroencephalogram is discussed. This problem is important in encephalographic studies of sleep and effects of different stimuli on the brain. Very often the Alpha voltage is tracked by passing the EEG signal through a bandpass filter centered at the Alpha frequency, which hopefully will filter out unwanted noise from the Alpha activity. Some alternative digital techniques are suggested and their performance is compared with the standard technique. These digital techniques can be used in an environment where an electroencephalograph is interfaced with a small digital computer via an A/D convertor. They have the advantage that statistical statements about their variability can sometimes be made so that the effect sought can be assessed correctly in the presence of random fluctuations.

  15. Identification of physiologically active substances as novel ligands for MRGPRD.

    PubMed

    Uno, Makiko; Nishimura, Satoko; Fukuchi, Keisuke; Kaneta, Yasuyuki; Oda, Yoko; Komori, Hironobu; Takeda, Shigeki; Haga, Tatsuya; Agatsuma, Toshinori; Nara, Futoshi

    2012-01-01

    Mas-related G-protein coupled receptor member D (MRGPRD) is a G protein-coupled receptor (GPCR) which belongs to the Mas-related GPCRs expressed in the dorsal root ganglia (DRG). In this study, we investigated two novel ligands in addition to beta-alanine: (1) beta-aminoisobutyric acid, a physiologically active substance, with which possible relation to tumors has been seen together with beta-alanine; (2) diethylstilbestrol, a synthetic estrogen hormone. In addition to the novel ligands, we found that transfection of MRGPRD leads fibroblast cells to form spheroids, which would be related to oncogenicity. To understand the MRGPRD novel character, oncogenicity, a large chemical library was screened in order to obtain MRGPRD antagonists to utilize in exploring the character. The antagonist in turn inhibited the spheroid proliferation that is dependent on MRGPRD signaling as well as MRGPRD signals activated by beta-alanine. The antagonist, a small-molecule compound we found in this study, is a potential anticancer agent.

  16. Identification of Physiologically Active Substances as Novel Ligands for MRGPRD

    PubMed Central

    Uno, Makiko; Nishimura, Satoko; Fukuchi, Keisuke; Kaneta, Yasuyuki; Oda, Yoko; Komori, Hironobu; Takeda, Shigeki; Haga, Tatsuya; Agatsuma, Toshinori; Nara, Futoshi

    2012-01-01

    Mas-related G-protein coupled receptor member D (MRGPRD) is a G protein-coupled receptor (GPCR) which belongs to the Mas-related GPCRs expressed in the dorsal root ganglia (DRG). In this study, we investigated two novel ligands in addition to beta-alanine: (1) beta-aminoisobutyric acid, a physiologically active substance, with which possible relation to tumors has been seen together with beta-alanine; (2) diethylstilbestrol, a synthetic estrogen hormone. In addition to the novel ligands, we found that transfection of MRGPRD leads fibroblast cells to form spheroids, which would be related to oncogenicity. To understand the MRGPRD novel character, oncogenicity, a large chemical library was screened in order to obtain MRGPRD antagonists to utilize in exploring the character. The antagonist in turn inhibited the spheroid proliferation that is dependent on MRGPRD signaling as well as MRGPRD signals activated by beta-alanine. The antagonist, a small-molecule compound we found in this study, is a potential anticancer agent. PMID:23091359

  17. Estrogen Receptors Alpha (ERα) and Beta (ERβ): Subtype-Selective Ligands and Clinical Potential

    PubMed Central

    Paterni, Ilaria; Granchi, Carlotta; Katzenellenbogen, John A.; Minutolo, Filippo

    2014-01-01

    Estrogen receptors alpha (ERα) and beta (ERβ) are nuclear transcription factors that are involved in the regulation of many complex physiological processes in humans. Modulation of these receptors by prospective therapeutic agents is currently being considered for prevention and treatment of a wide variety of pathological conditions, such as, cancer, metabolic and cardiovascular diseases, neurodegeneration, inflammation, and osteoporosis. This review provides an overview and update of compounds that have been recently reported as modulators of ERs, with a particular focus on their potential clinical applications. PMID:24971815

  18. Peroxisome proliferator-activated receptor alpha and the ketogenic diet.

    PubMed

    Cullingford, Tim

    2008-11-01

    Peroxisome proliferator-activated receptor alpha (PPARalpha) is a drug/fatty acid-activated trans cription factor involved in the starvation response, and is thus relevant to the ketogenic diet (KD). This article summarizes research indicating the role of PPARalpha in central and peripheral nervous system function with particular reference to downstream targets relevant to anticonvulsant action.

  19. [F-18]-(-,-)-FQNPe - an attractive ligand for evaluation of muscarinic-cholinergic neuron activity by PET

    SciTech Connect

    Luo, H.; McPherson, D.W.; Beets, A.L.; Knapp, F.F. Jr.

    1997-05-01

    The stereoisomers of 1-azabicyclo[2.2.2]oct-3-yl {alpha}-{alpha}-(1-fluoropentan-5-yl)-{alpha}-hydroxy-{alpha}-phenylacetate ({open_quotes}FQNPe{close_quotes}) have been resolved. (-,-)- receptors (K{sub i}, nM; ml, 0.3; m2, 0.1). [F-18]-(-,-)-FQNPe demonstrated high cerebral and myocardial uptake in rats in vivo. We now report significant blocking of [F-18]-(-.-)-FQNPe uptake in receptor-rich tissues in rats in vivo after (R)-QNB pretreatment and the absence of any TLC detectable FQNPe metabolites in tissue extracts. Rats were injected with (R)-QNB (3 mg/kg) 1 h prior to [F-18]-FQNPe injection (370-629 KBq). After 1 h, rats were sacrificed and tissues removed and counted. (R)-QNB significantly decreased FQNPe uptake in heart and all receptor-rich regions but not blood (Table; Mean % ID/g, n=5); C, control; Q, (R)-QNB; Hrt, heart; Cer, cerebellum; Pon, pons; Med, medulla; Cor, cortex; Stri, striatum; Hip, hippocampus; Th, thallamus; SuC, superior colliculi; InC, inferior colliculi. Tissues from untreated rats were Folch-extracted and 71-77% of activity was in organic extracts from brain and heart. TLC of organic extracts indicated a single radioactive component with R{sub f} of FQNPe. These combined results demonstrate that [F-18]-(-,-)-FQNPe does not appear to be metabolized in heart and brain, shows good receptor localization and is thus an attractive ligand for evaluation as a potential imaging agent by PET.

  20. DTG and (+)-3-PPP inhibit a ligand-activated hyperpolarization in mammalian neurons.

    PubMed

    Bobker, D H; Shen, K Z; Surprenant, A; Williams, J T

    1989-12-01

    The effects of three compounds with high affinity for the haloperidol-sensitive alpha-binding site were studied with intracellular recordings in the vitro neuronal preparations of the rat locus ceruleus, rat dorsal raphe and the guinea pig submucous plexus. Both (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine [(+)-3-PPP] and 1,3-di-o-tolylguanidine (DTG) inhibited the hyperpolarization induced by a ligand-activated potassium conductance. In the locus ceruleus, (+)-3-PPP and DTG produced a maximal 40 to 45% inhibition of the [Met5]enkephalin hyperpolarization, and had EC50 values of 6.6 and 2.2 microM, respectively. In the submucous plexus, the two compounds had a similar action on the alpha-2 adrenoceptor agonist UK14304 hyperpolarization, producing a maximal 50% inhibition with EC50 values of 140 and 32 nM, respectively. In addition, DTG inhibited the alpha-2-mediated inhibitory postsynaptic potential in both preparations. In contrast, (+)-3-PPP increased and prolonged the inhibitory postsynaptic potential. This action is qualitatively similar to the actions of cocaine on locus ceruleus and submucous plexus neurons. Haloperidol (1-10 microM) shared none of these actions. It is concluded that DTG and (+)-3-PPP are inhibitors of the opiate and alpha-2-mediated hyperpolarization at a postreceptor site, possibly the potassium channel. In addition, (+)-3-PPP, but not DTG, inhibits norepinephrine reuptake. None of these effects appear to be related to the sigma -binding site, because haloperidol acted as neither an agonist nor an antagonist.

  1. Cutting edge: nonglycosidic CD1d lipid ligands activate human and murine invariant NKT cells.

    PubMed

    Silk, Jonathan D; Salio, Mariolina; Reddy, B Gopal; Shepherd, Dawn; Gileadi, Uzi; Brown, James; Masri, S Hajar; Polzella, Paolo; Ritter, Gerd; Besra, Gurdyal S; Jones, E Yvonne; Schmidt, Richard R; Cerundolo, Vincenzo

    2008-05-15

    Invariant NKT cells (iNKT cells) recognize CD1d/glycolipid complexes. We demonstrate that the nonglycosidic compound threitolceramide efficiently activates iNKT cells, resulting in dendritic cell (DC) maturation and the priming of Ag-specific T and B cells. Threitolceramide-pulsed DCs are more resistant to iNKT cell-dependent lysis than alpha-galactosylceramide-pulsed DCs due to the weaker affinity of the human iNKT TCR for CD1d/ threitolceramide than CD1d/alpha-galactosylceramide complexes. iNKT cells stimulated with threitolceramide also recover more quickly from activation-induced anergy. Kinetic and functional experiments showed that shortening or lengthening the threitol moiety by one hydroxymethylene group modulates ligand recognition, as human and murine iNKT cells recognize glycerolceramide and arabinitolceramide differentially. Our data broaden the range of potential iNKT cell agonists. The ability of these compounds to assist the priming of Ag-specific immune responses while minimizing iNKT cell-dependent DC lysis makes them attractive adjuvants for vaccination strategies.

  2. Discovery of an Oxybenzylglycine Based Peroxisome Proliferator Activated Receptor Alpha Selective

    SciTech Connect

    Li, J.; Kennedy, L; Shi, Y; Tao, S; Ye, X; Chen, S; Wang, Y; Hernandez, A; Wang, W; et al.

    2010-01-01

    An 1,3-oxybenzylglycine based compound 2 (BMS-687453) was discovered to be a potent and selective peroxisome proliferator activated receptor (PPAR) {alpha} agonist, with an EC{sub 50} of 10 nM for human PPAR{alpha} and {approx}410-fold selectivity vs human PPAR{gamma} in PPAR-GAL4 transactivation assays. Similar potencies and selectivity were also observed in the full length receptor co-transfection assays. Compound 2 has negligible cross-reactivity against a panel of human nuclear hormone receptors including PPAR{delta}. Compound 2 demonstrated an excellent pharmacological and safety profile in preclinical studies and thus was chosen as a development candidate for the treatment of atherosclerosis and dyslipidemia. The X-ray cocrystal structures of the early lead compound 12 and compound 2 in complex with PPAR{alpha} ligand binding domain (LBD) were determined. The role of the crystal structure of compound 12 with PPAR{alpha} in the development of the SAR that ultimately resulted in the discovery of compound 2 is discussed.

  3. Dephosphorylation of alpha(s)- and beta-caseins and its effect on chaperone activity: a structural and functional investigation.

    PubMed

    Koudelka, Tomas; Hoffmann, Peter; Carver, John A

    2009-07-08

    Milk casein proteins can act as molecular chaperones: under conditions of stress, such as elevated temperature, molecular chaperones stabilize proteins from unfolding, aggregating, and precipitating. In this study, alpha(s)- and beta-caseins were dephosphorylated using alkaline phosphatase. A structural and functional investigation was undertaken to determine the effect of dephosphorylation on the chaperone activity of alpha(s)- and beta-caseins against two types of protein misfolding, i.e., amorphous aggregation and amyloid fibril assembly. The dephosphorylation of alpha(s)- and beta-caseins resulted in a decrease in the chaperone efficiency against both heat- and reduction-induced amorphously aggregating target proteins. In contrast, dephosphorylation had no effect on the chaperone activity of alpha(s)- and beta-caseins against the amyloid-forming target protein kappa-casein. Circular dichroism and fluorescence spectroscopic data indicated that the loss of negative charge associated with dephosphorylation led to an increase in ordered structure of alpha(s)- and beta-caseins. It is concluded that the flexible, dynamic, and relatively unstructured and amphiphatic nature of alpha(s)- and beta-caseins is important in their chaperone action.

  4. H-alpha response to geomagnetic disturbed activity at Arecibo.

    NASA Astrophysics Data System (ADS)

    Santos, Pedrina; Kerr, R.; Noto, J.; Brum, Christiano; Gonzalez, Sixto

    Configured with a spectral resolution of 0.0086 nm at 6563A, the low resolution Fabry-Perot Interferometer (FPI) installed at Arecibo Observatory sampled the geocoronal Balmer-alpha emission for sixty nights during new moon periods from September 2006 to September 2007. In this work two of these periods are analyzed according to the variability with the geomagnetic activity. With this purpose, the effect of the shadow height, local time and solar flux depen-dencies were found and isolated and only the possible variations due the geomagnetic activity were evaluated. The residuos of the relative H-alpha intensity and temperature are analyzed.

  5. Activation of peroxisome proliferator-activated receptor-{alpha} enhances fatty acid oxidation in human adipocytes

    SciTech Connect

    Lee, Joo-Young; Hashizaki, Hikari; Goto, Tsuyoshi; Sakamoto, Tomoya; Takahashi, Nobuyuki; Kawada, Teruo

    2011-04-22

    Highlights: {yields} PPAR{alpha} activation increased mRNA expression levels of adipocyte differentiation marker genes and GPDH activity in human adipocytes. {yields} PPAR{alpha} activation also increased insulin-dependent glucose uptake in human adipocytes. {yields} PPAR{alpha} activation did not affect lipid accumulation in human adipocytes. {yields} PPAR{alpha} activation increased fatty acid oxidation through induction of fatty acid oxidation-related genes in human adipocytes. -- Abstract: Peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}) is a key regulator for maintaining whole-body energy balance. However, the physiological functions of PPAR{alpha} in adipocytes have been unclarified. We examined the functions of PPAR{alpha} using human multipotent adipose tissue-derived stem cells as a human adipocyte model. Activation of PPAR{alpha} by GW7647, a potent PPAR{alpha} agonist, increased the mRNA expression levels of adipocyte differentiation marker genes such as PPAR{gamma}, adipocyte-specific fatty acid-binding protein, and lipoprotein lipase and increased both GPDH activity and insulin-dependent glucose uptake level. The findings indicate that PPAR{alpha} activation stimulates adipocyte differentiation. However, lipid accumulation was not changed, which is usually observed when PPAR{gamma} is activated. On the other hand, PPAR{alpha} activation by GW7647 treatment induced the mRNA expression of fatty acid oxidation-related genes such as CPT-1B and AOX in a PPAR{alpha}-dependent manner. Moreover, PPAR{alpha} activation increased the production of CO{sub 2} and acid soluble metabolites, which are products of fatty acid oxidation, and increased oxygen consumption rate in human adipocytes. The data indicate that activation of PPAR{alpha} stimulates both adipocyte differentiation and fatty acid oxidation in human adipocytes, suggesting that PPAR{alpha} agonists could improve insulin resistance without lipid accumulation in adipocytes. The expected

  6. Sigma ligand S14905 and locomotor activity in mice.

    PubMed

    Hascoet, M; Bourin, M; Payeur, R; Lombet, A; Peglion, J L

    1995-12-01

    The binding and locomotor profile of a new sigma ligand, S14905, (isobutyl-N-(1-indan-2yl-piperid-4-yl)N-methyl carbamate, furamate) was studied. The binding data revealed that S14905 has a high affinity for sigma receptors and very low affinity for both dopamine D1 and D2 receptors. We have demonstrated that this sigma ligand prevents the locomotor stimulation induced by morphine (32 and 64 mg/kg), cocaine (16 mg/kg), amphetamine (4 mg/kg) and adrafinil (32 mg/kg) at doses lower than those required to depress spontaneous locomotor activity. The antagonism observed in the present study seems to be more specific of morphine induced hyperlocomotion. The high affinity of this compound for sigma receptors makes it a good choice to study the role of this receptor in the CNS. In addition, S14905 does not directly block dopamine receptors but may modulate them in some manner, and would thus warrant further study as a potential atypical antipsychotic agent, and an antagonist for the hyperactivity induced by opiate drug.

  7. High-affinity benzodiazepine receptor ligands among benzodiazepines and betacarbolines with different intrinsic activity

    SciTech Connect

    Yliniemelae, A.; Gynther, J. ); Konschin, H.; Tylli, H. ); Rouvinen, J. )

    1989-01-01

    Structural and electrostatic features of diazepam, flumazenil, and methyl betacarboline-3-carboxylate (BCCM) have been investigated using the molecular superimposition method. These high-affinity benzodiazepine (BZ) receptor ligands are structurally unrelated and they have different intrinsic activity. These ligands are superimposed in such a way that common structural and electrostatic features essential for the high receptor binding affinity overlap. In addition to this binding pharmacophore, there are roughly three separate binding zones in the BZ receptor, one for each class of ligands. The intrinsic activity of BZ receptor ligands depends on the molecular structures and the way the ligand approaches the receptor.

  8. A Pseudopterane Diterpene Isolated From the Octocoral Pseudopterogorgia acerosa Inhibits the Inflammatory Response Mediated by TLR-Ligands and TNF-Alpha in Macrophages

    PubMed Central

    González, Yisett; Doens, Deborah; Santamaría, Ricardo; Ramos, Marla; Restrepo, Carlos M.; Barros de Arruda, Luciana; Lleonart, Ricardo; Gutiérrez, Marcelino; Fernández, Patricia L.

    2013-01-01

    Several diterpenoids isolated from terrestrial and marine environments have been identified as important anti-inflammatory agents. Although considerable progress has been made in the area of anti-inflammatory treatment, the search for more effective and safer compounds is a very active field of research. In this study we investigated the anti-inflammatory effects of a known pseudopterane diterpene (referred here as compound 1) isolated from the octocoral Pseudopterogorgia acerosa on the tumor necrosis factor- alpha (TNF-α) and TLRs- induced response in macrophages. Compound 1 inhibited the expression and secretion of the inflammatory mediators TNF-α, interleukin (IL)-6, IL-1β, nitric oxide (NO), interferon gamma-induced protein 10 (IP-10), ciclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS) and monocyte chemoattractant protein-1 (MCP-1) induced by LPS in primary murine macrophages. This effect was associated with the inhibition of IκBα degradation and subsequent activation of NFκB. Compound 1 also inhibited the expression of the co-stimulatory molecules CD80 and CD86, which is a hallmark of macrophage activation and consequent initiation of an adaptive immune response. The anti-inflammatory effect was not exclusive to LPS because compound 1 also inhibited the response of macrophages to TNF-α and TLR2 and TLR3 ligands. Taken together, these results indicate that compound 1 is an anti-inflammatory molecule, which modulates a variety of processes occurring in macrophage activation. PMID:24358331

  9. A pseudopterane diterpene isolated from the octocoral Pseudopterogorgia acerosa inhibits the inflammatory response mediated by TLR-ligands and TNF-alpha in macrophages.

    PubMed

    González, Yisett; Doens, Deborah; Santamaría, Ricardo; Ramos, Marla; Restrepo, Carlos M; Barros de Arruda, Luciana; Lleonart, Ricardo; Gutiérrez, Marcelino; Fernández, Patricia L

    2013-01-01

    Several diterpenoids isolated from terrestrial and marine environments have been identified as important anti-inflammatory agents. Although considerable progress has been made in the area of anti-inflammatory treatment, the search for more effective and safer compounds is a very active field of research. In this study we investigated the anti-inflammatory effects of a known pseudopterane diterpene (referred here as compound 1) isolated from the octocoral Pseudopterogorgia acerosa on the tumor necrosis factor- alpha (TNF-α) and TLRs- induced response in macrophages. Compound 1 inhibited the expression and secretion of the inflammatory mediators TNF-α, interleukin (IL)-6, IL-1β, nitric oxide (NO), interferon gamma-induced protein 10 (IP-10), ciclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS) and monocyte chemoattractant protein-1 (MCP-1) induced by LPS in primary murine macrophages. This effect was associated with the inhibition of IκBα degradation and subsequent activation of NFκB. Compound 1 also inhibited the expression of the co-stimulatory molecules CD80 and CD86, which is a hallmark of macrophage activation and consequent initiation of an adaptive immune response. The anti-inflammatory effect was not exclusive to LPS because compound 1 also inhibited the response of macrophages to TNF-α and TLR2 and TLR3 ligands. Taken together, these results indicate that compound 1 is an anti-inflammatory molecule, which modulates a variety of processes occurring in macrophage activation.

  10. alpha-Diimine Ligand Coordination and C H Bond Activation in the Reaction of Os3(CO)10(MeCN)2 with 6-R-2,2'-Bipyridine (where R = Et, Ph): X-ray Diffraction Structures of the Ortho-Metalated

    SciTech Connect

    Carrano, Carl J.; Wang, Xiaoping; Poola, Bhaskar; Powell, Cynthia B.; Richmond, Michael G.

    2009-01-01

    The reactivity of the labile cluster Os3(CO)10(MeCN)2 (1) with the monofunctionalized heterocyclic ligands 6-R-2,2 -bipyridine (where R = Et, Ph) has been investigated. The alkyl-substituted heterocycle 6-Et-2,2 -bipyridine reacts with 1 in refluxing CH2Cl2 to give an isomeric mixture of HOs3(CO)9(N2C12H11) due to cyclometalation of the side-chain ethyl group (2) and ortho metalation of the unsubstituted bipyridine ring (3). The solid-state structure of the latter cluster, HOs3(CO)9(N2C10H6-6-Et) (3), has unequivocally established the site of the C-H bond activation in the product. Treatment of 1 with the aryl-substituted ligand 6-Ph-2,2 -bipyridine proceeds similarly with ortho metalation at the ancillary phenyl group and the C-6 ortho site of the unsubstituted bipyridine ring, as verified by 1H NMR spectroscopy. The X-ray diffraction structure of the thermodynamically more stable bipyridine-metalated cluster HOs3(CO)9(N2C10H6-6-Ph) (5) has been determined. The course of these reactions is discussed with respect to our recent study involving the reaction of cluster 1 with the ligand 6-Me-2,2 -bipyridine. Graphical Abstract The reaction between the labile cluster Os3(CO)10(MeCN)2 (1) and the monofunctionalized heterocyclic ligand 6-Et-2,2 -bipyridine proceeds readily at room temperature to furnish an isomeric mixture of the cyclometalated and ortho-metalated hydride-bridged clusters HOs3(CO)9(N2C12H11) (2 and 3). Treatment of 1 with 6-Ph-2,2 -bipyridine also yields two distinct hydride-containing clusters that result from independent ortho-metalation paths involving the 6-phenyl substituent and unsubstituted bipyridine group. The bipyridine-derived ortho metalation attendant in the new clusters HOs3(CO)9(N2C10H6-6-Et) (3) and HOs3(CO)9(N2C10H6-6-Ph) (5) has been established by X-ray crystallography.

  11. Identification of putative ligand-binding sites of the integrin alpha 4 beta 1 (VLA-4, CD49d/CD29)

    PubMed Central

    Kamata, T; Puzon, W; Takada, Y

    1995-01-01

    Integrin alpha 4 beta 1 recognizes both fibronectin (CS-1 sequence) and vascular cell adhesion molecule-1 (VCAM-1). To localize the ligand-binding sites of alpha 4, we located the epitopes for function-blocking anti-alpha 4 monoclonal antibodies (mAbs), including those that recognize previously described (but not yet physically localized) functional epitopes (A, B1, B2 and C) using interspecies alpha 4 chimeras expressed in mammalian cells. Epitopes B1 and B2 were associated with ligand binding, and epitopes A and B2 with homotypic cellular aggregation. mAbs P4C2 (epitope B2), 20E4 and PS/2 were mapped within residues 108-182; mAbs HP2/1 (epitope B1), SG/73 and R1-2 within residues 195-268; mAbs HP1/3 (epitope A) and P4G9 within residues 1-52; and B5G10 (epitope C) within residues 269-548. The data suggest that residues 108-268, which do not include bivalent-cation-binding motifs, are related to VCAM-1 and CS-1 binding, and more N-terminal portions of alpha 4 (residues 1 and 52 and 108-182) to homotypic aggregation. Since mAbs PS/2 and HP2/1 block alpha 4 beta 7 binding to mucosal addressin cell adhesion molecule-1 (MAdCAM-1), the MAdCAM-1-binding site is close to, or overlapping with, VCAM-1- and CS-1-binding sites. The role of Asp-130 of beta 1 in the binding to VCAM-1 and CS-1 peptide was examined. Chinese hamster ovary (CHO) cells expressing beta 1 (D130A) (Asp-130 to Ala mutant of beta 1) and alpha 4 showed much less binding to both ligands than CHO cells expressing wild-type beta 1 and alpha 4 [a dominant negative effects of beta 1 (D130A)], suggesting that Asp-130 of beta 1 is critical for binding to both ligands and that the two ligand share common binding mechanisms [corrected]. Images Figure 2 Figure 3 Figure 5 PMID:7531439

  12. Estrogen receptor alpha is cell cycle-regulated and regulates the cell cycle in a ligand-dependent fashion.

    PubMed

    JavanMoghadam, Sonia; Weihua, Zhang; Hunt, Kelly K; Keyomarsi, Khandan

    2016-06-17

    Estrogen receptor alpha (ERα) has been implicated in several cell cycle regulatory events and is an important predictive marker of disease outcome in breast cancer patients. Here, we aimed to elucidate the mechanism through which ERα influences proliferation in breast cancer cells. Our results show that ERα protein is cell cycle-regulated in human breast cancer cells and that the presence of 17-β-estradiol (E2) in the culture medium shortened the cell cycle significantly (by 4.5 hours, P < 0.05) compared with unliganded conditions. The alterations in cell cycle duration were observed in the S and G2/M phases, whereas the G1 phase was indistinguishable under liganded and unliganded conditions. In addition, ERα knockdown in MCF-7 cells accelerated mitotic exit, whereas transfection of ERα-negative MDA-MB-231 cells with exogenous ERα significantly shortened the S and G2/M phases (by 9.1 hours, P < 0.05) compared with parental cells. Finally, treatment of MCF-7 cells with antiestrogens revealed that tamoxifen yields a slower cell cycle progression through the S and G2/M phases than fulvestrant does, presumably because of the destabilizing effect of fulvestrant on ERα protein. Together, these results show that ERα modulates breast cancer cell proliferation by regulating events during the S and G2/M phases of the cell cycle in a ligand-dependent fashion. These results provide the rationale for an effective treatment strategy that includes a cell cycle inhibitor in combination with a drug that lowers estrogen levels, such as an aromatase inhibitor, and an antiestrogen that does not result in the degradation of ERα, such as tamoxifen.

  13. Activation of the platelet collagen receptor integrin alpha(2)beta(1): its mechanism and participation in the physiological functions of platelets.

    PubMed

    Jung, S M; Moroi, M

    2000-10-01

    When platelets are stimulated by agonists, integrin alpha(2)beta(1) (GP Ia/IIa), one of the platelet collagen receptors, is activated to forms with high affinities for its ligand collagen. Here we describe our studies to characterize the binding kinetics of the activated integrin forms and the activation mechanism. Under low agonist concentrations, integrin alpha(2)beta(1) is activated through a mechanism involving ADP/ADP receptors; and under high agonist concentrations, multiple signaling pathways are involved in its activation. Such differences in mechanism at low and high agonist concentrations are also suggested in the activation of integrin alpha(IIb)beta(3), the platelet fibrinogen receptor. We describe our flow adhesion studies, from which evidence was obtained about the involvement of integrin alpha(2)beta(1) activation in the physiological function of platelets, adhesion and thrombus formation.

  14. [L-lysine-alpha-oxidase activity of some Trichoderma species].

    PubMed

    Smirnova, I P; Khaduev, S Kh

    1984-01-01

    Trichoderma cultures were tested for their ability to produce L-lysine-alpha-oxidase. The highest enzyme activity was manifested by T. harzianum (MGU), T. longibrachiatum Rifai VKM F-2025 and T. aureoviride Rifai VKM F-2026. The biosynthesis of the enzyme did not depend on the growth of the cultures and did not vary among the species.

  15. alpha-Glucosidase inhibitory activity of Mangifera indica bark.

    PubMed

    Prashanth, D; Amit, A; Samiulla, D S; Asha, M K; Padmaja, R

    2001-08-01

    The ethanolic extracts of Lawsonia inermis leaves, Holarrhena antidysenterica bark, Swertia chirata whole plant and Mangifera indica bark were tested (in-vitro) for alpha-glucosidase inhibitory activity. M. indica extract was found to be the most potent, with an IC(50) value of 314 microg/ml.

  16. Somatosensory Anticipatory Alpha Activity Increases to Suppress Distracting Input

    ERIC Educational Resources Information Center

    Haegens, Saskia; Luther, Lisa; Jensen, Ole

    2012-01-01

    Effective processing of sensory input in daily life requires attentional selection and amplification of relevant input and, just as importantly, attenuation of irrelevant information. It has been proposed that top-down modulation of oscillatory alpha band activity (8-14 Hz) serves to allocate resources to various regions, depending on task…

  17. Structural analysis of a chimeric bacterial alpha-amylase. High-resolution analysis of native and ligand complexes.

    PubMed

    Brzozowski, A M; Lawson, D M; Turkenburg, J P; Bisgaard-Frantzen, H; Svendsen, A; Borchert, T V; Dauter, Z; Wilson, K S; Davies, G J

    2000-08-08

    Several chimeric alpha-amylases genes were constructed by an in vivo recombination technique from the Bacillus amyloliquefaciens and Bacillus licheniformis genes. One of the fusion amylases (hereafter BA2), consisting of residues 1-300 from B. amyloliquefaciens and 301-483 from B. licheniformis, has been extensively studied by X-ray crystallography at resolutions between 2.2 and 1.7 A. The 3-dimensional structure of the native enzyme was solved by multiple isomorphous replacement, and refined at a resolution of 1.7 A. It consists of 483 amino acids, organized similarly to the known B. lichiniformis alpha-amylase structure [Machius et al. (1995) J. Mol. Biol. 246, 545-559], but features 4 bound calcium ions. Two of these form part of a linear cluster of three ions, the central ion being attributed to sodium. This cluster lies at the junction of the A and B domains with one calcium of the cluster structurally equivalent to the major Ca(2+) binding site of fungal alpha-amylases. The third calcium ion is found at the interface of the A and C domains. BA2 contains a fourth calcium site, not observed in the B. licheniformis alpha-amylase structure. It is found on the C domain where it bridges the two beta-sheets. Three acid residues (Glu261, Asp328, and Asp231) form an active site similar to that seen in other amylases. In the presence of TRIS buffer, a single molecule of TRIS occupies the -1 subsite of the enzyme where it is coordinated by the three active-center carboxylates. Kinetic data reveal that BA2 displays properties intermediate to those of its parents. Data for crystals soaked in maltooligosaccharides reveal the presence of a maltotriose binding site on the N-terminal face of the (beta/alpha)(8) barrel of the molecule, not previously described for any alpha-amylase structure, the biological function of which is unclear. Data for a complex soaked with the tetrasaccharide inhibitor acarbose, at 1.9 A, reveal a decasaccharide moiety, spanning the -7 to +3

  18. Purification of alpha-mannosidase activity from Indian lablab beans.

    PubMed

    Tulasi, R B; Nadimpalli, S K

    1997-04-01

    Seeds of Dolichos lablab var. typicus (Indian lablab beans) contain a glucose/ mannose specific lectin that was affinity purified on Sepharose mannose columns in our laboratory. The unbound fraction from this matrix showed alpha-mannosidase activity. In the present study this has been purified to homogeneity by a combination of ion-exchange, hydrophobic chromatography and gel filtration. Purified alpha-mannosidase had an apparent molecular weight of 195,000 +/- 5,000 with 4.5% carbohydrate. On SDS-PAGE under reducing conditions, the enzyme dissociated into two major bands corresponding to Mr 66,000 and Mr 44,000. An antibody to the well studied jack bean alpha-mannosidase cross-reacts with the enzyme from the lablab beans suggesting antigenic similarity between these two legume mannosidases.

  19. EEG alpha activity and hallucinatory experience during sensory deprivation.

    PubMed

    Hayashi, M; Morikawa, T; Hori, T

    1992-10-01

    The relationship between hallucinatory experiences under sensory deprivation and EEG alpha activities was studied. Each of seven male students lived alone in an air conditioned, soundproof dark room for 72 hours. When hallucinatory experiences occurred, the students pressed a button at once. If they could not press the button during the experience, they were required to press it two times when the hallucinatory experience was finished. Spectral analysis was performed on the consecutive EEG samples from just before button-presses to 10 min. before them, and the average alpha band amplitudes were obtained for the four epochs (0-.5, .5-2, 2-5, 5-10 min.). For the single button-presses, the amplitude of alpha band increased 2 min. before the button-presses. Right-hemisphere EEG activation was observed in the occipital area for the double button-presses. The results suggest an association between the hallucinatory experiences under sensory deprivation and the amount of EEG alpha activity.

  20. In vitro anticancer activity and biologically relevant metabolization of organometallic ruthenium complexes with carbohydrate-based ligands.

    PubMed

    Berger, Isabella; Hanif, Muhammad; Nazarov, Alexey A; Hartinger, Christian G; John, Roland O; Kuznetsov, Maxim L; Groessl, Michael; Schmitt, Frederic; Zava, Olivier; Biba, Florian; Arion, Vladimir B; Galanski, Markus; Jakupec, Michael A; Juillerat-Jeanneret, Lucienne; Dyson, Paul J; Keppler, Bernhard K

    2008-01-01

    The synthesis and in vitro anticancer activity of dihalogenido(eta6-p-cymene)(3,5,6-bicyclophosphite-alpha-D-glucofuranoside)ruthenium(II) complexes are described. The compounds were characterized by NMR spectroscopy and ESI mass spectrometry, and the molecular structures of dichlorido-, dibromido- and diiodido(eta6-p-cymene)(3,5,6-bicyclophosphite-1,2-O-isopropylidene-alpha-D-glucofuranoside)ruthenium(II) were determined by X-ray diffraction analysis. The complexes were shown to undergo aquation of the first halido ligand in aqueous solution, followed by hydrolysis of a P--O bond of the phosphite ligand, and finally formation of dinuclear species. The hydrolysis mechanism was confirmed by DFT calculations. The aquation of the complexes was markedly suppressed in 100 mM NaCl solution, and notably only very slow hydrolysis of the P--O bond was observed. The complexes showed affinity towards albumin and transferrin and monoadduct formation with 9-ethylguanine. In vitro studies revealed that the 3,5,6-bicyclophosphite-1,2-O-cyclohexylidene-alpha-D-glucofuranoside complex is the most cytotoxic compound in human cancer cell lines (IC50 values from 30 to 300 microM depending on the cell line).

  1. Identification of inhibitors against the potential ligandable sites in the active cholera toxin.

    PubMed

    Gangopadhyay, Aditi; Datta, Abhijit

    2015-04-01

    The active cholera toxin responsible for the massive loss of water and ions in cholera patients via its ADP ribosylation activity is a heterodimer of the A1 subunit of the bacterial holotoxin and the human cytosolic ARF6 (ADP Ribosylation Factor 6). The active toxin is a potential target for the design of inhibitors against cholera. In this study we identified the potential ligandable sites of the active cholera toxin which can serve as binding sites for drug-like molecules. By employing an energy-based approach to identify ligand binding sites, and comparison with the results of computational solvent mapping, we identified two potential ligandable sites in the active toxin which can be targeted during structure-based drug design against cholera. Based on the probe affinities of the identified ligandable regions, docking-based virtual screening was employed to identify probable inhibitors against these sites. Several indole-based alkaloids and phosphates showed strong interactions to the important residues of the ligandable region at the A1 active site. On the other hand, 26 top scoring hits were identified against the ligandable region at the A1 ARF6 interface which showed strong hydrogen bonding interactions, including guanidines, phosphates, Leucopterin and Aristolochic acid VIa. This study has important implications in the application of hybrid structure-based and ligand-based methods against the identified ligandable sites using the identified inhibitors as reference ligands, for drug design against the active cholera toxin.

  2. alpha-Tocopheryl phosphate – an active lipid mediator?

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The vitamin E (alpha-tocopherol, alphaT) derivative, alpha-tocopheryl phosphate (alphaTP), is detectable in small amounts in plasma, tissues, and cultured cells. Studies done in vitro and in vivo suggest that alphaT can become phosphorylated and alphaTP dephosphorylated, suggesting the existence of ...

  3. Alpha-band EEG activity in perceptual learning

    PubMed Central

    Bays, Brett C.; Visscher, Kristina M.; Le Dantec, Christophe C.; Seitz, Aaron R.

    2015-01-01

    In studies of perceptual learning (PL), subjects are typically highly trained across many sessions to achieve perceptual benefits on the stimuli in those tasks. There is currently significant debate regarding what sources of brain plasticity underlie these PL-based learning improvements. Here we investigate the hypothesis that PL, among other mechanisms, leads to task automaticity, especially in the presence of the trained stimuli. To investigate this hypothesis, we trained participants for eight sessions to find an oriented target in a field of near-oriented distractors and examined alpha-band activity, which modulates with attention to visual stimuli, as a possible measure of automaticity. Alpha-band activity was acquired via electroencephalogram (EEG), before and after training, as participants performed the task with trained and untrained stimuli. Results show that participants underwent significant learning in this task (as assessed by threshold, accuracy, and reaction time improvements) and that alpha power increased during the pre-stimulus period and then underwent greater desynchronization at the time of stimulus presentation following training. However, these changes in alpha-band activity were not specific to the trained stimuli, with similar patterns of posttraining alpha power for trained and untrained stimuli. These data are consistent with the view that participants were more efficient at focusing resources at the time of stimulus presentation and are consistent with a greater automaticity of task performance. These findings have implications for PL, as transfer effects from trained to untrained stimuli may partially depend on differential effort of the individual at the time of stimulus processing. PMID:26370167

  4. Peroxisome proliferator-activated receptor {alpha} agonists modulate Th1 and Th2 chemokine secretion in normal thyrocytes and Graves' disease

    SciTech Connect

    Antonelli, Alessandro; Ferrari, Silvia Martina; Frascerra, Silvia; Corrado, Alda; Pupilli, Cinzia; Bernini, Giampaolo; Benvenga, Salvatore; Ferrannini, Ele; Fallahi, Poupak

    2011-07-01

    Until now, no data are present about the effect of peroxisome proliferator-activated receptor (PPAR){alpha} activation on the prototype Th1 [chemokine (C-X-C motif) ligand (CXCL)10] (CXCL10) and Th2 [chemokine (C-C motif) ligand 2] (CCL2) chemokines secretion in thyroid cells. The role of PPAR{alpha} and PPAR{gamma} activation on CXCL10 and CCL2 secretion was tested in Graves' disease (GD) and control primary thyrocytes stimulated with interferon (IFN){gamma} and tumor necrosis factor (TNF){alpha}. IFN{gamma} stimulated both CXCL10 and CCL2 secretion in primary GD and control thyrocytes. TNF{alpha} alone stimulated CCL2 secretion, while had no effect on CXCL10. The combination of IFN{gamma} and TNF{alpha} had a synergistic effect both on CXCL10 and CCL2 chemokines in GD thyrocytes at levels comparable to those of controls. PPAR{alpha} activators inhibited the secretion of both chemokines (stimulated with IFN{gamma} and TNF{alpha}) at a level higher (for CXCL10, about 60-72%) than PPAR{gamma} agonists (about 25-35%), which were confirmed to inhibit CXCL10, but not CCL2. Our data show that CCL2 is modulated by IFN{gamma} and TNF{alpha} in GD and normal thyrocytes. Furthermore we first show that PPAR{alpha} activators inhibit the secretion of CXCL10 and CCL2 in thyrocytes, suggesting that PPAR{alpha} may be involved in the modulation of the immune response in the thyroid.

  5. alpha-MSH enhances activity-based anorexia.

    PubMed

    Hillebrand, Jacquelien J G; Kas, Martien J H; Adan, Roger A H

    2005-10-01

    Activity-based anorexia (ABA) is considered an animal model of anorexia nervosa (AN). In ABA, scheduled feeding in combination with voluntary access to running wheels, results in hyperactivity, hypophagia, body weight loss and activation of the HPA axis. Since stimulation of the melanocortin (MC) system has similar effects, this system is a candidate system involved in ABA. Here it is shown that chronic alpha-MSH treatment enhances ABA by increasing running wheel activity (RWA), decreasing food intake and increasing HPA axis activation.

  6. Derivatization of (+/-)-5-[(2-methylphenoxy)methyl]-2-amino-2-oxazoline, an imidazoline binding sites ligand, with (+)-(R)-alpha-methylbenzyl isocyanate for drug monitoring purposes.

    PubMed

    Matoga, Myriam; Forfar, Isabelle; Chaimbault, Corinne; Guillon, Jean; Péhourcq, Fabienne; Bosc, Jean-Jacques; Rettori, Marie-Claire; Jarry, Christian

    2002-12-01

    The derivatization of racemic 5-[(2-methylphenoxy)methyl]-2-amino-2-oxazoline, developed as an imidazoline binding sites ligand, with (+)-(R)-alpha-methylbenzyl isocyanate was performed in chloroform. The reaction led to two pairs of diastereomers, which were separated by RP-HPLC. A kinetic study of the derivatization reaction was achieved in order to establish conditions suitable for experimental drug monitoring.

  7. Temporary anion states of. pi. -ligand transition-metal carbonyls studied by means of electron transmission spectroscopy and x. cap alpha. calculations

    SciTech Connect

    Modelli, A.; Distefano, G.; Guerra, M.; Jones, D.

    1987-07-22

    The resonances observed in the electron transmission spectra of (benzene)chromium tricarbonyl, (cyclopentadienyl)manganese tricarbonyl, (1,3-butadiene)iron tricarbonyl, and (cyclopentadienyl)cobalt dicarbonyl have been assigned with the aid of MS X..cap alpha.. calculations. In contrast with previous theoretical results, the present calculations on the neutral states show a large net electronic charge transfer from the ..pi.. ligand to the metal.

  8. Statins enhance peroxisome proliferator-activated receptor gamma coactivator-1alpha activity to regulate energy metabolism.

    PubMed

    Wang, Wenxian; Wong, Chi-Wai

    2010-03-01

    Peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) serves as an inducible coactivator for a number of transcription factors to control energy metabolism. Insulin signaling through Akt kinase has been demonstrated to phosphorylate PGC-1alpha at serine 571 and downregulate its activity in the liver. Statins are 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors that reduce cholesterol synthesis in the liver. In this study, we found that statins reduced the active form of Akt and enhanced PGC-1alpha activity. Specifically, statins failed to activate an S571A mutant of PGC-1alpha. The activation of PGC-1alpha by statins selectively enhanced the expression of energy metabolizing enzymes and regulators including peroxisome proliferator-activated receptor alpha, acyl-CoA oxidase, carnitine palmitoyl transferase-1A, and pyruvate dehydrogenase kinase 4. Importantly, a constitutively active form of Akt partially reduced the statin-enhanced gene expression. Our study thus provides a plausible mechanistic explanation for the hypolipidemic effect of statin through elevating the rate of beta-oxidation and mitochondrial Kreb's cycle capacity to enhance fatty acid utilization while reducing the rate of glycolysis.

  9. Mutation of Pro-258 in transmembrane domain 6 constitutively activates the G protein-coupled alpha-factor receptor.

    PubMed Central

    Konopka, J B; Margarit, S M; Dube, P

    1996-01-01

    The alpha-factor pheromone receptor stimulates MATa yeast cells to undergo conjugation. The receptor contains seven transmembrane domains that function in ligand binding and in transducing a signal to the cytoplasmic receptor sequences to mediate G protein activation. A genetic screen was used to isolate receptor mutations that constitutively signal in the absence of alpha-factor. The Pro-258-->Leu (P258L) mutation caused constitutive receptor signaling that was equivalent to about 45% of the maximum level observed in wild-type cells stimulated with alpha-factor. Mutations of both Pro-258 and the adjacent Ser-259 to Leu increased constitutive signaling to > or = 90% of the maximum level. Since Pro-258 occurs in the central portion of transmembrane domain 6, and since proline residues are expected to cause a kink in alpha-helical domains, the P258L mutation is predicted to alter the structure of transmembrane domain 6. The P258L mutation did not result in a global distortion of receptor structure because alpha-factor bound to the mutant receptors with high affinity and induced even higher levels of signaling. These results suggest that sequences surrounding Pro-258 may be involved in ligand activation of the receptor. Conformational changes in transmembrane domain 6 may effect a change in the adjacent sequences in the third intracellular loop that are thought to function in G protein activation. Greater than 90% of all G protein-coupled receptors contain a proline residue at a similar position in transmembrane domain 6, suggesting that this aspect of receptor activation may be conserved in other receptors. Images Fig. 3 PMID:8692892

  10. The laminin-binding activity of the alpha 7 integrin receptor is defined by developmentally regulated splicing in the extracellular domain.

    PubMed Central

    Ziober, B L; Chen, Y; Kramer, R H

    1997-01-01

    The expression pattern of the laminin-binding alpha 7 beta 1 integrin is developmentally regulated in skeletal, cardiac, and smooth muscle. The X1/X2 alternative splicing in the extracellular domain of alpha 7 is found in the variable region between conserved alpha-chain homology repeat domains III and IV, a site implicated in ligand binding. To assess differences in X1/X2 isoform activity, we generated MCF-7 cell lines transfected with alpha 7-X1/X2 cDNAs. Transfectants expressing the alpha 7-X2 variant adhered rapidly to laminin 1, whereas those expressing alpha 7-X1 failed to attach. That alpha 7-X1 exists in an inactive state was established in assays using an activating beta 1 antibody that induced X1-dependent cell adhesion and spreading. Furthermore, the activation of alpha 7-X1 was cell type specific, and when expressed in HT1080 cells, the integrin was converted into a fully functional receptor capable of promoting adhesion. Thus, the expression of the alpha 7-X1/X2 integrin is a novel mechanism that regulates receptor affinity states in a cell-specific context and may modulate integrin-dependent events during muscle development and repair. Images PMID:9307969

  11. Diabetes or peroxisome proliferator-activated receptor alpha agonist increases mitochondrial thioesterase I activity in heart

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Peroxisome proliferator-activated receptor alpha (PPAR alpha) is a transcriptional regulator of the expression of mitochondrial thioesterase I (MTE-I) and uncoupling protein 3 (UCP3), which are induced in the heart at the mRNA level in response to diabetes. Little is known about the regulation of pr...

  12. Activation of the alpha 4 beta 1 integrin through the beta 1 subunit induces recognition of the RGDS sequence in fibronectin

    PubMed Central

    1994-01-01

    Lymphocyte attachment to fibronectin is mainly mediated by the interaction of alpha 5 beta 1 and alpha 4 beta 1 integrins with the RGD and CS-1/Hep II sites, respectively. We have recently shown that the anti-beta 1 mAb TS2/16 can convert the partly active alpha 4 beta 1 present on certain hemopoietic cells that recognizes CS-1 but not Hep II, to a high avidity form that binds both ligands. In this report we have studied whether mAb TS2/16 also affects alpha 4 beta 1 ligand specificity. Incubation of the B cell lines Ramos and Daudi (which lack alpha 5 beta 1) with mAb TS2/16 induced specific attachment to an 80-kD fragment which lacks CS-1 and Hep II and contains the RGD sequence. mAbs anti-alpha 4 and the synthetic peptides CS-1 and IDAPS inhibited adhesion to the 80-kD fragment thus implying alpha 4 beta 1 as the receptor for this fragment. Interestingly, the synthetic peptide GRGDSPC and a 15-kD peptic fibronectin fragment containing the RGD sequence also inhibited B cell adhesion to the 80-kD fragment. Because we have previously shown that RGD peptides do not affect the constitutive function of alpha 4 beta 1, we tested whether TS2/16- activated alpha 4 beta 1 acquired the capacity to recognize RGD. Indeed RGD peptides inhibited TS2/16-treated B cell adhesion to a 38-kD fragment containing CS-1 and Hep II but did not affect binding of untreated cells to this fragment. An anti-fibronectin mAb reactive with an epitope on or near the RGD sequence also efficiently inhibited cell adhesion to the 80-kD fragment, indicating that the RGD sequence is a novel adhesive ligand for activated alpha 4 beta 1. These results emphasize the role of alpha 4 beta 1 as a receptor with different ligand specificities according to the activation state, a fact that may be important for lymphocyte migration, localization, and function. PMID:7517944

  13. Activity of alpha-amylase inhibitors from Phaseolus coccineus on digestive alpha-amylases of the coffee berry borer.

    PubMed

    Valencia-Jiménez, Arnubio; Arboleda Valencia, Jorge W; Grossi-De-Sá, Maria Fátima

    2008-04-09

    Seeds of scarlet runner bean ( Phaseolus coccineus L.) were analyzed for alpha-amylase inhibitor (alpha-AI) activity. Through the use of polyclonal antibodies raised against pure alpha-AI-1 from common bean ( Phaseolus vulgaris L.), typical alpha-AlphaIota polypeptides (Mr 14-18 kDa) as well as a large polypeptide of Mr 32000 Da, usually referred to as "amylase inhibitor like", were detected. The inhibitor activity present in four accessions of P. coccineus was examined, both in semiquantitative zymograms allowing the separation of different isoforms and in quantitative assays against human salivary amylase, porcine pancreatic amylase, and coffee berry borer, Hypothenemus hampei Ferrari (Coleoptera: Scolytidae) amylase. Differential inhibition curves lead to the suggestion that the gene encoding one of the inhibitors in P. coccineus (in accession G35590) would be a good candidate for genetic engineering of coffee resistance toward the coffee berry borer. An in vitro proteolytic digestion treatment of pure alpha-AlphaIota-1 resulted in a rapid loss of the inhibitory activity, seriously affecting its natural capacity to interact with mammalian alpha-amylases.

  14. The inhibition of the human cholesterol 7alpha-hydroxylase gene (CYP7A1) promoter by fibrates in cultured cells is mediated via the liver x receptor alpha and peroxisome proliferator-activated receptor alpha heterodimer.

    PubMed

    Gbaguidi, G Franck; Agellon, Luis B

    2004-01-01

    In previous work, we showed that the binding of the liver x receptor alpha:peroxisome proliferator-activated receptor alpha (LXRalpha:PPARalpha) heterodimer to the murine Cyp7a1 gene promoter antagonizes the stimulatory effect of their respective ligands. In this study, we determined if LXRalpha:PPARalpha can also regulate human CYP7A1 gene promoter activity. Co-expression of LXRalpha and PPARalpha in McArdle RH7777 hepatoma cells decreased the activity of the human CYP7A1 gene promoter in response to fibrates and 25-hydroxycholesterol. In vitro, the human CYP7A1 Site I bound LXRalpha:PPARalpha, although with substantially less affinity compared with the murine Cyp7a1 Site I. The binding of LXRalpha:PPARalpha to human CYP7A1 Site I was increased in the presence of either LXRalpha or PPARalpha ligands. In HepG2 hepatoblastoma cells, fibrates and 25-hydroxycholesterol inhibited the expression of the endogenous CYP7A1 gene as well as the human CYP7A1 gene promoter when co-transfected with plasmids encoding LXRalpha and PPARalpha. However, a derivative of the human CYP7A1 gene promoter that contains a mutant form of Site I that does not bind LXRalpha:PPARalpha was not inhibited by WY 14,643 or 25-hydroxycholesterol in both McArdle RH7777 and HepG2 cells. The ligand-dependent recruitment of LXRalpha:PPARalpha heterodimer onto the human CYP7A1 Site I can explain the inhibition of the human CYP7A1 gene promoter in response to fibrates and 25-hydroxycholesterol.

  15. Synthesis, structure characterization and biological activity of selected metal complexes of sulfonamide Schiff base as a primary ligand and some mixed ligand complexes with glycine as a secondary ligand

    NASA Astrophysics Data System (ADS)

    Sharaby, Carmen M.; Amine, Mona F.; Hamed, Asmaa A.

    2017-04-01

    The current work reports synthesis of metal complexes and mixed ligand complexes of a novel sulfonamide Schiff base ligand (HL) resulted from the condensation of sulfametrole [N‧-(4-methoxy-1,2,5-thiadiazol-3-yl]sulfanilamide and acetyl-acetone as a primary ligand and glycine as a secondary ligand. The metal complexes and mixed ligand complexes of HL Schiff base ligand were synthesized and characterized using different physicochemical studies as elemental analyses, mass spectra, conductivity measurement, IR spectra, 1H NMR spectra, UV-vis Spectra, solid reflectance, magnetic susceptibility, thermal analyses (TGA and DTA) and their microbial and anticancer activities. The spectroscopic data of the complexes suggest their 1:2(L1:M) complex structures and 1:2:2(L1:L2:M) mixed ligand complex structures, where L1 = HL and L2 = glycine. Also, the spectroscopic studies suggested the octahedral structure for all complexes. The synthesized Schiff base, its metal and mixed ligand complexes were screened for their bacterial, antifungal and anticancer activity. The activity data show that the metal complexes and mixed ligand complexes exhibited promising microbial and anticancer activities than their parent HL Schiff base ligand, also the data show that the mixed ligand complexes more effective than the metal complexes.

  16. Thermodynamics of the ligandin function of human class Alpha glutathione transferase A1-1: energetics of organic anion ligand binding.

    PubMed Central

    Sayed, Yasien; Hornby, Judith A T; Lopez, Marimar; Dirr, Heini

    2002-01-01

    In addition to their catalytic functions, cytosolic glutathioneS-transferases (GSTs) are a major reserve of high-capacity binding proteins for a large variety of physiological and exogenous non-substrate compounds. This ligandin function has implicated GSTs in numerous ligand-uptake, -transport and -storage processes. The binding of non-substrate ligands to GSTs can inhibit catalysis. In the present study, the energetics of the binding of the non-substrate ligand 8-anilino-1-naphthalene sulphonate (ANS) to wild-type human class Alpha GST with two type-1 subunits (hGSTA1-1) and its DeltaPhe-222 deletion mutant were studied by isothermal titration calorimetry. The stoichiometry of binding to both proteins is one ANS molecule per GST subunit with a greater affinity for the wild-type (K(d)=65 microM) than for the DeltaPhe-222 mutant (K(d)=105 microM). ANS binding to the wild-type protein is enthalpically driven and it is characterized by a large negative heat-capacity change, DeltaC(p). The negative DeltaC(p) value for ANS binding indicates a specific interface with a significant hydrophobic component in the protein-ligand complex. The negatively charged sulphonate group of the anionic ligand is apparently not a major determinant of its binding. Phe-222 contributes to the binding affinity for ANS and the hydrophobicity of the binding site. PMID:11931663

  17. Activating STAT3 Alpha for Promoting Healing of Neurons

    NASA Technical Reports Server (NTRS)

    Conway, Greg

    2008-01-01

    A method of promoting healing of injured or diseased neurons involves pharmacological activation of the STAT3 alpha protein. Usually, injured or diseased neurons heal incompletely or not at all for two reasons: (1) they are susceptible to apoptosis (cell death); and (2) they fail to engage in axogenesis that is, they fail to re-extend their axons to their original targets (e.g., muscles or other neurons) because of insufficiency of compounds, denoted neurotrophic factors, needed to stimulate such extension. The present method (see figure) of treatment takes advantage of prior research findings to the effect that the STAT3 alpha protein has anti-apoptotic and pro-axogenic properties.

  18. Regulation of constitutive androstane receptor and its target genes by fasting, cAMP, hepatocyte nuclear factor alpha, and the coactivator peroxisome proliferator-activated receptor gamma coactivator-1alpha.

    PubMed

    Ding, Xunshan; Lichti, Kristin; Kim, Insook; Gonzalez, Frank J; Staudinger, Jeff L

    2006-09-08

    Animal studies reveal that fasting and caloric restriction produce increased activity of specific metabolic pathways involved in resistance to weight loss in liver. Evidence suggests that this phenomenon may in part occur through the action of the constitutive androstane receptor (CAR, NR1I3). Currently, the precise molecular mechanisms that activate CAR during fasting are unknown. We show that fasting coordinately induces expression of genes encoding peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha), CAR, cytochrome P-450 2b10 (Cyp2b10), UDP-glucuronosyltransferase 1a1 (Ugt1a1), sulfotransferase 2a1 (Sult2a1), and organic anion-transporting polypeptide 2 (Oatp2) in liver in mice. Treatments that elevate intracellular cAMP levels also produce increased expression of these genes in cultured hepatocytes. Our data show that PGC-1alpha interaction with hepatocyte nuclear factor 4alpha (HNF4alpha, NR2A1) directly regulates CAR gene expression through a novel and evolutionarily conserved HNF4-response element (HNF4-RE) located in its proximal promoter. Expression of PGC-1alpha in cells increases CAR expression and ligand-independent CAR activity. Genetic studies reveal that hepatic expression of HNF4alpha is required to produce fasting-inducible CAR expression and activity. Taken together, our data show that fasting produces increased expression of genes encoding key metabolic enzymes and an uptake transporter protein through a network of interactions involving cAMP, PGC-1alpha, HNF4alpha, CAR, and CAR target genes in liver. Given the recent finding that mice lacking CAR exhibit a profound decrease in resistance to weight loss during extended periods of caloric restriction, our findings have important implications in the development of drugs for the treatment of obesity and related diseases.

  19. Identification of Domains for Efficient Notch Signaling Activity in Immobilized Notch Ligand Proteins.

    PubMed

    Liu, Ledi; Wada, Hiroe; Matsubara, Natsuki; Hozumi, Katsuto; Itoh, Motoyuki

    2017-04-01

    Notch is a critical signaling pathway that controls cell fate and tissue homeostasis, but the functional characterization of Notch ligand domains that activate Notch receptors remains incomplete. Here, we established a method for immobilizing Notch ligand proteins onto beads to measure time-dependent Notch activity after the addition of Notch ligand-coated beads. A comparison between activities by the Notch ligand found on the cell surface to that of the ligand immobilized on beads showed that immobilized Notch ligand protein produces comparable signal activity during the first 10 h. Follow-up truncation studies showed that the N-terminal epidermal growth factor (EGF) repeat three region of delta like canonical Notch ligand 4 (DLL4) or jagged 1 (JAG1) is the minimum region for activating Notch signaling, and the DLL4 EGF repeat three domain may have a role in activation through a mechanism other than by increasing binding affinity. In addition, we found that reconstruction of the DLL4 delta and OSM-11 (DOS) motif (N257P) resulted in an increase in both binding affinity and signaling activity, which suggests that the role of the DOS motif is conserved among Notch ligands. Furthermore, active DLL4 protein on beads promoted T cell differentiation or inhibited B cell differentiation in vitro, whereas JAG1 proteins on beads did not have any effect. Taken together, our findings provide unambiguous evidence for the role of different Notch ligands and their domains in Notch signal activation, and may be potential tools for controlling Notch signaling activation. J. Cell. Biochem. 118: 785-796, 2017. © 2016 Wiley Periodicals, Inc.

  20. Ultraviolet-mediated antimycotic activity of alpha-terthienyl on Microsporum cookei.

    PubMed

    Mares, D; Fasulo, M P; Bruni, A

    1990-01-01

    Alpha-terthienyl (alpha-T) in the presence of UV-A irradiation reduced the growth rate of Microsporum cookei. In the dark, alpha-T accumulated in small diffuse vacuoles within the hyphae. After UV-A treatment, alpha-T caused damage to the membranes of the nucleus, mitochondria and endoplasmic reticulum. Plasmolytic and autolytic changes occurred resulting in plasma membrane breakage and cell wall aberrations. UV-A activated alpha-T would appear to target membrane proteins.

  1. The crystal structure of a TL/CD8{alpha}{alpha} complex at 2.1 {angstrom} resolution : implications for modulation of T cell activation and memory.

    SciTech Connect

    Liu, Y.; Xiong, Y.; Naidenko, O. V.; Liu, J.-H.; Zhang, R.; Joachimiak, A.; Kronenberg, M.; Cheroutre, H.; Reinherz, E. L.; Wang, J.-H.; Biosciences Division; Dana-Farber Cancer Inst.; Harvard Medical School; La Jolla Inst. of Allergy and Immunology

    2003-02-01

    TL is a nonclassical MHC class I molecule that modulates T cell activation through relatively high-affinity interaction with CD8{alpha}{alpha}. To investigate how the TL/CD8{alpha}{alpha} interaction influences TCR signaling, we characterized the structure of the TL/CD8{alpha}{alpha} complex using X-ray crystallography. Unlike antigen-presenting molecules, the TL antigen-binding groove is occluded by specific conformational changes. This feature eliminates antigen presentation, severely hampers direct TCR recognition, and prevents TL from participating in the TCR activation complex. At the same time, the TL/CD8{alpha}{alpha} interaction is strengthened through subtle structure changes in the TL {alpha}3 domain. Thus, TL functions to sequester and redirect CD8{alpha}{alpha} away from the TCR, modifying lck-dependent signaling.

  2. Cortical alpha activity predicts the confidence in an impending action

    PubMed Central

    Kubanek, Jan; Hill, N. Jeremy; Snyder, Lawrence H.; Schalk, Gerwin

    2015-01-01

    When we make a decision, we experience a degree of confidence that our choice may lead to a desirable outcome. Recent studies in animals have probed the subjective aspects of the choice confidence using confidence-reporting tasks. These studies showed that estimates of the choice confidence substantially modulate neural activity in multiple regions of the brain. Building on these findings, we investigated the neural representation of the confidence in a choice in humans who explicitly reported the confidence in their choice. Subjects performed a perceptual decision task in which they decided between choosing a button press or a saccade while we recorded EEG activity. Following each choice, subjects indicated whether they were sure or unsure about the choice. We found that alpha activity strongly encodes a subject's confidence level in a forthcoming button press choice. The neural effect of the subjects' confidence was independent of the reaction time and independent of the sensory input modeled as a decision variable. Furthermore, the effect is not due to a general cognitive state, such as reward expectation, because the effect was specifically observed during button press choices and not during saccade choices. The neural effect of the confidence in the ensuing button press choice was strong enough that we could predict, from independent single trial neural signals, whether a subject was going to be sure or unsure of an ensuing button press choice. In sum, alpha activity in human cortex provides a window into the commitment to make a hand movement. PMID:26283892

  3. Localization of the fourth membrane spanning domain as a ligand binding site in the human platelet. alpha. sub 2 -adrenergic receptor

    SciTech Connect

    Matsui, Hiroaki; Lefkowitz, R.J.; Caron, M.G.; Regan, J.W. )

    1989-05-02

    The human platelet {alpha}{sub 2}-adrenergic receptor is an integral membrane protein which binds epinephrine. The gene for this receptor has been cloned, and the primary structure is thus known. A model of its secondary structure predicts that the receptor has seven transmembrane spanning domains. By covalent labeling and peptide mapping, the authors have identified a region of the receptor that is directly involved with ligand binding. Partially purified preparations of the receptor were covalently radiolabeled with either of two specific photoaffinity ligands: ({sup 3}H)SKF 102229 (an antagonist) or p-azido({sup 3}H)clonidine (an agonist). The radiolabeled receptors were then digested with specific endopeptidases, and peptides containing the covalently bound radioligands were identified. Lysylendopeptidase treatment of ({sup 3}H)SKF 102229 labeled receptor yielded one peptide of M{sub r} 2400 as the product of a complete digest. Endopeptidase Arg-C gave a labeled peptide of M{sub r} 4000, which was further digested to the M{sub r} 2400 peptide by additional treatment with lysylendopeptidase. Using p-azido({sup 3}H)clonidine-labeled receptor, a similar M{sub r} 2400 peptide was obtained by lysylendopeptidase cleavage. This M{sub r} 2400 peptide corresponds to the fourth transmembrane spanning domain of the receptor. These data suggest that this region forms part of the ligand binding domain of the human platelet {alpha}{sub 2}-adrenergic receptor.

  4. Selective Electrocatalytic Activity of Ligand Stabilized Copper Oxide Nanoparticles

    SciTech Connect

    Kauffman, Douglas R; Ohodnicki, Paul R; Kail, Brian W; Matranga, Christopher

    2011-01-01

    Ligand stabilization can influence the surface chemistry of Cu oxide nanoparticles (NPs) and provide unique product distributions for electrocatalytic methanol (MeOH) oxidation and CO{sub 2} reduction reactions. Oleic acid (OA) stabilized Cu{sub 2}O and CuO NPs promote the MeOH oxidation reaction with 88% and 99.97% selective HCOH formation, respectively. Alternatively, CO{sub 2} is the only reaction product detected for bulk Cu oxides and Cu oxide NPs with no ligands or weakly interacting ligands. We also demonstrate that OA stabilized Cu oxide NPs can reduce CO{sub 2} into CO with a {approx}1.7-fold increase in CO/H{sub 2} production ratios compared to bulk Cu oxides. The OA stabilized Cu oxide NPs also show 7.6 and 9.1-fold increases in CO/H{sub 2} production ratios compared to weakly stabilized and non-stabilized Cu oxide NPs, respectively. Our data illustrates that the presence and type of surface ligand can substantially influence the catalytic product selectivity of Cu oxide NPs.

  5. DNA-binding activity of TNF-{alpha} inducing protein from Helicobacter pylori

    SciTech Connect

    Kuzuhara, T. Suganuma, M.; Oka, K.; Fujiki, H.

    2007-11-03

    Tumor necrosis factor-{alpha} (TNF-{alpha}) inducing protein (Tip{alpha}) is a carcinogenic factor secreted from Helicobacter pylori (H. pylori), mediated through both enhanced expression of TNF-{alpha} and chemokine genes and activation of nuclear factor-{kappa}B. Since Tip{alpha} enters gastric cancer cells, the Tip{alpha} binding molecules in the cells should be investigated. The direct DNA-binding activity of Tip{alpha} was observed by pull down assay using single- and double-stranded genomic DNA cellulose. The surface plasmon resonance assay, indicating an association between Tip{alpha} and DNA, revealed that the affinity of Tip{alpha} for (dGdC)10 is 2400 times stronger than that of del-Tip{alpha}, an inactive Tip{alpha}. This suggests a strong correlation between DNA-binding activity and carcinogenic activity of Tip{alpha}. And the DNA-binding activity of Tip{alpha} was first demonstrated with a molecule secreted from H. pylori.

  6. Synthesis and antimalarial activity of metal complexes of cross-bridged tetraazamacrocyclic ligands

    PubMed Central

    Hubin, Timothy J.; Amoyaw, Prince N. -A.; Roewe, Kimberly D.; Simpson, Natalie C.; Maples, Randall D.; Carder Freeman, TaRynn N.; Cain, Amy N.; Le, Justin G.; Archibald, Stephen J.; Khan, Shabana I.; Tekwani, Babu L.; Khan, M. O. Faruk

    2014-01-01

    Using transition metals such as manganese(II), iron(II), cobalt(II), nickel(II), copper(II), and zinc(II), several new metal complexes of cross-bridged tetraazamacrocyclic chelators namely, cyclen- and cyclam-analogs with benzyl groups, were synthesized and screened for in vitro antimalarial activity against chloroquine-resistant (W2) and chloroquine-sensitive (D6) strains of Plasmodium falciparum. The metal-free chelators tested showed little or no antimalarial activity. All the metal complexes of the dibenzyl cross-bridged cyclam ligand exhibited potent antimalarial activity. The Mn2+ complex of this ligand was the most potent with IC50s of 0.127 and 0.157 µM against the chloroquine-sensitive (D6) and chloroquine-resistant (W2) P. falciparum strains, respectively. In general, the dibenzyl hydrophobic ligands showed better antimalarial activity compared to the activity of monobenzyl ligands, potentially because of their higher lipophilicity and thus better cell penetration ability. The higher antimalarial activity displayed by the manganese complex for the cyclam ligand in comparison to that of the cyclen, correlates with the larger pocket of cyclam compared to that of cyclen which produces a more stable complex with the Mn2+. Few of the Cu2+ and Fe2+ complexes also showed improvement in activity but Ni2+, Co2+ and Zn2+ complexes did not show any improvement in activity upon the metal-free ligands for anti-malarial development. PMID:24857776

  7. Increased alpha 2-macroglobulin in diabetes: a hyperglycemia related phenomenon associated with reduced antithrombin III activity.

    PubMed

    Ceriello, A; Giugliano, D; Quatraro, A; Stante, A; Dello Russo, P; Torella, R

    1989-01-01

    Increased alpha 2-macroglobulin (alpha 2M) activity and concentration, and decreased antithrombin III (ATIII) plasma concentration are reported in diabetic subjects. In diabetes an inverse correlation between ATIII activity and blood glucose, HbA1, alpha 2M activity and alpha 2M concentration, and a direct correlation between both alpha 2M activity and alpha 2M concentration with blood glucose and HbA1 are found. Moreover, a direct correlation between alpha 2M activity and alpha 2M concentration fails. In both diabetic and normal subjects induced hyperglycemia increases alpha 2M activity and alpha 2M concentration reduces ATIII activity, while ATIII concentration is not affected. These data which show that hyperglycemia may increase alpha 2M molecule levels while altering only the biological function of ATIII, provide evidence that hyperglycemia may decrease, directly, the biological function of some proteins and may condition the levels of some risk factors for the development of diabetic complications such as alpha 2M.

  8. MULTIDENTATE TEREPHTHALAMIDATE AND HYDROXYPYRIDONATE LIGANDS: TOWARDS NEW ORALLY ACTIVE CHELATORS

    SciTech Connect

    Abergel, Rebecca J.; Raymond, Kenneth N.

    2011-07-13

    The limitations of current therapies for the treatment of iron overload or radioisotope contamination have stimulated efforts to develop new orally bioavailable iron and actinide chelators. Siderophore-inspired tetradentate, hexadentate and octadentate terephthalamidate and hydroxypyridonate ligands were evaluated in vivo as selective and efficacious iron or actinide chelating agents, with several metal loading and ligand assessment procedures, using {sup 59}Fe, {sup 238}Pu, and {sup 241}Am as radioactive tracers. The compounds presented in this study were compared to commercially available therapeutic sequestering agents [deferoxamine (DFO) for iron and diethylenetriaminepentaacetic acid (DPTA) for actinides] and are unrivaled in terms of affinity, selectivity and decorporation efficacy, which attests to the fact that high metal affinity may overcome the low bioavailability properties commonly associated to multidenticity.

  9. Effects of 5-HT-receptor and alpha 2-adrenoceptor ligands on the haemodynamic response to acute central hypovolaemia in conscious rabbits.

    PubMed Central

    Evans, R. G.; Haynes, J. M.; Ludbrook, J.

    1993-01-01

    1. We set out to elucidate the pharmacological mechanisms by which alpha 2-adrenoceptor and 5-HT-receptor ligands affect the haemodynamic response to acute central hypovolaemia in conscious rabbits. 2. Acute central hypovolaemia was produced by inflating an inferior vena caval cuff so that cardiac output fell at a constant rate of approximately 8.5% of its baseline level per min. 3. Drugs were administered into the fourth cerebral ventricle in either 154 mM NaCl (saline) or 20% w/v 2-hydroxypropyl-beta-cyclodextrin (beta-CDX). After vehicle treatments, the haemodynamic response to acute central hypovolaemia had the usual two phases. During Phase I, systemic vascular conductance fell in proportion to cardiac output so that mean arterial pressure fell by only 8 mmHg. Phase II commenced when cardiac output had fallen to approximately 60% of its baseline level, when vascular conductance rose abruptly and arterial pressure fell to < or = 40 mmHg. The haemodynamic response was not dependent on the vehicle used (saline or beta-CDX). 4. Methysergide delayed the occurrence of Phase II in a dose-dependent manner, and prevented it at a dose of 30- 600 nmol (geometric mean = 186 nmol). The effects and potency of methysergide were not dependent on the vehicle used, indicating that beta-CDX can be used as a vehicle for fourth ventricular administration of lipophilic drugs to conscious rabbits. Clonidine (10 nmol) reversed the effects of a critical dose of methysergide. 5. Phase II was also prevented by 8-hydroxy-2-(di-n-propylamino)tetralin (5-HT1A-selective agonist, geometric mean critical dose (range) = 13.1 (10-30) nmol), sumatriptan (5-HT1D-selective agonist, 72.1 (10-300) nmol), mesulergine (5-HT2/1C-selective antagonist, 173 (30-1000) nmol), idazoxan (alpha 2-adrenoceptor-selective antagonist, 548 (100-3000) nmol), and mianserin (5-HT2/1C-selective antagonist, 548 (100-3000) nmol). It was not affected by MDL 72222 (5-HT3-selective antagonist, 300 nmol) or ketanserin (5-HT2

  10. Phosphorylated nuclear receptor CAR forms a homodimer to repress its constitutive activity for ligand activation.

    PubMed

    Shizu, Ryota; Osabe, Makoto; Perera, Lalith; Moore, Rick; Sueyoshi, Tatsuya; Negishi, Masahiko

    2017-03-06

    Nuclear receptor CAR (NR1I3) regulates hepatic drug and energy metabolism as well as cell fate. Its activation can be a critical factor in drug-induced toxicity and disease development such as diabetes and tumors. CAR inactivates its constitutive activity by phosphorylation at threonine 38. Utilizing receptor for protein kinase 1 (RACK1) as the regulatory subunit, protein phosphatase PP2A dephosphorylates threonine 38 to activate CAR. Here we have demonstrated that CAR undergoes its homodimer-monomer conversion to regulate this dephosphorylation. By co-expressing two differently-tagged CAR proteins in Huh-7 cells, mouse primary hepatocytes and mouse livers, co-immunoprecipitation and two-dimensional gel electrophoresis revealed that CAR can form a homodimer in a configuration in which the PP2A/RACK1 binding site is buried within its dimer interface. Epidermal growth factor (EGF) was found to stimulate CAR homo-dimerization, thus constraining CAR in its inactive form. The agonistic ligand CITCO binds directly to the CAR homodimer and dissociates phosphorylated CAR into its monomer, exposing the PP2A/RACK1 binding site for dephosphorylation. Phenobarbital, which is not a CAR ligand, binds the EGF receptor, reversing the EGF signal to monomerize CAR for its indirect activation. Thus, the homodimer-monomer conversion is the underlying molecular mechanism that regulates CAR activation, by placing phosphorylated threonine 38 as the common target for both direct and in direct activation of CAR.

  11. Ligand Lone-Pair Influence on Hydrocarbon C-H Activation. A Computational Perspective

    SciTech Connect

    Ess, Daniel H.; Gunnoe, T. Brent; Cundari, Thomas R.; Goddard, William A.; Periana, Roy A.

    2010-12-03

    Mid to late transition metal complexes that break hydrocarbon C-H bonds by transferring the hydrogen to a heteroatom ligand while forming a metal-alkyl bond offer a promising strategy for C-H activation. Here we report a density functional (B3LYP, M06, and X3LYP) analysis of cis-(acac)2MX and TpM(L)X (M = Ir, Ru, Os, and Rh; acac = acetylacetonate, Tp = tris(pyrazolyl)borate; X = CH3, OH, OMe, NH2, and NMe2) systems for methane C-H bond activation reaction kinetics and thermodynamics. We address the importance of whether a ligand lone pair provides an intrinsic kinetic advantage through possible electronic dπ-pπ repulsions for M-OR and M-NR2 systems versus M-CH3 systems. This involves understanding the energetic impact of the X ligand group on ligand loss, C-H bond coordination, and C-H bond cleavage steps as well as understanding how the nucleophilicity of the ligand X group, the electrophilicity of the transition metal center, and cis-ligand stabilization effect influence each of these steps. We also explore how spectator ligands and second- versus third-row transition metal centers impact the energetics of each of these C-H activation steps.

  12. Endogenous IGF-I and alpha v beta3 integrin ligands regulate increased smooth muscle growth in TNBS-induced colitis.

    PubMed

    Hazelgrove, Krystina B; Flynn, Robert S; Qiao, Li-Ya; Grider, John R; Kuemmerle, John F

    2009-06-01

    Endogenous insulin-like growth factor-I (IGF-I) regulates intestinal smooth muscle growth by concomitantly stimulating proliferation and inhibiting apoptosis. IGF-I-stimulated growth is augmented by the alpha(v)beta(3) integrin ligands vitronectin and fibronectin. IGF-I expression in smooth muscle is increased in both TNBS-induced colitis and Crohn's disease. We hypothesized that intestinal inflammation increased vitronectin and fibronectin expression by smooth muscle and, along with IGF-I upregulation, increased intestinal muscle growth. Intestinal smooth muscle cells were examined 7 days following the induction of TNBS-induced colitis. Although alpha(v)beta(3) integrin expression was not altered by TNBS-induced colitis, vitronectin and fibronectin levels were increased by 80 +/- 10% and 90 +/- 15%, above control levels, respectively. Basal IGF-I receptor phosphorylation in inflamed muscle from TNBS-treated rats was increased by 86 +/- 8% over vehicle-treated controls. Basal ERK1/2, p70S6 kinase, and GSK-3beta phosphorylation in muscle cells of TNBS-treated rats were also increased by 140-180%. TNBS treatment increased basal muscle cell proliferation by 130 +/- 15% and decreased apoptosis by 20 +/- 2% compared with that in vehicle-treated controls. The changes in proliferation and apoptosis were reversed by an IGF-I receptor tyrosine kinase inhibitor or an alpha(v)beta(3) integrin antagonist. The results suggest that smooth muscle hyperplasia in TNBS-induced colitis partly results from the upregulation of endogenous IGF-I and ligands of alpha(v)beta(3) integrin that mediate increased smooth muscle cell proliferation and decreased apoptosis. This paper has identified one mechanism regulating smooth muscle hyperplasia, a feature of stricture formation that occurs in the chronically inflamed intestine of TNBS-induced colitis and potentially Crohn's disease.

  13. Nanoconjugation of PSMA-targeting ligands enhances perinuclear localization and improves efficacy of delivered alpha-particle emitters against tumor endothelial analogues

    PubMed Central

    Sempkowski, Michelle; Banerjee, Sangeeta Ray; Pomper, Martin G.; Bruchertseifer, Frank; Morgenstern, Alfred; Sofou, Stavroula

    2015-01-01

    This study aims to evaluate the effect on killing efficacy of the intracellular trafficking patterns of alpha-particle emitters by using different radionuclide carriers in the setting of targeted antivascular alpha-radiotherapy. Nanocarriers (lipid vesicles) targeted to the prostate-specific-membrane-antigen (PSMA), which is unique to human neovasculature for a variety of solid tumors, were loaded with the alpha-particle generator actinium-225 and were compared to a PSMA-targeted radiolabeled antibody. Actinium-225 emits a total of four alpha-particles per decay, providing highly lethal and localized irradiation of targeted cells with minimal exposure to surrounding healthy tissues. Lipid vesicles were derivatized with two types of PSMA-targeting ligands: a fully human PSMA antibody (mAb), and a urea-based, low-molecular-weight agent. Target selectivity and extent of internalization were evaluated on monolayers of human endothelial cells (HUVEC) induced to express PSMA in static incubation conditions and in a flow field. Both types of radiolabeled PSMA-targeted vesicles exhibit similar killing efficacy, which is greater than the efficacy of the radiolabeled control mAb when compared on the basis of delivered radioactivity per cell. Fluorescence confocal microscopy demonstrates that targeted vesicles localize closer to the nucleus, unlike antibodies which localize near the plasma membrane. In addition, targeted vesicles cause larger numbers of DNA double strand breaks per nucleus of treated cells compared to the radiolabeled mAb. These findings demonstrate that radionuclide carriers, such as PSMA-targeted lipid-nanocarriers, which localize close to the nucleus increase the probability of alpha-particle trajectories crossing the nuclei, and, therefore, enhance the killing efficacy of alpha-particle emitters. PMID:26586724

  14. Structural basis for PPAR partial or full activation revealed by a novel ligand binding mode

    NASA Astrophysics Data System (ADS)

    Capelli, Davide; Cerchia, Carmen; Montanari, Roberta; Loiodice, Fulvio; Tortorella, Paolo; Laghezza, Antonio; Cervoni, Laura; Pochetti, Giorgio; Lavecchia, Antonio

    2016-10-01

    The peroxisome proliferator-activated receptors (PPARs) are nuclear receptors involved in the regulation of the metabolic homeostasis and therefore represent valuable therapeutic targets for the treatment of metabolic diseases. The development of more balanced drugs interacting with PPARs, devoid of the side-effects showed by the currently marketed PPARγ full agonists, is considered the major challenge for the pharmaceutical companies. Here we present a structure-based virtual screening approach that let us identify a novel PPAR pan-agonist with a very attractive activity profile and its crystal structure in the complex with PPARα and PPARγ, respectively. In PPARα this ligand occupies a new pocket whose filling is allowed by the ligand-induced switching of the F273 side chain from a closed to an open conformation. The comparison between this pocket and the corresponding cavity in PPARγ provides a rationale for the different activation of the ligand towards PPARα and PPARγ, suggesting a novel basis for ligand design.

  15. Effect of axial ligands on the molecular configurations, stability, reactivity, and photodynamic activities of silicon phthalocyanines.

    PubMed

    Luan, Liqiang; Ding, Lanlan; Shi, Jiawei; Fang, Wenjuan; Ni, Yuxing; Liu, Wei

    2014-12-01

    To demonstrate the effect of axial ligands on the structure-activity relationship, a series of axially substituted silicon phthalocyanines (SiPcs) have been synthesized with changes to the axial ligands. The reactivity of the axial ligand upon shielding by the phthalocyanine ring current, along with their stability, photophysical, and photodynamic therapy (PDT) activities were compared and evaluated for the first time. As revealed by single-crystal XRD analysis, rotation of the axial -OMe ligands was observed in SiPc 3, which resulted in two molecular configurations coexisting synchronously in both the solid and solution states and causing a split of the phthalocyanine α protons in the (1)H NMR spectra that is significantly different from all SiPcs reported so far. The remarkable photostability, good singlet oxygen quantum yield, and efficient in vitro photodynamic activity synergistically show that compound 3 is one of the most promising photosensitizers for PDT.

  16. Tools and techniques to study ligand-receptor interactions and receptor activation by TNF superfamily members.

    PubMed

    Schneider, Pascal; Willen, Laure; Smulski, Cristian R

    2014-01-01

    Ligands and receptors of the TNF superfamily are therapeutically relevant targets in a wide range of human diseases. This chapter describes assays based on ELISA, immunoprecipitation, FACS, and reporter cell lines to monitor interactions of tagged receptors and ligands in both soluble and membrane-bound forms using unified detection techniques. A reporter cell assay that is sensitive to ligand oligomerization can identify ligands with high probability of being active on endogenous receptors. Several assays are also suitable to measure the activity of agonist or antagonist antibodies, or to detect interactions with proteoglycans. Finally, self-interaction of membrane-bound receptors can be evidenced using a FRET-based assay. This panel of methods provides a large degree of flexibility to address questions related to the specificity, activation, or inhibition of TNF-TNF receptor interactions in independent assay systems, but does not substitute for further tests in physiologically relevant conditions.

  17. Left temporal alpha-band activity reflects single word intelligibility

    PubMed Central

    Becker, Robert; Pefkou, Maria; Michel, Christoph M.; Hervais-Adelman, Alexis G.

    2013-01-01

    The electroencephalographic (EEG) correlates of degraded speech perception have been explored in a number of recent studies. However, such investigations have often been inconclusive as to whether observed differences in brain responses between conditions result from different acoustic properties of more or less intelligible stimuli or whether they relate to cognitive processes implicated in comprehending challenging stimuli. In this study we used noise vocoding to spectrally degrade monosyllabic words in order to manipulate their intelligibility. We used spectral rotation to generate incomprehensible control conditions matched in terms of spectral detail. We recorded EEG from 14 volunteers who listened to a series of noise vocoded (NV) and noise-vocoded spectrally-rotated (rNV) words, while they carried out a detection task. We specifically sought components of the EEG response that showed an interaction between spectral rotation and spectral degradation. This reflects those aspects of the brain electrical response that are related to the intelligibility of acoustically degraded monosyllabic words, while controlling for spectral detail. An interaction between spectral complexity and rotation was apparent in both evoked and induced activity. Analyses of event-related potentials showed an interaction effect for a P300-like component at several centro-parietal electrodes. Time-frequency analysis of the EEG signal in the alpha-band revealed a monotonic increase in event-related desynchronization (ERD) for the NV but not the rNV stimuli in the alpha band at a left temporo-central electrode cluster from 420–560 ms reflecting a direct relationship between the strength of alpha-band ERD and intelligibility. By matching NV words with their incomprehensible rNV homologues, we reveal the spatiotemporal pattern of evoked and induced processes involved in degraded speech perception, largely uncontaminated by purely acoustic effects. PMID:24416001

  18. Activation and Molecular Targets of Peroxisome Proliferator-Activated Receptor-γ Ligands in Lung Cancer

    PubMed Central

    Nemenoff, Raphael A.; Weiser-Evans, Mary; Winn, Robert A.

    2008-01-01

    Lung cancer is the leading cause of cancer death, and five-year survival remains poor, raising the urgency for new treatment strategies. Activation of PPARγ represents a potential target for both the treatment and prevention of lung cancer. Numerous studies have examined the effect of thiazolidinediones such as rosiglitazone and pioglitazone on lung cancer cells in vitro and in xenograft models. These studies indicate that activation of PPARγ inhibits cancer cell proliferation as well as invasiveness and metastasis. While activation of PPARγ can occur by direct binding of pharmacological ligands to the molecule, emerging data indicate that PPARγ activation can occur through engagement of other signal transduction pathways, including Wnt signaling and prostaglandin production. Data, both from preclinical models and retrospective clinical studies, indicate that activation of PPARγ may represent an attractive chemopreventive strategy. This article reviews the existing biological and mechanistic experiments focusing on the role of PPARγ in lung cancer, focusing specifically on nonsmall cell lung cancer. PMID:18509496

  19. Multiple, Ligand-Dependent Routes from the Active Site of Cytochrome P450 2C9

    SciTech Connect

    Cojocaru, Vlad; Winn, Peter J.; Wade, Rebecca C.

    2012-02-13

    The active site of liver-specific, drug-metabolizing cytochrome P450 (CYP) monooxygenases is deeply buried in the protein and is connected to the protein surface through multiple tunnels, many of which were found open in different CYP crystal structures. It has been shown that different tunnels could serve as ligand passage routes in different CYPs. However, it is not understood whether one CYP uses multiple routes for substrate access and product release and whether these routes depend on ligand properties. From 300 ns of molecular dynamics simulations of CYP2C9, the second most abundant CYP in the human liver we found four main ligand exit routes, the occurrence of each depending on the ligand type and the conformation of the F-G loop, which is likely to be affected by the CYP-membrane interaction. A non-helical F-G loop favored exit towards the putative membrane-embedded region. Important protein features that direct ligand exit include aromatic residues that divide the active site and whose motions control access to two pathways. The ligands interacted with positively charged residues on the protein surface through hydrogen bonds that appear to select for acidic substrates. The observation of multiple, ligand-dependent routes in a CYP aids understanding of how CYP mutations affect drug metabolism and provides new possibilities for CYP inhibition.

  20. Antitumor activity of tumor necrosis factor-alpha conjugated with polyvinylpyrrolidone on solid tumors in mice.

    PubMed

    Kamada, H; Tsutsumi, Y; Yamamoto, Y; Kihira, T; Kaneda, Y; Mu, Y; Kodaira, H; Tsunoda, S I; Nakagawa, S; Mayumi, T

    2000-11-15

    We attempted the development of a novel polymer conjugation to further improve the therapeutic potency of antitumor cytokines compared with PEGylation for clinical application. Compared with native tumor necrosis factor (TNF)-alpha in vitro, specific bioactivities of polyvinyl-pyrrolidone (PVP)-modified TNF-alphas (PVP-TNF-alphas) were decreased by increasing the degree of PVP attachment. PVP-TNF-alpha fraction 3, Mr 101,000, had the most effective antitumor activity of the various PVP-TNF-alphas in vivo. PVP-TNF-alpha fraction 3 had >200-fold higher antitumor effect than native TNF-alpha, and the antitumor activity of PVP-TNF-alpha fraction 3 was >2-fold higher than that of MPEG-TNF-alpha (Mr 108,000), which had the highest antitumor activity among the polyethylene glycol (PEG)-conjugated TNF-alphas. Additionally, a high dose of native TNF-alpha induced toxic side effects such as body weight reduction, piloerection. and tissue inflammation, whereas no side effects were observed after i.v. administration of PVP-TNF-alpha fraction 3. The plasma half-life of PVP-TNF-alpha fraction 3 (360 min) was about 80- and 3-fold longer than those of native TNF-alpha (4.6 mm) and MPEG-TNF-alpha (122 min), respectively. The mechanism of increased antitumor effect in vivo caused the prolongation of plasma half-life and increase in stability. These results suggested that PVP is a useful polymeric modifier for bioconjugation of TNF-alpha to increase its antitumor potency, and multifunctionally bioconjugated TNF-alpha may be a potentiated antitumor agent for clinical use.

  1. Spectroscopic studies on ligand-enzyme interactions: complexation of alpha-chymotrypsin with 4',6-diamidino-2-phenylindole (DAPI).

    PubMed

    Banerjee, Debapriya; Srivastava, Sachin Kumar; Pal, Samir Kumar

    2008-02-14

    In the present study, the interaction of two structurally related proteolytic enzymes trypsin and alpha-chymotrypsin (CHT) with 4',6-Diamidino-2-phenylindole (DAPI) has been addressed. The binding of DAPI to CHT has been characterized by steady-state and picosecond time-resolved spectroscopic techniques. Enzymatic activity of CHT and simultaneous binding of the well-known inhibitor proflavin (PF) in the presence of DAPI clearly rule out the possibility of DAPI binding at the catalytic site of the enzyme. The spectral overlap between the emission of DAPI and absorption of PF offers the opportunity to explore the binding site of DAPI using Förster resonance energy transfer (FRET). FRET studies between DAPI and PF indicate that DAPI is bound to CHT with its transition dipole nearly perpendicular to that of PF. Competitive binding of DAPI with another fluorescent probe 2,6-p-toluidinonaphthalene sulfonate (TNS), having a well-defined binding site, indicates that DAPI and TNS bind at the same hydrophobic site of the enzyme CHT. The difference in the interactions of two well-studied, structurally similar enzymes with the same molecule may find its application in the design of specific substrate mimics or inhibitors of the enzymes.

  2. Role of the essential yeast protein PSU1 in p6anscriptional enhancement by the ligand-dependent activation function AF-2 of nuclear receptors.

    PubMed Central

    Gaudon, C; Chambon, P; Losson, R

    1999-01-01

    Nuclear receptors (NRs) can function as ligandinducible transregulators in both mammalian and yeast cells, indicating that important features of transcriptional control have been conserved throughout evolution. We report here the isolation and characterization of an essential yeast protein of unknown function, PSU1, which exhibits properties expected for a co-activator/mediator of the ligand-dependent activation function AF-2 present in the ligand-binding domain (LBD, region E) of NRs. PSU1 interacts in a ligand-dependent manner with the LBD of several NRs, including retinoic acid (RARalpha), retinoid X (RXRalpha), thyroid hormone (TRalpha), vitamin D3 (VDR) and oestrogen (ERalpha) receptors. Importantly, both in yeast and in vitro, these interactions require the integrity of the AF-2 activating domain. When tethered to a heterologous DNA-binding domain, PSU1 can activate transcription on its own. By using yeast reporter cells that express PSU1 conditionally, we show that PSU1 is required for transactivation by the AF-2 of ERalpha. Taken together these data suggest that in yeast, PSU1 is involved in ligand-dependent transactivation by NRs. Sequence analysis revealed that in addition to a highly conserved motif found in a family of MutT-related proteins, PSU1 contains several alpha-helical leucine-rich motifs sharing the consensus sequence LLxPhiL (x, any amino acid; Phi, hydrophobic amino acid) in regions that elicit either transactivation or NR-binding activity. PMID:10205176

  3. A shed NKG2D ligand that promotes natural killer cell activation and tumor rejection

    PubMed Central

    Deng, Weiwen; Gowen, Benjamin G.; Zhang, Li; Wang, Lin; Lau, Stephanie; Iannello, Alexandre; Xu, Jianfeng; Rovis, Tihana L.; Xiong, Na; Raulet, David H.

    2016-01-01

    Immune cells, including natural killer (NK) cells, recognize transformed cells and eliminate them in a process termed immunosurveillance. It is thought that tumor cells evade immunosurveillance by shedding membrane ligands that bind to the NKG2D activating receptor on NK cells and/or T cells, and desensitize these cells. In contrast, we show that in mice, shedding of MULT1, a high affinity NKG2D ligand, causes NK cell activation and tumor rejection. Recombinant soluble MULT1 stimulated tumor rejection in mice. Soluble MULT1 functions, at least in part, by competitively reversing a global desensitization of NK cells imposed by engagement of membrane NKG2D ligands on tumor-associated cells, such as myeloid cells. The results overturn conventional wisdom that soluble ligands are inhibitory, and suggest a new approach for cancer immunotherapy. PMID:25745066

  4. The glucocorticoid receptor hormone binding domain mediates transcriptional activation in vitro in the absence of ligand.

    PubMed Central

    Schmitt, J; Stunnenberg, H G

    1993-01-01

    We show that recombinant rat glucocorticoid receptor (vvGR) expressed using vaccinia virus is indistinguishable from authentic GR with respect to DNA and hormone binding. In the absence of hormone, vvGR is mainly found in the cytoplasm in a complex with heat shock protein 90. Upon incubation with ligand, vvGR is released from this complex and translocated to the nucleus. Thus, the ligand binding domain displays the known biochemical properties. However, in vitro, transcription from a synthetic promoter and from the mouse mammary tumor virus (MMTV) promoter is enhanced by recombinant GR in a ligand independent manner. Both transactivation domains contribute to the transcriptional activity, additively on a synthetic promoter and cooperatively on the MMTV promoter. We thus provide the first evidence that in vitro the hormone binding domain has a transcriptional activity even in the absence of ligand. Images PMID:8392705

  5. Mechanism of activation of a hafnium pyridyl-amide olefin polymerization catalyst: ligand modification by monomer.

    PubMed

    Froese, Robert D J; Hustad, Phillip D; Kuhlman, Roger L; Wenzel, Timothy T

    2007-06-27

    We have investigated the olefin polymerization mechanism of hafnium catalysts supported by a pyridyl-amide ligand with an ortho-metalated naphthyl group. Ethylene-alpha-olefin copolymers from these catalysts have broad molecular weight distributions that can be fit to a bimodal distribution. We propose a unique mechanism to explain this behavior involving monomer modification of the catalyst, which generates multiple catalyst species when multiple monomers are present. More specifically, we present evidence that the hafnium alkyl cation initially undergoes monomer insertion into the Hf-naphthyl bond, which permanently modifies the ligand to generate new highly active olefin polymerization catalysts. Under ethylene/octene copolymerization conditions, a plurality of new catalysts is formed in relative proportion to the respective monomer concentrations. Due to the asymmetry of the metal complex, two "ethylene-inserted" and eight "octene-inserted" isomers are possible, but it is a useful approximation to consider only one of each in the polymerization behavior. Consequently, gel permeation chromatography data for the polymers can be fit to a bimodal distribution having a continuous shift from a predominantly low molecular weight fraction to predominantly higher molecular weight fraction as [octene]/[ethylene] is increased. Theoretical calculations show that such insertions into the Hf-aryl bond have lower barriers than corresponding insertions into the Hf-alkyl bond. The driving forces for this insertion into the Hf-aryl bond include elimination of an eclipsing H-H interaction and formation of a stabilizing Hf-arene interaction. These new "monomer-inserted catalysts" have no beta-agostic interaction, very weak olefin binding, and olefin-insertion transition states which differ on the two sides by more than 4 kcal/mol. Thus, the barrier to site epimerization is very low and high polymerization rates are possible even when the chain wags prior to every insertion

  6. Peroxisome proliferator-activated receptor alpha controls hepatic heme biosynthesis through ALAS1.

    PubMed

    Degenhardt, Tatjana; Väisänen, Sami; Rakhshandehroo, Maryam; Kersten, Sander; Carlberg, Carsten

    2009-05-01

    Heme is an essential prosthetic group of proteins involved in oxygen transport, energy metabolism and nitric oxide production. ALAS1 (5-aminolevulinate synthase) is the rate-limiting enzyme in heme synthesis in the liver and is highly regulated to adapt to the metabolic demand of the hepatocyte. In the present study, we describe human hepatic ALAS1 as a new direct target for the nuclear receptor peroxisome proliferator-activated receptor alpha (PPARalpha). In primary human hepatocytes and in HepG2 cells, PPARalpha agonists induced an increase in ALAS1 mRNA levels, which was abolished by PPARalpha silencing. These effects are mediated by two functional PPAR binding sites at positions -9 and -2.3 kb relative to the ALAS1 transcription start site. PPARalpha ligand treatment also up-regulated the mRNA levels of the genes ALAD (5-aminolevulinate dehydratase), UROS (uroporphyrinogen III synthase), UROD (uroporphyrinogen decarboxylase), CPOX (coproporphyrinogen oxidase) and PPOX (protoporphyrinogen oxidase) encoding for enzymes controlling further steps in heme biosynthesis. In HepG2 cells treated with PPARalpha agonists and in mouse liver upon fasting, the association of PPARalpha, its partner retinoid X receptor, PPARgamma co-activator 1alpha and activated RNA polymerase II with the transcription start site region of all six genes was increased, leading to higher levels of the metabolite heme. In conclusion, these data strongly support a role of PPARalpha in the regulation of human ALAS1 and of five additional genes of the pathway, consequently leading to increased heme synthesis.

  7. Phentolamine inhibits exocytosis of glucagon by Gi2 protein-dependent activation of calcineurin in rat pancreatic alpha -cells.

    PubMed

    Høy, M; Bokvist, K; Xiao-Gang, W; Hansen, J; Juhl, K; Berggren, P O; Buschard, K; Gromada, J

    2001-01-12

    Capacitance measurements were used to investigate the molecular mechanisms by which imidazoline compounds inhibit glucagon release in rat pancreatic alpha-cells. The imidazoline compound phentolamine reversibly decreased depolarization-evoked exocytosis >80% without affecting the whole-cell Ca(2+) current. During intracellular application through the recording pipette, phentolamine produced a concentration-dependent decrease in the rate of exocytosis (IC(50) = 9.7 microm). Another imidazoline compound, RX871024, exhibited similar effects on exocytosis (IC(50) = 13 microm). These actions were dependent on activation of pertussis toxin-sensitive G(i2) proteins but were not associated with stimulation of ATP-sensitive K(+) channels or adenylate cyclase activity. The inhibitory effect of phentolamine on exocytosis resulted from activation of the protein phosphatase calcineurin and was abolished by cyclosporin A and deltamethrin. Exocytosis was not affected by intracellular application of specific alpha(2), I(1), and I(2) ligands. Phentolamine reduced glucagon release (IC(50) = 1.2 microm) from intact islets by 40%, an effect abolished by pertussis toxin, cyclosporin A, and deltamethrin. These data suggest that imidazoline compounds inhibit glucagon secretion via G(i2)-dependent activation of calcineurin in the pancreatic alpha-cell. The imidazoline binding site is likely to be localized intracellularly and probably closely associated with the secretory granules.

  8. Automated docking of {alpha}-(1,4)- and {alpha}-(1,6)-linked glucosyl trisaccharides in the glucoamylase active site

    SciTech Connect

    Countinho, P.M.; Reilly, P.J.; Dowd, M.K.

    1998-06-01

    Low-energy conformers of five {alpha}-(1,4)- and {alpha}-(1,6)-linked glucosyl trisaccharides were flexibly docked into the glucoamylase active site using AutoDock 2.2. To ensure that all significant conformational space was searched, the starting trisaccharide conformers for docking were all possible combinations of the corresponding disaccharide low-energy conformers. All docked trisaccharides occupied subsites {minus}1 and +1 in very similar modes to those of corresponding nonreducing-end disaccharides. For linear substrates, full binding at subsite +2 occurred only when the substrate reducing end was {alpha}-(1,4)-linked, with hydrogen-bonding with the hydroxy-methyl group being the only polar interaction there. Given the absence of other important interactions at this subsite, multiple substrate conformations are allowed. For the one docked branched substrate, steric hindrance in the {alpha}-(1,6)-glycosidic oxygen suggests that the active-site residues have to change position for hydrolysis to occur. Subsite +1 of the glucoamylase active site allows flexibility in binding but, at least in Aspergillus glucoamylases, subsite +2 selectively binds substrates {alpha}-(1,4)-linked between subsites +1 and +2. Enzyme engineering to limit substrate flexibility at subsite +2 could improve glucoamylase industrial properties.

  9. The nonpsychotropic cannabinoid cannabidiol modulates and directly activates alpha-1 and alpha-1-Beta glycine receptor function.

    PubMed

    Ahrens, Jörg; Demir, Reyhan; Leuwer, Martin; de la Roche, Jeanne; Krampfl, Klaus; Foadi, Nilufar; Karst, Matthias; Haeseler, Gertrud

    2009-01-01

    Loss of inhibitory synaptic transmission within the dorsal horn of the spinal cord plays a key role in the development of chronic pain following inflammation or nerve injury. Inhibitory postsynaptic transmission in the adult spinal cord involves mainly glycine. Cannabidiol is a nonpsychotropic plant constituent of Cannabis sativa. As we hypothesized that non-CB receptor mechanisms of cannabidiol might contribute to its anti-inflammatory and neuroprotective effects, we investigated the interaction of cannabidiol with strychnine-sensitive alpha(1 )and alpha(1)beta glycine receptors by using the whole-cell patch clamp technique. Cannabidiol showed a positive allosteric modulating effect in a low micromolar concentration range (EC(50) values: alpha(1) = 12.3 +/- 3.8 micromol/l and alpha(1)beta = 18.1 +/- 6.2 micromol/l). Direct activation of glycine receptors was observed at higher concentrations above 100 micromol/l (EC(50) values: alpha(1) = 132.4 +/- 12.3 micromol/l and alpha(1)beta = 144.3 +/- 22.7 micromol/l). These in vitro results suggest that strychnine-sensitive glycine receptors may be a target for cannabidiol mediating some of its anti-inflammatory and neuroprotective properties.

  10. Identification of mangiferin as a potential Glucokinase activator by structure-based virtual ligand screening

    PubMed Central

    Min, Qiuxia; Cai, Xinpei; Sun, Weiguang; gao, Fei; Li, Zhimei; Zhang, Qian; Wan, Luo-Sheng; Li, Hua; Chen, Jiachun

    2017-01-01

    The natural product mangiferin (compound 7) has been identified as a potential glucokinase activator by structure-based virtual ligand screening. It was proved by enzyme activation experiment and cell-based assays in vitro, with potency in micromolar range. Meanwhile, this compound showed good antihyperglycemic activity in db/db mice without obvious side effects such as excessive hypoglycaemia. PMID:28317897

  11. Differential ligand-dependent interactions between the AF-2 activating domain of nuclear receptors and the putative transcriptional intermediary factors mSUG1 and TIF1.

    PubMed Central

    vom Baur, E; Zechel, C; Heery, D; Heine, M J; Garnier, J M; Vivat, V; Le Douarin, B; Gronemeyer, H; Chambon, P; Losson, R

    1996-01-01

    Using a yeast two-hybrid system we report the isolation of a novel mouse protein, mSUG1, that interacts with retinoic acid receptor alpha (RAR alpha) both in yeast cells and in vitro in a ligand- and AF-2 activating domain (AF-2 AD)-dependent manner and show that it is a structural and functional homologue of the essential yeast protein SUG1. mSUG1 also efficiently interacts with other nuclear receptors, including oestrogen (ER), thyroid hormone (TR), Vitamin D3 (VDR) and retinoid X (RXR) receptors. By comparing the interaction properties of these receptors with mSUG1 and TIF1, we demonstrate that: (i) RXR alpha efficiently interacts with TIF1, but not with mSUG1, whereas TR alpha interacts much more efficiently with mSUG1 than with TIF1, and RAR alpha, VDR and ER efficiently interact with mSUG1 and TIF1; (ii) the amphipathic alpha-helix core of the AF-2 AD is differentially involved in interactions of RAR alpha with mSUG1 and TIF1; (iii) the AF-2 AD cores of RAR alpha and ER are similarly involved in their interaction with TIF1, but not with mSUG1. Thus, the interaction interfaces between the different receptors and either mSUG1 or TIF1 may vary depending on the nature of the receptor and the putative mediator of its AF-2 function. We discuss the possibility that mSUG1 and TIF1 may mediate the transcriptional activity of the AF-2 of nuclear receptors through different mechanisms. Images PMID:8598193

  12. Pharmacophore modeling improves virtual screening for novel peroxisome proliferator-activated receptor-gamma ligands

    PubMed Central

    Lewis, Stephanie N.; Garcia, Zulma; Hontecillas, Raquel; Bassaganya-Riera, Josep; Bevan, David R.

    2015-01-01

    Peroxisome proliferator-activated receptor-gamma (PPARγ) is a nuclear hormone receptor involved in regulating various metabolic and immune processes. The PPAR family of receptors possesses a large binding cavity that imparts promiscuity of ligand binding not common to other nuclear receptors. This feature increases the challenge of using computational methods to identify PPAR ligands that will dock favorably into a structural model. Utilizing both ligand- and structure-based pharmacophore methods, we sought to improve agonist prediction by grouping ligands according to pharmacophore features, and pairing models derived from these features with receptor structures for docking. For 22 of the 33 receptor structures evaluated we observed an increase in true positive rate (TPR) when screening was restricted to compounds sharing molecular features found in rosiglitazone. A combination of structure models used for docking resulted in a higher TPR (40%) when compared to docking with a single structure model (less than 20%). Prediction was also improved when specific protein-ligand interactions between the docked ligands and structure models were given greater weight than the calculated free energy of binding. A large-scale screen of compounds using a marketed drug database verified the predictive ability of the selected structure models. This study highlights the steps necessary to improve screening for PPARγ ligands using multiple structure models, ligand-based pharmacophore data, evaluation of protein-ligand interactions, and comparison of docking datasets. The unique combination of methods presented here holds potential for more efficient screening of compounds with unknown affinity for PPARγ that could serve as candidates for therapeutic development. PMID:25616366

  13. Pharmacophore modeling improves virtual screening for novel peroxisome proliferator-activated receptor-gamma ligands

    NASA Astrophysics Data System (ADS)

    Lewis, Stephanie N.; Garcia, Zulma; Hontecillas, Raquel; Bassaganya-Riera, Josep; Bevan, David R.

    2015-05-01

    Peroxisome proliferator-activated receptor-gamma (PPARγ) is a nuclear hormone receptor involved in regulating various metabolic and immune processes. The PPAR family of receptors possesses a large binding cavity that imparts promiscuity of ligand binding not common to other nuclear receptors. This feature increases the challenge of using computational methods to identify PPAR ligands that will dock favorably into a structural model. Utilizing both ligand- and structure-based pharmacophore methods, we sought to improve agonist prediction by grouping ligands according to pharmacophore features, and pairing models derived from these features with receptor structures for docking. For 22 of the 33 receptor structures evaluated we observed an increase in true positive rate (TPR) when screening was restricted to compounds sharing molecular features found in rosiglitazone. A combination of structure models used for docking resulted in a higher TPR (40 %) when compared to docking with a single structure model (<20 %). Prediction was also improved when specific protein-ligand interactions between the docked ligands and structure models were given greater weight than the calculated free energy of binding. A large-scale screen of compounds using a marketed drug database verified the predictive ability of the selected structure models. This study highlights the steps necessary to improve screening for PPARγ ligands using multiple structure models, ligand-based pharmacophore data, evaluation of protein-ligand interactions, and comparison of docking datasets. The unique combination of methods presented here holds potential for more efficient screening of compounds with unknown affinity for PPARγ that could serve as candidates for therapeutic development.

  14. Rapid broad-spectrum analgesia through activation of peroxisome proliferator-activated receptor-alpha.

    PubMed

    LoVerme, Jesse; Russo, Roberto; La Rana, Giovanna; Fu, Jin; Farthing, Jesse; Mattace-Raso, Giuseppina; Meli, Rosaria; Hohmann, Andrea; Calignano, Antonio; Piomelli, Daniele

    2006-12-01

    Severe pain remains a major area of unmet medical need. Here we report that agonists of the nuclear receptor PPAR-alpha (peroxisome proliferator-activated receptor-alpha) suppress pain behaviors induced in mice by chemical tissue injury, nerve damage, or inflammation. The PPAR-alpha agonists GW7647 [2-(4-(2-(1-cyclohexanebutyl)-3-cyclohexylureido)ethyl)phenylthio)-2-methylpropionic acid], Wy-14643 [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid], and palmitoylethanolamide (PEA) reduced nocifensive behaviors elicited in mice by intraplantar (i.pl.) injection of formalin or i.p. injection of magnesium sulfate. These effects were absent in PPAR-alpha-null mice yet occurred within minutes of agonist administration in wild-type mice, suggesting that they were mediated through a transcription-independent mechanism. Consistent with this hypothesis, blockade of calcium-operated IK(ca) (K(Ca)3.1) and BK(ca) (K(Ca)1.1) potassium channels prevented the effects of GW7647 and PEA in the formalin test. Three observations suggest that PPAR-alpha agonists may inhibit nocifensive responses by acting on peripheral PPAR-alpha. (i) PEA reduced formalin-induced pain at i.pl. doses that produced no increase in systemic PEA levels; (ii) PPAR-alpha was expressed in dorsal root ganglia neurons of wild-type but not PPAR-alpha-null mice; and (ii) GW7647 and PEA prevented formalin-induced firing of spinal cord nociceptive neurons in rats. In addition to modulating nociception, GW7647 and PEA reduced hyperalgesic responses in the chronic constriction injury model of neuropathic pain; these effects were also contingent on PPAR-alpha expression and were observed following either acute or subchronic PPAR-alpha agonist administration. Finally, acute administration of GW7647 and PEA reduced hyperalgesic responses in the complete Freund's adjuvant and carrageenan models of inflammatory pain. Our results suggest that PPAR-alpha agonists may represent a novel class of analgesics.

  15. Cyclin C regulates adipogenesis by stimulating transcriptional activity of CCAAT/enhancer binding protein alpha.

    PubMed

    Song, Ziyi; Xiaoli, Alus M; Zhang, Quanwei; Zhang, Yi; Yang, Ellen S T; Wang, Sven; Chang, Rui; Zhang, Zhengdong D; Yang, Gongshe; Strich, Randy; Pessin, Jeffrey E; Yang, Fajun

    2017-03-28

    Brown adipose tissue (BAT) is important for maintaining energy homeostasis and adaptive thermogenesis in rodents and humans. As disorders arising from dysregulated energy metabolism, such as obesity and metabolic diseases, have increased, so has interest in the molecular mechanisms in adipocyte biology. Using a functional screen, we identified cyclin C (CycC), a conserved subunit of the Mediator complex, as a novel regulator for brown adipocyte formation. siRNA-mediated CycC knockdown (KD) in brown preadipocytes impaired the early transcriptional program of differentiation, and genetic knockout (KO) of CycC completely blocked the differentiation process. RNA-seq analyses of CycC-KD revealed a critical role of CycC in activating genes co-regulated by peroxisome proliferator activated receptor gamma (PPARγ) and CCAAT/enhancer binding protein alpha (C/EBPα). Overexpression of PPARγ2 or addition of the PPARγ ligand rosiglitazone rescued the defects in CycC-KO brown preadipocytes, and efficiently activated the PPARγ-responsive promoters in both wild-type (WT) and CycC-KO cells, suggesting that CycC is not essential for PPARγ transcriptional activity. In contrast, CycC-KO significantly reduced C/EBPα-dependent gene expression. Unlike for PPARγ, overexpression of C/EBPα could not induce C/EBPα target gene expression in CycC-KO cells or rescue the CycC-KO defects in brown adipogenesis, suggesting that CycC is essential for C/EBPα-mediated gene activation. CycC physically interacted with C/EBPα and this interaction was required for C/EBPα transactivation domain activity. Consistent with the role of C/EBPα in white adipogenesis, CycC-KD also inhibited differentiation of 3T3-L1 cells into white adipocytes. Together, these data indicate that CycC activates adipogenesis by stimulating the transcriptional activity of C/EBPα.

  16. Alpha 1-antitrypsin activity is markedly decreased in Wegener's granulomatosis.

    PubMed

    Mota, Ali; Sahebghadam Lotfi, Abbas; Jamshidi, Ahmad-Reza; Najavand, Saeed

    2014-04-01

    Alpha 1-antitrypsin (A1AT) is the most abundant proteinase inhibitor in plasma and the main inhibitor of Proteinase 3, the target antigen of antineutrophil cytoplasmic antibodies (ANCAs) that predominant in Wegeners' granulomatosis. Α1AT deficiency correlated with ANCA-associated vasculitis. This study explores the trypsin inhibitory capacity (TIC), specific activity, and phenotypic deficiency of Α1AT in Wegener's granulomatosis. Twenty-seven WG patients were studied. ANCA was tested by IIF and ELISA. Serum a1-anti-trypsin levels were quantified in WG patients and healthy controls by immunoturbidimetric assay. Serum TIC was assessed by the enzymatic colorimetric assay. Phenotypes of A1AT were detected by Isoelectric Focusing. A1AT concentration was equivalent in patients and controls; however, serum TIC (P = 0.001) and specific activity of A1AT (P = 0.001) were dramatically lower in WG patients. Five patients had deficient phenotypes of A1AT: MZ (n = 3), MS (n = 1) and SS (n = 1). This was correlated with an increase in the prevalence of deficient phenotypes of A1AT in WG (P = 0.01). Trypsin inhibitory capacity and specific activity of A1AT were decreased in WG patients and may be involve in disease pathogenesis and can worsen the clinical manifestations. This A1AT deficiency probably resulted from oxidative inactivation and/or enzymatic degradation of A1AT. This could result in localized deficiency of A1AT in vessel wall interfaces and lead to severe disease.

  17. Heterogeneity of alpha1 receptors associated with vascular smooth muscle: evidence from functional and ligand binding studies

    SciTech Connect

    Babich, M.; Pedigo, N.W.; Butler, B.T.; Piascik, M.T.

    1987-08-10

    The nature of the alpha1 receptor associated with rabbit aorta has been examined in functional and receptor binding studies. In isolated aortic rings the dose-response curve for (-)metaraminol was not parallel to that of (-)epinephrine, (-)norepinephrine or (-)phenylephrine. Following inactivation of a portion of the alpha receptors with phenoxybenzamine, the occupancy versus response relationship for metaraminol, in contrast to the other test agonists, was biphasic. In microsomes prepared from aorta, metaraminol bound to two classes of sites labelled by the selective alpha1 antagonist (TH) prazosin. Norepinephrine also bound to two sites on the alpha receptor in all three preparations tested. The Scatchard plot of (TH)prazosin binding to microsomes prepared from frozen aorta was curvilinear. Estimates of the affinities and site densities were 49.6 +/- 15.3 pM and 44.8 +/- 11.8 pmol/gm protein and 1.0 +/- 0.2 nM and 43.8 +/- 17.4 pmol/gm for the high and low affinity sites, respectively. These data are consistent with the idea that there are subtypes of the alpha1 receptor. 33 references, 5 figures.

  18. Ligands Raise the Constraint That Limits Constitutive Activation in G Protein-coupled Opioid Receptors*

    PubMed Central

    Vezzi, Vanessa; Onaran, H. Ongun; Molinari, Paola; Guerrini, Remo; Balboni, Gianfranco; Calò, Girolamo; Costa, Tommaso

    2013-01-01

    Using a cell-free bioluminescence resonance energy transfer strategy we compared the levels of spontaneous and ligand-induced receptor-G protein coupling in δ (DOP) and μ (MOP) opioid receptors. In this assay GDP can suppress spontaneous coupling, thus allowing its quantification. The level of constitutive activity was 4–5 times greater at the DOP than at the MOP receptor. A series of opioid analogues with a common peptidomimetic scaffold displayed remarkable inversions of efficacy in the two receptors. Agonists that enhanced coupling above the low intrinsic level of the MOP receptor were inverse agonists in reducing the greater level of constitutive coupling of the DOP receptor. Yet the intrinsic activities of such ligands are identical when scaled over the GDP base line of both receptors. This pattern is in conflict with the predictions of the ternary complex model and the “two state” extensions. According to this theory, the order of spontaneous and ligand-induced coupling cannot be reversed if a shift of the equilibrium between active and inactive forms raises constitutive activation in one receptor type. We propose that constitutive activation results from a lessened intrinsic barrier that restrains spontaneous coupling. Any ligand, regardless of its efficacy, must enhance this constraint to stabilize the ligand-bound complexed form. PMID:23836900

  19. Ligands raise the constraint that limits constitutive activation in G protein-coupled opioid receptors.

    PubMed

    Vezzi, Vanessa; Onaran, H Ongun; Molinari, Paola; Guerrini, Remo; Balboni, Gianfranco; Calò, Girolamo; Costa, Tommaso

    2013-08-16

    Using a cell-free bioluminescence resonance energy transfer strategy we compared the levels of spontaneous and ligand-induced receptor-G protein coupling in δ (DOP) and μ (MOP) opioid receptors. In this assay GDP can suppress spontaneous coupling, thus allowing its quantification. The level of constitutive activity was 4-5 times greater at the DOP than at the MOP receptor. A series of opioid analogues with a common peptidomimetic scaffold displayed remarkable inversions of efficacy in the two receptors. Agonists that enhanced coupling above the low intrinsic level of the MOP receptor were inverse agonists in reducing the greater level of constitutive coupling of the DOP receptor. Yet the intrinsic activities of such ligands are identical when scaled over the GDP base line of both receptors. This pattern is in conflict with the predictions of the ternary complex model and the "two state" extensions. According to this theory, the order of spontaneous and ligand-induced coupling cannot be reversed if a shift of the equilibrium between active and inactive forms raises constitutive activation in one receptor type. We propose that constitutive activation results from a lessened intrinsic barrier that restrains spontaneous coupling. Any ligand, regardless of its efficacy, must enhance this constraint to stabilize the ligand-bound complexed form.

  20. Cell cycle regulation and p53 activation by protein phosphatase 2C alpha.

    PubMed

    Ofek, Paula; Ben-Meir, Daniella; Kariv-Inbal, Zehavit; Oren, Moshe; Lavi, Sara

    2003-04-18

    Protein phosphatase 2C (PP2C) dephosphorylates a broad range of substrates, regulating stress response and growth-related pathways in both prokaryotes and eukaryotes. We now demonstrate that PP2C alpha, a major mammalian isoform, inhibits cell growth and activates the p53 pathway. In 293 cell clones, in which PP2C alpha expression is regulated by a tetracycline-inducible promoter, PP2C alpha overexpression led to G(2)/M cell cycle arrest and apoptosis. Furthermore, PP2C alpha induced the expression of endogenous p53 and the p53-responsive gene p21. Activation of the p53 pathway by PP2C alpha took place both in cells harboring endogenous p53, as well as in p53-null cells transfected with exogenous p53. Induction of PP2C alpha resulted in an increase in the overall levels of p53 protein as well as an augmentation of p53 transcription activity. The dephosphorylation activity of PP2C alpha is essential to the described phenomena, as none of these effects was detected when an enzymatically inactive PP2C alpha mutant was overexpressed. p53 plays an important role in PP2C alpha-directed cell cycle arrest and apoptosis because perturbation of p53 expression in human 293 cells by human papillomavirus E6 led to a significant increase in cell survival. The role of PP2C alpha in p53 activation is discussed.

  1. Contribution of mucosal maltase-glucoamylase activities to mouse small intestinal starch alpha-glucogenesis.

    PubMed

    Quezada-Calvillo, Roberto; Robayo-Torres, Claudia C; Opekun, Antone R; Sen, Partha; Ao, Zihua; Hamaker, Bruce R; Quaroni, Andrea; Brayer, Gary D; Wattler, Sigrid; Nehls, Michael C; Sterchi, Erwin E; Nichols, Buford L

    2007-07-01

    Digestion of starch requires activities provided by 6 interactive small intestinal enzymes. Two of these are luminal endo-glucosidases named alpha-amylases. Four are exo-glucosidases bound to the luminal surface of enterocytes. These mucosal activities were identified as 4 different maltases. Two maltase activities were associated with sucrase-isomaltase. Two remaining maltases, lacking other identifying activities, were named maltase-glucoamylase. These 4 activities are better described as alpha-glucosidases because they digest all linear starch oligosaccharides to glucose. Because confusion persists about the relative roles of these 6 enzymes, we ablated maltase-glucoamylase gene expression by homologous recombination in Sv/129 mice. We assayed the alpha-glucogenic activities of the jejunal mucosa with and without added recombinant pancreatic alpha-amylase, using a range of food starch substrates. Compared with wild-type mucosa, null mucosa or alpha-amylase alone had little alpha-glucogenic activity. alpha-Amylase amplified wild-type and null mucosal alpha-glucogenesis. alpha-Amylase amplification was most potent against amylose and model resistant starches but was inactive against its final product limit-dextrin and its constituent glucosides. Both sucrase-isomaltase and maltase-glucoamylase were active with limit-dextrin substrate. These mucosal assays were corroborated by a 13C-limit-dextrin breath test. In conclusion, the global effect of maltase-glucoamylase ablation was a slowing of rates of mucosal alpha-glucogenesis. Maltase-glucoamylase determined rates of digestion of starch in normal mice and alpha-amylase served as an amplifier for mucosal starch digestion. Acarbose inhibition was most potent against maltase-glucoamylase activities of the wild-type mouse. The consortium of 6 interactive enzymes appears to be a mechanism for adaptation of alpha-glucogenesis to a wide range of food starches.

  2. SENESCENCE-ASSOCIATED DECLINE IN HEPATIC PEROXISOMAL ENZYME ACTIVITIES CORRESPONDS WITH DIMINISHED LEVELS OF RETINOID X RECEPTOR ALPHA, BUT NOT PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR ALPHA1

    EPA Science Inventory

    Abstract

    Aging is associated with alterations in hepatic peroxisomal metabolism and susceptibility to hepatocarcinogenecity produced by agonists of peroxisome proliferator-activated receptor alpha (PPARa). Mechanisms involved in these effects are not well understood. Howev...

  3. Activation of bean (Phaseolus vulgaris) [alpha]-amylase inhibitor requires proteolytic processing of the proprotein

    SciTech Connect

    Pueyo, J.J.; Hunt, D.C.; Chrispeels, M.J. )

    1993-04-01

    Seeds of the common bean (Phaseolus vulgaris) contain a plant defense protein that inhibits the [alpha]-amylases of mammals and insects. This [alpha]-amylase inhibitor ([alpha]Al) is synthesized as a proprotein on the endoplasmic reticulum and is proteolytically processed after arrival in the protein storage vacuoles to polypeptides of relative molecular weight (M[sub r]) 15,000 to 18,000. The authors report two types of evidence that proteolytic processing is linked to activation of the inhibitory activity. First, by surveying seed extracts of wild accessions of P. vulgaris and other species in the genus Phaseolus, they found that antibodies to [alpha]Al recognize large (M[sub r] 30,000-35,000) polypeptides as well as typical [alpha]Al processing products (M[sub r] 15,000-18,000). [alpha]Al activity was found in all extracts that had the typical [alpha]Al processed polypeptides, but was absent from seed extracts that lacked such polypeptides. Second, they made a mutant [alpha]Al in which asparagine-77 is changed to aspartic acid-77. This mutation slows down the proteolytic processing of pro-[alpha]Al when the gene is expressed in tobacco. When pro-[alpha]Al was separated from mature [alpha]Al by gel filtration, pro-[alpha]Al was found not to have [alpha]-amylase inhibitory activity. The authors interpret these results to mean that formation of the active inhibitor is causally related to proteolytic processing of the proprotein. They suggest that the polypeptide cleavage removes a conformation constraint on the precursor to produce the biochemically active molecule. 43 refs., 5 figs., 1 tab.

  4. Evidence that activation of nuclear peroxisome proliferator-activated receptor alpha (PPARα) modulates sleep homeostasis in rats.

    PubMed

    Murillo-Rodríguez, Eric; Guzmán, Khalil; Arankowsky-Sandoval, Gloria; Salas-Crisóstomo, Mireille; Jiménez-Moreno, Ramsés; Arias-Carrión, Oscar

    2016-10-01

    The peroxisome proliferator-activated receptor alpha (PPARα) is a member of the nuclear receptor superfamily that has been suggested as a modulator of several physiological functions. The PPARα recognizes as an endogenous ligand the anorexic lipid mediator oleoylethanolamide (OEA) which displays wake-inducing properties. Despite that recent evidence indicates that activation of PPARα by synthetic agonists such as Wy14643 enhances waking as well as the extracellular contents of wake-related neurotransmitters, the role of PPARα in sleep recovery after prolonged waking has not been fully described. Thus, the aim of this study was to characterize if PPARα regulates sleep rebound after total sleep deprivation (TSD). We report that after 6h of TSD activation of PPARα by pharmacological systemic administration of OEA (10, 20 or 30mg/Kg, i.p.) promoted alertness by blocking the sleep rebound after TSD. Besides, wake-linked compounds such as dopamine, norepinephrine, serotonin, or adenosine collected from nucleus accumbens were enhanced after TSD in OEA-treated animals. These sleep and neurochemical results were mimicked after injection of PPARα agonist Wy14643 (10, 20, 30mg/Kg, i.p.). However, similar findings from the sham of vehicle groups were observed if PPARα antagonist MK-886 was administered to rats (10, 20, 30mg/Kg, i.p.). Our results strengthened the hypothesis that PPARα might modulate sleep and neurochemical homeostasis after sleep deprivation.

  5. Highly active chromium-based selective ethylene tri-/tetramerization catalysts supported by PNPO phosphazane ligands.

    PubMed

    Zhou, Yusheng; Wu, Hongfei; Xu, Sheng; Zhang, Xuejun; Shi, Min; Zhang, Jun

    2015-05-28

    Novel Cr(iii) catalysts supported by PNPO phosphazane ligands of the type Ph2PN(R)P(Ph)OAr have been prepared, all of which, upon activation with MMAO-3A, are highly active in ethylene tri-/tetramerization with considerable selectivity. The effect of ligand substitution on the catalytic performance has been examined. The Cr precatalyst supported by the PNPO phosphazane ligand with an N-cyclohexyl achieved high activity of 316.7 kg (g Cr h(-1))(-1) and a high total selectivity of 85.1% towards valuable 1-hexene (45.7%) and 1-octene (39.4%) using chlorobenzene as the solvent at 35 bar and 40 °C. In methylcyclohexane, the precatalyst supported by [Ph2PN((i)Pr)P(Ph)OPh] exhibited a higher 1-octene selectivity (54.0%) with a considerable activity of 73.3 kg (g Cr h(-1))(-1) at 35 bar and 40 °C. With the fine-tuned ligand backbone, such a PNPO phosphazane-based catalyst system provides a mode for precise understanding of the impact of ligand variations on catalytic performance.

  6. Integrin alpha1beta1 controls reactive oxygen species synthesis by negatively regulating epidermal growth factor receptor-mediated Rac activation.

    PubMed

    Chen, Xiwu; Abair, Tristin D; Ibanez, Maria R; Su, Yan; Frey, Mark R; Dise, Rebecca S; Polk, D Brent; Singh, Amar B; Harris, Raymond C; Zent, Roy; Pozzi, Ambra

    2007-05-01

    Integrins control many cell functions, including generation of reactive oxygen species (ROS) and regulation of collagen synthesis. Mesangial cells, found in the glomerulus of the kidney, are able to produce large amounts of ROS via the NADPH oxidase. We previously demonstrated that integrin alpha1-null mice develop worse fibrosis than wild-type mice following glomerular injury and this is due, in part, to excessive ROS production by alpha1-null mesangial cells. In the present studies, we describe the mechanism whereby integrin alpha1-null mesangial cells produce excessive ROS. Integrin alpha1-null mesangial cells have constitutively increased basal levels of activated Rac1, which result in its increased translocation to the cell membrane, excessive ROS production, and consequent collagen IV deposition. Basal Rac1 activation is a direct consequence of ligand-independent increased epidermal growth factor receptor (EGFR) phosphorylation in alpha1-null mesangial cells. Thus, our study demonstrates that integrin alpha1beta1-EGFR cross talk is a key step in negatively regulating Rac1 activation, ROS production, and excessive collagen synthesis, which is a hallmark of diseases characterized by irreversible fibrosis.

  7. Endothelial Lu/BCAM glycoproteins are novel ligands for red blood cell alpha4beta1 integrin: role in adhesion of sickle red blood cells to endothelial cells.

    PubMed

    El Nemer, Wassim; Wautier, Marie-Paule; Rahuel, Cécile; Gane, Pierre; Hermand, Patricia; Galactéros, Frédéric; Wautier, Jean-Luc; Cartron, Jean-Pierre; Colin, Yves; Le Van Kim, Caroline

    2007-04-15

    The Lutheran (Lu) blood group and basal cell adhesion molecule (BCAM) antigens are both carried by 2 glycoprotein isoforms of the immunoglobulin superfamily representing receptors for the laminin alpha(5) chain. In addition to red blood cells, Lu/BCAM proteins are highly expressed in endothelial cells. Abnormal adhesion of red blood cells to the endothelium could potentially contribute to the vaso-occlusive episodes in sickle cell disease. Considering the presence of integrin consensus-binding sites in Lu/BCAM proteins, we investigated their potential interaction with integrin alpha(4)beta(1), the unique integrin expressed on immature circulating sickle red cells. Using cell adhesion assays under static and flow conditions, we demonstrated that integrin alpha(4)beta(1) expressed on transfected cells bound to chimeric Lu-Fc protein. We showed that epinephrine-stimulated sickle cells, but not control red cells, adhered to Lu-Fc via integrin alpha(4)beta(1) under flow conditions. Antibody-mediated activation of integrin alpha(4)beta(1) induced adhesion of sickle red cells to primary human umbilical vein endothelial cells; this adhesion was inhibited by soluble Lu-Fc and vascular cell adhesion molecule-1 (VCAM-1)-Fc proteins. This novel interaction between integrin alpha(4)beta(1) in sickle red cells and endothelial Lu/BCAM proteins could participate in sickle cell adhesion to endothelium and potentially play a role in vaso-occlusive episodes.

  8. Conditional expression of constitutively active estrogen receptor {alpha} in chondrocytes impairs longitudinal bone growth in mice

    SciTech Connect

    Ikeda, Kazuhiro; Tsukui, Tohru; Imazawa, Yukiko; Horie-Inoue, Kuniko; Inoue, Satoshi

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer Conditional transgenic mice expressing constitutively active estrogen receptor {alpha} (caER{alpha}) in chondrocytes were developed. Black-Right-Pointing-Pointer Expression of caER{alpha} in chondrocytes impaired longitudinal bone growth in mice. Black-Right-Pointing-Pointer caER{alpha} affects chondrocyte proliferation and differentiation. Black-Right-Pointing-Pointer This mouse model is useful for understanding the physiological role of ER{alpha}in vivo. -- Abstract: Estrogen plays important roles in the regulation of chondrocyte proliferation and differentiation, which are essential steps for longitudinal bone growth; however, the mechanisms of estrogen action on chondrocytes have not been fully elucidated. In the present study, we generated conditional transgenic mice, designated as caER{alpha}{sup ColII}, expressing constitutively active mutant estrogen receptor (ER) {alpha} in chondrocytes, using the chondrocyte-specific type II collagen promoter-driven Cre transgenic mice. caER{alpha}{sup ColII} mice showed retardation in longitudinal growth, with short bone lengths. BrdU labeling showed reduced proliferation of hypertrophic chondrocytes in the proliferating layer of the growth plate of tibia in caER{alpha}{sup ColII} mice. In situ hybridization analysis of type X collagen revealed that the maturation of hypertrophic chondrocytes was impaired in caER{alpha}{sup ColII} mice. These results suggest that ER{alpha} is a critical regulator of chondrocyte proliferation and maturation during skeletal development, mediating longitudinal bone growth in vivo.

  9. Ligand-induced adhesion to activated endothelium and to vascular cell adhesion molecule-1 in lymphocytes transfected with the N-formyl peptide receptor.

    PubMed

    Honda, S; Campbell, J J; Andrew, D P; Engelhardt, B; Butcher, B A; Warnock, R A; Ye, R D; Butcher, E C

    1994-04-15

    Binding of FMLP to the neutrophil N-formyl peptide receptor (FPR) transmits signals through pertussis toxin-sensitive G proteins triggering Ca2+ flux, superoxide production, granule exocytosis, and neutrophil aggregation and adhesion involving the beta 2 (CD18) integrins. Expression of the FPR in mouse fibroblasts or human kidney cells has been shown to confer an N-formyl peptide-inducible Ca2+ flux in transfectants. Here we demonstrate that the transfected receptor can also support ligand-induced alterations in cellular adhesion. We established stable transfectants of mouse L1-2 pre-B cells with cDNA for human FPR (L1-2 FPR cells). The transfectants bind N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein with 1.4 x 10(5) sites per cell and a dissociation constant of 3.3 nM. Stimulation with FMLP induces a transient Ca2+ flux. FMLP also triggers adhesion of L1-2 FPR cells to TNF-alpha- or LPS-activated bEnd3 cells (mouse brain-derived endothelial cells) and to purified mouse VCAM-1. Binding is inhibited by Abs to VCAM-1 and to the alpha-chain of its lymphocyte receptor (the alpha 4 beta 1 integrin, VLA-4). Stimulation with FMLP does not induce a change in cell surface expression of alpha 4. Induced adhesion to VCAM-1 is rapid, detectable at the earliest times measurable (30 to 60 s after FMLP addition), and is inhibited by pertussis toxin. We conclude that FPR can mediate integrin activation not only in neutrophils but also in lymphocytes, and can trigger rapid adhesion via lymphocyte alpha 4 beta 1. The adhesion of lymphocytes is critical to their migration and targeting; our results suggest the possibility of manipulating adhesive responses through expression of chemoattractant receptors in lymphoid cells engineered for cellular therapy, allowing targeted adhesion and potentially migration in response to locally administered ligands.

  10. Spatial correspondence of brain alpha activity component in fMRI and EEG

    NASA Astrophysics Data System (ADS)

    Jeong, Jeong-Won; Kim, Sung-Heon; Singh, Manbir

    2005-04-01

    This paper presents a new approach to investigate the spatial correlation of brain alpha activity in functional magnetic resonance imaging (fMRI) and electroencephalography (EEG). To avoid potential problems of simultaneous fMRI and EEG acquisitions in imaging brain alpha activity, data from each modality were acquired separately under a "three conditions" setup where one of the conditions involved closing eyes and relaxing, thus making it conducive to generation of alpha activity. The other two conditions -- eyes open in a lighted room or engaged in a mental arithmetic task, were designed to attenuate alpha activity. Using the Mixture Density Independent Component Analysis (MD-ICA) that incorporates flexible non-linearity functions into the conventional ICA framework, we could identify the spatiotemporal components of fMRI activations and EEG activities associated with the alpha rhythm. The sources of the individual EEG alpha activity component were localized by a Maximum Entropy (ME) method that solves an inverse problem in the framework of a classical four-sphere head model. The resulting dipole sources of EEG alpha activity were spatially transformed to 3D MRIs of the subject and compared to fMRI ICA-determined alpha activity maps.

  11. High activity of alpha-glycerophosphate oxidation by human placental mitochondria.

    PubMed

    Swierczyński, J; Scislowski, P; Aleksandrowicz, Z

    1976-03-11

    Human term placental mitochondria oxidize alpha-glycerophosphate at an unusually high rate as compared to other substrates. The apparent Km both for oxidation and alpha-glycerophosphate dehydrogenase (EC 1.1.99.5) activity of DL-alpha glycerophosphate determined in a medium containing 2mM EDTA and 5 mM MgSO4 was approx. 0.7 mM. EDTA inhibited the alpha-glycerophosphate oxidation if the later was used at low concentrations. A subsequent addition of MgSO4 or CaCl2 restored the original activity. EDTA had no effect on mitochondrial respiration at high concentration of alpha-glycerophosphate. Possible physiological role of relatively high activity of human placental mitochondrial alpha-glycerophosphate dehydrogenase is discussed.

  12. A semisynthetic Eph receptor tyrosine kinase provides insight into ligand-induced kinase activation

    PubMed Central

    Singla, Nikhil; Erdjument-Bromage, Hediye; Himanen, Juha P.; Muir, Tom W.; Nikolov, Dimitar B.

    2011-01-01

    SUMMARY We have developed a methodology for generating milligram amounts of functional Eph tyrosine kinase receptor using the protein engineering approach of expressed protein ligation. Stimulation with ligand induces efficient autophosphorylation of the semisynthetic Eph construct. The in vitro phosphorylation of key Eph tyrosine residues upon ligand-induced activation was monitored via time-resolved, quantitative phosphoproteomics, suggesting a precise and unique order of phosphorylation of the Eph tyrosines in the kinase activation process. To our knowledge, this work represents the first reported semisynthesis of a receptor tyrosine kinase and provides a potentially general method for producing single-pass membrane proteins for structural and biochemical characterization. PMID:21439481

  13. Alpha-Amylase Activity in Blood Increases after Pharmacological, But Not Psychological, Activation of the Adrenergic System

    PubMed Central

    Nater, Urs M.; La Marca, Roberto; Erni, Katja; Ehlert, Ulrike

    2015-01-01

    Background & Aim Alpha-amylase in both blood and saliva has been used as a diagnostic parameter. While studies examining alpha-amylase activity in saliva have shown that it is sensitive to physiological and psychological challenge of the adrenergic system, no challenge studies have attempted to elucidate the role of the adrenergic system in alpha-amylase activity in blood. We set out to examine the impact of psychological and pharmacological challenge on alpha-amylase in blood in two separate studies. Methods In study 1, healthy subjects were examined in a placebo-controlled, double-blind paradigm using yohimbine, an alpha2-adrenergic antagonist. In study 2, subjects were examined in a standardized rest-controlled psychosocial stress protocol. Alpha-amylase activity in blood was repeatedly measured in both studies. Results Results of study 1 showed that alpha-amylase in blood is subject to stronger increases after injection of yohimbine compared to placebo. In study 2, results showed that there was no significant effect of psychological stress compared to rest. Conclusions Alpha-amylase in blood increases after pharmacological activation of the adrenergic pathways suggesting that sympathetic receptors are responsible for these changes. Psychological stress, however, does not seem to have an impact on alpha-amylase in blood. Our findings provide insight into the mechanisms underlying activity changes in alpha-amylase in blood in healthy individuals. PMID:26110636

  14. Novel alpha-melanocyte stimulating hormone peptide analogues with high candidacidal activity.

    PubMed

    Grieco, Paolo; Rossi, Claudia; Colombo, Gualtiero; Gatti, Stefano; Novellino, Ettore; Lipton, James M; Catania, Anna

    2003-02-27

    alpha-Melanocyte stimulating hormone (alpha-MSH) is an endogenous linear tridecapeptide with potent antiinflammatory effects. We recently demonstrated that alpha-MSH and its C-terminal sequence Lys-Pro-Val (alpha-MSH (11-13)) have antimicrobial effects against two major and representative pathogens: Staphylococcus aureus and Candida albicans. In an attempt to improve the candidacidal activity of alpha-MSH and to better understand the peptide structure-antifungal activity relations, we designed and synthesized novel peptide analogues. Because previous data suggested that antimicrobial effects of alpha-MSH were receptor-mediated, we chose to focus on the sequence alpha-MSH (6-13), which contains the invariant core sequence His-Phe-Arg-Trp (6-9) that is important for binding to the known melanocortin receptors and also contains the sequence Lys-Pro-Val (11-13) that is known to be important for antimicrobial activity. In this structure-activity study, we discovered several compounds that have greater candidacidal activity than alpha-MSH. The peptide [d-Nal-7,Phe-12]-alpha-MSH (6-13) was the most potent of the analogues tested. The present results are very encouraging because they show the great potential of these peptides as a truly novel class of candidacidal compounds.

  15. Modulation of the ligand-independent activation of the human estrogen receptor by hormone and antihormone.

    PubMed Central

    Smith, C L; Conneely, O M; O'Malley, B W

    1993-01-01

    It has been previously demonstrated that several members of the steroid receptor superfamily may be activated by the neurotransmitter dopamine in the apparent absence of cognate ligand. We have examined wild-type and mutant human estrogen receptors (ERs, [Gly400]ER and [Val400]ER, respectively) for their abilities to activate ER-dependent transcription of a transgene in a ligand-independent manner. In cells expressing the wild-type ER, dopamine was nearly as effective as 17 beta-estradiol at inducing the chloramphenicol acetyltransferase activity of the reporter gene in a dose-dependent manner; simultaneous addition of suboptimal concentrations of 17 beta-estradiol and dopamine stimulated transcription more than either compound alone. Dopamine alone was unable to induce gene expression in cells expressing [Val400]ER mutant receptors, but concomitant treatment with 17 beta-estradiol produced a synergistic increase in transcription, suggesting that the ligand may alter the mutant receptor's conformation such that it can be activated subsequently by a dopaminergic signaling mechanism. In the presence of the antiestrogen ICI 164,384, dopamine-stimulated gene expression was undetectable in cells expressing either form of ER. However, simultaneous treatment of cells expressing wild-type ER with trans-4-hydroxytamoxifen and dopamine resulted in transgene expression that was additive in nature compared to either compound alone; similar treatment of cells expressing [Val400]ER produced a synergistic increase. Our results suggest that ligand and ligand-independent activation of the ER initiate from distinct pathways and that the latter may occur in a variety of target tissues subject to modulation by receptor ligands. Images Fig. 5 PMID:8327492

  16. Structure- and conformation-activity studies of nociceptin/orphanin FQ receptor dimeric ligands

    PubMed Central

    Pacifico, Salvatore; Carotenuto, Alfonso; Brancaccio, Diego; Novellino, Ettore; Marzola, Erika; Ferrari, Federica; Cerlesi, Maria Camilla; Trapella, Claudio; Preti, Delia; Salvadori, Severo; Calò, Girolamo; Guerrini, Remo

    2017-01-01

    The peptide nociceptin/orphanin FQ (N/OFQ) and the N/OFQ receptor (NOP) constitute a neuropeptidergic system that modulates various biological functions and is currently targeted for the generation of innovative drugs. In the present study dimeric NOP receptor ligands with spacers of different lengths were generated using both peptide and non-peptide pharmacophores. The novel compounds (12 peptide and 7 nonpeptide ligands) were pharmacologically investigated in a calcium mobilization assay and in the mouse vas deferens bioassay. Both structure- and conformation-activity studies were performed. Results demonstrated that dimerization did not modify the pharmacological activity of both peptide and non-peptide pharmacophores. Moreover, when dimeric compounds were obtained with low potency peptide pharmacophores, dimerization recovered ligand potency. This effect depends on the doubling of the C-terminal address sequence rather than the presence of an additional N-terminal message sequence or modifications of peptide conformation. PMID:28383520

  17. Computation of Rate Constants for Diffusion of Small Ligands to and from Buried Protein Active Sites.

    PubMed

    Wang, P-H; De Sancho, D; Best, R B; Blumberger, J

    2016-01-01

    The diffusion of ligands to actives sites of proteins is essential to enzyme catalysis and many cellular signaling processes. In this contribution we review our recently developed methodology for calculation of rate constants for diffusion and binding of small molecules to buried protein active sites. The diffusive dynamics of the ligand obtained from molecular dynamics simulation is coarse grained and described by a Markov state model. Diffusion and binding rate constants are then obtained either from the reactive flux formalism or by fitting the time-dependent population of the Markov state model to a phenomenological rate law. The method is illustrated by applications to diffusion of substrate and inhibitors in [NiFe] hydrogenase, CO-dehydrogenase, and myoglobin. We also discuss a recently developed sensitivity analysis that allows one to identify hot spots in proteins, where mutations are expected to have the strongest effects on ligand diffusion rates.

  18. Human NK cells in acute myeloid leukaemia patients: analysis of NK cell-activating receptors and their ligands.

    PubMed

    Sanchez-Correa, Beatriz; Morgado, Sara; Gayoso, Inmaculada; Bergua, Juan M; Casado, Javier G; Arcos, Maria Jose; Bengochea, Maria Luisa; Duran, Esther; Solana, Rafael; Tarazona, Raquel

    2011-08-01

    Natural killer (NK) cell activation is strictly regulated to ensure that healthy cells are preserved, but tumour-transformed or virus-infected cells are recognized and eliminated. To carry out this selective killing, NK cells have an ample repertoire of receptors on their surface. Signalling by inhibitory and activating receptors by interaction with their ligands will determine whether the NK cell becomes activated and kills the target cell. Here, we show reduced expression of NKp46, NKp30, DNAM-1, CD244 and CD94/NKG2C activating receptors on NK cells from acute myeloid leukaemia patients. This reduction may be induced by chronic exposure to their ligands on leukaemic blasts. The analysis of ligands for NK cell-activating receptors showed that leukaemic blasts from the majority of patients express ligands for NK cell-activating receptors. DNAM-1 ligands are frequently expressed on blasts, whereas the expression of the NKG2D ligand MICA/B is found in half of the patients and CD48, a ligand for CD244, in only one-fourth of the patients. The decreased expression of NK cell-activating receptors and/or the heterogeneous expression of ligands for major receptors on leukaemic blasts can lead to an inadequate tumour immunosurveillance by NK cells. A better knowledge of the activating receptor repertoire on NK cells and their putative ligands on blasts together with the possibility to modulate their expression will open new possibilities for the use of NK cells in immunotherapy against leukaemia.

  19. beta-Naphthoflavone protects from peritonitis by reducing TNF-alpha-induced endothelial cell activation.

    PubMed

    Hsu, Sheng-Yao; Liou, Je-Wen; Cheng, Tsung-Lin; Peng, Shih-Yi; Lin, Chi-Chen; Chu, Yuan-Yuan; Luo, Wei-Cheng; Huang, Zheng-Kai; Jiang, Shinn-Jong

    2015-12-01

    β-Naphthoflavone (β-NF), a ligand of the aryl hydrocarbon receptor, has been shown to possess anti-oxidative properties. We investigated the anti-oxidative and anti-inflammatory potential of β-NF in human microvascular endothelial cells treated with tumor necrosis factor-alpha (TNF-α). Pretreatment with β-NF significantly inhibited TNF-α-induced intracellular reactive oxygen species, translocation of p67(phox), and TNF-α-induced monocyte binding and transmigration. In addition, β-NF significantly inhibited TNF-α-induced ICAM-1 and VCAM-1 expression. The mRNA expression levels of the inflammatory cytokines TNF-α and IL-6 were reduced by β-NF, as was the infiltration of white blood cells, in a peritonitis model. The inhibition of adhesion molecules was associated with suppressed nuclear translocation of NF-κB p65 and Akt, and suppressed phosphorylation of ERK1/2 and p38. The translocation of Egr-1, a downstream transcription factor involved in the MEK-ERK signaling pathway, was suppressed by β-NF treatment. Our findings show that β-NF inhibits TNF-α-induced NF-kB and ERK1/2 activation and ROS generation, thereby suppressing the expression of adhesion molecules. This results in reduced adhesion and transmigration of leukocytes in vitro and prevents the infiltration of leukocytes in a peritonitis model. Our findings also suggest that β-NF might prevent TNF-α-induced inflammation.

  20. Enhanced calcium cycling and contractile function in transgenic hearts expressing constitutively active G alpha o* protein.

    PubMed

    Zhu, Ming; Gach, Agnieszka A; Liu, GongXin; Xu, Xiaomei; Lim, Chee Chew; Zhang, Julie X; Mao, Lan; Chuprun, Kurt; Koch, Walter J; Liao, Ronglih; Koren, Gideon; Blaxall, Burns C; Mende, Ulrike

    2008-03-01

    In contrast to the other heterotrimeric GTP-binding proteins (G proteins) Gs and Gi, the functional role of G o is still poorly defined. To investigate the role of G alpha o in the heart, we generated transgenic mice with cardiac-specific expression of a constitutively active form of G alpha o1* (G alpha o*), the predominant G alpha o isoform in the heart. G alpha o expression was increased 3- to 15-fold in mice from 5 independent lines, all of which had a normal life span and no gross cardiac morphological abnormalities. We demonstrate enhanced contractile function in G alpha o* transgenic mice in vivo, along with increased L-type Ca2+ channel current density, calcium transients, and cell shortening in ventricular G alpha o*-expressing myocytes compared with wild-type controls. These changes were evident at baseline and maintained after isoproterenol stimulation. Expression levels of all major Ca2+ handling proteins were largely unchanged, except for a modest reduction in Na+/Ca2+ exchanger in transgenic ventricles. In contrast, phosphorylation of the ryanodine receptor and phospholamban at known PKA sites was increased 1.6- and 1.9-fold, respectively, in G alpha o* ventricles. Density and affinity of beta-adrenoceptors, cAMP levels, and PKA activity were comparable in G alpha o* and wild-type myocytes, but protein phosphatase 1 activity was reduced upon G alpha o* expression, particularly in the vicinity of the ryanodine receptor. We conclude that G alpha o* exerts a positive effect on Ca2+ cycling and contractile function. Alterations in protein phosphatase 1 activity rather than PKA-mediated phosphorylation might be involved in hyperphosphorylation of key Ca2+ handling proteins in hearts with constitutive G alpha o activation.

  1. Activation of peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}) suppresses postprandial lipidemia through fatty acid oxidation in enterocytes

    SciTech Connect

    Kimura, Rino; Takahashi, Nobuyuki; Murota, Kaeko; Yamada, Yuko; Niiya, Saori; Kanzaki, Noriyuki; Murakami, Yoko; Moriyama, Tatsuya; Goto, Tsuyoshi; Kawada, Teruo

    2011-06-24

    Highlights: {yields} PPAR{alpha} activation increased mRNA expression levels of fatty acid oxidation-related genes in human intestinal epithelial Caco-2 cells. {yields} PPAR{alpha} activation also increased oxygen consumption rate and CO{sub 2} production and decreased secretion of triglyceride and ApoB from Caco-2 cells. {yields} Orally administration of bezafibrate increased mRNA expression levels of fatty acid oxidation-related genes and CO{sub 2} production in small intestinal epithelial cells. {yields} Treatment with bezafibrate decreased postprandial serum concentration of triglyceride after oral injection of olive oil in mice. {yields} It suggested that intestinal lipid metabolism regulated by PPAR{alpha} activation suppresses postprandial lipidemia. -- Abstract: Activation of peroxisome proliferator-activated receptor (PPAR)-{alpha} which regulates lipid metabolism in peripheral tissues such as the liver and skeletal muscle, decreases circulating lipid levels, thus improving hyperlipidemia under fasting conditions. Recently, postprandial serum lipid levels have been found to correlate more closely to cardiovascular diseases than fasting levels, although fasting hyperlipidemia is considered an important risk of cardiovascular diseases. However, the effect of PPAR{alpha} activation on postprandial lipidemia has not been clarified. In this study, we examined the effects of PPAR{alpha} activation in enterocytes on lipid secretion and postprandial lipidemia. In Caco-2 enterocytes, bezafibrate, a potent PPAR{alpha} agonist, increased mRNA expression levels of fatty acid oxidation-related genes, such as acyl-CoA oxidase, carnitine palmitoyl transferase, and acyl-CoA synthase, and oxygen consumption rate (OCR) and suppressed secretion levels of both triglycerides and apolipoprotein B into the basolateral side. In vivo experiments revealed that feeding high-fat-diet containing bezafibrate increased mRNA expression levels of fatty acid oxidation-related genes and

  2. CHARMM Force Field Parameterization of Peroxisome Proliferator-Activated Receptor γ Ligands

    PubMed Central

    Mottin, Melina; Souza, Paulo C. T.; Ricci, Clarisse G.; Skaf, Munir S.

    2016-01-01

    The peroxisome proliferator-activated receptor γ (PPARγ) ligands are important therapeutic drugs for the treatment of type 2 diabetes, obesity and cardiovascular diseases. In particular, partial agonists and non-agonists are interesting targets to reduce glucose levels, presenting few side effects in comparison to full agonists. In this work, we present a set of CHARMM-based parameters of a molecular mechanics force field for two PPARγ ligands, GQ16 and SR1664. GQ16 belongs to the thiazolidinedione class of drugs and it is a PPARγ partial agonist that has been shown to promote the “browning” of white adipose tissue. SR1664 is the precursor of the PPARγ non-agonist class of ligands that activates PPARγ in a non-classical manner. Here, we use quantum chemical calculations consistent with the CHARMM protocol to obtain bonded and non-bonded parameters, including partial atomic charges and effective torsion potentials for both molecules. The newly parameterized models were evaluated by examining the behavior of GQ16 and SR1664 free in water and bound to the ligand binding pocket of PPARγ using molecular dynamics simulations. The potential parameters derived here are readily transferable to a variety of pharmaceutical compounds and similar PPARγ ligands. PMID:28025495

  3. Identification and functional analysis of ligands for natural killer cell activating receptors in colon carcinoma.

    PubMed

    Zhang, Zhang; Su, Tao; He, Liang; Wang, Hongtao; Ji, Gang; Liu, Xiaonan; Zhang, Yun; Dong, Guanglong

    2012-01-01

    Natural killer (NK) cells play important roles in the immune defense against tumor cells. The function of NK cells is determined by a balance between activating and inhibitory signals. DNAX accessory molecule-1 (DNAM-1) and NK group 2 member D (NKG2D) are major NK cell activating receptors, which transduce activating signals after binding their ligands CD155, CD112 and major histocompatibility complex class I-related chains A and B (MICA/B). However, the expression and functions of these ligands in colon carcinoma are still elusive. Here, we show the higher expression of CD155, CD112 and MICA/B in colon carcinoma tissues, although no correlations between the ligands expression and patient clinicopathological parameters were found. The subsequent cytotoxicity assay indicated that NK cells effectively kill colon carcinoma cells. Functional blocking of these ligands and/or receptors with antibodies led to significant inhibition of NK cell cytotoxicity. Importantly, expression of DNAM-1 and NKG2D was reduced in NK cells of colon cancer patients, and this reduction could directly suppress the activation of NK cells. Moreover, colon cancer patients have higher serum concentrations of sCD155 and sMICA/B (soluble ligands, secreted or shed from cells) than those in healthy donors (sCD155, 127.82 ± 44.12 vs. 63.67 ± 22.30 ng/ml; sMICA, 331.51 ± 65.23 vs. 246.74 ± 20.76 pg/ml; and sMICB, 349.42 ± 81.69 vs. 52.61 ± 17.56 pg/ml). The up-regulation of these soluble ligands may down-regulate DNAM-1 and NKG2D on NK cells, ultimately leading to the inhibition of NK cytotoxicity. Colon cancer might be a promising target for NK cell-based adoptive immunotherapy.

  4. Biochemical and Cellular Analysis Reveals Ligand Binding Specificities, a Molecular Basis for Ligand Recognition, and Membrane Association-dependent Activities of Cripto-1 and Cryptic.

    PubMed

    Aykul, Senem; Parenti, Anthony; Chu, Kit Yee; Reske, Jake; Floer, Monique; Ralston, Amy; Martinez-Hackert, Erik

    2017-03-10

    Transforming growth factor β (TGF-β) pathways are key determinants of cell fate in animals. Their basic mechanism of action is simple. However, to produce cell-specific responses, TGF-β pathways are heavily regulated by secondary factors, such as membrane-associated EGF-CFC family proteins. Cellular activities of EGF-CFC proteins have been described, but their molecular functions, including how the mammalian homologs Cripto-1 and Cryptic recognize and regulate TGF-β family ligands, are less clear. Here we use purified human Cripto-1 and mouse Cryptic produced in mammalian cells to show that these two EGF-CFC homologs have distinct, highly specific ligand binding activities. Cripto-1 interacts with BMP-4 in addition to its known partner Nodal, whereas Cryptic interacts only with Activin B. These interactions depend on the integrity of the protein, as truncated or deglycosylated Cripto-1 lacked BMP-4 binding activity. Significantly, Cripto-1 and Cryptic blocked binding of their cognate ligands to type I and type II TGF-β receptors, indicating that Cripto-1 and Cryptic contact ligands at their receptor interaction surfaces and, thus, that they could inhibit their ligands. Indeed, soluble Cripto-1 and Cryptic inhibited ligand signaling in various cell-based assays, including SMAD-mediated luciferase reporter gene expression, and differentiation of a multipotent stem cell line. But in agreement with previous work, the membrane bound form of Cripto-1 potentiated signaling, revealing a critical role of membrane association for its established cellular activity. Thus, our studies provide new insights into the mechanism of ligand recognition by this enigmatic family of membrane-anchored TGF-β family signaling regulators and link membrane association with their signal potentiating activities.

  5. How to build optically active alpha-amino acids.

    PubMed

    Calmes, M; Daunis, J

    1999-01-01

    Various methodologies published in the literature dealing with alpha-amino carboxylic acid asymmetric synthesis are presented in a digest form. In each case, only some recent or most typical works are mentioned.

  6. PGC-1alpha activates CYP7A1 and bile acid biosynthesis.

    PubMed

    Shin, Dong-Ju; Campos, Jose A; Gil, Gregorio; Osborne, Timothy F

    2003-12-12

    Cholesterol 7-alpha-hydroxylase (CYP7A1) is the key enzyme that commits cholesterol to the neutral bile acid biosynthesis pathway and is highly regulated. In the current studies, we have uncovered a role for the transcriptional co-activator PGC-1alpha in CYP7A1 gene transcription. PGC-1alpha plays a vital role in adaptive thermogenesis in brown adipose tissue and stimulates genes important to mitochondrial function and oxidative metabolism. It is also involved in the activation of hepatic gluconeogenesic gene expression during fasting. Because the mRNA for CYP7A1 was also induced in mouse liver by fasting, we reasoned that PGC-1alpha might be an important co-activator for CYP7A1. Here we show that PGC-1alpha and CYP7A1 are also co-induced in livers of mice in response to streptozotocin induced diabetes. Additionally, infection of cultured HepG2 cells with a recombinant adenovirus expressing PGC-1alpha directly activates CYP7A1 gene expression and increases bile acid biosynthesis as well. Furthermore, we show that PGC-1alpha activates the CYP7A1 promoter directly in transient transfection assays in cultured cells. Thus, PGC-1alpha is a key activator of CYP7A1 and bile acid biosynthesis and is likely responsible for the fasting and diabetes dependent induction of CYP7A1. PGC-1alpha has already been shown to be a critical activator of several other oxidative processes including adaptive thermogenesis and fatty acid oxidation. Our studies provide further evidence of the fundamental role played by PGC-1alpha in oxidative metabolism and define PGC-1alpha as a link between diabetes and bile acid metabolism.

  7. Inhibition of pea chloroplast DNA helicase unwinding and ATPase activities by DNA-interacting ligands.

    PubMed

    Tuteja, N; Phan, T N

    1998-03-27

    DNA helicases unwind the duplex DNA in an ATP dependent manner and thus play an essential role in DNA replication, repair, recombination and transcription. Any DNA-interacting ligand which will modulate DNA helicase activity may interrupt practically all kinds of DNA transactions. There are no studies on the effect of various cytotoxic DNA-interacting ligands on organelle helicases. We have determined the effect of camptothecin, VP-16 (etoposide), ellipticine, genistein, novobiocin, m-AMSA, actinomycin C1, ethidium bromide, daunorubicin and nogalamycin on unwinding and ATPase activities of purified chloroplast DNA helicase from pea (Pisum sativum). Our study has shown that DNA-intercalating ligands actinomycin C1, ethidium bromide, daunorubicin and nogalamycin were inhibiting the DNA unwinding activity with an apparent Ki of 2.9 microM, 3.0 microM, 1.4 microM and 1.0 microM, respectively. These four inhibitors also inhibited the ATPase activity of pea chloroplast DNA helicase. These results indicate that the intercalation of the inhibitors into DNA generates a complex that impedes the translocation of chloroplast DNA helicase, resulting in both inhibition of unwinding activity and ATP hydrolysis. This study would be useful for understanding the mechanism of organelle DNA helicase unwinding and the mechanism by which these DNA-interacting ligands inhibit cellular function.

  8. Synthesis, characterization and antimicrobial activities of mixed ligand transition metal complexes with isatin monohydrazone Schiff base ligands and heterocyclic nitrogen base

    NASA Astrophysics Data System (ADS)

    Devi, Jai; Batra, Nisha

    2015-01-01

    Mixed ligand complexes of Co(II), Ni(II), Cu(II) and Zn(II) with various uninegative tridentate ligands derived from isatin monohydrazone with 2-hydroxynapthaldehyde/substituted salicylaldehyde and heterocyclic nitrogen base 8-hydroxyquinoline have been synthesized and characterized by elemental analysis, conductometric studies, magnetic susceptibility and spectroscopic techniques (IR, UV-VIS, NMR, mass and ESR). On the basis of these characterizations, it was revealed that Schiff base ligands existed as monobasic tridentate ONO bonded to metal ion through oxygen of carbonyl group, azomethine nitrogen and deprotonated hydroxyl oxygen and heterocyclic nitrogen base 8-hydroxyquinoline existed as monobasic bidentate ON bonded through oxygen of hydroxyl group and nitrogen of quinoline ring with octahedral or distorted octahedral geometry around metal ion. All the compounds have been tested in vitro against various pathogenic Gram positive bacteria, Gram negative bacteria and fungi using different concentrations (25, 50, 100, 200 μg/mL) of ligands and their complexes. Comparative study of antimicrobial activity of ligands, and their mixed complexes indicated that complexes exhibit enhanced activity as compared to free ligands and copper(II) Cu(LIV)(Q)ṡH2O complex was found to be most potent antimicrobial agent.

  9. Affinophoresis of pea lectin and fava bean lectin with an anionic affinophore, bearing rho-aminophenyl-alpha-D-mannoside as an affinity ligand.

    PubMed

    Shimura, K; Kasai, K

    1987-07-29

    Affinophoresis is an electrophoretic separation technique for biological polymers with the aid of an affinophore, which is a macromolecular polyelectrolyte bearing affinity ligands. The affinophore migrates rapidly in an electric field, and consequently the electrophoretic mobility of molecules having an affinity for the ligand is specifically changed. An anionic affinophore-bearing mannosyl residue was synthesized for the affinophoresis of lectins. rho-Aminophenyl-alpha-D-mannopyranoside and aminomethanesulphonic acid were coupled to about one-tenth and one-fifth, respectively, of the carboxyl groups of succinyl-poly-L-lysine with an average degree of polymerization of 120 by the use of a water-soluble carbodiimide. Extracts of seeds of pea (Pisum sativum) or fava bean (Vicia fava) were subjected to two-dimensional agarose gel electrophoresis, in which the first dimension was ordinary agarose gel electrophoresis and the second dimension was affinophoresis with the affinophore. The separated proteins were stained with Coomassie Blue R250. The lectins in both seed extracts were separated from a diagonal line formed by other proteins in the extracts. About 10 ng of the separated pea lectin was detected on a nitrocellulose blot by immunostaining with a horseradish peroxidase-conjugated second antibody.

  10. Inhibition of type 1 and type 2 5alpha-reductase activity by free fatty acids, active ingredients of Permixon.

    PubMed

    Raynaud, Jean Pierre; Cousse, Henri; Martin, Pierre Marie

    2002-10-01

    In different cell systems, the lipido-sterolic extract of Serenoa repens (LSESr, Permixon inhibits both type 1 and type 2 5alpha-reductase activity (5alphaR1 and 5alphaR2). LSESr is mainly constituted of fatty acids (90+/-5%) essentially as free fatty acids (80%). Among these free fatty acids, the main components are oleic and lauric acids which represent 65% and linoleic and myristic acids 15%. To evaluate the inhibitory effect of the different components of LSESr on 5alphaR1 or 5alphaR2 activity, the corresponding type 1 and type 2 human genes have been cloned and expressed in the baculovirus-directed insect cell expression system Sf9. The cells were incubated at pH 5.5 (5alphaR2) and pH 7.4 (5alphaR1) with 1 or 3nM testosterone in presence or absence of various concentrations of LSESr or of its different components. Dihydrotestosterone formation was measured with an automatic system combining HPLC and an on-line radiodetector. The inhibition of 5alphaR1 and 5alphaR2 activity was only observed with free fatty acids: esterified fatty acids, alcohols as well as sterols assayed were inactive. A specificity of the fatty acids in 5alphaR1 or 5alphaR2 inhibition has been found. Long unsaturated chains (oleic and linolenic) were active (IC(50)=4+/-2 and 13+/-3 microg/ml, respectively) on 5alphaR1 but to a much lesser extent (IC(50)>100 and 35+/-21 microg/ml, respectively) on 5alphaR2. Palmitic and stearic acids were inactive on the two isoforms. Lauric acid was active on 5alphaR1 (IC(50)=17+/-3 microg/ml) and 5alphaR2 (IC(50)=19+/-9 microg/ml). The inhibitory activity of myristic acid was evaluated on 5alphaR2 only and found active on this isoform (IC(50)=4+/-2 microg/ml). The dual inhibitory activity of LSESr on 5alpha-reductase type 1 and type 2 can be attributed to its high content in free fatty acids.

  11. Role of ligand-dependent GR phosphorylation and half-life in determination of ligand-specific transcriptional activity.

    PubMed

    Avenant, Chanel; Ronacher, Katharina; Stubsrud, Elisabeth; Louw, Ann; Hapgood, Janet P

    2010-10-07

    A central question in glucocorticoid mechanism of action via the glucocorticoid receptor (GR) is what determines ligand-selective transcriptional responses. Using a panel of 12 GR ligands, we show that the extent of GR phosphorylation at S226 and S211, GR half-life and transcriptional response, occur in a ligand-selective manner. While GR phosphorylation at S226 was shown to inhibit maximal transcription efficacy, phosphorylation at S211 is required for maximal transactivation, but not for transrepression efficacy. Both ligand-selective GR phosphorylation and half-life correlated with efficacy for transactivation and transrepression. For both expressed and endogenous GR, in two different cell lines, agonists resulted in the greatest extent of phosphorylation and the greatest extent of GR downregulation, suggesting a link between these functions. However, using phosphorylation-deficient GR mutants we established that phosphorylation of the GR at S226 or S211 does not determine the rank order of ligand-selective GR transactivation. These results are consistent with a model whereby ligand-selective GR phosphorylation and half-life are a consequence of upstream events, such as ligand-specific GR conformations, which are maintained in the phosphorylation mutants.

  12. Assessment of alpha activity of building materials commonly used in West Bengal, India.

    PubMed

    Ghosh, Dipak; Deb, Argha; Bera, Sukumar; Sengupta, Rosalima; Patra, Kanchan Kumar

    2008-02-01

    This paper, reports for the first time, an extensive study of alpha activity of all widely used building materials (plaster of Paris, stone chips, marble, white cement, mosaic stone, limestone, sand, granite, cement brick, asbestos, red brick, cement tile, ceramic tile and ceramics) in West Bengal, India. The alpha activities have been measured using Solid State Nuclear Track Detector (SSNTD), a very sensitive detector for alpha particles. The samples were collected from local markets of Kolkata. The measured average alpha activities ranged from 22.7+/-2.5 to 590.6+/-16.8Bqkg(-1). The alpha activity of ceramic tiles was highest and provides additional data to estimate the effect of environmental radiation exposure on human health.

  13. Adsorption of carbon monoxide on activated carbon tin ligand

    NASA Astrophysics Data System (ADS)

    Mohamad, A. B.; Iyuke, S. E.; Daud, W. R. W.; Kadhum, A. A. H.; Fisal, Z.; Al-Khatib, M. F.; Shariff, A. M.

    2000-09-01

    Activated carbon was impregnated with 34.57% SnCl 2·2H 2O salt and then dried at 180°C to produce AC-SnO 2 to improve its adsorptive interaction with CO. Besides the fact that activated carbon has its original different pore sizes for normal gas phase CO adsorption (as in the case of pure carbon), the impregnated carbon has additional CO adsorption ability due to the presence of O -(ads) on the active sites. AC-SnO 2 proved to be a superior adsorber of CO than pure carbon when used for H 2 purification in a PSA system. Discernibly, the high adsorptive selectivity of AC-SnO 2 towards gas phase CO portrays a good future for the applicability of this noble adsorbent, since CO has become a notorious threat to the global ecosystem due to the current level of air pollution.

  14. Antitumor and antiparasitic activity of novel ruthenium compounds with polycyclic aromatic ligands.

    PubMed

    Miserachs, Helena Guiset; Cipriani, Micaella; Grau, Jordi; Vilaseca, Marta; Lorenzo, Julia; Medeiros, Andrea; Comini, Marcelo A; Gambino, Dinorah; Otero, Lucía; Moreno, Virtudes

    2015-09-01

    Five novel ruthenium(II)-arene complexes with polycyclic aromatic ligands were synthesized, comprising three compounds of the formula [RuCl(η(6)-p-cym)(L)][PF6], where p-cym = 1-isopropyl-4-methylbenzene and L are the bidentate aromatic ligands 1,10-phenanthroline-5,6-dione, 1, 5-amine-1,10-phenanthroline, 4, or 5,6-epoxy-5,6-dihydro-phenanthroline, 5. In the other two complexes [RuCl2(η(6)-p-cym)(L')], the metal is coordinated to a monodentate ligand L', where L' is phenanthridine, 2, or 9-carbonylanthracene, 3. All compounds were fully characterized by mass spectrometry and elemental analysis, as well as NMR and IR spectroscopic techniques. Obtained ruthenium compounds as well as their respective ligands were tested for their antiparasitic and antitumoral activities. Even though all compounds showed lower Trypanosoma brucei activity than the free ligands, they also resulted less toxic on mammalian cells. Cytotoxicity assays on HL60 cells showed a moderate antitumoral activity for all ruthenium compounds. Compound 1 was the most potent antitumoral (IC50 = 1.26±0.78 μM) and antiparasitic (IC50 = 0.19 ± 0.05 μM) agent, showing high selectivity towards the parasites (selectivity index >100). As complex 1 was the most promising antitumoral compound, its interaction with ubiquitin as potential target was also studied. In addition, obtained ruthenium compounds were found to bind DNA, and they are thought to interact with this macromolecule mainly through intercalation of the aromatic ligand.

  15. New ligands with affinity for the alpha4beta2 subtype of nicotinic acetylcholine receptors. Synthesis, receptor binding, and 3D-QSAR modeling.

    PubMed

    Audouze, Karine; Nielsen, Elsebet Østergaard; Olsen, Gunnar M; Ahring, Philip; Jørgensen, Tino Dyhring; Peters, Dan; Liljefors, Tommy; Balle, Thomas

    2006-06-01

    A new series of piperazines, diazepanes, diazocanes, diazabicyclononanes, and diazabicyclodecanes with affinity for the alpha4beta2 subtype of nicotinic acetylcholine receptors were synthesized on the basis of results from a previous computational study. A predictive 3D-QSAR model was developed using the GRID/GOLPE approach (R2 = 0.94, Q2 = 0.83, SDEP = 0.34). The SAR was interpreted in terms of contour maps of the PLS coefficients and in terms of a homology model of the alpha4beta2 subtype of the nicotinic acetylcholine receptors. The results reveal that hydrogen bonding from both hydrogens on the protonated amine and from the pyridine nitrogen to a water molecule as well as van der Waals interactions between the substituent bearing the protonated amine and the receptor is of importance for ligand affinity. The combination of 3D-QSAR and homology modeling proved successful for the interpretation of structure-affinity relationships as well as the validation of the individual modeling approaches.

  16. Variability of the Lyman alpha flux with solar activity

    SciTech Connect

    Lean, J.L.; Skumanich, A.

    1983-07-01

    A three-component model of the solar chromosphere, developed from ground based observations of the Ca II K chromospheric emission, is used to calculate the variability of the Lyman alpha flux between 1969 and 1980. The Lyman alpha flux at solar minimum is required in the model and is taken as 2.32 x 10/sup 11/ photons/cm/sup 2//s. This value occurred during 1975 as well as in 1976 near the commencement of solar cycle 21. The model predicts that the Lyman alpha flux increases to as much as 5 x 10/sup 11/ photons/cm/sup 2//s at the maximum of the solar cycle. The ratio of the average fluxes for December 1979 (cycle maximum) and July 1976 (cycle minimum) is 1.9. During solar maximum the 27-day solar rotation is shown to cause the Lyman alpha flux to vary by as much as 40% or as little as 5%. The model also shows that the Lyman alpha flux varies over intermediate time periods of 2 to 3 years, as well as over the 11-year sunspot cycle. We conclude that, unlike the sunspot number and the 10.7-cm radio flux, the Lyman alpha flux had a variability that was approximately the same during each of the past three cycles. Lyman alpha fluxes calculated by the model are consistent with measurements of the Lyman alpha flux made by 11 of a total of 14 rocket experiments conducted during the period 1969--1980. The model explains satisfactorily the absolute magnitude, long-term trends, and the cycle variability seen in the Lyman alpha irradiances by the OSO 5 satellite experiment. The 27-day variability observed by the AE-E satellite experiment is well reproduced. However, the magntidue of the AE-E 1 Lyman alpha irradiances are higher than the model calculations by between 40% and 80%. We suggest that the assumed calibration of the AE-E irradiances is in error.

  17. Synthesis and antitumor activity of a series of osmium(VI) nitrido complexes bearing quinolinolato ligands.

    PubMed

    Tang, Quan; Ni, Wen-Xiu; Leung, Chi-Fai; Man, Wai-Lun; Lau, Kenneth King-Kwan; Liang, Yimin; Lam, Yun-Wah; Wong, Wai-Yeung; Peng, Shie-Ming; Liu, Gui-Jian; Lau, Tai-Chu

    2013-11-04

    A series of osmium(VI) nitrido complexes supported by quinolinolato ligands have been prepared and they exhibit promising in vitro anti-cancer activities. These results establish that Os(VI)≡N is a potentially versatile and promising platform for the design of a variety of high-valent anti-cancer drugs.

  18. Chemical Genetics: receptor-ligand pairs for rapid manipulation of neuronal activity

    PubMed Central

    Wulff, Peer; Arenkiel, Benjamin R.

    2012-01-01

    Towards the functional dissection of neuronal circuits, a number of new genetic tools have been developed that enable rapid and reversible manipulation of genetically defined neuronal subtypes in intact mammalian brain circuits. Alongside the breakthrough technology of optogenetics, receptor-ligand pairs provide complementary approaches to modulate neuronal activity using chemical-genetics. PMID:22119143

  19. Tumor escape mechanisms: Potential role of soluble HLA antigens and NK cells activating ligands

    PubMed Central

    Campoli, Michael; Ferrone, Soldano

    2009-01-01

    The crucial role played by HLA antigens and natural killer (NK) cell activating ligands in the interactions of malignant cells with components of the host's immune system has stimulated interest in the characterization of their expression by malignant cells. Convincing evidence generated by the immunohistochemical staining of surgically removed malignant lesions with monoclonal antibodies (mAb) recognizing HLA antigens and NK cell activating ligands indicates that the surface expression of these molecules is frequently altered on malignant cells. These changes appear to have clinical significance, since in some types of malignant disease they are associated with the histopathological characteristics of the lesions as well as with disease free interval and survival. These associations have been suggested to reflect the effect of HLA antigen and NK cell activating ligand abnormalities on the interactions of tumor cells with antigen-specific cytotoxic T lymphocytes (CTL) and with NK cells. Nevertheless, there are examples in which disease progresses in the face of appropriate HLA antigen and/or NK cell activating ligand as well as tumor antigen expression by malignant cells and of functional antigen-specific CTL in the investigated patient. In such scenarios, it is likely that the tumor microenvironment is unfavorable for CTL and NK cell activity and contributes to tumor immune escape. Many distinct escape mechanisms have been shown to protect malignant cells from immune recognition and destruction in the tumor microenvironment. In this paper, following the description of the structural and functional characteristics of soluble HLA antigens and NK cell activating ligands, we will review changes in their serum level in malignant disease and discuss their potential role in the escape mechanisms utilized by tumor cells to avoid recognition and destruction. PMID:18700879

  20. Quantitative control of active targeting of nanocarriers to tumor cells through optimization of folate ligand density.

    PubMed

    Tang, Zhaomin; Li, Dan; Sun, Huili; Guo, Xing; Chen, Yuping; Zhou, Shaobing

    2014-09-01

    The active targeting delivery system has been widely studied in cancer therapy by utilizing folate (FA) ligands to generate specific interaction between nanocarriers and folate receptors (FRs) on tumor cell. However, there is little work that has been published to investigate the influence of the definite density of the FA ligands on the active targeting of nanocarriers. In this study, we have combined magnetic-guided iron oxide nanoparticles with FA ligands, adjusted the FA ligand density and then studied the resulting effects on the active targeting ability of this dual-targeting drug delivery system to tumor cells. We have also optimized the FA ligand density of the drug delivery system for their active targeting to FR-overexpressing tumor cells in vitro. Prussian blue staining, semi-thin section of cells observed with transmission electron microscopy (TEM) and inductively coupled plasma-atomic emission spectroscopy (ICP-AES) have shown that the optimal FA density is from 2.3 × 10(18) to 2.5 × 10(18) per gram nanoparticles ((g·NPs)(-1)). We have further tried to qualitatively and quantitatively control the active targeting and delivering of drugs to tumors on 4T1-bearing BALB/c mice. As expected, the in vivo experimental results have also demonstrated that the FA density of the magnetic nanoparticles (MNPs) could be optimized for a more easily binding to tumor cells via the multivalent linkages and more readily internalization through the FR-mediated endocytosis. Our study can provide a strategy to quantitatively control the active targeting of nanocarriers to tumor cells for cancer therapy.

  1. Voltage clustering in redox-active ligand complexes: mitigating electronic communication through choice of metal ion

    SciTech Connect

    Zarkesh, Ryan A.; Ichimura, Andrew S.; Monson, Todd C.; Tomson, Neil C.; Anstey, Mitchell R.

    2016-02-01

    We used the redox-active bis(imino)acenapthene (BIAN) ligand to synthesize homoleptic aluminum, chromium, and gallium complexes of the general formula (BIAN)3M. The resulting compounds were characterized using X-ray crystallography, NMR, EPR, magnetic susceptibility and cyclic voltammetry measurements and modeled using both DFT and ab initio wavefunction calculations to compare the orbital contributions of main group elements and transition metals in ligand-based redox events. Ultimately, complexes of this type have the potential to improve the energy density and electrolyte stability of grid-scale energy storage technologies, such as redox flow batteries, through thermodynamically-clustered redox events.

  2. Voltage clustering in redox-active ligand complexes: mitigating electronic communication through choice of metal ion

    DOE PAGES

    Zarkesh, Ryan A.; Ichimura, Andrew S.; Monson, Todd C.; ...

    2016-02-01

    We used the redox-active bis(imino)acenapthene (BIAN) ligand to synthesize homoleptic aluminum, chromium, and gallium complexes of the general formula (BIAN)3M. The resulting compounds were characterized using X-ray crystallography, NMR, EPR, magnetic susceptibility and cyclic voltammetry measurements and modeled using both DFT and ab initio wavefunction calculations to compare the orbital contributions of main group elements and transition metals in ligand-based redox events. Ultimately, complexes of this type have the potential to improve the energy density and electrolyte stability of grid-scale energy storage technologies, such as redox flow batteries, through thermodynamically-clustered redox events.

  3. O2 activation by metal-ligand cooperation with Ir(I) PNP pincer complexes.

    PubMed

    Feller, Moran; Ben-Ari, Eyal; Diskin-Posner, Yael; Carmieli, Raanan; Weiner, Lev; Milstein, David

    2015-04-15

    A unique mode of molecular oxygen activation, involving metal-ligand cooperation, is described. Ir pincer complexes [((t)BuPNP)Ir(R)] (R = C6H5 (1), CH2COCH3 (2)) react with O2 to form the dearomatized hydroxo complexes [((t)BuPNP*)Ir(R)(OH)] ((t)BuPNP* = deprotonated (t)BuPNP ligand), in a process which utilizes both O-atoms. Experimental evidence, including NMR, EPR, and mass analyses, indicates a binuclear mechanism involving an O-atom transfer by a peroxo intermediate.

  4. Cognitive improvement by activation of alpha7 nicotinic acetylcholine receptors: from animal models to human pathophysiology.

    PubMed

    Thomsen, Morten S; Hansen, Henrik H; Timmerman, Daniel B; Mikkelsen, Jens D

    2010-01-01

    Agonists and positive allosteric modulators of the alpha(7) nicotinic acetylcholine receptor (nAChR) are currently being developed for the treatment of cognitive disturbances in patients with schizophrenia or Alzheimer's disease. This review describes the neurobiological properties of the alpha nAChR and the cognitive effects of alpha(7) nAChR activation, focusing on the translational aspects in the development of these drugs. The functional properties and anatomical localization of the alpha(7) nAChR makes it well suited to modulate cognitive function. Accordingly, systemic administration of alpha(7) nAChR agonists improves learning, memory, and attentional function in variety of animal models, and pro-cognitive effects of alpha(7) nAChR agonists have recently been demonstrated in patients with schizophrenia or Alzheimer's disease. The alpha(7) nAChR desensitizes rapidly in vitro, and this has been a major concern in the development of alpha(7) nAChR agonists as putative drugs. Our review of the existing literature shows that development of tolerance to the behavioral effects of alpha(7) nAChR agonists does not occur in animal models or humans. However, the long-term memory-enhancing effects seen in animal models are not mimicked in healthy humans and schizophrenic patients, where attentional improvement predominates. This discrepancy may result from inherent differences in testing methods or from species differences in the level of expression of alpha(7) nAChRs in limbic brain regions, and may hamper preclinical evaluation of alpha(7) nAChR activation. It is therefore important to consider the translational power of the animal models used before entering into a clinical evaluation of the pro-cognitive effects of alpha(7) nAChR activation.

  5. Tumor cell alpha-N-acetylgalactosaminidase activity and its involvement in GcMAF-related macrophage activation.

    PubMed

    Mohamad, Saharuddin B; Nagasawa, Hideko; Uto, Yoshihiro; Hori, Hitoshi

    2002-05-01

    Alpha-N-acetyl galactosaminidase (alpha-NaGalase) has been reported to accumulate in serum of cancer patients and be responsible for deglycosylation of Gc protein, which is a precursor of GcMAF-mediated macrophage activation cascade, finally leading to immunosuppression in advanced cancer patients. We studied the biochemical characterization of alpha-NaGalase from several human tumor cell lines. We also examined its effect on the potency of GcMAF to activate mouse peritoneal macrophage to produce superoxide in GcMAF-mediated macrophage activation cascade. The specific activity of alpha-NaGalases from human colon tumor cell line HCT116, human hepatoma cell line HepG2, and normal human liver cells (Chang liver cell line) were evaluated using two types of substrates; GalNAc-alpha-PNP (exo-type substrate) and Gal-beta-GalNAc-alpha-PNP (endo-type substrate). Tumor-derived alpha-NaGalase having higher activity than normal alpha-NaGalase, had higher substrate specificity to the exo-type substrate than to the endo-type substrate, and still maintained its activity at pH 7. GcMAF enhance superoxide production in mouse macrophage, and pre-treatment of GcMAF with tumor cell lysate reduce the activity. We conclude that tumor-derived alpha-NaGalase is different in biochemical characterization compared to normal alpha-NaGalase from normal Chang liver cells. In addition, tumor cell-derived alpha-NaGalase decreases the potency of GcMAF on macrophage activation.

  6. Binding of a soluble alpha beta T-cell receptor to superantigen/major histocompatibility complex ligands.

    PubMed Central

    Kappler, J; White, J; Kozono, H; Clements, J; Marrack, P

    1994-01-01

    The genes for the alpha and beta chains of a murine T-cell receptor were truncated just prior to the portions encoding the transmembrane regions and introduced into baculovirus by recombination. Insect cells infected with the virus secreted a soluble form of the receptor that could be purified to homogeneity. This soluble receptor reacted with a set of six monoclonal antibodies originally raised to different epitopes on the natural transmembrane-region-containing receptor and bound with appropriate specificity to a cell surface complex of the human major histocompatibility complex class II molecule DR1 with the bacterial superantigen staphylococcal enterotoxin B. Images PMID:8078904

  7. The first dipeptide ligand of translocator protein: Design and anxiolytic activity.

    PubMed

    Gudasheva, T A; Deeva, O A; Mokrov, G V; Yarkov, S A; Yarkova, M A; Seredenin, S B

    2015-01-01

    On the basis of the structure of Alpidem, a pyrazolopyrimidine ligand of the translocator protein (TSPO), a dipeptide TSPO ligand, N-carbobenzoxy-L-tryptophanyl-L-isoleucine amide (GD-23), was designed and synthesized using our own original peptide design strategy. This compound exhibited anxiolytic activity in BALB/cAnN mice in the "open-field" test and in outbred CD1 mice in the "elevated plus maze" test. The stereoselectivity of the anxiolytic effect of GD-23 is demonstrated. The results of this study suggest that GD-23 is a ligand of the translocator protein, and its structure can become the basis for creating anxiolytics with a fundamentally new mechanism of action.

  8. Biologically active monoiodinated alpha-MSH derivatives for receptor binding studies using human melanoma cells

    SciTech Connect

    Eberle, A.N.; Verin, V.J.; Solca, F.; Siegrist, W.; Kueenlin, C.B.; Bagutti, C.; Stutz, S.; Girard, J. , University Hospital, Basel )

    1991-01-01

    Three different monoiodinated radioligands of alpha-MSH (alpha-melanocyte-stimulating hormone) were compared in a binding assay with human D10 melanoma cells: (Tyr(125I)2)-alpha-MSH, (Tyr(125I)2,NIe4)-alpha-MSH, and (Tyr(125I)2,NIe4,D-Phe7)-alpha-MSH. They were prepared either by the classical chloramine T method or by the Enzymobead method. A simple and rapid purification scheme was developed consisting of a primary separation on reversed-phase C18 silica cartridges immediately after the iodination, followed by HPLC purification before each binding experiment. Biological testing of the three radioligands showed that they all retained high melanotropic activity in the B16 melanin assay and the Anolis melanophore assay. However, in human D10 melanoma cells, (Tyr(125I)2,NIe4)-alpha-MSH led to a high degree of non-specific binding to the cells which could not be displaced by excess alpha-MSH and only partially by (NIe4)-alpha-MSH. The (Tyr(125I)2,NIe4,D-Phe7)-alpha-MSH tracer gave similar results but with a much lower proportion of non-specific binding. On the other hand, (Tyr(125I)2)-alpha-MSH proved to be an excellent radioligand whose non-specific binding to the D10 cells was not higher than 20% of the total binding.

  9. Interaction of iodine with 2-hydroxypropyl-alpha-cyclodextrin and its bactericidal activity.

    PubMed

    Tomono, K; Goto, H; Suzuki, T; Ueda, H; Nagai, T; Watanabe, J

    2002-11-01

    To obtain an effective iodine solution, the use of 2-hydroxypropyl-alpha-cyclodextrin (2-HP-alpha-CD) as solubilizer was examined in comparison with alpha-cyclodextrin (alpha-CD), beta-cyclodextrin (beta-CD), potassium iodide (KI), and polyvinylpyrrolidone (PVP). The stability constants for inclusion of iodine with cyclodextrin and KI were ascertained by the solubility method. The apparent stability constants increased in the following order: KI < beta-CD < alpha-CD < 2-HP-alpha-CD. This order was nearly in accordance with that of the stabilization ability. The largest volatile depression effect was exhibited by 2HP-alpha-CD. The measurement of the minimum inhibitory concentration (MC) using Escherichia coli NIH-J-2 and Staphylococcus aureus FDA209P suggested that the bactericidal activity of the iodine/2-HP-alpha-CD system was the same as that of the iodine/alpha-CD, iodine/beta-CD, and iodine/PVP systems. The present results suggest that the combination of 2-HP-alpha-CD and iodine is useful for a stable and effective iodine solution.

  10. Hypoxia-inducible factor 1 mediates hypoxia-induced cardiomyocyte lipid accumulation by reducing the DNA binding activity of peroxisome proliferator-activated receptor {alpha}/retinoid X receptor

    SciTech Connect

    Belanger, Adam J.; Luo Zhengyu; Vincent, Karen A.; Akita, Geoffrey Y.; Cheng, Seng H.; Gregory, Richard J.; Jiang Canwen

    2007-12-21

    In response to cellular hypoxia, cardiomyocytes adapt to consume less oxygen by shifting ATP production from mitochondrial fatty acid {beta}-oxidation to glycolysis. The transcriptional activation of glucose transporters and glycolytic enzymes by hypoxia is mediated by hypoxia-inducible factor 1 (HIF-1). In this study, we examined whether HIF-1 was involved in the suppression of mitochondrial fatty acid {beta}-oxidation in hypoxic cardiomyocytes. We showed that either hypoxia or adenovirus-mediated expression of a constitutively stable hybrid form (HIF-1{alpha}/VP16) suppressed mitochondrial fatty acid metabolism, as indicated by an accumulation of intracellular neutral lipid. Both treatments also reduced the mRNA levels of muscle carnitine palmitoyltransferase I which catalyzes the rate-limiting step in the mitochondrial import of fatty acids for {beta}-oxidation. Furthermore, adenovirus-mediated expression of HIF-1{alpha}/VP16 in cardiomyocytes under normoxic conditions also mimicked the reduction in the DNA binding activity of peroxisome proliferator-activated receptor {alpha} (PPAR{alpha})/retinoid X receptor (RXR), in the presence or absence of a PPAR{alpha} ligand. These results suggest that HIF-1 may be involved in hypoxia-induced suppression of fatty acid metabolism in cardiomyocytes by reducing the DNA binding activity of PPAR{alpha}/RXR.

  11. Frontal Alpha EEG Asymmetry Before and After Behavioral Activation Treatment for Depression

    PubMed Central

    Gollan, Jackie K.; Hoxha, Denada; Chihade, Dietta; Pflieger, Mark E.; Rosebrock, Laina; Cacioppo, John

    2015-01-01

    Background Mid-frontal and mid-lateral (F3/F4 and F7/F8) EEG asymmetry has been associated with motivation and affect. We examined alpha EEG asymmetry in depressed and healthy participants before and after Behavioral Activation treatment for depression; examined the association between alpha EEG asymmetry and motivational systems and affect; and evaluated the utility of alpha EEG asymmetry in predicting remission. Methods Depressed (n = 37) and healthy participants (n = 35) were assessed before and after treatment using a clinical interview, a task to measure baseline EEG, and questionnaires of behavioral activation and inhibition, avoidance, and affect. Results Alpha EEG asymmetry was significantly higher in depressed than healthy participants at pre-treatment, positively correlated with negative affect and behavioral inhibition, and inversely correlated with lower behavioral activation sensitivity. Conclusions Heightened alpha EEG asymmetry in depressed participants was significantly associated with increased behavioral inhibition and negative emotion and was independent of clinical remission. PMID:24674708

  12. The anti-HIV activity of the phytochemical alpha-terthienyl.

    PubMed

    Hudson, J B; Harris, L; Teeple, A; Towers, G H

    1993-01-01

    The plant trithiophene, alpha-terthienyl (alpha T), was evaluated for activity against the human immunodeficiency virus (HIV-1). Antiviral activity specifically required long wavelength light (UVA, 320-400 nm). The compound had little or no activity in visible light or in the dark. The anti-HIV effect was UVA-dose dependent and was proportional to the concentration of alpha T, according to several parameters of virus infectivity and replication. The efficacy was decreased to some extent by the presence of bovine serum in the reactions; but under optimal conditions 0.1 microgram/ml. alpha T (3 x 10(-7) M) could inactivate 10(4)-10(5) infectious particles. In contrast poliovirus and Coxsackievirus infectivity were relatively resistant to alpha T + UVA.

  13. [Study of the effect of Pb2+ on alpha-amylase activity by spectroscopy].

    PubMed

    Hong, Fa-shui

    2003-06-01

    The activity of alpha-amylase from porcine pancreas was enhanced under the treatment by Pb2+ at low concentration (0.5-4 mumol.L-1), but was inhibited by Pb2+ at high concentration (above 4 mumol.L-1). Pb2+ at high concentration could competitively displace Ca2+ from alpha-amylase. The EXAFS demonstrated that Pb2+ was bound to the active site of alpha-amylase, the coordination atom was oxygen, the coordination number was 2, and the Pb-O bond length was 0.234 nm. Circular dichroism spectra showed that the secondary structure of trypsin was greatly changed by Pb2+ at high concentration, as alpha-helix, beta-turn and random coil contents decreased, while beta-sheet, aromatic and disulfide bond contents increased. It was suggested that Pb2+ was bound to result in an alpha-amylase conformational change, and the enzyme activity decreased.

  14. Molecular Dynamics Simulations Reveal the Mechanisms of Allosteric Activation of Hsp90 by Designed Ligands

    NASA Astrophysics Data System (ADS)

    Vettoretti, Gerolamo; Moroni, Elisabetta; Sattin, Sara; Tao, Jiahui; Agard, David A.; Bernardi, Anna; Colombo, Giorgio

    2016-04-01

    Controlling biochemical pathways through chemically designed modulators may provide novel opportunities to develop therapeutic drugs and chemical tools. The underlying challenge is to design new molecular entities able to act as allosteric chemical switches that selectively turn on/off functions by modulating the conformational dynamics of their target protein. We examine the origins of the stimulation of ATPase and closure kinetics in the molecular chaperone Hsp90 by allosteric modulators through atomistic molecular dynamics (MD) simulations and analysis of protein-ligand interactions. In particular, we focus on the cross-talk between allosteric ligands and protein conformations and its effect on the dynamic properties of the chaperone’s active state. We examine the impact of different allosteric modulators on the stability, structural and internal dynamics properties of Hsp90 closed state. A critical aspect of this study is the development of a quantitative model that correlates Hsp90 activation to the presence of a certain compound, making use of information on the dynamic adaptation of protein conformations to the presence of the ligand, which allows to capture conformational states relevant in the activation process. We discuss the implications of considering the conformational dialogue between allosteric ligands and protein conformations for the design of new functional modulators.

  15. Molecular Dynamics Simulations Reveal the Mechanisms of Allosteric Activation of Hsp90 by Designed Ligands

    PubMed Central

    Vettoretti, Gerolamo; Moroni, Elisabetta; Sattin, Sara; Tao, Jiahui; Agard, David A.; Bernardi, Anna; Colombo, Giorgio

    2016-01-01

    Controlling biochemical pathways through chemically designed modulators may provide novel opportunities to develop therapeutic drugs and chemical tools. The underlying challenge is to design new molecular entities able to act as allosteric chemical switches that selectively turn on/off functions by modulating the conformational dynamics of their target protein. We examine the origins of the stimulation of ATPase and closure kinetics in the molecular chaperone Hsp90 by allosteric modulators through atomistic molecular dynamics (MD) simulations and analysis of protein-ligand interactions. In particular, we focus on the cross-talk between allosteric ligands and protein conformations and its effect on the dynamic properties of the chaperone’s active state. We examine the impact of different allosteric modulators on the stability, structural and internal dynamics properties of Hsp90 closed state. A critical aspect of this study is the development of a quantitative model that correlates Hsp90 activation to the presence of a certain compound, making use of information on the dynamic adaptation of protein conformations to the presence of the ligand, which allows to capture conformational states relevant in the activation process. We discuss the implications of considering the conformational dialogue between allosteric ligands and protein conformations for the design of new functional modulators. PMID:27032695

  16. Structural basis for PPAR partial or full activation revealed by a novel ligand binding mode

    PubMed Central

    Capelli, Davide; Cerchia, Carmen; Montanari, Roberta; Loiodice, Fulvio; Tortorella, Paolo; Laghezza, Antonio; Cervoni, Laura; Pochetti, Giorgio; Lavecchia, Antonio

    2016-01-01

    The peroxisome proliferator-activated receptors (PPARs) are nuclear receptors involved in the regulation of the metabolic homeostasis and therefore represent valuable therapeutic targets for the treatment of metabolic diseases. The development of more balanced drugs interacting with PPARs, devoid of the side-effects showed by the currently marketed PPARγ full agonists, is considered the major challenge for the pharmaceutical companies. Here we present a structure-based virtual screening approach that let us identify a novel PPAR pan-agonist with a very attractive activity profile and its crystal structure in the complex with PPARα and PPARγ, respectively. In PPARα this ligand occupies a new pocket whose filling is allowed by the ligand-induced switching of the F273 side chain from a closed to an open conformation. The comparison between this pocket and the corresponding cavity in PPARγ provides a rationale for the different activation of the ligand towards PPARα and PPARγ, suggesting a novel basis for ligand design. PMID:27708429

  17. Acetylation of pregnane X receptor protein determines selective function independent of ligand activation

    SciTech Connect

    Biswas, Arunima; Pasquel, Danielle; Tyagi, Rakesh Kumar; Mani, Sridhar

    2011-03-18

    Research highlights: {yields} Pregnane X receptor (PXR), a major regulatory protein, is modified by acetylation. {yields} PXR undergoes dynamic deacetylation upon ligand-mediated activation. {yields} SIRT1 partially mediates PXR deacetylation. {yields} PXR deacetylation per se induces lipogenesis mimicking ligand-mediated activation. -- Abstract: Pregnane X receptor (PXR), like other members of its class of nuclear receptors, undergoes post-translational modification [PTM] (e.g., phosphorylation). However, it is unknown if acetylation (a major and common form of protein PTM) is observed on PXR and, if it is, whether it is of functional consequence. PXR has recently emerged as an important regulatory protein with multiple ligand-dependent functions. In the present work we show that PXR is indeed acetylated in vivo. SIRT1 (Sirtuin 1), a NAD-dependent class III histone deacetylase and a member of the sirtuin family of proteins, partially mediates deacetylation of PXR. Most importantly, the acetylation status of PXR regulates its selective function independent of ligand activation.

  18. TRIM5{alpha} association with cytoplasmic bodies is not required for antiretroviral activity

    SciTech Connect

    Song, Byeongwoon; Diaz-Griffero, Felipe; Park, Do Hyun; Rogers, Thomas; Stremlau, Matthew; Sodroski, Joseph . E-mail: joseph_sodroski@dfci.harvard.edu

    2005-12-20

    The tripartite motif (TRIM) protein, TRIM5{alpha}, restricts infection by particular retroviruses. Many TRIM proteins form cytoplasmic bodies of unknown function. We investigated the relationship between cytoplasmic body formation and the structure and antiretroviral activity of TRIM5{alpha}. In addition to diffuse cytoplasmic staining, the TRIM5{alpha} proteins from several primate species were located in cytoplasmic bodies of different sizes; by contrast, TRIM5{alpha} from spider monkeys did not form cytoplasmic bodies. Despite these differences, all of the TRIM5{alpha} proteins exhibited the ability to restrict infection by particular retroviruses. Treatment of cells with geldanamycin, an Hsp90 inhibitor, resulted in disappearance or reduction of the TRIM5{alpha}-associated cytoplasmic bodies, yet exerted little effect on the restriction of retroviral infection. Studies of green fluorescent protein-TRIM5{alpha} fusion proteins indicated that no TRIM5{alpha} domain is specifically required for association with cytoplasmic bodies. Apparently, the formation of cytoplasmic bodies is not required for the antiretroviral activity of TRIM5{alpha}.

  19. Direct activation of Ca2+ channels by palmitoyl carnitine, a putative endogenous ligand.

    PubMed Central

    Spedding, M.; Mir, A. K.

    1987-01-01

    degrees C (42 +/- 5 mumol l-1). Palmitoyl carnitine interacted selectively with the Ca2+ channel, in that effects on ligand binding to alpha-adrenoceptors, beta-adrenoceptors and 5-HT1A receptors occurred only at 5-10 fold higher concentrations. 5 It is concluded that palmitoyl carnitine, at concentrations which have previously been shown to occur in the cytoplasm during myocardial ischaemia, may interact directly with Ca2+ channels and may therefore be considered as an endogenous modulator of channel function. The site of action differs from that of other agents. PMID:2445406

  20. Anticancer Activity and Modes of Action of (arene) ruthenium(II) Complexes Coordinated to C-, N-, and O-ligands.

    PubMed

    Biersack, Bernhard

    2016-01-01

    An overview of anticancer active (arene)ruthenium(II) complexes coordinated to period 2 element-based ligand systems, i.e., carbon-, nitrogen-, and oxygen-coordinated ligands, is provided in this mini-review. A bridge is forged from the large group of anticancer active ruthenium compounds with monodentate and chelating nitrogen ligands via complexes of O,O-chelating ligands to organometallic ruthenium derivatives coordinated to carbon. (Arene)ruthenium(II) complexes with reduced side-effects and enhanced efficacy against cancer are highlighted. Pertinent literature is covered up to 2014.

  1. Cyanide does more to inhibit heme enzymes, than merely serving as an active-site ligand

    SciTech Connect

    Parashar, Abhinav; Venkatachalam, Avanthika; Gideon, Daniel Andrew; Manoj, Kelath Murali

    2014-12-12

    Highlights: • Cyanide (CN) is a well-studied toxic principle, known to inhibit heme-enzymes. • Inhibition is supposed to result from CN binding at the active site as a ligand. • Diverse heme enzymes’ CN inhibition profiles challenge prevailing mechanism. • Poor binding efficiency of CN at low enzyme concentrations and ligand pressures. • CN-based diffusible radicals cause ‘non-productive electron transfers’ (inhibition). - Abstract: The toxicity of cyanide is hitherto attributed to its ability to bind to heme proteins’ active site and thereby inhibit their activity. It is shown herein that the long-held interpretation is inadequate to explain several observations in heme-enzyme reaction systems. Generation of cyanide-based diffusible radicals in heme-enzyme reaction milieu could shunt electron transfers (by non-active site processes), and thus be detrimental to the efficiency of oxidative outcomes.

  2. Structural Differences between Active Forms of Plasminogen Activator Inhibitor Type 1 Revealed by Conformationally Sensitive Ligands*

    PubMed Central

    Li, Shih-Hon; Gorlatova, Natalia V.; Lawrence, Daniel A.; Schwartz, Bradford S.

    2008-01-01

    Plasminogen activator inhibitor type 1 (PAI-1) is a serine protease inhibitor (serpin) in which the reactive center loop (RCL) spontaneously inserts into a central β-sheet, β-sheet A, resulting in inactive inhibitor. Available x-ray crystallographic studies of PAI-1 in an active conformation relied on the use of stabilizing mutations. Recently it has become evident that these structural models do not adequately explain the behavior of wild-type PAI-1 (wtPAI-1) in solution. To probe the structure of native wtPAI-1, we used three conformationally sensitive ligands: the physiologic cofactor, vitronectin; a monoclonal antibody, 33B8, that binds preferentially to RCL-inserted forms of PAI-1; and RCL-mimicking peptides that insert into β-sheet A. From patterns of interaction with wtPAI-1 and the stable mutant, 14-1B, we propose a model of the native conformation of wtPAI-1 in which the bottom of the central sheet is closed, whereas the top of the β-sheet A is open to allow partial insertion of the RCL. Because the incorporation of RCL-mimicking peptides into wtPAI-1 is accelerated by vitronectin, we further propose that vitronectin alters the conformation of the RCL to allow increased accessibility to β-sheet A, yielding a structural hypothesis that is contradictory to the current structural model of PAI-1 in solution and its interaction with vitronectin. PMID:18436534

  3. Ligand-dependent intersubunit association with the insulin receptor complex activates its intrinsic kinase activity

    SciTech Connect

    Boeni-Schnetzler, M.; Kaligian, A.; DelVecchio, R.; Pilch, P.F.

    1988-05-15

    Insulin receptor halves (..cap alpha beta..) were obtained upon selective reduction of the holoreceptor (..cap alpha../sub 2/..beta../sub 2/) and were isolated in concentrated form. Autophosphorylation of concentrated ..cap alpha beta.. receptor halves can be stimulated by insulin an average of 4.0-fold, whereas nonreduced holoreceptor can be stimulated 5.4-fold. If ..cap alpha beta.. half-receptors are immobilized on wheat germ agglutinin-agarose, no insulin-stimulated autophosphorylation is observed, whereas immobilized holoreceptor retains insulin responsiveness. Treatment of ..cap alpha beta.. half-receptors with glutathione in the presence of insulin results in reoxidation to the holoreceptor form (..cap alpha../sub 2/..beta../sub 2/) with an efficiency of 60-70% as visualized by immunoblotting, thus providing evidence that two ..cap alpha beta.. halves are in close physical proximity. This reoxidation reaction, which is evident prior to autophosphorylation, is rapid and strictly dependent on the presence of insulin, consistent with the hypothesis that insulin promotes the association of two ..cap alpha beta.. halves. Furthermore, the insulin-induced reoxidation reaction and the insulin-induced autophosphorylation show the same dose dependence suggesting that the noncovalent association of ..cap alpha beta.. half-receptors upon insulin binding is a prerequisite for insulin-stimulated autophosphorylation in concentrated ..gamma beta.. half-receptor preparations. If the ..cap alpha beta.. half-receptor forms are phosphorylated in the presence of an anti-phosphotyrosine antibody and separated from nonphosphorylated ..cap alpha beta.. receptors, we observe that the phosphorylated ..cap alpha beta.. receptor halves contain bound insulin.

  4. Immune Activation Resulting from NKG2D/Ligand Interaction Promotes Atherosclerosis

    PubMed Central

    Xia, Mingcan; Guerra, Nadia; Sukhova, Galina K.; Yang, Kangkang; Miller, Carla K.; Shi, Guo-Ping; Raulet, David H.; Xiong, Na

    2012-01-01

    Background The interplay between the immune system and abnormal metabolic conditions sustains and propagates a vicious feedback cycle of chronic inflammation and metabolic dysfunction that is critical for atherosclerotic progression. It is well established that abnormal metabolic conditions, such as dyslipidemia and hyperglycemia, cause various cellular stress responses that induce tissue inflammation and immune cell activation, which in turn exacerbate the metabolic dysfunction. However, molecular events linking these processes are not well understood. Methods and Results Tissues and organs of humans and mice with hyperglycemia and hyperlipidemia were examined for expression of ligands for NKG2D, a potent immune activating receptor expressed by several types of immune cells, and the role of NKG2D in atherosclerosis and metabolic diseases was probed using mice lacking NKG2D or by blocking NKG2D with monoclonal antibodies. NKG2D ligands were upregulated in multiple organs, particularly atherosclerotic aortae and inflamed livers. Ligand upregulation was induced in vitro by abnormal metabolites associated with metabolic dysfunctions. Using ApoE-/- mouse models we demonstrated that preventing NKG2D functions resulted in a dramatic reduction in plaque formation, suppressed systemic and organ inflammation mediated by multiple immune cell types, and alleviated abnormal metabolic conditions. Conclusions The NKG2D/ligand interaction is a critical molecular link in the vicious cycle of chronic inflammation and metabolic dysfunction that promotes atherosclerosis and might be a useful target for therapeutic intervention in the disease. PMID:22104546

  5. Syntheses, characterization, biological activities and photophysical properties of lanthanides complexes with a tetradentate Schiff base ligand

    NASA Astrophysics Data System (ADS)

    Taha, Ziyad A.; Ajlouni, Abdulaziz M.; Al Momani, Waleed; Al-Ghzawi, Abeer A.

    2011-10-01

    A tetradentate Schiff base ligand L (N,N'-bis(1-naphthaldimine)-o-phenylenediamine) was prepared from the condensation of 2-hydroxy-1-naphthaldehyde with o-phenylenediamine in a molar ratio of 2:1. New eight lanthanide metal complexes [Ln L(NO 3) 2(H 2O) x](NO 3) {Ln(III) = Nd, Dy, Sm, Pr, Gd, Tb, La and Er, x = 0 for Nd, Sm, 1 for La, Gd, Pr, Nd, Dy, and 2 for Tb} were prepared. The characterization and nature of bonding of these complexes were elucidated by elemental analysis, spectral analysis ( 1H NMR, FT-IR, UV-vis), molar conductivity measurements, luminescence spectra and thermogravimetric studies. Analytical and spectral data revealed that the ligand L coordinates to the central Ln(III) ions by its two imine nitrogen atoms and two phenolic oxygen atoms with 1:1 stoichiometry. Under the excitation with 329 nm at room temperature, Tb and Dy complexes exhibited characteristic luminescence of the central metal ions attributed to efficient energy transfer from the ligand to the metal center. Most of Ln(III) complexes found to exhibit antibacterial activities against a number of pathogenic bacteria. We found that the antioxident activity of Ln(III) complexes on DPPH rad is concentration dependent and higher than that of the free ligand L.

  6. EEG Alpha and Beta Activity in Normal and Deaf Subjects.

    ERIC Educational Resources Information Center

    Waldron, Manjula; And Others

    Electroencephalogram and task performance data were collected from three groups of young adult males: profoundly deaf Ss who signed from an early age, profoundly deaf Ss who only used oral (speech and speedreading) methods of communication, and normal hearing Ss. Alpha and Beta brain wave patterns over the Wernicke's area were compared across…

  7. The alpha1-fetoprotein locus is activated by a nuclear receptor of the Drosophila FTZ-F1 family.

    PubMed

    Galarneau, L; Paré, J F; Allard, D; Hamel, D; Levesque, L; Tugwood, J D; Green, S; Bélanger, L

    1996-07-01

    The alpha1-fetoprotein (AFP) gene is located between the albumin and alpha-albumin genes and is activated by transcription factor FTF (fetoprotein transcription factor), presumed to transduce early developmental signals to the albumin gene cluster. We have identified FTF as an orphan nuclear receptor of the Drosophila FTZ-F1 family. FTF recognizes the DNA sequence 5'-TCAAGGTCA-3', the canonical recognition motif for FTZ-F1 receptors. cDNA sequence homologies indicate that rat FTF is the ortholog of mouse LRH-1 and Xenopus xFF1rA. Rodent FTF is encoded by a single-copy gene, related to the gene encoding steroidogenic factor 1 (SF-1). The 5.2-kb FTF transcript is translated from several in-frame initiator codons into FTF isoforms (54 to 64 kDa) which appear to bind DNA as monomers, with no need for a specific ligand, similar KdS (approximately equal 3 x 10(-10) M), and similar transcriptional effects. FTF activates the AFP promoter without the use of an amino-terminal activation domain; carboxy-terminus-truncated FTF exerts strong dominant negative effects. In the AFP promoter, FTF recruits an accessory trans-activator which imparts glucocorticoid reactivity upon the AFP gene. FTF binding sites are found in the promoters of other liver-expressed genes, some encoding liver transcription factors; FTF, liver alpha1-antitrypsin promoter factor LFB2, and HNF-3beta promoter factor UF2-H3beta are probably the same factor. FTF is also abundantly expressed in the pancreas and may exert differentiation functions in endodermal sublineages, similar to SF-1 in steroidogenic tissues. HepG2 hepatoma cells seem to express a mutated form of FTF.

  8. The alpha1-fetoprotein locus is activated by a nuclear receptor of the Drosophila FTZ-F1 family.

    PubMed Central

    Galarneau, L; Paré, J F; Allard, D; Hamel, D; Levesque, L; Tugwood, J D; Green, S; Bélanger, L

    1996-01-01

    The alpha1-fetoprotein (AFP) gene is located between the albumin and alpha-albumin genes and is activated by transcription factor FTF (fetoprotein transcription factor), presumed to transduce early developmental signals to the albumin gene cluster. We have identified FTF as an orphan nuclear receptor of the Drosophila FTZ-F1 family. FTF recognizes the DNA sequence 5'-TCAAGGTCA-3', the canonical recognition motif for FTZ-F1 receptors. cDNA sequence homologies indicate that rat FTF is the ortholog of mouse LRH-1 and Xenopus xFF1rA. Rodent FTF is encoded by a single-copy gene, related to the gene encoding steroidogenic factor 1 (SF-1). The 5.2-kb FTF transcript is translated from several in-frame initiator codons into FTF isoforms (54 to 64 kDa) which appear to bind DNA as monomers, with no need for a specific ligand, similar KdS (approximately equal 3 x 10(-10) M), and similar transcriptional effects. FTF activates the AFP promoter without the use of an amino-terminal activation domain; carboxy-terminus-truncated FTF exerts strong dominant negative effects. In the AFP promoter, FTF recruits an accessory trans-activator which imparts glucocorticoid reactivity upon the AFP gene. FTF binding sites are found in the promoters of other liver-expressed genes, some encoding liver transcription factors; FTF, liver alpha1-antitrypsin promoter factor LFB2, and HNF-3beta promoter factor UF2-H3beta are probably the same factor. FTF is also abundantly expressed in the pancreas and may exert differentiation functions in endodermal sublineages, similar to SF-1 in steroidogenic tissues. HepG2 hepatoma cells seem to express a mutated form of FTF. PMID:8668203

  9. Alpha 2-adrenergic receptors influence tyrosine hydroxylase activity in retinal dopamine neurons.

    PubMed

    Iuvone, P M; Rauch, A L

    1983-12-12

    Dopamine (DA) is a putative neurotransmitter in a population of interneurons in the mammalian retina that are activated by photic stimulation. Pharmacological studies were conducted to determine if alpha 2-adrenergic receptors influence the activity of retinal tyrosine hydroxylase (TH), a biochemical indicator of changes in the activity of the DA-containing neurons. TH activity was low in dark-adapted retinas and high in light-exposed retinas. Systemic administration of the alpha 2-adrenoceptor antagonists, yohimbine and piperoxane, to dark-adapted rats significantly stimulated TH activity. This effect was apparently mediated locally within the retina because the response could also be elicited by direct injection of yohimbine into the vitreous. The dose-response relationships for the effects of alpha 2-adrenoceptor antagonists on retinal TH activity were similar to those for the effects on brain noradrenergic neurons, where alpha 2-adrenoceptors have been shown to be involved in the autoregulation of neuronal activity. Clonidine, an alpha 2-adrenoceptor agonist, had no effect when administered alone to dark-adapted rats, but it attenuated the stimulatory effect of yohimbine. In contrast, clonidine decreased TH activity of light-exposed retinas, an effect that was reversed by yohimbine. These observations suggest that alpha 2-adrenoceptors influence the activity of retinal DA-containing neurons.

  10. Evidence for a Dual Role of an Active Site Histidine in [alpha]-Amino-[beta]-carboxymuconate-[epsilon]-semialdehyde Decarboxylase

    SciTech Connect

    Huo, Lu; Fielding, Andrew J.; Chen, Yan; Li, Tingfeng; Iwaki, Hiroaki; Hosler, Jonathan P.; Chen, Lirong; Hasegawa, Yoshie; Que, Jr., Lawrence; Liu, Aimin

    2012-10-09

    The previously reported crystal structures of {alpha}-amino-{beta}-carboxymuconate-{epsilon}-semialdehyde decarboxylase (ACMSD) show a five-coordinate Zn(II)(His){sub 3}(Asp)(OH{sub 2}) active site. The water ligand is H-bonded to a conserved His228 residue adjacent to the metal center in ACMSD from Pseudomonas fluorescens (PfACMSD). Site-directed mutagenesis of His228 to tyrosine and glycine in this study results in a complete or significant loss of activity. Metal analysis shows that H228Y and H228G contain iron rather than zinc, indicating that this residue plays a role in the metal selectivity of the protein. As-isolated H228Y displays a blue color, which is not seen in wild-type ACMSD. Quinone staining and resonance Raman analyses indicate that the blue color originates from Fe(III)-tyrosinate ligand-to-metal charge transfer. Co(II)-substituted H228Y ACMSD is brown in color and exhibits an electron paramagnetic resonance spectrum showing a high-spin Co(II) center with a well-resolved {sup 59}Co (I = 7/2) eight-line hyperfine splitting pattern. The X-ray crystal structures of as-isolated Fe-H228Y (2.8 {angstrom}) and Co-substituted (2.4 {angstrom}) and Zn-substituted H228Y (2.0 {angstrom} resolution) support the spectroscopic assignment of metal ligation of the Tyr228 residue. The crystal structure of Zn-H228G (2.6 {angstrom}) was also determined. These four structures show that the water ligand present in WT Zn-ACMSD is either missing (Fe-H228Y, Co-H228Y, and Zn-H228G) or disrupted (Zn-H228Y) in response to the His228 mutation. Together, these results highlight the importance of His228 for PfACMSD's metal specificity as well as maintaining a water molecule as a ligand of the metal center. His228 is thus proposed to play a role in activating the metal-bound water ligand for subsequent nucleophilic attack on the substrate.

  11. Ligand-binding domains of nuclear receptors facilitate tight control of split CRISPR activity

    PubMed Central

    Nguyen, Duy P.; Miyaoka, Yuichiro; Gilbert, Luke A.; Mayerl, Steven J.; Lee, Brian H.; Weissman, Jonathan S.; Conklin, Bruce R.; Wells, James A.

    2016-01-01

    Cas9-based RNA-guided nuclease (RGN) has emerged to be a versatile method for genome editing due to the ease of construction of RGN reagents to target specific genomic sequences. The ability to control the activity of Cas9 with a high temporal resolution will facilitate tight regulation of genome editing processes for studying the dynamics of transcriptional regulation or epigenetic modifications in complex biological systems. Here we show that fusing ligand-binding domains of nuclear receptors to split Cas9 protein fragments can provide chemical control over split Cas9 activity. The method has allowed us to control Cas9 activity in a tunable manner with no significant background, which has been challenging for other inducible Cas9 constructs. We anticipate that our design will provide opportunities through the use of different ligand-binding domains to enable multiplexed genome regulation of endogenous genes in distinct loci through simultaneous chemical regulation of orthogonal Cas9 variants. PMID:27363581

  12. The disintegrin/metalloprotease ADAM 10 is essential for Notch signalling but not for alpha-secretase activity in fibroblasts.

    PubMed

    Hartmann, Dieter; de Strooper, Bart; Serneels, Lutgarde; Craessaerts, Katleen; Herreman, An; Annaert, Wim; Umans, Lieve; Lübke, Torben; Lena Illert, Anna; von Figura, Kurt; Saftig, Paul

    2002-10-01

    The metalloprotease ADAM 10 is an important APP alpha-secretase candidate, but in vivo proof of this is lacking. Furthermore, invertebrate models point towards a key role of the ADAM 10 orthologues Kuzbanian and sup-17 in Notch signalling. In the mouse, this function is, however, currently attributed to ADAM 17/TACE, while the role of ADAM 10 remains unknown. We have created ADAM 10-deficient mice. They die at day 9.5 of embryogenesis with multiple defects of the developing central nervous system, somites, and cardiovascular system. In situ hybridization revealed a reduced expression of the Notch target gene hes-5 in the neural tube and an increased expression of the Notch ligand dll-1, supporting an important role for ADAM 10 in Notch signalling in the vertebrates as well. Since the early lethality precluded the establishment of primary neuronal cultures, APPs alpha generation was analyzed in embryonic fibroblasts and found to be preserved in 15 out of 17 independently generated ADAM 10-deficient fibroblast cell lines, albeit at a quantitatively more variable level than in controls, whereas a severe reduction was found in only two cases. The variability was not due to differences in genetic background or to variable expression of the alternative alpha-secretase candidates ADAM 9 and ADAM 17. These results indicate, therefore, either a regulation between ADAMs on the post-translational level or that other, not yet known, proteases are able to compensate for ADAM 10 deficiency. Thus, the observed variability, together with recent reports on tissue-specific expression patterns of ADAMs 9, 10 and 17, points to the existence of tissue-specific 'teams' of different proteases exerting alpha-secretase activity.

  13. T cell selection and differential activation on structurally related HLA-DR4 ligands.

    PubMed

    Gebe, J A; Novak, E J; Kwok, W W; Farr, A G; Nepom, G T; Buckner, J H

    2001-09-15

    Plasticity of TCR interactions during CD4(+) T cell activation by an MHC-peptide complex accommodates variation in the peptide or MHC contact sites in which recognition of an altered ligand by the T cell can modify the T cell response. To explore the contribution of this form of TCR cross-recognition in the context of T cell selection on disease-associated HLA molecules, we have analyzed the relationship between TCR recognition of the DRB1*0401- and DRB1*0404-encoded HLA class II molecules associated with rheumatoid arthritis. Thymic reaggregation cultures demonstrated that CD4(+) T cells selected on either DRB1*0401 or DRB1*0404 could be subsequently activated by the other MHC molecule. Using HLA tetramer technology we identify hemagglutinin residue 307-319-specific T cells restricted by DRB1*0401, but activated by hemagglutinin residues 307-319, in the context of DRB1*0404. One such clone exhibits an altered cytokine profile upon activation with the alternative MHC ligand. This altered phenotype persists when both class II molecules are present. These findings directly demonstrate that T cells selected on an MHC class II molecule carry the potential for activation on altered self ligands when encountering Ags presented on a related class II molecule. In individuals heterozygous for these alleles the possibility of TCR cross-recognition could lead to an aberrant immune response.

  14. Integrin beta3 regions controlling binding of murine mAb 7E3: implications for the mechanism of integrin alphaIIbbeta3 activation.

    PubMed

    Artoni, Andrea; Li, JiHong; Mitchell, Beau; Ruan, Jian; Takagi, Junichi; Springer, Timothy A; French, Deborah L; Coller, Barry S

    2004-09-07

    Abciximab, a derivative of the murine mAb 7E3, protects against ischemic complications of percutaneous coronary interventions by inhibiting ligand binding to the alphaIIbbeta3 receptor. In this study we identified regions on integrin beta3 that control 7E3 binding. Murine/human amino acid substitutions were created in two regions of the betaA domain that previous studies found to influence 7E3 binding: the C177-C184 loop and K125-N133. The T182N substitution and a K125Q mutation reduced 7E3 binding to human beta3 in complex with alphaIIb. The introduction of both the human C177-C184 region and human W129 into murine beta3 was necessary and sufficient to permit 7E3 binding to the human alphaIIb/murine beta3 complex. Although we cannot exclude allosteric effects, we propose that 7E3 binds between C177-C184 and W129, which are within 15 A of each other in the crystal structure and close to the beta3 metal ion-dependent adhesion site. We previously demonstrated that 7E3 binds more rapidly to activated than unactivated platelets. Because it has been proposed that alphaIIbbeta3 changes from a bent to an extended conformation upon activation, we hypothesized that 7E3 binds less well to the bent than the extended conformation. In support of this hypothesis we found that 7E3 bound less well to an alphaIIbbeta3 construct locked in a bent conformation, and unlocking the conformation restored 7E3 binding. Thus, our data are consistent with alphaIIbbeta3 existing in variably bent conformations in equilibrium with each other on unactivated platelets, and activation resulting in alphaIIbbeta3 adopting a more extended conformation.

  15. Alpha-interferon suppresses food intake and neuronal activity of the lateral hypothalamus.

    PubMed

    Reyes-Vázquez, C; Prieto-Gómez, B; Dafny, N

    1994-12-01

    Alpha-interferon (alpha-IFN) treatment in humans induces anorexic effects. However, the mechanisms and sites of action are unknown. Rats implanted with an intracerebroventricular (i.c.v.) cannula for local injection, and semi-microelectrodes in the lateral hypothalamic (LH) area for neuronal recording were used. The animals were kept in metabolic cages, and food and water intake was measured daily at 7:00 and 19:00 hr for 35 days, including: 5 days before the experiment; 10 days during daily alpha-IFN application (either i.p. 1500 I.U./gbw, or i.c.v. 1500 and 150 I.U./animal) and/or a vehicle control group; and 20 days post drug treatment. The unitary activity recording from the LH area was made before (30 min), during (10 min) and after (200 min) the alpha-IFN applications. alpha-IFN elicited a reversible dose-related decrease of both food intake and body weight. This decrease in food intake following alpha-IFN injections was correlated with a depression of LH neuronal electrical activity. Since direct brain application (i.c.v.) and systemic (i.p.) alpha-IFN treatment elicited identical responses, it is possible to assume that alpha-IFN suppresses food intake by a direct action on CNS sites including the LH neurons.

  16. AMPK activation regulates apoptosis, adipogenesis, and lipolysis by eIF2{alpha} in adipocytes

    SciTech Connect

    Dagon, Yossi; Avraham, Yosefa; Berry, Elliot M. . E-mail: Berry@md.huji.ac.il

    2006-02-03

    AMP-activated protein kinase (AMPK) is a metabolic master switch regulating glucose and lipid metabolism. Recently, AMPK has been implicated in the control of adipose tissue content. Yet, the nature of this action is controversial. We examined the effect on F442a adipocytes of the AMPK activator-AICAR. Activation of AMPK induced dose-dependent apoptotic cell death, inhibition of lipolysis, and downregulatation key adipogenic genes, such as peroxisome proliferator-activated receptor (PPAR{gamma}) and CCAAT/enhancer-binding protein alpha (C/EBP{alpha}). We have identified the {alpha}-subunit of the eukaryotic initiation factor-2 (eIF2{alpha}) as a target gene which is phosphorylated following AICAR treatment. Such phosphorylation is one of the best-characterized mechanisms for downregulating protein synthesis. 2-Aminopurine (2-AP), an inhibitor of eIF2{alpha} kinases, could overcome the apoptotic effect of AICAR, abolishing the reduction of PPAR{gamma} and C/EBP{alpha} and the lipolytic properties of AMPK. Thus, AMPK may diminish adiposity via reduction of fat cell number through eIF2{alpha}-dependent translation shutdown.

  17. Pravastatin activates activator protein 2 alpha to argument the angiotensin II-induced abdominal aortic aneurysms.

    PubMed

    Ma, Hui; Liang, Wen-Jing; Shan, Mei-Rong; Wang, Xue-Qing; Zhou, Sheng-Nan; Chen, Yuan; Guo, Tao; Li, Peng; Yu, Hai-Ya; Liu, Chao; Yin, Ya-Ling; Wang, Yu-Lin; Dong, Bo; Pang, Xin-Yan; Wang, Shuang-Xi

    2017-02-04

    We have previously reported that activation of AMP-activated kinase alpha 2 (AMPKα2) by nicotine or angiotensin II (AngII) instigates formation of abdominal aortic aneurysms (AAA) in Apoe-/- mice. Statins, used to treat hyperlipidemia widely, activate AMPK in vascular cells. We sought to examine the effects of pravastatin on AAA formation and uncover the molecular mechanism. The AAA model was induced by AngII and evaluated by incidence, elastin degradation, and maximal abdominal aortic diameter in Apoe-/- mice. The phosphorylated levels of AMPKα2 and activator protein 2 alpha (AP-2α) were examined in cultured vascular smooth muscle cells (VSMCs) or in mice. We observed that pravastatin (50 mg/kg/day, 8 weeks) remarkably increased the AngII-induced AAA incidence in mice. In VSMCs, pravastatin increased the levels of pAMPK, pAP-2α, and MMP2 in both basal and AngII-stressed conditions, which were abolished by tempol and compound C. Pravastatin-upregulated MMP2 was abrogated by AMPKα2 or AP-2α siRNA. Lentivirus-mediated gene silence of AMPKα2 or AP-2α abolished pravastatin-worsened AAA formations in AngII-infused Apoe-/- mice. Clinical investigations demonstrated that both AMPKα2 and AP-2α phosphorylations were increased in AAA patients or human subjects taking pravastatin. In conclusion, pravastatin promotes AAA formation through AMPKα2-dependent AP-2α activations.

  18. Identification of a locality in snake venom alpha-neurotoxins with a significant compositional similarity to marine snail alpha-conotoxins: implications for evolution and structure/activity.

    PubMed

    Dufton, M J; Bladon, P; Harvey, A L

    1989-10-01

    alpha-neurotoxins from elapid snake venoms and alpha-conotoxins from marine snails bind specifically and with high affinity to nicotinic cholinoceptors. Although both types of toxin are polypeptides, there is more than a fourfold difference in size between the two and no clear sequence homology is evident. A systematic computer search of the three-dimensional structure of erabutoxin b (an alpha-neurotoxin from the false sea snake Laticauda semifasciata) was performed to identify the locality that most closely matched the amino acid compositions of the smaller alpha-conotoxins (from the marine snails Conus magus and Conus geographus). The area of greatest similarity centered on residue position 25 of erabutoxin b, a locale that is conserved throughout the snake alpha-neurotoxins and their homologues. Six proteins unrelated to erabutoxin b were compared to the alpha-conotoxins to show that the extent of the erabutoxin b/alpha-conotoxin match was too high to be coincidental. Homologues of erabutoxin b, namely alpha-cobratoxin from Naja naja siamensis and cytotoxin VII4 from Naja mossambica mossambica, were also analyzed. The extent of the matching with the alpha-conotoxins decreased in the series erabutoxin b greater than alpha-cobratoxin greater than cytotoxin VII4, and this also relates the order of similarity to the pharmacological properties of the alpha-conotoxins. The alpha-conotoxin-like area of the snake alpha-neurotoxins is peripheral to the site previously considered important for binding to the cholinoceptor, even though it seems to represent the focus of evolutionary convergence between the two types of neurotoxin. The area of resemblance does, however, have strong associations with the conformational behavior of the snake toxins. Hence, the outcome of this study has important consequences for the current ideas on snake alpha-neurotoxin structure/activity relationships and the evolutionary origins of neurotoxicity.

  19. The region of CQQQKPQRRP of PGC-1{alpha} interacts with the DNA-binding complex of FXR/RXR{alpha}

    SciTech Connect

    Kanaya, Eiko; Jingami, Hisato . E-mail: jingami@mfour.med.kyoto-u.ac.jp

    2006-04-14

    PGC-1{alpha} co-activates transcription by several nuclear receptors. To study the interaction among PGC-1{alpha}, RXR{alpha}/FXR, and DNA, we performed electrophoresis mobility shift assays. The RXR{alpha}/FXR proteins specifically bound to DNA containing the IR-1 sequence in the absence of ligand. When the fusion protein of GST-PGC-1{alpha} was added to the mixture of RXR{alpha}/FXR/DNA, the ligand-influenced retardation of the mobility was observed. The ligand for RXR{alpha} (9-cis-retinoic acid) was necessary for this retardation, whereas, the ligand for FXR, chenodeoxycholic acid, barely had an effect. The results obtained using truncated PGC-1{alpha} proteins suggested that two regions are necessary for PGC-1{alpha} to interact with the DNA-binding complex of RXR{alpha}/FXR. One is the region of the second leucine-rich motif, and the other is that of the amino acid sequence CQQQKPQRRP, present between the second and third leucine-rich motifs. The results obtained with the SPQSS mutation for KPQRR suggested that the basic amino acids are important for the interaction.

  20. The intrinsically liganded cyclic nucleotide-binding homology domain promotes KCNH channel activation.

    PubMed

    Zhao, Yaxian; Goldschen-Ohm, Marcel P; Morais-Cabral, João H; Chanda, Baron; Robertson, Gail A

    2017-02-01

    Channels in the ether-à-go-go or KCNH family of potassium channels are characterized by a conserved, C-terminal domain with homology to cyclic nucleotide-binding homology domains (CNBhDs). Instead of cyclic nucleotides, two amino acid residues, Y699 and L701, occupy the binding pocket, forming an "intrinsic ligand." The role of the CNBhD in KCNH channel gating is still unclear, however, and a detailed characterization of the intrinsic ligand is lacking. In this study, we show that mutating both Y699 and L701 to alanine, serine, aspartate, or glycine impairs human EAG1 channel function. These mutants slow channel activation and shift the conductance-voltage (G-V) relation to more depolarized potentials. The mutations affect activation and the G-V relation progressively, indicating that the gating machinery is sensitive to multiple conformations of the CNBhD. Substitution with glycine at both sites (GG), which eliminates the side chains that interact with the binding pocket, also reduces the ability of voltage prepulses to populate more preactivated states along the activation pathway (i.e., the Cole-Moore effect), as if stabilizing the voltage sensor in deep resting states. Notably, deletion of the entire CNBhD (577-708, ΔCNBhD) phenocopies the GG mutant, suggesting that GG is a loss-of-function mutation and the CNBhD requires an intrinsic ligand to exert its functional effects. We developed a kinetic model for both wild-type and ΔCNBhD mutant channels that describes all our observations on activation kinetics, the Cole-Moore shift, and G-V relations. These findings support a model in which the CNBhD both promotes voltage sensor activation and stabilizes the open pore. The intrinsic ligand is critical for these functional effects.

  1. Double N,B-Type Bidentate Boryl Ligands Enabling a Highly Active Iridium Catalyst for C-H Borylation.

    PubMed

    Wang, Guanghui; Xu, Liang; Li, Pengfei

    2015-07-01

    Boryl ligands hold promise in catalysis due to their very high electron-donating property. In this communication double N,B-type boryl anions were designed as bidentate ligands to promote an sp(2) C-H borylation reaction. A symmetric pyridine-containing tetraaminodiborane(4) compound (1) was readily prepared as the ligand precursor that could be used, in combination with [Ir(OMe)(COD)]2, to in situ generate a highly active catalyst for a broad range of (hetero)arene substrates including highly electron-rich and/or sterically hindered ones. This work provides the first example of a bidentate boryl ligand in supporting homogeneous organometallic catalysis.

  2. Peroxisome Proliferator-Activated Receptor γ (PPARγ) and Ligand Choreography: Newcomers Take the Stage.

    PubMed

    Garcia-Vallvé, Santiago; Guasch, Laura; Tomas-Hernández, Sarah; del Bas, Josep Maria; Ollendorff, Vincent; Arola, Lluís; Pujadas, Gerard; Mulero, Miquel

    2015-07-23

    Thiazolidinediones (TZDs), such as rosiglitazone and pioglitazone, are peroxisome proliferator-activated receptor γ (PPARγ) full agonists that have been widely used in the treatment of type 2 diabetes mellitus. Despite the demonstrated beneficial effect of reducing glucose levels in the plasma, TZDs also induce several adverse effects. Consequently, the search for new compounds with potent antidiabetic effects but fewer undesired effects is an active field of research. Interestingly, the novel proposed mechanisms for the antidiabetic activity of PPARγ agonists, consisting of PPARγ Ser273 phosphorylation inhibition, ligand and receptor mutual dynamics, and the presence of an alternate binding site, have recently changed the view regarding the optimal characteristics for the screening of novel PPARγ ligands. Furthermore, transcriptional genomics could bring essential information about the genome-wide effects of PPARγ ligands. Consequently, facing the new mechanistic scenario proposed for these compounds is essential for resolving the paradoxes among their agonistic function, antidiabetic activities, and side effects and should allow the rational development of better and safer PPARγ-mediated antidiabetic drugs.

  3. Synthesis, characterization and anti-proliferative activity of Cd(II) complexes with NNN type pyrazole-based ligand and pseudohalide ligands as coligand.

    PubMed

    Hopa, Cigdem; Yildirim, Hatice; Kara, Hulya; Kurtaran, Raif; Alkan, Mahir

    2014-01-01

    Cd(II) complexes of tridentate nitrogen donor ligand, 2,6-bis(3,4,5-trimethylpyrazolyl)pyridine (btmpp), Cd(btmpp)X2 (X:Cl, ONO or N(CN)2) have been synthesized and characterized by elemental and spectral (FT-IR, (1)H NMR, (13)C NMR, UV-Vis) analyses, differential thermal analysis and single crystal X-ray diffraction studies. The molecular structure of reported complex 1, revealed distorted square-pyramidal geometry around Cadmium. Complexes 1-3 and corresponding ligand were tested for cytotoxic activity against the human carcinoma cell lines HEP3B (hepatocellular carcinoma), PC3 (prostate adenocarcinoma), MCF7 (breast adenocarcinoma) and Saos2 (osteosarcoma). The results show that, complexes are more cytotoxic than the free ligand and complex 2 is the most cytotoxic complex for PC3.

  4. Synthesis, characterization and anti-proliferative activity of Cd(II) complexes with NNN type pyrazole-based ligand and pseudohalide ligands as coligand

    NASA Astrophysics Data System (ADS)

    Hopa, Cigdem; Yildirim, Hatice; Kara, Hulya; Kurtaran, Raif; Alkan, Mahir

    2014-03-01

    Cd(II) complexes of tridentate nitrogen donor ligand, 2,6-bis(3,4,5-trimethylpyrazolyl)pyridine (btmpp), Cd(btmpp)X2 (X:Cl, ONO or N(CN)2) have been synthesized and characterized by elemental and spectral (FT-IR, 1H NMR, 13C NMR, UV-Vis) analyses, differential thermal analysis and single crystal X-ray diffraction studies. The molecular structure of reported complex 1, revealed distorted square-pyramidal geometry around Cadmium. Complexes 1-3 and corresponding ligand were tested for cytotoxic activity against the human carcinoma cell lines HEP3B (hepatocellular carcinoma), PC3 (prostate adenocarcinoma), MCF7 (breast adenocarcinoma) and Saos2 (osteosarcoma). The results show that, complexes are more cytotoxic than the free ligand and complex 2 is the most cytotoxic complex for PC3.

  5. Conservation of mouse alpha A-crystallin promoter activity in chicken lens epithelial cells.

    PubMed

    Donovan, D M; Sax, C M; Klement, J F; Li, X; Chepelinsky, A B; Piatigorsky, J

    1992-10-01

    Previous transfection experiments have shown that 162 base pairs (bp) of the 5' flanking sequence of the chicken alpha A-crystallin gene are required for promoter activity in primary chicken lens epithelial cells (PLE), while only 111 bp of the 5' flanking sequence are needed for activity of the mouse alpha A-crystallin promoter in transfected chicken PLE cells or in a SV40 T-antigen-transformed transfected mouse lens epithelial cell line (alpha TN4-1). The effect of site-directed mutations covering positions -111 to -34 of the mouse alpha A-crystallin promoter fused to the bacterial chloramphenicol acetyltransferase (CAT) gene was compared in transfected chicken PLE cells and mouse alpha TN4-1 cells; selected mutations were also examined in a nontransformed rabbit lens epithelial cell line (N/N1003A). In general, the same mutations reduced promoter activity in the transfected lens cells from all three species, although differences were noted. The mutations severely affected regions -111/-106 and -69/-40 regions in all the transfected cells examined; by contrast, mutations at positions -105/-99 and -87/-70 had a somewhat greater effect in the chicken PLE than the mouse alpha TN4-1 cells, while mutations of the -93/-88 sequence reduced expression in the alpha TN4-1 but not the PLE cells. A partial cDNA with sequence similarity to alpha A-CRYPB1 of the mouse has been isolated from a chicken lens library; mouse alpha A-CRYBP1 is a putative transcription factor which binds to the -66/-55 sequence of the mouse alpha A-crystallin promoter.(ABSTRACT TRUNCATED AT 250 WORDS)

  6. 8-Iso-prostaglandin f(2alpha) reduces trophoblast invasion and matrix metalloproteinase activity.

    PubMed

    Staff, A C; Ranheim, T; Henriksen, T; Halvorsen, B

    2000-06-01

    Preeclampsia is a common pregnancy complication in the latter half of gestation diagnosed by hypertension and proteinuria. A key feature of preeclampsia is an altered placentation with reduced trophoblast invasion. Normal placentation requires controlled invasion of trophoblasts into the maternal uterine wall, with secretion of specific proteolytic enzymes able to degrade basement membranes and extracellular matrix, such as the matrix metalloproteinases (MMPs). 8-Iso-prostaglandin F(2alpha) (8-iso-PGF(2alpha)) is a marker of oxidative stress in vivo and is biologically active. We have recently reported an elevated content of free 8-iso-PGF(2alpha) in preeclamptic gestational tissue at delivery. Assuming an elevated level of 8-iso-PGF(2alpha) during the invasion period of the pregnancy, we hypothesized that 8-iso-PGF(2alpha) could reduce invasion of JAR cells, a choriocarcinoma cell line. We investigated JAR cell invasion with 2 types of Transwell assays and demonstrated that 8-iso-PGF(2alpha) (10 micromol/L) resulted in reduced cell invasion in both the colorimetric and radioactivity Transwell assays (P<0.01). Zymograms revealed reduced MMP-2 and MMP-9 activity in conditioned media from JAR cells incubated with 8-iso-PGF(2alpha) (10 micromol/L) (P<0.02). 8-Iso-PGF(2alpha) (10 micromol/L) also reduced the collagenase type IV activity in the conditioned media of JAR cells (P=0.04). No effects on MMP-2 and MMP-9 mRNA levels were observed after incubation with 8-iso-PGF(2alpha) (10 micromol/L), whereas protein levels were significantly decreased (P<0.02), suggesting a posttranscriptional regulation. We hypothesize a potential role for 8-iso-PGF(2alpha) in the reduced trophoblast invasion in preeclampsia.

  7. Developmental expression of trout egg polysialoglycoproteins and the prerequisite alpha 2,6-, and alpha 2,8-sialyl and alpha 2,8-polysialyltransferase activities required for their synthesis during oogenesis.

    PubMed

    Kitazume, S; Kitajima, K; Inoue, S; Inoue, Y; Troy, F A

    1994-04-08

    The developmental expression of the alpha 2,6- and alpha 2,8-linked sialic acid (Sia) residues in trout egg polysialoglycoproteins (PSGPs) was studied by correlating the temporal expression of these sugar residues, and the prerequisite sialyltransferases responsible for their synthesis, during oogenesis. The following new findings are reported. 1) Disialylated glycoproteins were identified in ovaries 4-6 months prior to ovulation. Three months prior to ovulation, a second more highly sialylated glycoprotein appeared. Structural studies confirmed that the two glycoproteins were discrete molecular species, designated PSGP(low Sia) and PSGP(high Sia), which differed only in their Sia content. PSGP(low Sia) contained mostly disialyl (Sia alpha 2,8-Sia alpha 2,6-) side chains, whereas PSGP(high Sia) contained alpha 2,8-linked oligo/polySia side chains ranging in length from 2 to over 20 Sia residues. The average degree of polymerization ([DP]av) was 6. 2) Biosynthetic studies using CMP-[14C]Neu5Ac indicated that three sialyltransferase activities were responsible for synthesis of the polysialyl residues of PSGPs: (i) alpha-N-acetylgalactosaminide alpha 2,6-sialyltransferase (alpha 2,6-ST), which catalyzed formation of the Sia residues alpha 2,6-linked to the proximal GalNAc residues in asialo-PSGP; (ii) alpha 2,6-sialoside alpha 2,8-sialyltransferase (alpha 2,8-ST or "initiase"), which catalyzed transfer of the first alpha 2,8-Sia residue to the alpha 2,6-linked Sia residue; and (iii) an alpha 2,8-polysialyltransferase (alpha 2,8-polyST or "polymerase"), responsible for synthesis of the alpha 2,8-linked poly/oligo Sia chains in PSGP(high Sia). Expression of these enzyme activities increased in accordance with the developmental appearance of each PSGP. 3) Structural characterization of the [14C]Sia-labeled side chains of each PSGP at different stages of development confirmed that synthesis of the disialyl unit containing a single alpha 2,8-Sia residue occurred before

  8. K-RAS(V12) Induces Autocrine Production of EGFR Ligands and Mediates Radioresistance Through EGFR-Dependent Akt Signaling and Activation of DNA-PKcs

    SciTech Connect

    Minjgee, Minjmaa; Toulany, Mahmoud; Kehlbach, Rainer; Giehl, Klaudia; Rodemann, H. Peter

    2011-12-01

    Purpose: It is known that postirradiation survival of tumor cells presenting mutated K-RAS is mediated through autocrine activation of epidermal growth factor receptor (EGFR). In this study the molecular mechanism of radioresistance of cells overexpressing mutated K-RAS(V12) was investigated. Methods and Materials: Head-and-neck cancer cells (FaDu) presenting wild-type K-RAS were transfected with empty vector or vector expressing mutated K-RAS(V12). The effect of K-RAS(V12) on autocrine production of EGFR ligands, activation of EGFR downstream pathways, DNA damage repair, and postirradiation survival was analyzed. Results: Conditioned medium collected from K-RAS(V12)-transfected cells enhanced activation of the phosphatidylinositol-3-kinase-Akt pathway and increased postirradiation survival of wild-type K-RAS parental cells when compared with controls. These effects were reversed by amphiregulin (AREG)-neutralizing antibody. In addition, secretion of the EGFR ligands AREG and transforming growth factor {alpha} was significantly increased upon overexpression of K-RAS(V12). Expression of mutated K-RAS(V12) resulted in an increase in radiation-induced DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylation at S2056. This increase was accompanied by increased repair of DNA double-strand breaks. Abrogation of DNA-PKcs phosphorylation by serum depletion or AREG-neutralizing antibody underscored the role of autocrine production of EGFR ligands, namely, AREG, in regulating DNA-PKcs activation in K-RAS mutated cells. Conclusions: These data indicate that radioresistance of K-RAS mutated tumor cells is at least in part due to constitutive production of EGFR ligands, which mediate enhanced repair of DNA double-strand breaks through the EGFR-phosphatidylinositol-3-kinase-Akt cascade.

  9. The active analog approach applied to the pharmacophore identification of benzodiazepine receptor ligands

    NASA Astrophysics Data System (ADS)

    Tebib, Souhail; Bourguignon, Jean-Jacques; Wermuth, Camille-Georges

    1987-07-01

    Applied to seven potent benzodiazepine-receptor ligands belonging to chemically different classes, the active analog approach allowed the stepwise identification of the pharmacophoric pattern associated with the recognition by the benzodiazepine receptor. A unique pharmacophore model was derived which involves six critical zones: (a) a π-electron rich aromatic (PAR) zone; (b) two electron-rich zones δ1 and δ2 placed at 5.0 and 4.5 Å respectively from the reference centroid in the PAR zone; (c) a freely rotating aromatic ring (FRA) region; (d) an out-of-plane region (OPR), strongly associated with agonist properties; and (e) an additional hydrophobic region (AHR). The model accommodates all presently known ligands of the benzodiazepine receptor, identifies sensitivity to steric hindrance close to the δ1 zone, accounts for R and S differential affinities and distinguishes requirements for agonist versus non-agonist activity profiles.

  10. Ligand Biological Activity Predictions Using Fingerprint-Based Artificial Neural Networks (FANN-QSAR)

    PubMed Central

    Myint, Kyaw Z.; Xie, Xiang-Qun

    2015-01-01

    This chapter focuses on the fingerprint-based artificial neural networks QSAR (FANN-QSAR) approach to predict biological activities of structurally diverse compounds. Three types of fingerprints, namely ECFP6, FP2, and MACCS, were used as inputs to train the FANN-QSAR models. The results were benchmarked against known 2D and 3D QSAR methods, and the derived models were used to predict cannabinoid (CB) ligand binding activities as a case study. In addition, the FANN-QSAR model was used as a virtual screening tool to search a large NCI compound database for lead cannabinoid compounds. We discovered several compounds with good CB2 binding affinities ranging from 6.70 nM to 3.75 μM. The studies proved that the FANN-QSAR method is a useful approach to predict bioactivities or properties of ligands and to find novel lead compounds for drug discovery research. PMID:25502380

  11. Short term integrative meditation improves resting alpha activity and stroop performance.

    PubMed

    Fan, Yaxin; Tang, Yi-Yuan; Tang, Rongxiang; Posner, Michael I

    2014-12-01

    Our previous research showed that short term meditation training reduces the time to resolve conflict in the flanker task. Studies also show that resting alpha increases with long term meditation practice. The aim of this study is to determine whether short term meditation training both increases resting alpha activity and reduces the time to resolve conflict in the Stroop task and whether these two effects are related. Forty-three Chinese undergraduates were randomly assigned an experiment group given 5 days meditation training using integrative body-mind training (IBMT) and a relaxation training control. After training, only the IBMT group showed decreased conflict reaction time (RT), and increased resting mean alpha power. Moreover, the higher the enhancement of resting alpha power, the stronger the improvement of conflict RT. The results indicate that short term meditation diffusely enhances alpha and improves the ability to deal with conflict and moreover these two effects are positively related.

  12. Expression and enzymatic activity of recombinant cytochrome P450 17 alpha-hydroxylase in Escherichia coli.

    PubMed Central

    Barnes, H J; Arlotto, M P; Waterman, M R

    1991-01-01

    When the cDNA encoding bovine microsomal 17 alpha-hydroxylase cytochrome P450 (P45017 alpha) containing modifications within the first seven codons which favor expression in Escherichia coli is placed in a highly regulated tac promoter expression plasmid, as much as 16 mg of spectrally detectable P45017 alpha per liter of culture can be synthesized and integrated into E. coli membranes. The known enzymatic activities of bovine P45017 alpha can be reconstituted by addition of purified rat liver NADPH-cytochrome P450 reductase to isolated E. coli membrane fractions containing the recombinant P45017 alpha enzyme. Surprisingly, it is found that E. coli contain an electron-transport system that can substitute for the mammalian microsomal NADPH-cytochrome P450 reductase in supporting both the 17 alpha-hydroxylase and 17,20-lyase activities of P45017 alpha. Thus, not only can E. coli express this eukaryotic membrane protein at relatively high levels, but as evidenced by metabolism of steroids added directly to the cells, the enzyme is catalytically active in vivo. These studies establish E. coli as an efficacious heterologous expression system for structure-function analysis of the cytochrome P450 system. Images PMID:1829523

  13. Synthesis, characterization and biological activities of mixed ligand Zr(IV) complexes.

    PubMed

    Malghe, Yuvraj S; Prabhu, Rakesh C; Raut, Rajesh W

    2009-01-01

    Mixed ligand ternary Zr(IV) complexes of type [M(Q)2LNO3xH2O] have been synthesized using 8-hydroxyquinoline (HQ) as a primary ligand and N- and/O-donor amino acids (HL) such as L-serine, L-alanine and glycine as secondary ligands. These complexes were characterized on the basis of elemental analysis, conductance measurement, spectral and thermal studies. The molar conductance study of the complexes in DMF solvent signifies their non-electrolytic nature whereas the thermal analyses specify presence of a coordinated water molecule. The complexes were tested for antifungal and antibacterial activity by using agar well diffusion bioassay. The antibacterial activity was tested against the pathogenic bacteria Staphylococcus aureus and Enterococcus faecium. The results obtained were evaluated with antibacterial standard vancomycin. The antifungal activity was tested against Candida albicans, Candida krusei, Aspergillus fumigatus and the results obtained were compared with antifungal standard amphotericin B. The complexes were also screened for cytotoxicity studies against Ehrlich ascites cells and Daltons lymphoma ascites cells and show very low cytotoxicity.

  14. Biologically active macromolecular forms of oxytocin. [8-Lysine]oxytocin as a suitable ligand.

    PubMed Central

    Snell, C R; Smyth, D G

    1977-01-01

    [8-Lysine]oxytocin was synthesized on a solid support and possessed an oxytocic activity of 100 +/- 6 units mumol on the isolated rat uterus. The epsilon-carbamoyl, epsilon-3-carboxypropionyl and epsilon-3-carboxybutryl derivatives were prepared and had uterotonic activities of 400, 55 and 50 units/mumol respectively. [8-Lysine]oxytocin was coupled unambiguously through the epsilon-amino group to the carboxyl groups of carboxymethylated dextrans or epsilon-3-carboxypropionly-gelatin. The macromolecular oxytocins were water-soluble and retained signigicant oxytocic activity. [8-Lysine]oxytocin should prove a useful ligand for affinity chromatography of oxytocin-binding proteins. PMID:889573

  15. Tumor necrosis factor alpha-induced angiogenesis depends on in situ platelet-activating factor biosynthesis

    PubMed Central

    1994-01-01

    Tumor necrosis factor (TNF) alpha, a potent inhibitor of endothelial cell growth in vitro, is angiogenic in vivo. Therefore, it was suggested that the angiogenic properties of this agent might be consequent to the production of secondary mediators. Since TNF-alpha stimulates the synthesis of platelet-activating factor (PAF) by monocytes and endothelial cells, we investigated the possible involvement of PAF in the angiogenic effect of TNF-alpha. Angiogenesis was studied in a murine model in which Matrigel was used as a vehicle for the delivery of mediators. In this model the angiogenesis induced by TNF-alpha was shown to be inhibited by WEB 2170, a specific PAF receptor antagonist. Moreover, in mice injected with TNF-alpha, PAF was detected within the Matrigel, 6 and 24 h after TNF-alpha injection. The synthesis of PAF within the Matrigel was concomitant with the early migration of endothelial cells and infiltration of monocytes. No infiltration of lymphocytes or polymorphonuclear leukocytes was observed. Synthetic PAF as well as PAF extracted and purified from mice challenged with TNF-alpha induced a rapid angiogenic response, inhibited by WEB 2170. These results suggest that the angiogenic effect of TNF-alpha is, at least in part, mediated by PAF synthesized from monocytes and/or endothelial cells infiltrating the Matrigel plug. PMID:7516414

  16. Mutation of valine residue unique to alpha subunit of Gs abolishes activation.

    PubMed

    Devic, E; Journot, L; Pantaloni, C; Bockaert, J; Audigier, Y

    1994-08-19

    We recently characterized a decapeptide sequence (residues 367-376) that is important for the membrane association of the activated alpha subunit of Gs. We report here that when this sequence is replaced by the cognate sequence of Gi1 alpha subunit, the chimeric protein (Gsis alpha) still interacts with the membrane but cannot be activated, regardless of the mode of activation. Construction of various chimeras demonstrates that the single replacement of valine 367 by threonine, the cognate residue of Gi1 alpha subunit, fully reproduces the loss of activation. Analysis of nucleotide interaction reveals that the mutant V367T Gs alpha protein poorly binds GDP or GTP. On the other hand, the conservative change of valine to isoleucine does not alter activation. Interestingly, members of the Gs and G12 classes have a valine and an isoleucine, respectively, at this position, whereas members of the Gi or Gq class contain a threonine residue. The evolutionary relationship between the different classes suggests that the presence of a hydrophobic or a hydrophilic residue is not fortuitous in these alpha subunits and might provide distinctive structural and/or functional properties.

  17. Glucocorticoid receptor (GR) {beta} has intrinsic, GR{alpha}-independent transcriptional activity

    SciTech Connect

    Kino, Tomoshige; Manoli, Irini; Kelkar, Sujata; Wang, Yonghong; Su, Yan A.; Chrousos, George P.

    2009-04-17

    The human glucocorticoid receptor (GR) gene produces C-terminal GR{beta} and GR{alpha} isoforms through alternative use of specific exons 9{beta} and {alpha}, respectively. We explored the transcriptional activity of GR{beta} on endogenous genes by developing HeLa cells stably expressing EGFP-GR{beta} or EGFP. Microarray analyses revealed that GR{beta} had intrinsic gene-specific transcriptional activity, regulating mRNA expression of a large number of genes negatively or positively. Majority of GR{beta}-responsive genes was distinct from those modulated by GR{alpha}, while GR{beta} and GR{alpha} mutually modulated each other's transcriptional activity in a subpopulation of genes. We did not observe in HCT116 cells nuclear translocation of GR{beta} and activation of this receptor by RU 486, a synthetic steroid previously reported to bind GR{beta} and to induce nuclear translocation. Our results indicate that GR{beta} has intrinsic, GR{alpha}-independent, gene-specific transcriptional activity, in addition to its previously reported dominant negative effect on GR{alpha}-induced transactivation of GRE-driven promoters.

  18. Unlike PPAR{gamma}, PPAR{alpha} or PPAR{beta}/{delta} activation does not promote human monocyte differentiation toward alternative macrophages

    SciTech Connect

    Bouhlel, Mohamed Amine; Brozek, John; Derudas, Bruno; Zawadzki, Christophe; Jude, Brigitte; Staels, Bart; Chinetti-Gbaguidi, Giulia

    2009-08-28

    Macrophages adapt their response to micro-environmental signals. While Th1 cytokines promote pro-inflammatory M1 macrophages, Th2 cytokines promote an 'alternative' anti-inflammatory M2 macrophage phenotype. Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors expressed in macrophages where they control the inflammatory response. It has been shown that PPAR{gamma} promotes the differentiation of monocytes into anti-inflammatory M2 macrophages in humans and mice, while a role for PPAR{beta}/{delta} in this process has been reported only in mice and no data are available for PPAR{alpha}. Here, we show that in contrast to PPAR{gamma}, expression of PPAR{alpha} and PPAR{beta}/{delta} overall does not correlate with the expression of M2 markers in human atherosclerotic lesions, whereas a positive correlation with genes of lipid metabolism exists. Moreover, unlike PPAR{gamma}, PPAR{alpha} or PPAR{beta}/{delta} activation does not influence human monocyte differentiation into M2 macrophages in vitro. Thus, PPAR{alpha} and PPAR{beta}/{delta} do not appear to modulate the alternative differentiation of human macrophages.

  19. Alpha-particle activity of apollo 11 samples.

    PubMed

    Richardson, K A; McCkay, D S; Greenwood, W R; Foss, T H

    1970-01-30

    Nine polishled thin sectionis have been exposed to nulclear track plates, three have been counted by alplia-particle spectrometry, and one has been examined by electron mocroprobe. Interpretation of the results is in a preliminary stage. Alpha track distribiutioni in the autoradiograph of a breccia forms a network that appears related to the rims of accretionary lapilli comiiposinig the breccia. Thorium in a coarse-grained crystalline rock is concenitrated in micron-sized, zirconium-rich crystals. Alplia count rates agree with what would be predicted from previously reported thorium and uranium contents of the same rocks, suggesting secular equilibriunm for the thorium and uranium decay series.

  20. Seasonal changes in the synthesis of the neurosteroid 7alpha-hydroxypregnenolone stimulating locomotor activity in newts.

    PubMed

    Haraguchi, Shogo; Matsunaga, Masahiro; Koyama, Teppei; Do Rego, Jean-Luc; Tsutsui, Kazuyoshi

    2009-04-01

    We recently found that the newt brain actively produces 7alpha-hydroxypregnenolone, a novel amphibian neurosteroid stimulating locomotor activity. It is well known that locomotor activity of male newts increases during the breeding period. To understand the physiological role of 7alpha-hydroxypregnenolone, we investigated seasonal changes in 7alpha-hydroxypregnenolone synthesis in the brain of male newts. Interestingly, 7alpha-hydroxypregnenolone synthesis in the brain showed marked changes during the annual breeding cycle, with a maximal level in the breeding period when locomotor activity of male newts increases. These results suggest that 7alpha-hydroxypregnenolone induces seasonal locomotor changes in male newts.

  1. Hypertonic saline attenuates TNF-alpha-induced NF-kappaB activation in pulmonary epithelial cells.

    PubMed

    Nydam, Trevor L; Moore, Ernest E; McIntyre, Robert C; Wright, Franklin L; Gamboni-Robertson, Fabia; Eckels, Phillip C; Banerjee, Anirban

    2009-05-01

    Resuscitation with hypertonic saline (HTS) attenuates acute lung injury (ALI) and modulates postinjury hyperinflammation. TNF-alpha-stimulated pulmonary epithelium is a major contributor to hemorrhage-induced ALI. We hypothesized that HTS would inhibit TNF-alpha-induced nuclear factor (NF)-kappaB proinflammatory signaling in pulmonary epithelial cells. Therefore, we pretreated human pulmonary epithelial cells (A549) with hypertonic medium (180 mM NaCl) for 30 min, followed by TNF-alpha stimulation (10 ng/mL). Key regulatory steps and protein concentrations in this pathway were assessed for significant alterations. Hypertonic saline significantly reduced TNF-alpha-induced intercellular adhesion molecule 1 levels and NF-kappaB nuclear localization. The mechanism is attenuated phosphorylation and delayed degradation of IkappaB alpha. Hypertonic saline did not alter TNF-alpha-induced p38 mitogen-activated protein kinase phosphorylation or constitutive vascular endothelial growth factor expression, suggesting that the observed inhibition is not a generalized suppression of protein phosphorylation or cellular function. These results show that HTS inhibits TNF-alpha-induced NF-kappaB activation in the pulmonary epithelium and, further, our understanding of its beneficial effects in hemorrhage-induced ALI.

  2. Copper (II) complexes possessing alkyl-substituted polypyridyl ligands: Structural characterization and in vitro antitumor activity.

    PubMed

    Angel, Noah R; Khatib, Raneen M; Jenkins, Julia; Smith, Michelle; Rubalcava, Justin M; Le, Brian Khoa; Lussier, Daniel; Chen, Zhuo Georgia; Tham, Fook S; Wilson, Emma H; Eichler, Jack F

    2017-01-01

    In an effort to find alternatives to the antitumor drug cisplatin, a series of copper (II) complexes possessing alkyl-substituted polypyridyl ligands have been synthesized. Eight new complexes are reported herein: μ-dichloro-bis{2,9-di-sec-butyl-1,10-phenanthrolinechlorocopper(II)} {[((di-sec-butyl)phen)ClCu(μ-Cl)2CuCl((di-sec-butyl)phen)]}(1), 2-sec-butyl-1,10-phenanthrolinedichlorocopper(II) {([mono-sec-butyl)phen) CuCl2} (2), 2,9-di-n-butyl-1,10-phenanthrolinedichlorocopper(II) {[(di-n-butyl)phen) CuCl2}(3), 2-n-butyl-1,10-phenanthrolinedichlorocopper(II) {[(mono-n-butyl)phen) CuCl2} (4), 2,9-di-methyl-1,10-phenanthrolineaquadichlorocopper(II) {[(di-methyl)phen) Cu(H2O)Cl2}(5), μ-dichloro-bis{6-sec-butyl-2,2'-bipyridinedichlorocopper(II)} {((mono-sec-butyl)bipy) ClCu(μ-Cl)2CuCl((mono-sec-butyl)bipy)} (6), 6,6'-di-methyl-2,2'-bipyridinedichlorocopper(II) {(6,6'-di-methyl)bipy) CuCl2} (7), and 4,4'-dimethyl-2,2'-bipyridinedichlorocopper(II) {(4,4'-di-methyl)bipy) CuCl2} (8). These complexes have been characterized via elemental analysis, UV-vis spectroscopy, and mass spectrometry. Single crystal X-ray diffraction experiments revealed the complexes synthesized with the (di-sec-butyl)phen ligand (1) and (mono-sec-butyl)bipy ligand (6) crystallized as dimers in which two copper(II) centers are bridged by two chloride ligands. Conversely, complexes 2, 7, and 8 were isolated as monomeric species possessing distorted tetrahedral geometries, and the [((di-methyl)phen)Cu(H2O)Cl2] (5) complex was isolated as a distorted square pyramidal monomer possessing a coordinating aqua ligand. Compounds 1-8 were evaluated for their in vitro antitumor efficacy. Compounds 1, 5, and 7 in particular were found to exhibit remarkable activity against human derived lung cancer cells, yet this class of copper(II) compounds had minimal cytotoxic effect on non-cancerous cells. In vitro control experiments indicate the activity of the copper(II) complexes most likely does not arise from the

  3. A novel partial agonist of peroxisome proliferator-activated receptor-gamma (PPARgamma) recruits PPARgamma-coactivator-1alpha, prevents triglyceride accumulation, and potentiates insulin signaling in vitro.

    PubMed

    Burgermeister, Elke; Schnoebelen, Astride; Flament, Angele; Benz, Jörg; Stihle, Martine; Gsell, Bernard; Rufer, Arne; Ruf, Armin; Kuhn, Bernd; Märki, Hans Peter; Mizrahi, Jacques; Sebokova, Elena; Niesor, Eric; Meyer, Markus

    2006-04-01

    Partial agonists of peroxisome proliferator-activated receptor-gamma (PPARgamma), also termed selective PPARgamma modulators, are expected to uncouple insulin sensitization from triglyceride (TG) storage in patients with type 2 diabetes mellitus. These agents shall thus avoid adverse effects, such as body weight gain, exerted by full agonists such as thiazolidinediones. In this context, we describe the identification and characterization of the isoquinoline derivative PA-082, a prototype of a novel class of non-thiazolidinedione partial PPARgamma ligands. In a cocrystal with PPARgamma it was bound within the ligand-binding pocket without direct contact to helix 12. The compound displayed partial agonism in biochemical and cell-based transactivation assays and caused preferential recruitment of PPARgamma-coactivator-1alpha (PGC1alpha) to the receptor, a feature shared with other selective PPARgamma modulators. It antagonized rosiglitazone-driven transactivation and TG accumulation during de novo adipogenic differentiation of murine C3H10T1/2 mesenchymal stem cells. The latter effect was mimicked by overexpression of wild-type PGC1alpha but not its LXXLL-deficient mutant. Despite failing to promote TG loading, PA-082 induced mRNAs of genes encoding components of insulin signaling and adipogenic differentiation pathways. It potentiated glucose uptake and inhibited the negative cross-talk of TNFalpha on protein kinase B (AKT) phosphorylation in mature adipocytes and HepG2 human hepatoma cells. PGC1alpha is a key regulator of energy expenditure and down-regulated in diabetics. We thus propose that selective recruitment of PGC1alpha to favorable PPARgamma-target genes provides a possible molecular mechanism whereby partial PPARgamma agonists dissociate TG accumulation from insulin signaling.

  4. Naringenin enhances NK cell lysis activity by increasing the expression of NKG2D ligands on Burkitt's lymphoma cells.

    PubMed

    Kim, Jeong Hwa; Lee, Jae Kwon

    2015-11-01

    Natural killer (NK) cells are capable of identifying and killing tumor cells as well as virus infected cells without pre-sensitization. NK cells express activating and inhibitory receptors, and can distinguish between normal and tumor cells. The present study was designed to demonstrate the importance of the expression level of NKG2D ligands on the Burkitt's lymphoma cell line, Raji, in enhancing NK cell cytolytic activity. Various flavonoids were used as stimulants to enhance the expression of NKG2D ligands. NK cell lysis activity against Raji was not changed by pre-treatment of Raji with luteolin, kaempferol, taxifolin and hesperetin. However, treatment of Raji with naringenin showed increased sensitivity to NK cell lysis than untreated control cells. The activity of naringenin was due to enhanced NKG2D ligand expression. These results provide evidence that narigenin's antitumor activity may be due to targeting of NKG2D ligand expression and suggests a possible immunotherapeutic role for cancer treatment.

  5. Syntheses, crystal structures, anticancer activities of three reduce Schiff base ligand based transition metal complexes

    NASA Astrophysics Data System (ADS)

    Chang, Hui-Qin; Jia, Lei; Xu, Jun; Zhu, Tao-Feng; Xu, Zhou-Qing; Chen, Ru-Hua; Ma, Tie-Liang; Wang, Yuan; Wu, Wei-Na

    2016-02-01

    Three nickel(II) complexes, [Ni2(L1)2(tren)2(H2O)](ClO4)3 (1), [NiL2(tren)2](ClO4)·2.5H2O (2), [NiL2(tren)2]I·1.5H2O·CH3OH (3) based on amino acid reduced Schiff ligands are synthesized and characterized by physico-chemical and spectroscopic methods. The results show that in all complexes, the amino acid ligand is deprotonated and acts as an anionic ligand. In the dinuclear complex 1, each Ni(II) atom has a distorted octahedron geometry while with different coordination environment. However, the complexes 2 and 3 are mononuclear, almost with the same coordination environment. Furthermore, in vitro experiments are carried out, including MTT assay, Annexin V/PI flow cytometry and western blotting, to assess whether the complexes have antitumor effect. And the results show that all the three complexes have moderate anticancer activity towards human hepatic cancer (HepG2), human cervical cancer (HeLa) and human prostate (PC3) cell lines, in a concentration dependent way. The complex 1 exhibit higher cytotoxicity than the other two complexes and can induce human hepatic cancer cell (HepG2) to cell apoptosis by activating caspase 3.

  6. Photoaffinity ligands in the study of cytochrome p450 active site structure.

    PubMed

    Gartner, Carlos Augusto

    2003-04-01

    While photoaffinity ligands have been widely used to probe the structures of many receptors and nucleic acid binding proteins, their effective use in the study of cytochrome p450 structure is less established. Nevertheless, significant advances in this field have been made since the technique was first applied to p450cam in 1979. In several cases, especially studies involving p450s of the 1A and 2B families, peptides covalently modified with photoaffinity ligands have been isolated and characterized. Some of these peptides were predicted by molecular modeling to line substrate binding regions of the enzymes. Other data obtained from such studies were more difficult to reconcile with theory. This review addresses the status of photoaffinity labeling as a tool for studying cytochrome p450 structure. In addition, potential future directions in this field are discussed, including the development of heme-directed agents and validation of their effectiveness as photoaffinity ligands using sperm whale myoglobin as a test protein. The potential for hydroxyaromatic compounds to serve as photoactivated probes of active site nucleophiles is also discussed. This class of compounds and its derivatives has long been known in the fields of photochemistry and photophysics to be precursors of reactive radicals and quinone methides that are likely to serve as effective active site probes of the p450s.

  7. A categorical structure-activity relationship analysis of GPR119 ligands.

    PubMed

    Kumar, Pritesh; Carrasquer, Carl A; Carter, Arren; Song, Zhao-Hui; Cunningham, Albert R

    2014-01-01

    The categorical structure-activity relationship (cat-SAR) expert system has been successfully used in the analysis of chemical compounds that cause toxicity. Herein we describe the use of this fragment-based approach to model ligands for the G protein-coupled receptor 119 (GPR119). Using compounds that are known GPR119 agonists and compounds that we have confirmed experimentally that are not GPR119 agonists, four distinct cat-SAR models were developed. Using a leave-one-out validation routine, the best GPR119 model had an overall concordance of 99%, a sensitivity of 99%, and a specificity of 100%. Our findings from the in-depth fragment analysis of several known GPR119 agonists were consistent with previously reported GPR119 structure-activity relationship (SAR) analyses. Overall, while our results indicate that we have developed a highly predictive cat-SAR model that can be potentially used to rapidly screen for prospective GPR119 ligands, the applicability domain must be taken into consideration. Moreover, our study demonstrates for the first time that the cat-SAR expert system can be used to model G protein-coupled receptor ligands, many of which are important therapeutic agents.

  8. Spectral, XRD, SEM and biological activities of transition metal complexes of polydentate ligands containing thiazole moiety

    NASA Astrophysics Data System (ADS)

    Neelakantan, M. A.; Marriappan, S. S.; Dharmaraja, J.; Jeyakumar, T.; Muthukumaran, K.

    2008-11-01

    Metal complexes of o-vanillidene-2-aminobenzothiazole have been prepared and characterized by elemental and spectral (vibrational, electronic, 1H NMR and EPR) data as well as magnetic susceptibility measurements and thermo gravimetric analysis (TG/DTA). The low molar conductance values reveal the non-electrolytic nature of these complexes. The elemental analysis suggests that the stoichiometry to be 1:2 (metal:ligand). Magnetic susceptibility data coupled with electronic spectra suggest that two ligands coordinate to each metal atom by phenolic oxygen and imino nitrogen to form high spin octahedral complex with Co(II), Mn(II) and Ni(II). The fifth and sixth position of metal ion is satisfied with water molecules. The thermal behaviour (TG/DTA) of the synthesised complexes shows that the complexes loss water molecules in the first step followed by decomposition of the ligand. Spin Hamiltonian parameters predict a distorted tetrahedral geometry for the copper complex. XRD and SEM analysis provide the crystalline nature and the morphology of the metal complexes. The in vitro biological activity of the metal chelates is tested against the Gram positive bacteria ( Bacillus amyloliquifacians) and gram negative bacteria ( Pseudomonas species), fungus ( Aspergillus niger) and yeast ( Sacchromyces cereviaceae). Most of the metal chelates exhibited higher biological activities.

  9. NK cell activating receptor ligand expression in lymphangioleiomyomatosis is associated with lung function decline

    PubMed Central

    Osterburg, Andrew R.; Nelson, Rebecca L.; Yaniv, Benyamin Z.; Foot, Rachel; Donica, Walter R.F.; Nashu, Madison A.; Liu, Huan; Wikenheiser-Brokamp, Kathryn A.; Moss, Joel; McCormack, Francis X.; Borchers, Michael T.

    2016-01-01

    Lymphangioleiomyomatosis (LAM) is a rare lung disease of women that leads to progressive cyst formation and accelerated loss of pulmonary function. Neoplastic smooth muscle cells from an unknown source metastasize to the lung and drive destructive remodeling. Given the role of NK cells in immune surveillance, we postulated that NK cell activating receptors and their cognate ligands are involved in LAM pathogenesis. We found that ligands for the NKG2D activating receptor UL-16 binding protein 2 (ULBP2) and ULBP3 are localized in cystic LAM lesions and pulmonary nodules. We found elevated soluble serum ULBP2 (mean = 575 pg/ml ± 142) in 50 of 100 subjects and ULBP3 in 30 of 100 (mean = 8,300 pg/ml ± 1,515) subjects. LAM patients had fewer circulating NKG2D+ NK cells and decreased NKG2D surface expression. Lung function decline was associated with soluble NKG2D ligand (sNKG2DL) detection. The greatest rate of decline forced expiratory volume in 1 second (FEV1, –124 ± 30 ml/year) in the 48 months after enrollment (NHLBI LAM Registry) occurred in patients expressing both ULBP2 and ULBP3, whereas patients with undetectable sNKG2DL levels had the lowest rate of FEV1 decline (–32.7 ± 10 ml/year). These data suggest a role for NK cells, sNKG2DL, and the innate immune system in LAM pathogenesis. PMID:27734028

  10. Expression of enzymatically active, recombinant barley alpha-glucosidase in yeast and immunological detection of alpha-glucosidase from seed tissue.

    PubMed

    Tibbot, B K; Henson, C A; Skadsen, R W

    1998-10-01

    An alpha-glucosidase cDNA clone derived from barley aleurone tissue was expressed in Pichia pastoris and Escherichia coli. The gene was fused with the N-terminal region of the Saccharomyces cerevisiae alpha-factor secretory peptide and placed under control of the Pichia AOX1 promoter in the vector pPIC9. Enzymatically active, recombinant alpha-glucosidase was synthesized and secreted from the yeast upon induction with methanol. The enzyme hydrolyzed maltose > trehalose > nigerose > isomaltose. Maltase activity occurred over the pH range 3.5-6.3 with an optimum at pH 4.3, classifying the enzyme as an acid alpha-glucosidase. The enzyme had a Km of 1.88 mM and Vmax of 0.054 micromol/min on maltose. The recombinant alpha-glucosidase expressed in E. coli was used to generate polyclonal antibodies. The antibodies detected 101 and 95 kDa forms of barley alpha-glucosidase early in seed germination. Their levels declined sharply later in germination, as an 81 kDa alpha-glucosidase became prominent. Synthesis of these proteins also occurred in isolated aleurones after treatment with gibberellin, and this was accompanied by a 14-fold increase in alpha-glucosidase enzyme activity.

  11. Control of immune ligands by members of a cytomegalovirus gene expansion suppresses natural killer cell activation

    PubMed Central

    Fielding, Ceri A; Weekes, Michael P; Nobre, Luis V; Ruckova, Eva; Wilkie, Gavin S; Paulo, Joao A; Chang, Chiwen; Suárez, Nicolás M; Davies, James A; Antrobus, Robin; Stanton, Richard J; Aicheler, Rebecca J; Nichols, Hester; Vojtesek, Borek; Trowsdale, John; Davison, Andrew J; Gygi, Steven P

    2017-01-01

    The human cytomegalovirus (HCMV) US12 family consists of ten sequentially arranged genes (US12-21) with poorly characterized function. We now identify novel natural killer (NK) cell evasion functions for four members: US12, US14, US18 and US20. Using a systematic multiplexed proteomics approach to quantify ~1300 cell surface and ~7200 whole cell proteins, we demonstrate that the US12 family selectively targets plasma membrane proteins and plays key roles in regulating NK ligands, adhesion molecules and cytokine receptors. US18 and US20 work in concert to suppress cell surface expression of the critical NKp30 ligand B7-H6 thus inhibiting NK cell activation. The US12 family is therefore identified as a major new hub of immune regulation. DOI: http://dx.doi.org/10.7554/eLife.22206.001 PMID:28186488

  12. Cholinesterase inhibitory activity of chlorophenoxy derivatives-Histamine H3 receptor ligands.

    PubMed

    Łażewska, Dorota; Jończyk, Jakub; Bajda, Marek; Szałaj, Natalia; Więckowska, Anna; Panek, Dawid; Moore, Caitlin; Kuder, Kamil; Malawska, Barbara; Kieć-Kononowicz, Katarzyna

    2016-08-15

    In recent years, multitarget-directed ligands have become an interesting strategy in a search for a new treatment of Alzheimer's disease. Combination of both: a histamine H3 receptor antagonist/inverse agonist and a cholinesterases inhibitor in one molecule could provide a new therapeutic opportunity. Here, we present biological evaluation of histamine H3 receptor ligands-chlorophenoxyalkylamine derivatives against cholinesterases: acetyl- and butyrylcholinesterase. The target compounds showed cholinesterase inhibitory activity in a low micromolar range. The most potent in this group was 1-(7-(4-chlorophenoxy)heptyl)homopiperidine (18) inhibiting the both enzymes (EeAChE IC50=1.93μM and EqBuChE IC50=1.64μM). Molecular modeling studies were performed to explain the binding mode of 18 with histamine H3 receptor as well as with cholinesterases.

  13. Differences in EEG Alpha Activity between Gifted and Non-Identified Individuals: Insights into Problem Solving.

    ERIC Educational Resources Information Center

    Jausovec, Norbert

    1997-01-01

    This study examined differences in electroencephalography (EEG) alpha activity between gifted and nongifted Slovenian student-teachers (N=17 each). Gifted students showed greater left hemisphere activation than nongifted subjects in relaxed states, but lower activation during problem solving. The same pattern was observed in overall hemispheric…

  14. Structural basis for iloprost as a dual peroxisome proliferator-activated receptor alpha/delta agonist.

    PubMed

    Jin, Lihua; Lin, Shengchen; Rong, Hui; Zheng, Songyang; Jin, Shikan; Wang, Rui; Li, Yong

    2011-09-09

    Iloprost is a prostacyclin analog that has been used to treat many vascular conditions. Peroxisome proliferator-activated receptors (PPARs) are ligand-regulated transcription factors with various important biological effects such as metabolic and cardiovascular physiology. Here, we report the crystal structures of the PPARα ligand-binding domain and PPARδ ligand-binding domain bound to iloprost, thus providing unambiguous evidence for the direct interaction between iloprost and PPARs and a structural basis for the recognition of PPARα/δ by this prostacyclin analog. In addition to conserved contacts for all PPARα ligands, iloprost also initiates several specific interactions with PPARs using its unique structural groups. Structural and functional studies of receptor-ligand interactions reveal strong functional correlations of the iloprost-PPARα/δ interactions as well as the molecular basis of PPAR subtype selectivity toward iloprost ligand. As such, the structural mechanism may provide a more rational template for designing novel compounds targeting PPARs with more favorable pharmacologic impact based on existing iloprost drugs.

  15. Monitoring Solution Structures of Peroxisome Proliferator-Activated Receptor β/δ upon Ligand Binding

    PubMed Central

    Schwarz, Rico; Tänzler, Dirk; Ihling, Christian H.; Sinz, Andrea

    2016-01-01

    Peroxisome proliferator-activated receptors (PPARs) have been intensively studied as drug targets to treat type 2 diabetes, lipid disorders, and metabolic syndrome. This study is part of our ongoing efforts to map conformational changes in PPARs in solution by a combination of chemical cross-linking and mass spectrometry (MS). To our best knowledge, we performed the first studies addressing solution structures of full-length PPAR-β/δ. We monitored the conformations of the ligand-binding domain (LBD) as well as full-length PPAR-β/δ upon binding of two agonists. (Photo-) cross-linking relied on (i) a variety of externally introduced amine- and carboxyl-reactive linkers and (ii) the incorporation of the photo-reactive amino acid p-benzoylphenylalanine (Bpa) into PPAR-β/δ by genetic engineering. The distances derived from cross-linking experiments allowed us to monitor conformational changes in PPAR-β/δ upon ligand binding. The cross-linking/MS approach proved highly advantageous to study nuclear receptors, such as PPARs, and revealed the interplay between DBD (DNA-binding domain) and LDB in PPAR-β/δ. Our results indicate the stabilization of a specific conformation through ligand binding in PPAR-β/δ LBD as well as full-length PPAR-β/δ. Moreover, our results suggest a close distance between the N- and C-terminal regions of full-length PPAR-β/δ in the presence of GW1516. Chemical cross-linking/MS allowed us gaining detailed insights into conformational changes that are induced in PPARs when activating ligands are present. Thus, cross-linking/MS should be added to the arsenal of structural methods available for studying nuclear receptors. PMID:26992147

  16. Impact of protecting ligands on surface structure and antibacterial activity of silver nanoparticles.

    PubMed

    Padmos, J Daniel; Boudreau, Robert T M; Weaver, Donald F; Zhang, Peng

    2015-03-31

    Silver nanoparticles (Ag NPs) have attracted much attention in the past decade because of their unique physicochemical properties and notable antibacterial activities. In particular, thiol-protected Ag NPs have come to the forefront of metal nanoparticle studies, as they have been shown to possess high stability and interesting structure-property relationships. However, a clear correlation between thiol-protecting ligands, the resulting Ag NP surface structure, and their antibacterial properties has yet to be determined. Here, a multielement (Ag and S), multi-edge (Ag K-edge, Ag L3-edge, S K-edge) X-ray absorption spectroscopy (XAS) methodology was used to identify the structure and composition of Ag NPs protected with cysteine. XAS characterization was carried out on similar-sized Ag NPs protected with poly(vinylpyrrolidone) (PVP), in order to provide a valid comparison of the ligand effect on surface structure. The PVP-Ag NPs showed a metallic Ag surface and composition, consistent with metal NPs protected by weak protecting ligands. On the other hand, the Cys-Ag NPs exhibited a distinct surface shell of silver sulfide, which is remarkably different than previously studied Cys-Ag NPs. The minimum inhibitory concentration (MIC) of both types of Ag NPs against Gram-positive (+) and Gram-negative (-) bacteria were tested, including Staphylococcus aureus (+), Escherichia coli (-), and Pseudomonas aeruginosa (-). It was found that the MICs of the Cys-Ag NPs were significantly lower than the PVP-Ag NPs for each bacteria, implicating the influence of the sulfidized surface structure. Overall, this work shows the effect of ligand on the surface structure of Ag NPs, as well as the importance of surface structure in controlling antibacterial activity.

  17. FEF-Controlled Alpha Delay Activity Precedes Stimulus-Induced Gamma-Band Activity in Visual Cortex.

    PubMed

    Popov, Tzvetan; Kastner, Sabine; Jensen, Ole

    2017-04-12

    Recent findings in the visual system of nonhuman primates have demonstrated an important role of gamma-band activity (40-100 Hz) in the feedforward flow of sensory information, whereas feedback control appears to be established dynamically by oscillations in the alpha (8-13 Hz) and beta (13-18 Hz) bands (van Kerkoerle et al., 2014; Bastos et al., 2015). It is not clear, however, how alpha oscillations are controlled and how they interact with the flow of visual information mediated by gamma-band activity. Using noninvasive human MEG recordings in subjects performing a visuospatial attention task, we show that fluctuations in alpha power during a delay period in a spatial attention task preceded subsequent stimulus-driven gamma-band activity. Importantly, these interactions correlated with behavioral performance. Using Granger analysis, we further show that the right frontal-eye field (rFEF) exerted feedback control of the visual alpha oscillations. Our findings suggest that alpha oscillations controlled by the FEF route cortical information flow by modulating gamma-band activity.SIGNIFICANCE STATEMENT Visual perception relies on a feedforward flow of information from sensory regions, which is modulated by a feedback drive. We have identified the neuronal dynamics supporting integration of the feedforward and feedback information. Alpha oscillations in early visual regions reflect feedback control when spatial attention is allocated and this control is exercised by the right frontal eye field. Importantly, the alpha-band activity predicted both performance and activity in the gamma band. In particular, gamma activity was modulated by the phase of the alpha oscillations. These findings provide novel insight into how the brain operates as a network and suggest that the integration of feedforward and feedback information is implemented by cross-frequency interactions between slow and fast neuronal oscillations.

  18. Synthesis, spectroscopic, coordination and biological activities of some organometallic complexes derived from thio-Schiff base ligands

    PubMed Central

    Abou-Hussein, Azza A.; Linert, Wolfgang

    2014-01-01

    Two series of mono- and binuclear complexes cyclic or acyclic thio-ferocine Schiff base ligands, derived from the condensation of 2-aminobenzenthiol (L) with monoacetyl ferrocene in the molar ratio 1:1 or in the molar ratio 1:2 for diacetyl ferocine have been prepared. The condensation reactions yield the corresponding Schiff Base ligands, HLa-Maf and H2Lb-Daf. The chelation of the ligands to metal ions occurs through the sulfur of the thiol group as well as the nitrogen atoms of the azomethine group of the ligands. HLa-Maf acts as monobasic bidentate or dibasic tetradentate, while H2Lb-Daf behaves as twice negatively cargend tetradentate ligand. The structures of these ligands were elucidated by elemental analysis, infrared, ultraviolet–visible spectra, as well as 1H NMR spectra. Reactions of the Schiff bases ligands with ruthenium(III), oxovanadium(IV) and dioxouranium(VI) afforded the corresponding transition metal complexes. The properties of the newly prepared complexes were analyse by elemental analyses, infrared, electronic spectra, 1H NMR as well as the magnetic susceptibility and conductivity measurement. The metal complexes exhibits different geometrical arrangements such as octahedral and square pyramidal coordination. Schiff base ligands and their metal complexes were tested against two pathogenic bacteria as Gram-positive and Gram-negative bacteria as well as one kind of fungi to study their biological activity. All the complexes exhibit antibacterial and antifungal activities against these organisms. PMID:24070648

  19. Synthesis, spectroscopic, coordination and biological activities of some organometallic complexes derived from thio-Schiff base ligands

    NASA Astrophysics Data System (ADS)

    Abou-Hussein, Azza A.; Linert, Wolfgang

    2014-01-01

    Two series of mono- and binuclear complexes cyclic or acyclic thio-ferocine Schiff base ligands, derived from the condensation of 2-aminobenzenthiol (L) with monoacetyl ferrocene in the molar ratio 1:1 or in the molar ratio 1:2 for diacetyl ferocine have been prepared. The condensation reactions yield the corresponding Schiff Base ligands, HLa-Maf and H2Lb-Daf. The chelation of the ligands to metal ions occurs through the sulfur of the thiol group as well as the nitrogen atoms of the azomethine group of the ligands. HLa-Maf acts as monobasic bidentate or dibasic tetradentate, while H2Lb-Daf behaves as twice negatively cargend tetradentate ligand. The structures of these ligands were elucidated by elemental analysis, infrared, ultraviolet-visible spectra, as well as 1H NMR spectra. Reactions of the Schiff bases ligands with ruthenium(III), oxovanadium(IV) and dioxouranium(VI) afforded the corresponding transition metal complexes. The properties of the newly prepared complexes were analyse by elemental analyses, infrared, electronic spectra, 1H NMR as well as the magnetic susceptibility and conductivity measurement. The metal complexes exhibits different geometrical arrangements such as octahedral and square pyramidal coordination. Schiff base ligands and their metal complexes were tested against two pathogenic bacteria as Gram-positive and Gram-negative bacteria as well as one kind of fungi to study their biological activity. All the complexes exhibit antibacterial and antifungal activities against these organisms.

  20. Impairments in Background and Event-Related Alpha-Band Oscillatory Activity in Patients with Schizophrenia

    PubMed Central

    Abeles, Ilana Y.; Gomez-Ramirez, Manuel

    2014-01-01

    Studies show that patients with schizophrenia exhibit impaired responses to sensory stimuli, especially at the early stages of neural processing. In particular, patients’ alpha-band (8–14 Hz) event-related desynchronization (ERD) and visual P1 event-related potential (ERP) component tend to be significantly reduced, with P1 ERP deficits greater for visual stimuli biased towards the magnocellular system. In healthy controls, studies show that pre-stimulus alpha (background alpha) plays a pivotal role in sensory processing and behavior, largely by shaping the neural responses to incoming stimuli. Here, we address whether patients’ ERD and P1 deficits stem from impairments in pre-stimulus alpha mechanisms. To address this question we recorded electrophysiological activity in patients with schizophrenia and healthy controls while they engaged in a visual discrimination task with low, medium, and high contrast stimuli. The results revealed a significant decrease in patients’ ERDs, which was largely driven by reductions in pre-stimulus alpha. These reductions were most prominent in right-hemispheric areas. We also observed a systematic relationship between pre-stimulus alpha and the P1 component across different contrast levels. However, this relationship was only observed in healthy controls. Taken together, these findings highlight a substantial anomaly in patients’ amplitude-based alpha background activity over visual areas. The results provide further support that pre-stimulus alpha activity plays an active role in perception by modulating the neural responses to incoming sensory inputs, a mechanism that seems to be compromised in schizophrenia. PMID:24646909

  1. Monitoring gross alpha and beta activity in liquids by using ZnS(Ag) scintillation detectors

    SciTech Connect

    Stevanato, L.; Cester, D.; Filippi, D.; Lunardon, M.; Mistura, G.; Moretto, S.; Viesti, G.; Badocco, D.; Pastore, P.; Romanini, F.

    2015-07-01

    In this work the possibility of monitoring gross alpha and beta activity in liquids using EJ-444 was investigated. Specific tests were carried out to determine the change of the detector properties in water tests. Possible protecting coating is also proposed and tested. Alpha/beta real-time monitoring in liquids is a goal of the EU project TAWARA{sub R}TM. (authors)

  2. Vitamin C suppresses TNF alpha-induced NF kappa B activation by inhibiting I kappa B alpha phosphorylation.

    PubMed

    Cárcamo, Juan M; Pedraza, Alicia; Bórquez-Ojeda, Oriana; Golde, David W

    2002-10-29

    Extracellular stimuli signal for activation of the transcription factor NFkappaB, leading to gene expression regulating processes involved in immune responses, inflammation, and cell survival. Tumor necrosis factor-alpha (TNFalpha) activates NFkappaB via a well-defined kinase pathways involving NFkappaB-inducing kinase (NIK), which activates downstream multisubunit IkappaB kinases (IKK). IKK in turn phosphorylates IkappaB, the central regulator of NFkappaB function. We found that intracellular vitamin C inhibits TNFalpha-induced activation of NFkappaB in human cell lines (HeLa, monocytic U937, myeloid leukemia HL-60, and breast MCF7) and primary endothelial cells (HUVEC) in a dose-dependent manner. Vitamin C is an important antioxidant, and most cells accumulate ascorbic acid (AA) intracellularly by transporting the oxidized form of the vitamin, dehydroascorbic acid (DHA). Because ascorbic acid is a strong pro-oxidant in the presence of transition metals in vitro, we loaded cells with vitamin C by incubating them with DHA. Vitamin C-loaded cells showed significantly decreased TNFalpha-induced nuclear translocation of NFkappaB, NFkappaB-dependent reporter transcription, and IkappaBalpha phosphorylation. Our data point to a mechanism of vitamin C suppression of NFkappaB activation by inhibiting TNFalpha-induced activation of NIK and IKKbeta kinases independent of p38 MAP kinase. These results suggest that intracellular vitamin C can influence inflammatory, neoplastic, and apoptotic processes via inhibition of NFkappaB activation.

  3. Prothymosin-alpha preconditioning activates TLR4-TRIF signaling to induce protection of ischemic retina.

    PubMed

    Halder, Sebok Kumar; Matsunaga, Hayato; Ishii, Ken J; Ueda, Hiroshi

    2015-12-01

    Prothymosin-alpha protects the brain and retina from ischemic damage. Although prothymosin-alpha contributes to toll-like receptor (TLR4)-mediated immnunopotentiation against viral infection, the beneficial effects of prothymosin-alpha-TLR4 signaling in protecting against ischemia remain to be elucidated. In this study, intravitreal administration of prothymosin-alpha 48 h before induction of retinal ischemia prevented retinal cellular damage as evaluated by histology, and retinal functional deficits as evaluated by electroretinography. Prothymosin-alpha preconditioning completely prevented the ischemia-induced loss of ganglion cells with partial survival of bipolar and photoreceptor cells, but not amacrine cells, in immunohistochemistry experiments. Prothymosin-alpha treatment in the absence of ischemia caused mild activation, proliferation, and migration of retinal microglia, whereas the ischemia-induced microglial activation was inhibited by prothymosin-alpha preconditioning. All these preventive effects of prothymosin-alpha preconditioning were abolished in TLR4 knock-out mice and by pre-treatments with anti-TLR4 antibodies or minocycline, a microglial inhibitor. Prothymosin-alpha preconditioning inhibited the retinal ischemia-induced up-regulation of TLR4-related injury genes, and increased expression of TLR4-related protective genes. Furthermore, the prothymosin-alpha preconditioning-induced prevention of retinal ischemic damage was abolished in TIR-domain-containing adapter-inducing interferon-β knock-out mice, but not in myeloid differentiation primary response gene 88 knock-out mice. Taken together, the results of this study suggest that prothymosin-alpha preconditioning selectively drives TLR4-TIR-domain-containing adapter-inducing interferon-β signaling and microglia in the prevention of retinal ischemic damage. We propose the following mechanism for prothymosin-alpha (ProTα) preconditioning-induced retinal prevention against ischemia: Pro

  4. Inhibition of neutrophil activation by alpha1-acid glycoprotein.

    PubMed Central

    Costello, M J; Gewurz, H; Siegel, J N

    1984-01-01

    We report that alpha1-acid glycoprotein (AAG), a naturally occurring human plasma protein and acute phase reactant of uncertain biological function, inhibits human neutrophil aggregation and superoxide anion generation induced by a variety of stimuli including zymosan treated serum, formyl-methionyl-leucyl-phenylalanine and phorbol myristate acetate. Inhibition was transient, directly proportional to the glycoprotein concentration and inversely proportional to the concentration of the stimulus added. Desialyzation, resulting in the removal of a substantial portion of the molecule's negative charge, did not alter the effectiveness of AAG. Removal of the penultimate galactose residues from desialyzed AAG resulted in a slight but significant reversal of inhibition, suggesting that the heteropolysaccharide units of AAG may be important for inhibition of cellular function. We therefore suggest that the acute phase glycoprotein AAG may be a significant modulator of neutrophil as well as platelet and lymphocyte function during inflammation. PMID:6321072

  5. Identification of a novel inducible cell-surface ligand of CD5 on activated lymphocytes

    PubMed Central

    1996-01-01

    CD5 is a 67-kD glycoprotein that is expressed on most T lymphocytes and on a subset of mature B cells. Although its physiologic function is unknown, several lines of evidence suggest that CD5 may play a role in the regulation of T cell activation and in T cell-antigen presenting cell interactions. Using a CD5-immunoglobulin fusion protein (CD5Rg, for receptorglobulin) we have uncovered a new CD5 ligand (CD5L) expressed on the surface of activated splenocytes. Stimulation of murine splenocytes with anti-CD3 and anti-CD28 antibodies induce transient expression of CD5L on B lymphocytes that lasts for approximately 72 h. Binding of CD5Rg to activated splenocytes is trypsin resistant and independent of divalent cations. However, it is pronase sensitive and dependent on N-linked glycosylation of CD5, since treatment of CD5Rg with PNGaseF on N-glycanase completely abrogates its ability to bind activated splenocytes. It addition to splenocytes, CD5L is expressed on activated murine T cell clones. Immunoprecipitation, antibody, and recombinant protein blocking studies indicate that CD5L is distinct from CD72, which has been proposed to be a CD5 ligand. To determine whether CD5-CD5L interaction might play a role in vivo, we tested the effect of CD5Rg in a murine model of antibody-mediated membranous glomerulonephritis. Injection of CD5Rg was found to abrogate development of the disease. Taken together, our results help identify a novel ligand of CD5 and propose a role for CD5 in the regulation of immune responses. PMID:9064341

  6. Peroxisome Proliferator-Activated Receptor-γ and Its Ligands in the Treatment of Tumors in the Nervous System.

    PubMed

    Shen, Yun; Lu, Yun; Yu, Fang; Zhu, Chuntie; Wang, Hua; Wang, Jing

    2016-01-01

    The peroxisome proliferator-activated receptor -γ (PPARγ) has been identified in a wide range of cancers, including brain, breast, colon, stomach and lung cancers. It belongs to the thyroid/ steroid hormone receptors superfamily. Binding with their special ligands, PPARγ plays important roles in regulating transcription of their target genes. PPARγ activation suppresses the growth of the tumor cells, implicating the anti-tumor potential of PPARγ ligand. Tumors in the nervous system are among the most devastating cancers. This review highlights key advances in understanding the effects of PPARγ ligands in the treatment of tumors in the nervous system.

  7. Synthesis and Antiproliferative Activity of New Ruthenium Complexes with Ethacrynic-Acid-Modified Pyridine and Triphenylphosphine Ligands.

    PubMed

    Agonigi, Gabriele; Riedel, Tina; Zacchini, Stefano; Păunescu, Emilia; Pampaloni, Guido; Bartalucci, Niccolò; Dyson, Paul J; Marchetti, Fabio

    2015-07-06

    Pyridine- and phosphine-based ligands modified with ethacrynic acid (a broad acting glutathione transferase inhibitor) were prepared and coordinated to ruthenium(II)-arene complexes and to a ruthenium(III) NAMI-A type complex. All the compounds (ligands and complexes) were fully characterized by analytical and spectroscopic methods and, in one case, by single-crystal X-ray diffraction. The in vitro anticancer activity of the compounds was studied, with the compounds displaying moderate cytotoxicity toward the human ovarian cancer cell lines. All the complexes led to similar levels of residual GST activity in the different cell lines, irrespective of the stability of the Ru-ligand bond.

  8. Hyperglycemia-conditioned increase in alpha-2-macroglobulin in healthy normal subjects: a phenomenon correlated with deficient antithrombin III activity.

    PubMed

    Ceriello, A; Quatraro, A; Dello Russo, P; Marchi, E; Barbanti, M; Giugliano, D

    1989-01-01

    Induced hyperglycemia in normal subjects increases alpha 2-macroglobulin (alpha 2M) activity and alpha 2M concentration and reduces antithrombin III (ATIII) activity, while it does not affect ATIII plasma concentration. Hyperglycemia-determined variations in ATIII activity and alpha 2M molecules are correlated in an inverse and parallel fashion. A compensatory role for the increase in alpha 2M in the regulation of the coagulation system may be hypothesized. Moreover, these data provide evidence that hyperglycemia may decrease, directly, the biological function of some proteins and may influence the levels of some risk factors for the development of complications in diabetes.

  9. G-protein alpha-s and -12 subunits are involved in androgen-stimulated PI3K activation and androgen receptor transactivation in prostate cancer cells

    PubMed Central

    Liu, Jianjun; Youn, Hyewon; Yang, Jun; Du, Ningchao; Liu, Jihong; Liu, Hongwei; Li, Benyi

    2011-01-01

    BACKGROUND The androgen receptor (AR) is a ligand-dependent transcription factor that mediates androgenic hormone action in cells. We recently demonstrated the involvement of phosphoinositide 3-OH kinase (PI3K) p110beta in AR transactivation and gene expression. In this study, we determined the upstream signals that lead to PI3K/p110beta activation and AR transactivation after androgen stimulation. METHODS Human prostate cancer LAPC-4 and 22Rv1 cell lines were used for the experiments. AR transactivation was assessed using an androgen responsive element-driven luciferase (ARE-LUC) assay. Cell proliferation was examined using BrdU incorporation and MTT assays. Target genes were silenced using small interfering RNA (siRNA) approach. Gene expression was evaluated at the mRNA level (real-time RT-PCR) and protein level (Western blot). PI3K kinase activities were measured using immunoprecipitation-based in vitro kinase assay. The AR-DNA binding activity was determined using Chromatin-immunoprecipitation (ChIP) assay. RESULTS First, at the cellular plasma membrane, disrupting the integrity of caveolae microdomain with methyl-β- cyclodextrin (M-β-CD) abolished androgen-induced AR transactivation and gene expression. Then, knocking down caveolae structural proteins caveolin-1 or -2 with the gene-specific siRNAs significantly reduced androgen-induced AR transactivation. Next, silencing Gαs and Gα12 genes but not other G-proteins blocked androgen-induced AR transactivation and cell proliferation. Consistently, overexpression of Gαs or Gα12 active mutants enhanced androgen-induced AR transactivation, of which Gαs active mutant sensitized the AR to castration-level of androgen (R1881). Most interestingly, knocking down Gαs but not Gα12 subunit significantly suppressed androgen-stimulated PI3K p110beta activation. However, chromatin-immunoprecipitation (ChIP) analysis revealed that both Gαs or Gα12 subunits are involved in androgen-induced AR interaction with the AR

  10. Activation of syndecan-1 ectodomain shedding by Staphylococcus aureus alpha-toxin and beta-toxin.

    PubMed

    Park, Pyong Woo; Foster, Timothy J; Nishi, Eiichiro; Duncan, Sheila J; Klagsbrun, Michael; Chen, Ye

    2004-01-02

    Exploitation of host components by microbes to promote their survival in the hostile host environment has been a recurring theme in recent years. Available data indicate that bacterial pathogens activate ectodomain shedding of host cell surface molecules to enhance their virulence. We reported previously that several major bacterial pathogens activate ectodomain shedding of syndecan-1, the major heparan sulfate proteoglycan of epithelial cells. Here we define the molecular basis of how Staphylococcus aureus activates syndecan-1 shedding. We screened mutant S. aureus strains devoid of various toxin and protease genes and found that only strains lacking both alpha-toxin and beta-toxin genes do not stimulate shedding. Mutations in the agr global regulatory locus, which positively regulates expression of alpha- and beta-toxins and other exoproteins, also abrogated the capacity to stimulate syndecan-1 shedding. Furthermore, purified S. aureus alpha- and beta-toxins, but not enterotoxin A and toxic shock syndrome toxin-1, rapidly potentiated shedding in a concentration-dependent manner. These results establish that S. aureus activates syndecan-1 ectodomain shedding via its two virulence factors, alpha- and beta-toxins. Toxin-activated shedding was also selectively inhibited by antagonists of the host cell shedding mechanism, indicating that alpha- and beta-toxins shed syndecan-1 ectodomains through stimulation of the host cell's shedding machinery. Interestingly, beta-toxin, but not alpha-toxin, also enhanced ectodomain shedding of syndecan-4 and heparin-binding epidermal growth factor. Because shedding of these ectodomains has been implicated in promoting bacterial pathogenesis, activation of ectodomain shedding by alpha-toxin and beta-toxin may be a previously unknown virulence mechanism of S. aureus.

  11. Phenylpiperazinylalkylamino substituted pyridazinones as potent alpha(1) adrenoceptor antagonists.

    PubMed

    Barlocco, D; Cignarella, G; Piaz, V D; Giovannoni, M P; De Benedetti, P G; Fanelli, F; Montesano, F; Poggesi, E; Leonardi, A

    2001-07-19

    QSAR models have been used for designing a series of compounds characterized by a N-phenylpiperazinylalkylamino moiety linked to substituted pyridazinones, which have been synthesized. Measurements of the binding affinities of the new compounds toward the alpha(1a)-, alpha(1b)-, and alpha(1d)-AR cloned subtypes as well as the 5-HT(1A) receptor have been done validating, at least in part, the estimations of the theoretical models. This study provides insight into the structure activity relationships of the alpha(1)-ARs ligands and their alpha(1)-AR/5-HT(1A) selectivity.

  12. Dynamics of the Ligand Binding Domain Layer during AMPA Receptor Activation

    PubMed Central

    Baranovic, Jelena; Chebli, Miriam; Salazar, Hector; Carbone, Anna L.; Faelber, Katja; Lau, Albert Y.; Daumke, Oliver; Plested, Andrew J.R.

    2016-01-01

    Ionotropic glutamate receptors are postsynaptic tetrameric ligand-gated channels whose activity mediates fast excitatory transmission. Glutamate binding to clamshell-shaped ligand binding domains (LBDs) triggers opening of the integral ion channel, but how the four LBDs orchestrate receptor activation is unknown. Here, we present a high-resolution x-ray crystal structure displaying two tetrameric LBD arrangements fully bound to glutamate. Using a series of engineered metal ion trapping mutants, we showed that the more compact of the two assemblies corresponds to an arrangement populated during activation of full-length receptors. State-dependent cross-linking of the mutants identified zinc bridges between the canonical active LBD dimers that formed when the tetramer was either fully or partially bound by glutamate. These bridges also stabilized the resting state, consistent with the recently published full-length apo structure. Our results provide insight into the activation mechanism of glutamate receptors and the complex conformational space that the LBD layer can sample. PMID:26910426

  13. Ligand-induced changes in the location of actin, myosin, 95K (alpha- actinin), and 120K protein in amebae of Dictyostelium discoideum

    PubMed Central

    1985-01-01

    In this study we investigated concanavalin A (Con A) induced changes in the locations of actin, myosin, 120K, and 95K (alpha-actinin) to determine the extent to which actin and myosin are reorganized during capping and the roles that 120K and 95K might play in this reorganization. We observed the location of each protein by indirect immunofluorescence using affinity purified antibodies. Four morphological states were distinguished in vegetative Dictyostelium amebae: ameboid cells before Con A binding, patched cells, capped cells, and ameboid cells with caps. The location of each protein was distinct in ameboid cells both before and after capping Actin and 120K were found in the cell cortex usually associated with surface projections, and myosin and 95K were diffusely distributed. Myosin was excluded from surface projections in ameboid cells. During patching, all four proteins were localized below Con A patches. During capping, actin, myosin, and 95K protein moved with the Con A patches into the cap whereas 120K protein was excluded from the cap. During the late stages of cap formation actin and myosin were progressively lost from the cap, and 120K became concentrated in new actin-filled projections that formed away from the cap. However, 95K remained tightly associated with the cap. Poisoning cells with sodium azide inhibited capping but not patching of ligand. In azide-poisoned cells, myosin and 95K did not co-patch with Con A, whereas copatching of 120K and actin with Con A occurred as usual. Our results support the hypothesis that capping is an actomyosin-mediated motile event that involves a sliding interaction between actin filaments, which are anchored through the membrane to ligand patches, and myosin in the cortex. They are also consistent with a role for 120K in the formation of surface projections by promoting growth and/or cross-linking of actin filaments within projections, and with a role for 95K in regulating actomyosin-mediated contractility, earlier

  14. Specific alpha-galactosidase inhibitors, N-methylcalystegines--structure/activity relationships of calystegines from Lycium chinense.

    PubMed

    Asano, N; Kato, A; Miyauchi, M; Kizu, H; Tomimori, T; Matsui, K; Nash, R J; Molyneux, R J

    1997-09-01

    An examination of the roots of Lycium chinense (Solanaceae) has resulted in the discovery of 14 calystegines, a cycloheptane bearing an amino group and three hydroxyl groups, and two polyhydroxylated piperidine alkaloids. Calystegines A7 and B5, in addition to the previously known calystegines A3, A5, A6, B1, B2, B3, B4, C1, C2 and N1, were isolated and determined as 1alpha,2beta,4alpha-trihydroxy-nortropane and 1alpha,2alpha,4alpha,7alpha-tetrahydroxy-nort ropane, respectively. L. chinense also had two polyhydroxytropanes bearing a methyl group on the nitrogen atom, unlike the previously reported nortropane alkaloids. They were established as N-methylcalystegines B2 and C1, and their N-methyl groups were found to be axially oriented from NOE experiments. 1Beta-amino-3beta,4beta,5alpha-trihydroxycyclohepta ne was also present in L. chinense and may be a biosynthetic precursor of the calystegines that occur in this plant. Two polyhydroxypiperidine alkaloids, fagomine and 6-deoxyfagomine, were isolated. Calystegine B2 is a potent competitive inhibitor of almond beta-glucosidase (Ki = 1.9 microM) and coffee bean alpha-galactosidase (Ki = 0.86 microM), while N-methylcalystegine B2 was a more potent competitive inhibitor of the latter enzyme (Ki = 0.47 microM) than the parent compound but showed a marked lack of inhibitory activities towards most other glycosidases. Since this compound is a very specific inhibitor of alpha-galactosidase and inhibits rat liver lysosomal alpha-galactosidase with a Ki of 1.8 microM, it may provide a useful experimental model for the lysosomal storage disorder, Fabry's disease. The addition of a hydroxyl group at C6exo, as in calystegines B1 and C1, enhances the inhibitory potential towards beta-glucosidase and beta-galactosidase but markedly lowers or abolishes inhibition towards alpha-galactosidase. Hence, the N-methylation of calystegine C1 did not enhance its inhibition of alpha-galactosidase. The chemical N-methylation of calystegines

  15. Occipital alpha activity during stimulus processing gates the information flow to object-selective cortex.

    PubMed

    Zumer, Johanna M; Scheeringa, René; Schoffelen, Jan-Mathijs; Norris, David G; Jensen, Ole

    2014-10-01

    Given the limited processing capabilities of the sensory system, it is essential that attended information is gated to downstream areas, whereas unattended information is blocked. While it has been proposed that alpha band (8-13 Hz) activity serves to route information to downstream regions by inhibiting neuronal processing in task-irrelevant regions, this hypothesis remains untested. Here we investigate how neuronal oscillations detected by electroencephalography in visual areas during working memory encoding serve to gate information reflected in the simultaneously recorded blood-oxygenation-level-dependent (BOLD) signals recorded by functional magnetic resonance imaging in downstream ventral regions. We used a paradigm in which 16 participants were presented with faces and landscapes in the right and left hemifields; one hemifield was attended and the other unattended. We observed that decreased alpha power contralateral to the attended object predicted the BOLD signal representing the attended object in ventral object-selective regions. Furthermore, increased alpha power ipsilateral to the attended object predicted a decrease in the BOLD signal representing the unattended object. We also found that the BOLD signal in the dorsal attention network inversely correlated with visual alpha power. This is the first demonstration, to our knowledge, that oscillations in the alpha band are implicated in the gating of information from the visual cortex to the ventral stream, as reflected in the representationally specific BOLD signal. This link of sensory alpha to downstream activity provides a neurophysiological substrate for the mechanism of selective attention during stimulus processing, which not only boosts the attended information but also suppresses distraction. Although previous studies have shown a relation between the BOLD signal from the dorsal attention network and the alpha band at rest, we demonstrate such a relation during a visuospatial task, indicating

  16. Decreased electroencephalogram alpha band [8-13 Hz] power in amyotrophic lateral sclerosis patients: a study of alpha activity in an awake relaxed state.

    PubMed

    Santhosh, Jayashree; Bhatia, Manvir; Sahu, Shweta; Anand, Sneh

    2005-03-01

    An attempt was made to quantitatively analyze the alpha activity in the awake relaxed state of Amyotrophic Lateral Sclerosis (ALS) patients and was compared with normals. ALS patients showed significantly low amplitude with a corresponding alpha band (8-13 Hz) power reduction, in both hemispheres though the change was more prominent in the left hemisphere. A review of the literature revealed no studies done on alpha oscillations in ALS patients; hence the results may have important implications for the interpretation of resting state brain activities.

  17. The signal peptide of the IgE receptor alpha-chain prevents surface expression of an immunoreceptor tyrosine-based activation motif-free receptor pool.

    PubMed

    Platzer, Barbara; Fiebiger, Edda

    2010-05-14

    The high affinity receptor for IgE, Fc epsilon receptor I (FcepsilonRI), is an activating immune receptor and key regulator of allergy. Antigen-mediated cross-linking of IgE-loaded FcepsilonRI alpha-chains induces cell activation via immunoreceptor tyrosine-based activation motifs in associated signaling subunits, such as FcepsilonRI gamma-chains. Here we show that the human FcepsilonRI alpha-chain can efficiently reach the cell surface by itself as an IgE-binding receptor in the absence of associated signaling subunits when the endogenous signal peptide is swapped for that of murine major histocompatibility complex class-I H2-K(b). This single-chain isoform of FcepsilonRI exited the endoplasmic reticulum (ER), trafficked to the Golgi and, subsequently, trafficked to the cell surface. Mutational analysis showed that the signal peptide regulates surface expression in concert with other described ER retention signals of FcepsilonRI-alpha. Once the FcepsilonRI alpha-chain reached the cell surface by itself, it formed a ligand-binding receptor that stabilized upon IgE contact. Independently of the FcepsilonRI gamma-chain, this single-chain FcepsilonRI was internalized after receptor cross-linking and trafficked into a LAMP-1-positive lysosomal compartment like multimeric FcepsilonRI. These data suggest that the single-chain isoform is capable of shuttling IgE-antigen complexes into antigen loading compartments, which plays an important physiologic role in the initiation of immune responses toward allergens. We propose that, in addition to cytosolic and transmembrane ER retention signals, the FcepsilonRI alpha-chain signal peptide contains a negative regulatory signal that prevents expression of an immunoreceptor tyrosine-based activation motif-free IgE receptor pool, which would fail to induce cell activation.

  18. New Ru(II) Complex for Dual Activity: Photoinduced Ligand Release and (1)O2 Production.

    PubMed

    Loftus, Lauren M; White, Jessica K; Albani, Bryan A; Kohler, Lars; Kodanko, Jeremy J; Thummel, Randolph P; Dunbar, Kim R; Turro, Claudia

    2016-03-07

    The new complex [Ru(pydppn)(biq)(py)](2+) (1) undergoes both py photodissociation in CH3CN with Φ500 =0.0070(4) and (1)O2 production with ΦΔ =0.75(7) in CH3 OH from a long-lived (3) ππ* state centered on the pydppn ligand (pydppn=3-(pyrid-2-yl)benzo[i]dipyrido[3,2-a:2',3'-c]phenazine; biq = 2,2'-biquinoline; py=pyridine). This represents an order of magnitude decrease in the Φ500 compared to the previously reported model compound [Ru(tpy)(biq)(py)](2+) (3) (tpy=2,2':6',2''-terpyridine) that undergoes only ligand exchange. The effect on the quantum yields by the addition of a second deactivation pathway through the low-lying (3) ππ* state necessary for dual reactivity was investigated using ultrafast and nanosecond transient absorption spectroscopy, revealing a significantly shorter (3) MLCT lifetime in 1 relative to that of the model complex 3. Due to the structural similarities between the two compounds, the lower values of Φ500 and ΦΔ compared to that of [Ru(pydppn)(bpy)(py)](2+) (2) (bpy=2,2'-bipyridine) are attributed to a competitive excited state population between the (3) LF states involved in ligand dissociation and the long-lived (3) ππ* state in 1. Complex 1 represents a model compound for dual activity that may be applied to photochemotherapy.

  19. Modulation of Opioid Receptor Ligand Affinity and Efficacy Using Active and Inactive State Receptor Models

    PubMed Central

    Anand, Jessica P.; Purington, Lauren C.; Pogozheva, Irina D.; Traynor, John R.; Mosberg, Henry I.

    2012-01-01

    Mu opioid receptor (MOR) agonists are widely used for the treatment of pain; however chronic use results in the development of tolerance and dependence. It has been demonstrated that co-administration of a MOR agonist with a delta opioid receptor (DOR) antagonist maintains the analgesia associated with MOR agonists, but with reduced negative side effects. Using our newly refined opioid receptor models for structure-based ligand design, we have synthesized several pentapeptides with tailored affinity and efficacy profiles. In particular, we have obtained pentapeptides 8, Tyr-c(S-S)[DCys-1Nal-Nle-Cys]NH2, and 12, Tyr-c(S-S)[DCys-1Nal-Nle-Cys]OH, which demonstrates high affinity and full agonist behavior at MOR, high affinity but very low efficacy for DOR, and minimal affinity for the kappa opioid receptor (KOR). Functional properties of these peptides as MOR agonists/DOR antagonists lacking undesired KOR activity make them promising candidates for future in vivo studies of MOR/DOR interactions. Subtle structural variation of 12, by substituting D-Cys5 for L-Cys5, generated analog 13 which maintains low nanomolar MOR and DOR affinity, but which displays no efficacy at either receptor. These results demonstrate the power and utility of accurate receptor models for structure-based ligand design, as well as the profound sensitivity of ligand function on its structure. PMID:22882801

  20. Synthesis and Activity of Dafachronic Acid Ligands for the C. elegans DAF-12 Nuclear Hormone Receptor

    PubMed Central

    Sharma, Kamalesh K.; Wang, Zhu; Motola, Daniel L.; Cummins, Carolyn L.; Mangelsdorf, David J.; Auchus, Richard J.

    2009-01-01

    The nuclear hormone receptor DAF-12 from Caenorhabditis elegans is activated by dafachronic acids, which derive from sterols upon oxidation by DAF-9, a cytochrome P450. DAF-12 activation is a critical checkpoint in C. elegans for acquisition of reproductive competence and for entry into adulthood rather than dauer diapause. Previous studies implicated the (25S)-Δ7-dafachronic acid isomer as the most potent compound, but the (25S)-Δ4-isomer was also identified as an activator of DAF-12. To explore the tolerance of DAF-12 for structural variations in the ligand and to enable further studies requiring large amounts of ligands for DAF-12 and homologs in other nematodes, we synthesized (25R)- and (25S)-isomers of five dafachronic acids differing in A/B-ring configurations. Both the (25S)- and (25R)-Δ7-dafachronic acids are potent transcriptional activators in a Gal4-transactivation assay using HEK-293 cells, with EC50 values of 23 and 33 nm, respectively, as are (25S)- and (25R)-Δ4-dafachronic acids, with EC50 values of 23 and 66 nm, respectively. The (25S)- and (25R)-Δ5-isomers were much less potent, with EC50 values approaching 1000 nm, and saturated 5α- and 5β-dafachronic acids showed mostly intermediate potencies. Rescue assays using daf- 9-null mutants confirmed the results from transactivation experiments, but this in vivo assay accentuated the greater potencies of the (25S)-epimers, particularly for the (25S)-Δ7-isomer. We conclude that DAF-12 accommodates a large range of structural variation in ligand geometry, but (25S)-Δ7-dafachronic acid is the most potent and probably biologically relevant isomer. Potency derives more from the A/B-ring configuration than from the stereochemistry at C-25. PMID:19196833

  1. Threonine-497 is a critical site for permissive activation of protein kinase C alpha.

    PubMed

    Cazaubon, S; Bornancin, F; Parker, P J

    1994-07-15

    Phosphorylation of the region containing Thr-494, Thr-495 and Thr-497, present in the catalytic domain of protein kinase C alpha (PKC alpha), is a preliminary event necessary for subsequent PKC activation [Cazaubon and Parker (1993) J. Biol. Chem. 268, 17559-17563]. To define the essential residues in this region, various combinations of alanine substitutions for threonine residues 494, 495 and 497 have been tested. These mutations yielded expressed polypeptides of 76 and 80 kDa in ratios that vary from 100% 80 kDa (wild-type kinase, active) to 100% 76 kDa (AAA mutant, inactive) with the hierarchy being wild-type PKC alpha (TTT), ATT, AAT, TTA, ATA, TAA, AAA (the nomenclature indicates the location of alanine residues substituted for Thr-494, Thr-495 and Thr-497 respectively). Only the mutants retaining Thr-497 displayed kinase activity in vitro. The results overall indicate that Thr-497 plays the dominant role in the regulation of PKC alpha activity but that in the wild-type protein, Thr-495 may also be important. Consistent with the need for phosphorylation in this region, an intrinsically active PKC alpha could be produced in bacteria by exchanging Thr-495 for a glutamic acid residue.

  2. Interferon-. alpha. selectively activates the. beta. isoform of protein kinase C through phosphatidylcholine hydrolysis

    SciTech Connect

    Pfeffer, L.M.; Saltiel, A.R. ); Strulovici, B. )

    1990-09-01

    The early events that occur after interferon binds to discrete cell surface receptors remain largely unknown. Human leukocyte interferon (interferon-{alpha}) rapidly increases the binding of ({sup 3}H)phorbol dibutyrate to intact HeLa cells a measure of protein kinase C activation, and induces the selective translocation of the {beta} isoform of protein kinase C from the cytosol to the particulate fraction of HeLa cells. The subcellular distribution of the {alpha} and {epsilon} isoforms is unaffected by interferon-{alpha} treatment. Activation of protein kinase C by phorbol esters mimics the inhibitory action of interferon-{alpha} on HeLa cell proliferation and down-regulation of protein kinase C blocks the induction of antiviral activity by interferon-{alpha} in HeLa cells. Increased phosphatidylcholine hydrolysis and phosphorylcholine production is accompanied by diacylglycerol production in response to interferon. However, inositol phospholipid turnover and free intracellular calcium concentration are unaffected. These results suggest that the transient increase in diacylglycerol, resulting from phosphatidylcholine hydrolysis, may selectively activate the {beta} isoform of protein kinase C. Moreover, the activation of protein kinase C is a necessary element in interferon action on cells.

  3. AutoDock-GIST: Incorporating Thermodynamics of Active-Site Water into Scoring Function for Accurate Protein-Ligand Docking.

    PubMed

    Uehara, Shota; Tanaka, Shigenori

    2016-11-23

    Water plays a significant role in the binding process between protein and ligand. However, the thermodynamics of water molecules are often underestimated, or even ignored, in protein-ligand docking. Usually, the free energies of active-site water molecules are substantially different from those of waters in the bulk region. The binding of a ligand to a protein causes a displacement of these waters from an active site to bulk, and this displacement process substantially contributes to the free energy change of protein-ligand binding. The free energy of active-site water molecules can be calculated by grid inhomogeneous solvation theory (GIST), using molecular dynamics (MD) and the trajectory of a target protein and water molecules. Here, we show a case study of the combination of GIST and a docking program and discuss the effectiveness of the displacing gain of unfavorable water in protein-ligand docking. We combined the GIST-based desolvation function with the scoring function of AutoDock4, which is called AutoDock-GIST. The proposed scoring function was assessed employing 51 ligands of coagulation factor Xa (FXa), and results showed that both scoring accuracy and docking success rate were improved. We also evaluated virtual screening performance of AutoDock-GIST using FXa ligands in the directory of useful decoys-enhanced (DUD-E), thus finding that the displacing gain of unfavorable water is effective for a successful docking campaign.

  4. Interleukin-1 alpha has antiallodynic and antihyperalgesic activities in a rat neuropathic pain model.

    PubMed

    Mika, Joanna; Korostynski, Michal; Kaminska, Dorota; Wawrzczak-Bargiela, Agnieszka; Osikowicz, Maria; Makuch, Wioletta; Przewlocki, Ryszard; Przewlocka, Barbara

    2008-09-15

    Nerve injury and the consequent release of interleukins (ILs) are processes implicated in pain transmission. To study the potential role of IL-1 in the pathogenesis of allodynia and hyperalgesia, IL-1alpha and comparative IL-1beta, IL-6, and IL-10 mRNA levels were quantified using competitive RT-PCR of the lumbar spinal cord and dorsal root ganglia (DRG; L5-L6) three and seven days after chronic constriction injury (CCI) in rats. Microglial and astroglial activation in the ipsilateral spinal cord and DRG were observed after injury. In naive and CCI-exposed rats, IL-1alpha mRNA and protein were not detected in the spinal cord. IL-1beta and IL-6 mRNAs were strongly ipsilaterally elevated on day seven after CCI. In the ipsilateral DRG, IL-1alpha, IL-6, and IL-10 mRNA levels were increased on days three and seven; IL-1beta was elevated only on day seven. Western blot analysis revealed both the presence of IL-1alpha proteins (45 and 31 kDa) in the DRG and the down-regulation of these proteins after CCI. Intrathecal administration of IL-1alpha (50-500 ng) in naive rats did not influence nociceptive transmission, but IL-1beta (50-500 ng) induced hyperalgesia. In rats exposed to CCI, an IL-1alpha or IL-1 receptor antagonist dose-dependently attenuated symptoms of neuropathic pain; however, no effect of IL-1beta was observed. In sum, the first days after CCI showed a high abundance of IL-1alpha in the DRG. Together with the antiallodynic and antihyperalgesic effects observed after IL-1alpha administration, this finding indicates an important role for IL-1alpha in the development of neuropathic pain symptoms.

  5. Three dimensional structure of porcine pancreatic alpha-amylase at 2.9 A resolution. Role of calcium in structure and activity.

    PubMed Central

    Buisson, G; Duée, E; Haser, R; Payan, F

    1987-01-01

    The crystal structure of porcine pancreatic alpha-amylase (PPA) has been solved at 2.9 A resolution by X-ray crystallographic methods. The enzyme contains three domains. The larger, in the N-terminal part, consists of 330 amino acid residues. This central domain has the typical parallel-stranded alpha-beta barrel structure (alpha beta)8, already found in a number of other enzymes like triose phosphate isomerase and pyruvate kinase. The C-terminal domain forms a distinct globular unit where the chain folds into an eight-stranded antiparallel beta-barrel. The third domain lies between a beta-strand and a alpha-helix of the central domain, in a position similar to those found for domain B in triose phosphate isomerase and pyruvate kinase. It is essentially composed of antiparallel beta-sheets. The active site is located in a cleft within the N-terminal central domain, at the carboxy-end of the beta-strands of the (alpha beta)8 barrel. Binding of various substrate analogues to the enzyme suggests that the amino acid residues involved in the catalytic reaction are a pair of aspartic acids. A number of other residues surround the substrate and seem to participate in its binding via hydrogen bonds and hydrophobic interactions. The 'essential' calcium ion has been located near the active site region and between two domains, each of them providing two calcium ligands. On the basis of sequence comparisons this calcium binding site is suggested to be a common structural feature of all alpha-amylases. It represents a new type of calcium-protein interaction pattern.(ABSTRACT TRUNCATED AT 250 WORDS) Images Fig. 1. Fig. 5. Fig. 7. PMID:3502087

  6. Three dimensional structure of porcine pancreatic alpha-amylase at 2.9 A resolution. Role of calcium in structure and activity.

    PubMed

    Buisson, G; Duée, E; Haser, R; Payan, F

    1987-12-20

    The crystal structure of porcine pancreatic alpha-amylase (PPA) has been solved at 2.9 A resolution by X-ray crystallographic methods. The enzyme contains three domains. The larger, in the N-terminal part, consists of 330 amino acid residues. This central domain has the typical parallel-stranded alpha-beta barrel structure (alpha beta)8, already found in a number of other enzymes like triose phosphate isomerase and pyruvate kinase. The C-terminal domain forms a distinct globular unit where the chain folds into an eight-stranded antiparallel beta-barrel. The third domain lies between a beta-strand and a alpha-helix of the central domain, in a position similar to those found for domain B in triose phosphate isomerase and pyruvate kinase. It is essentially composed of antiparallel beta-sheets. The active site is located in a cleft within the N-terminal central domain, at the carboxy-end of the beta-strands of the (alpha beta)8 barrel. Binding of various substrate analogues to the enzyme suggests that the amino acid residues involved in the catalytic reaction are a pair of aspartic acids. A number of other residues surround the substrate and seem to participate in its binding via hydrogen bonds and hydrophobic interactions. The 'essential' calcium ion has been located near the active site region and between two domains, each of them providing two calcium ligands. On the basis of sequence comparisons this calcium binding site is suggested to be a common structural feature of all alpha-amylases. It represents a new type of calcium-protein interaction pattern.(ABSTRACT TRUNCATED AT 250 WORDS)

  7. Pre- and postsynaptic alpha-adrenoceptor blocking activity of raubasine in the rat vas deferens.

    PubMed Central

    Demichel, P.; Gomond, P.; Roquebert, J.

    1981-01-01

    1 The actions of raubasine, yohimbine and corynanthine at pre- and postsynaptic alpha-adrenoceptors were studied in the rat vas deferens. 2 Low frequency electrical stimulation of the isolated vas deferens of the rat produced regular contractions that were inhibited by low concentrations of clonidine. This inhibition was presynaptic in origin and involved alpha-adrenoceptors. 3 Presynaptic alpha-adrenoceptor antagonist activity was assessed by studying the effect of increasing antagonist concentrations on cumulative clonidine dose-response curves on the stimulated vas deferens. 4 Postsynaptic alpha-adrenoceptor antagonist activity in the isolated vas deferens was assessed by comparing control cumulative noradrenaline dose-response curves in the absence and in the presence of increasing concentrations of antagonists. 5 The results indicate that raubasine and corynanthine preferentially block postsynaptic alpha-adrenoceptors. Yohimbine is more potent in blocking pre- than postsynaptic alpha-adrenoceptors. The ration of the pre/postsynaptic potency declines in the order yohimbine less than raubasine less than corynanthine. PMID:6118191

  8. Drosophila Crumbs prevents ectopic Notch activation in developing wings by inhibiting ligand-independent endocytosis.

    PubMed

    Nemetschke, Linda; Knust, Elisabeth

    2016-12-01

    Many signalling components are apically restricted in epithelial cells, and receptor localisation and abundance is key for morphogenesis and tissue homeostasis. Hence, controlling apicobasal epithelial polarity is crucial for proper signalling. Notch is a ubiquitously expressed, apically localised receptor, which performs a plethora of functions; therefore, its activity has to be tightly regulated. Here, we show that Drosophila Crumbs, an evolutionarily conserved polarity determinant, prevents Notch endocytosis in developing wings through direct interaction between the two proteins. Notch endocytosis in the absence of Crumbs results in the activation of the ligand-independent, Deltex-dependent Notch signalling pathway, and does not require the ligands Delta and Serrate or γ-secretase activity. This function of Crumbs is not due to general defects in apicobasal polarity, as localisation of other apical proteins is unaffected. Our data reveal a mechanism to explain how Crumbs directly controls localisation and trafficking of the potent Notch receptor, and adds yet another aspect of Crumbs regulation in Notch pathway activity. Furthermore, our data highlight a close link between the apical determinant Crumbs, receptor trafficking and tissue homeostasis.

  9. Transcriptional activation by the thyroid hormone receptor through ligand-dependent receptor recruitment and chromatin remodelling.

    PubMed

    Grøntved, Lars; Waterfall, Joshua J; Kim, Dong Wook; Baek, Songjoon; Sung, Myong-Hee; Zhao, Li; Park, Jeong Won; Nielsen, Ronni; Walker, Robert L; Zhu, Yuelin J; Meltzer, Paul S; Hager, Gordon L; Cheng, Sheue-yann

    2015-04-28

    A bimodal switch model is widely used to describe transcriptional regulation by the thyroid hormone receptor (TR). In this model, the unliganded TR forms stable, chromatin-bound complexes with transcriptional co-repressors to repress transcription. Binding of hormone dissociates co-repressors and facilitates recruitment of co-activators to activate transcription. Here we show that in addition to hormone-independent TR occupancy, ChIP-seq against endogenous TR in mouse liver tissue demonstrates considerable hormone-induced TR recruitment to chromatin associated with chromatin remodelling and activated gene transcription. Genome-wide footprinting analysis using DNase-seq provides little evidence for TR footprints both in the absence and presence of hormone, suggesting that unliganded TR engagement with repressive complexes on chromatin is, similar to activating receptor complexes, a highly dynamic process. This dynamic and ligand-dependent interaction with chromatin is likely shared by all steroid hormone receptors regardless of their capacity to repress transcription in the absence of ligand.

  10. Two tridentate Schiff base ligands and their mononuclear cobalt (III) complexes: Synthesis, characterization, antibacterial and antifungal activities.

    PubMed

    Gungor, Elif; Celen, Selma; Azaz, Dilek; Kara, Hulya

    2012-08-01

    Two Schiff base ligands (HL1, HL2) and their Co(III) complexes, [Co(HL1)(L1)] (1) and [Co(HL2)(L2)] (2) [where HL1=2-((E)-(2-hydroxyethylimino)methyl)-4-chlorophenol and HL2=2-((E)-(2-hydroxyethylimino)methyl)-4-bromophenol] were synthesized and characterized using spectroscopic methods. The crystal structures of 1 and 2 have been re-determined by single crystal diffraction at 100K. The ligands and their Co(III) complexes were screened for antibacterial and antifungal activities by the disc diffusion, microdilution broth and single spore culture techniques. The antimicrobial activity of the Co(III) complexes and the free ligands exhibit antimicrobial properties and the Co(III) complexes show enhanced inhibitory activity compared with their parent ligand.

  11. In vitro screening of 200 pesticides for agonistic activity via mouse peroxisome proliferator-activated receptor (PPAR){alpha} and PPAR{gamma} and quantitative analysis of in vivo induction pathway

    SciTech Connect

    Takeuchi, Shinji; Matsuda, Tadashi; Kobayashi, Satoshi; Takahashi, Tetsuo; Kojima, Hiroyuki . E-mail: kojima@iph.pref.hokkaido.jp

    2006-12-15

    Peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors and key regulators of lipid metabolism and cell differentiation. However, there have been few studies reporting on a variety of environmental chemicals, which may interact with these receptors. In the present study, we characterized mouse PPAR{alpha} and PPAR{gamma} agonistic activities of 200 pesticides (29 organochlorines, 11 diphenyl ethers, 56 organophosphorus pesticides, 12 pyrethroids, 22 carbamates, 11 acid amides, 7 triazines, 8 ureas and 44 others) by in vitro reporter gene assays using CV-1 monkey kidney cells. Three of the 200 pesticides, diclofop-methyl, pyrethrins and imazalil, which have different chemical structures, showed PPAR{alpha}-mediated transcriptional activities in a dose-dependent manner. On the other hand, none of the 200 pesticides showed PPAR{gamma} agonistic activity at concentrations {<=} 10{sup -5} M. To investigate the in vivo effects of diclofop-methyl, pyrethrins and imazalil, we examined the gene expression of PPAR{alpha}-inducible cytochrome P450 4As (CYP4As) in the liver of female mice intraperitoneally injected with these compounds ({<=} 300 mg/kg). RT-PCR revealed significantly high induction levels of CYP4A10 and CYP4A14 mRNAs in diclofop-methyl- and pyrethrins-treated mice, whereas imazalil induced almost no gene expressions of CYP4As. In particular, diclofop-methyl induced as high levels of CYP4A mRNAs as WY-14643, a potent PPAR{alpha} agonist. Thus, most of the 200 pesticides tested do not activate PPAR{alpha} or PPAR{gamma} in in vitro assays, but only diclofop-methyl and pyrethrins induce PPAR{alpha} agonistic activity in vivo as well as in vitro.

  12. Active Trafficking of Alpha 1 Antitrypsin across the Lung Endothelium

    PubMed Central

    Lockett, Angelia D.; Brown, Mary Beth; Santos-Falcon, Nieves; Rush, Natalia I.; Oueini, Houssam; Oberle, Amber J.; Bolanis, Esther; Fragoso, Miryam A.; Petrusca, Daniela N.; Serban, Karina A.; Schweitzer, Kelly S.; Presson Jr., Robert G.

    2014-01-01

    The homeostatic lung protective effects of alpha-1 antitrypsin (A1AT) may require the transport of circulating proteinase inhibitor across an intact lung endothelial barrier. We hypothesized that uninjured pulmonary endothelial cells transport A1AT to lung epithelial cells. Purified human A1AT was rapidly taken up by confluent primary rat pulmonary endothelial cell monolayers, was secreted extracellularly, both apically and basolaterally, and was taken up by adjacent rat lung epithelial cells co-cultured on polarized transwells. Similarly, polarized primary human lung epithelial cells took up basolaterally-, but not apically-supplied A1AT, followed by apical secretion. Evidence of A1AT transcytosis across lung microcirculation was confirmed in vivo by two-photon intravital microscopy in mice. Time-lapse confocal microscopy indicated that A1AT co-localized with Golgi in the endothelium whilst inhibition of the classical secretory pathway with tunicamycin significantly increased intracellular retention of A1AT. However, inhibition of Golgi secretion promoted non-classical A1AT secretion, associated with microparticle release. Polymerized A1AT or A1AT supplied to endothelial cells exposed to soluble cigarette smoke extract had decreased transcytosis. These results suggest previously unappreciated pathways of A1AT bidirectional uptake and secretion from lung endothelial cells towards the alveolar epithelium and airspaces. A1AT trafficking may determine its functional bioavailablity in the lung, which could be impaired in individuals exposed to smoking or in those with A1AT deficiency. PMID:24743137

  13. Contribution of mucosal maltase-glucoamylase activities to mouse small intestinal starch alpha-glucogenesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Digestion of starch requires activities provided by 6 interactive small intestinal enzymes. Two of these are luminal endo-glucosidases named alpha-amylases. Four are exo-glucosidases bound to the luminal surface of enterocytes. These mucosal activities were identified as 4 different maltases. Two ma...

  14. Developmental toxicity of perfluorononanoic acid is dependent on peroxisome proliferator activated receptor-alpha.

    EPA Science Inventory

    Perfluorononanoic acid (PFNA) is one of the predominant perfluoroalkyl acids in the environment and in tissues of humans and wildlife. PFNA strongly activates the mouse and human peroxisome proliferator-activated receptor-alpha (PPARα) in vitro and negatively impacts development ...

  15. The influence of ligand-activated LXR on primary human trophoblasts

    PubMed Central

    Larkin, Jacob C.; Sears, Sarah B.; Sadovsky, Yoel

    2014-01-01

    Introduction The Liver X Receptors (LXRs) are critical transcriptional regulators of cellular metabolism that promote cholesterol efflux and lipogenesis in response to excess intracellular cholesterol. In contrast, the Sterol Response Element Binding Protein-2 (SREBP2) promotes the synthesis and uptake of cholesterol. Oxysterols are products of cholesterol oxidation that accumulate in conditions associated with increased cellular levels of reactive oxygen species, such as hypoxia and oxidative stress, activating LXR and inhibiting SREBP2. While hypoxia and oxidative stress are commonly implicated in placental injury, the impact of the transcriptional regulation of cholesterol homeostasis on placental function is not well characterized. Methods We measured the effects of the synthetic LXR ligand T0901317 and the endogenous oxysterol 25-hydroxycholesterol (25OHC) on differentiation, cytotoxicity, progesterone synthesis, lipid droplet formation, and gene expression in primary human trophoblasts. Results Exposure to T0901317 promoted lipid droplet formation and inhibited differentiation, while 25OHC induced trophoblast toxicity, promoted hCG and progesterone release at lower concentrations with inhibition at higher concentrations, and had no effect on lipid droplet formation. The discrepant effect of these ligands was associated with distinct changes in expression of LXR and SREBP2 target genes, with upregulation of ABCA1 following 25OHC and T090317 exposure, exclusive activation of the lipogenic LXR targets SREBP1c, ACC1 and FAS by T0901317, and exclusive inhibition of the SREBP2 targets LDLR and HMGCR by 25OHC. Conclusion These findings implicate cholesterol oxidation as a determinant of trophoblast function and activity, and suggest that placental gene targets and functional pathways are selectively regulated by specific LXR ligands. PMID:25255963

  16. Dendrimers and Polyamino-Phenolic Ligands: Activity of New Molecules Against Legionella pneumophila Biofilms

    PubMed Central

    Andreozzi, Elisa; Barbieri, Federica; Ottaviani, Maria F.; Giorgi, Luca; Bruscolini, Francesca; Manti, Anita; Battistelli, Michela; Sabatini, Luigia; Pianetti, Anna

    2016-01-01

    Legionnaires’ disease is a potentially fatal pneumonia caused by Legionella pneumophila, an aquatic bacterium often found within the biofilm niche. In man-made water systems microbial biofilms increase the resistance of legionella to disinfection, posing a significant threat to public health. Disinfection methods currently used in water systems have been shown to be ineffective against legionella over the long-term, allowing recolonization by the biofilm-protected microorganisms. In this study, the anti-biofilm activity of previously fabricated polyamino-phenolic ligands and polyamidoamine dendrimers was investigated against legionella mono-species and multi-species biofilms formed by L. pneumophila in association with other bacteria that can be found in tap water (Aeromonas hydrophila, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae). Bacterial ability to form biofilms was verified using a crystal violet colorimetric assay and testing cell viability by real-time quantitative PCR and Plate Count assay. The concentration of the chemicals tested as anti-biofilm agents was chosen based on cytotoxicity assays: the highest non-cytotoxic chemical concentration was used for biofilm inhibition assays, with dendrimer concentration 10-fold higher than polyamino-phenolic ligands. While Macrophen and Double Macrophen were the most active substances among polyamino-phenolic ligands, dendrimers were overall twofold more effective than all other compounds with a reduction up to 85 and 73% of legionella and multi-species biofilms, respectively. Chemical interaction with matrix molecules is hypothesized, based on SEM images and considering the low or absent anti-microbial activity on planktonic bacteria showed by flow cytometry. These data suggest that the studied compounds, especially dendrimers, could be considered as novel molecules in the design of research projects aimed at the development of efficacious anti-biofilm disinfection treatments of water systems

  17. Diurnal changes in the synthesis of the neurosteroid 7alpha-hydroxypregnenolone stimulating locomotor activity in newts.

    PubMed

    Koyama, Teppei; Haraguchi, Shogo; Vaudry, Hubert; Tsutsui, Kazuyoshi

    2009-04-01

    We recently identified 7alpha-hydroxypregnenolone as a novel amphibian neurosteroid stimulating locomotor activity in newts. Because male newts show marked diurnal changes in locomotor activity, we hypothesized that 7alpha-hydroxypregnenolone may be a key factor for the induction of diurnal changes in locomotor activity in male newts. In this study, we found diurnal changes in 7alpha-hydroxypregnenolone synthesis in the brain of male newts, which paralleled locomotor activity. Interestingly, the production of 7alpha-hydroxypregnenolone in the male newt brain increased during the dark phase when locomotor activity of males was high.

  18. Phosphorylation of the alpha-subunit of Na,K-ATPase from duck salt glands by cAMP-dependent protein kinase inhibits the enzyme activity.

    PubMed

    Murtazina, D A; Petukhov, S P; Rubtsov, A M; Storey, K B; Lopina, O D

    2001-08-01

    Although it was shown earlier that phosphorylation of Na,K-ATPase by cAMP-dependent protein kinase (PKA) occurs in intact cells, the purified enzyme in vitro is phosphorylated by PKA only after treatment by detergent. This is accompanied by an unfortunate side effect of the detergent that results in complete loss of Na,K-ATPase activity. To reveal the effect of Na,K-ATPase phosphorylation by PKA on the enzyme activity in vitro, the effects of different detergents and ligands on the stoichiometry of the phosphorylation and activity of Na,K-ATPase from duck salt glands (alpha1beta1-isoenzyme) were comparatively studied. Chaps was shown to cause the least inhibition of the enzyme. In the presence of 0.4% Chaps at 1 : 10 protein/detergent ratio in medium containing 100 mM KCl and 0.3 mM ATP, PKA phosphorylates serine residue(s) of the Na,K-ATPase with stoichiometry 0.6 mol Pi/mol of alpha-subunit. Phosphorylation of Na,K-ATPase by PKA in the presence of the detergent inhibits the Na,K-ATPase. A correlation was found between the inclusion of P(i) into the alpha-subunit and the loss of activity of the Na,K-ATPase.

  19. Increased antiviral activity of microscale-purified HuIFN alpha 8 (human interferon alpha 8) over HuIFN alpha 2b in Hep-2 cells challenged with Mengo virus.

    PubMed

    García, Julio César Sánchez; Ariza, Alejandro Miranda; Lasa, Alexis Musacchio; González, Luis Javier; Perez, Vladimir Besada

    2007-11-01

    Human proteins are not routinely expressed at high levels in Escherichia coli for, among other reasons, different codon usage. Several purification procedures have been applied to recover recombinant proteins for further biological characterization. However, the vast majority involve costly chromatography procedures. In the present study, both (Hu)IFN(alpha 2b) (human interferon alpha 2b) and (Hu)IFN(alpha 8) were expressed efficiently in E. coli BL21-codonplus-RIL. Subsequently, both recombinant proteins were purified to homogeneity by passive elution from reverse-stained SDS/PAGE gels, a cost-effective purification procedure. After purification, both recovered proteins were biologically active. The (Hu)IFN(alpha 8) subtype induced 1.46-fold more antiviral activity than (Hu)IFN(alpha 2b) using Hep-2 human laryngeal carcinoma cell challenged with Mengo virus.

  20. Fulvestrant regulates epidermal growth factor (EGF) family ligands to activate EGF receptor (EGFR) signaling in breast cancer cells.

    PubMed

    Zhang, Xihong; Diaz, Michael R; Yee, Douglas

    2013-06-01

    Estrogen receptor-α (ER) targeted therapies are routinely used to treat breast cancer. However, patient responses are limited by resistance to endocrine therapy. Breast cancer cells resistant to the pure steroidal ER antagonist fulvestrant (fulv) demonstrate increased activation of epidermal growth factor receptor (EGFR) family members and downstream ERK signaling. In this study, we investigated the effects of fulv on EGFR signaling and ligand regulation in several breast cancer cell lines. EGFR/HER2/HER3 phosphorylation and ERK1,2 activation were seen after 24-48 h after fulvestrant treatment in ER-positive breast cancer cell lines. 4-Hydroxy-tamoxifen and estradiol did not cause EGFR activation. Fulvestrant did not affect EGFR expression. Cycloheximide abolished the ability of fulv to activate EGFR suggesting the autocrine production of EGFR ligands might be responsible for fulvestrant induced EGFR signaling. qRT-PCR results showed fulv differentially regulated EGFR ligands; HB-EGF mRNA was increased, while amphiregulin and epiregulin mRNAs were decreased. Fulvestrant induced EGFR activation and upregulation of EGFR ligands were ER dependent since fulv treatment in C4-12, an ER-negative cell line derivative of MCF-7 cells, did not result in EGFR activation or change in ligand mRNA levels. ER downregulation by siRNA induced similar EGFR activation and regulation of EGFR ligands as fulvestrant. Neutralizing HB-EGF antibody blocked fulv-induced EGFR activation. Combination of fulv and EGFR family tyrosine kinase inhibitors (erlotinib and lapatinib) significantly decreased EGFR signaling and cell survival. In conclusion, fulvestrant-activated EGFR family members accompanied by ER dependent upregulation of HB-EGF within 48 h. EGF receptor or ligand inhibition might enhance or prolong the therapeutic effects of targeting ER by fulvestrant in breast cancer.

  1. Phosphatidic Acid Induces Ligand-independent Epidermal Growth Factor Receptor Endocytic Traffic through PDE4 Activation

    PubMed Central

    Norambuena, Andrés; Metz, Claudia; Jung, Juan E.; Silva, Antonia; Otero, Carolina; Cancino, Jorge; Retamal, Claudio; Valenzuela, Juan C.; Soza, Andrea

    2010-01-01

    Endocytosis modulates EGFR function by compartmentalizing and attenuating or enhancing its ligand-induced signaling. Here we show that it can also control the cell surface versus intracellular distribution of empty/inactive EGFR. Our previous observation that PKA inhibitors induce EGFR internalization prompted us to test phosphatidic acid (PA) generated by phospholipase D (PLD) as an endogenous down-regulator of PKA activity, which activates rolipram-sensitive type 4 phosphodiesterases (PDE4) that degrade cAMP. We found that inhibition of PA hydrolysis by propranolol, in the absence of ligand, provokes internalization of inactive (neither tyrosine-phosphorylated nor ubiquitinated) EGFR, accompanied by a transient increase in PA levels and PDE4s activity. This EGFR internalization is mimicked by PA micelles and is strongly counteracted by PLD2 silencing, rolipram or forskolin treatment, and PKA overexpression. Accelerated EGFR endocytosis seems to be mediated by clathrin-dependent and -independent pathways, leading to receptor accumulation in juxtanuclear recycling endosomes, also due to a decreased recycling. Internalized EGFR can remain intracellular without degradation for several hours or return rapidly to the cell surface upon discontinuation of the stimulus. This novel regulatory mechanism of EGFR, also novel function of signaling PA, can transmodulate receptor accessibility in response to heterologous stimuli. PMID:20554760

  2. Retinal ligand mobility explains internal hydration and reconciles active rhodopsin structures.

    PubMed

    Leioatts, Nicholas; Mertz, Blake; Martínez-Mayorga, Karina; Romo, Tod D; Pitman, Michael C; Feller, Scott E; Grossfield, Alan; Brown, Michael F

    2014-01-21

    Rhodopsin, the mammalian dim-light receptor, is one of the best-characterized G-protein-coupled receptors, a pharmaceutically important class of membrane proteins that has garnered a great deal of attention because of the recent availability of structural information. Yet the mechanism of rhodopsin activation is not fully understood. Here, we use microsecond-scale all-atom molecular dynamics simulations, validated by solid-state (2)H nuclear magnetic resonance spectroscopy, to understand the transition between the dark and metarhodopsin I (Meta I) states. Our analysis of these simulations reveals striking differences in ligand flexibility between the two states. Retinal is much more dynamic in Meta I, adopting an elongated conformation similar to that seen in the recent activelike crystal structures. Surprisingly, this elongation corresponds to both a dramatic influx of bulk water into the hydrophobic core of the protein and a concerted transition in the highly conserved Trp265(6.48) residue. In addition, enhanced ligand flexibility upon light activation provides an explanation for the different retinal orientations observed in X-ray crystal structures of active rhodopsin.

  3. Debunking the Idea that Ligand Efficiency Indices Are Superior to pIC50 as QSAR Activities.

    PubMed

    Sheridan, Robert P

    2016-11-28

    Several papers have appeared in which a ligand efficiency index instead of pIC50 is used as the activity in QSAR. The claim is that better fits and predictions are obtained with ligand efficiency. We show on both public-domain and in-house data sets that the apparent superiority is a statistical artifact that occurs when ligand efficiency indices are correlated with the physical property included in their definition (number of non-hydrogens, ALOGP, TPSA, etc.) and when the property is easier to predict than the original pIC50.

  4. Constitutive activation of integrin alpha 4 beta 1 defines a unique stage of human thymocyte development

    PubMed Central

    1994-01-01

    Our understanding of thymocyte development and of the positive and negative selection events involved in shaping the repertoire of mature T lymphocytes has been greatly facilitated by the use of transgenic and gene knockout animals. Much less is known about the factors that control the homing and population of the thymus by T cell precursors and the subsequent migration of developing thymocytes through the thymic architecture. As the integrins represent a candidate group of cell surface receptors that may regulate thymocyte development, we have analyzed the expression and function of alpha 4 beta 1 and alpha 5 beta 1 on human thymocytes. A major portion of double positive (CD4+ CD8+) human thymocytes express alpha 4 beta 1 in a constitutively active form and adhere to fibronectin and vascular cell adhesion molecule 1. alpha 4 beta 1 expression is similar on adherent and nonadherent populations, thus, activity reflects the receptor state and not simple expression. The adherent cells are immature, expressing high levels of CD4/CD8 and low levels of CD3 and CD69. In contrast, nonadherent cells possess the phenotype of thymocytes after positive selection, expressing intermediate levels of CD4 and/or CD8 and high levels of CD3 and CD69. The adherent population fails to respond to activation with anti-CD3 and fibronectin, whereas nonadherents exhibit an alpha 5 beta 1- dependent proliferation. Differential regulation of alpha 4 beta 1 and alpha 5 beta 1 receptors may provide a mechanism controlling cellular traffic, differentiation, and positive selection of thymocytes. PMID:8163937

  5. Relative activities and stabilities of mutant Escherichia coli tryptophan synthase alpha subunits.

    PubMed Central

    Lim, W K; Shin, H J; Milton, D L; Hardman, J K

    1991-01-01

    In vitro mutagenesis of the Escherichia coli trpA gene has yielded 66 mutant tryptophan synthase alpha subunits containing single amino acid substitutions at 49 different residue sites and 29 double and triple amino acid substitutions at 16 additional sites, all within the first 121 residues of the protein. The 66 singly altered mutant alpha subunits encoded from overexpression vectors have been examined for their ability to support growth in trpA mutant host strains and for their enzymatic and stability properties in crude extracts. With the exception of mutant alpha subunits altered at catalytic residue sites Glu-49 and Asp-60, all support growth; this includes those (48 of 66) that have no enzymatic defects and those (18 of 66) that do. The majority of the enzymatically defective mutant alpha subunits have decreased capacities for substrate (indole-3-glycerol phosphate) utilization, typical of the early trpA missense mutants isolated by in vivo selection methods. These defects vary in severity from complete loss of activity for mutant alpha subunits altered at residue positions 49 and 60 to those, altered elsewhere, that are partially (up to 40 to 50%) defective. The complete inactivation of the proteins altered at the two catalytic residue sites suggest that, as found via in vitro site-specific mutagenesis of the Salmonella typhimurium tryptophan synthetase alpha subunit, both residues probably also participate in a push-pull general acid-base catalysis of indole-3-glycerol phosphate breakdown for the E. coli enzyme as well. Other classes of mutant alpha subunits include some novel types that are defective in their functional interaction with the other tryptophan synthetase component, the beta 2 subunit. Also among the mutant alpha subunits, 19 were found altered at one or another of the 34 conserved residue sites in this portion of the alpha polypeptide sequence; surprisingly, 10 of these have wild-type enzymatic activity, and 16 of these can satisfy growth

  6. Computational approaches to screen candidate ligands with anti- Parkinson's activity using R programming.

    PubMed

    Jayadeepa, R M; Niveditha, M S

    2012-01-01

    It is estimated that by 2050 over 100 million people will be affected by the Parkinson's disease (PD). We propose various computational approaches to screen suitable candidate ligand with anti-Parkinson's activity from phytochemicals. Five different types of dopamine receptors have been identified in the brain, D1-D5. Dopamine receptor D3 was selected as the target receptor. The D3 receptor exists in areas of the brain outside the basal ganglia, such as the limbic system, and thus may play a role in the cognitive and emotional changes noted in Parkinson's disease. A ligand library of 100 molecules with anti-Parkinson's activity was collected from literature survey. Nature is the best combinatorial chemist and possibly has answers to all diseases of mankind. Failure of some synthetic drugs and its side effects have prompted many researches to go back to ancient healing methods which use herbal medicines to give relief. Hence, the candidate ligands with anti-Parkinson's were selected from herbal sources through literature survey. Lipinski rules were applied to screen the suitable molecules for the study, the resulting 88 molecules were energy minimized, and subjected to docking using Autodock Vina. The top eleven molecules were screened according to the docking score generated by Autodock Vina Commercial drug Ropinirole was computed similarly and was compared with the 11 phytochemicals score, the screened molecules were subjected to toxicity analysis and to verify toxic property of phytochemicals. R Programming was applied to remove the bias from the top eleven molecules. Using cluster analysis and Confusion Matrix two phytochemicals were computationally selected namely Rosmarinic acid and Gingkolide A for further studies on the disease Parkinson's.

  7. Structural basis for activation of alpha-boranophosphate nucleotide analogues targeting drug-resistant reverse transcriptase.

    PubMed

    Meyer, P; Schneider, B; Sarfati, S; Deville-Bonne, D; Guerreiro, C; Boretto, J; Janin, J; Véron, M; Canard, B

    2000-07-17

    AIDS chemotherapy is limited by inadequate intracellular concentrations of the active triphosphate form of nucleoside analogues, leading to incomplete inhibition of viral replication and the appearance of drug-resistant virus. Drug activation by nucleoside diphosphate kinase and inhibition of HIV-1 reverse transcriptase were studied comparatively. We synthesized analogues with a borano (BH(3)(-)) group on the alpha-phosphate, and found that they are substrates for both enzymes. X-ray structures of complexes with nucleotide diphosphate kinase provided a structural basis for their activation. The complex with d4T triphosphate displayed an intramolecular CH.O bond contributing to catalysis, and the R(p) diastereoisomer of thymidine alpha-boranotriphosphate bound like a normal substrate. Using alpha-(R(p))-boranophosphate derivatives of the clinically relevant compounds AZT and d4T, the presence of the alpha-borano group improved both phosphorylation by nucleotide diphosphate kinase and inhibition of reverse transcription. Moreover, repair of blocked DNA chains by pyrophosphorolysis was reduced significantly in variant reverse transcriptases bearing substitutions found in drug-resistant viruses. Thus, the alpha-borano modification of analogues targeting reverse transcriptase may be of generic value in fighting viral drug resistance.

  8. Alpha-mannosidase activity in stallion epididymal fluid and spermatozoa.

    PubMed

    Retamal, C A; Dias, A J B; Brasil, F C; Lanzana, F R; López, M L

    2012-07-15

    The expression of α-D-mannosidase activity was fluorometrically and electrophoretically assessed in spermatozoa, epididymal fluid and homogenates of stallion epididymal tissue. Enzyme activity had regional differences; it was higher (P<0.05) in samples from the cauda epididymal region than in samples from the proximal caput region (largely composed of efferent ducts). Based on enzyme activity, as a function of pH of the assay substrate, electrophoretic analysis in native and native/SDS-PAGE conditions, and the effect of inhibitors or activators, we inferred the presence of at least two catalytically active forms of α-D-mannosidase. The neutral form of the enzyme (α-mannosidase II) was activated by Co2+, whereas the acid form (optimum pH 3.5 to 4.0) was sensitive to swainsonine (an inhibitor of α-mannosidase I), stabilized or stimulated by Zn2+, and not activated by Co2+ (activator of the neutral form). The activity of the acid form of the enzyme was highest in the epididymal fluid, where it seemed to be mainly in a secretory form. This form of the enzyme may have a role in plasma membrane remodeling associated with sperm maturation. In contrast, the activity of α-mannosidase II was higher in mature spermatozoa. It has been postulated that α-mannosidase II may act as a receptor in the recognition and binding of the complementary carbohydrate moieties present on the zona pellucida. With non-denaturing electrophoresis, α-D-mannosidase had an electrophoretic mobility of 0.35 and 0.24. When resolved by 1D and 2D SDS-PAGE (under denaturing conditions) the enzyme had a major protein band of molecular weight 154 kDa in spermatozoa and epididymal samples. Based on its properties under native conditions, we inferred that this enzyme might interact with other proteins and form transitory aggregates.

  9. Differential neural activation of vascular alpha-adrenoceptors in oral tissues of cats.

    PubMed

    Koss, Michael C

    2002-04-05

    The aim of this study was to determine the relative contribution of alpha(1)- and alpha(2)-adrenoceptors involved in sympathetic-evoked vasoconstrictor responses in tissues perfused by the lingual arterial circulation in pentobarbital anesthetized cats. Blood flow in the lingual artery was measured by ultrasonic flowmetry. Laser-Doppler flowmetry was utilized to measure oral tissue vasoconstrictor responses in the maxillary gingiva and from the surface of the tongue. Electrical stimulation of the preganglionic superior cervical sympathetic nerve resulted in frequency-dependent blood flow decreases at all three sites. These responses were stable over time and were uniformly antagonized by administration of phentolamine (0.3 - 3.0 mg kg(-1)). The selective alpha(1)-adrenoceptor antagonist, prazosin (10 - 300 microg kg(-1)), attenuated vasoconstriction in the lingual artery and gingiva, but was ineffective in blocking vasoconstriction in the tongue. Subsequent administration of rauwolscine (300 microg kg(-1)) antagonized remaining vasoconstrictor responses. In contrast, rauwolscine (10 - 300 microg kg(-1)), given alone, blocked evoked vasoconstriction in the tongue, and was without effect on gingival or lingual artery vasoconstrictor responses. Subsequent administration of prazosin (300 microg kg(-1)) largely antagonized remaining neurally elicited responses. These results suggest that neural vasoconstrictor responses in some regional vascular beds in the cat oral cavity are mediated by both alpha(1)- and alpha(2)-adrenoceptors. In contrast, tongue surface vasoconstrictor responses to sympathetic nerve activation appear to be mediated primarily by alpha(2)-adrenoceptors.

  10. Synthesis and biological activity of small peptides as NOP and opioid receptors' ligands: view on current developments.

    PubMed

    Naydenova, Emilia; Todorov, Petar; Zamfirova, Rositza

    2015-01-01

    The heptadecapeptide nociceptin, also called orphanin FQ (N/OFQ), is the endogenous agonist of the N/OFQ peptide receptor (NOP receptor) and is involved in several central nervous system pathways, such as nociception, reward, tolerance, and feeding. The discovery of small molecule ligands for NOP is being actively pursued for several therapeutic applications. This review presents overview of the several recently reported NOP ligands (agonists and antagonists), with an emphasis of the structural features that may be important for modulating the intrinsic activity of these ligands. In addition, a brief account on the characterization of newly synthesized ligands of NOP receptor with aminophosphonate moiety and β-tryptophan analogues will be presented.

  11. A Series of Diamagnetic Pyridine Monoimine Rhenium Complexes with Different Degrees of Metal-to-Ligand Charge Transfer: Correlating (13) C NMR Chemical Shifts with Bond Lengths in Redox-Active Ligands.

    PubMed

    Sieh, Daniel; Kubiak, Clifford P

    2016-07-18

    A set of pyridine monoimine (PMI) rhenium(I) tricarbonyl chlorido complexes with substituents of different steric and electronic properties was synthesized and fully characterized. Spectroscopic (NMR and IR) and single-crystal X-ray diffraction analyses of these complexes showed that the redox-active PMI ligands are neutral and that the overall electronic structure is little affected by the choices of the substituent at the ligand backbone. One- and two-electron reduction products were prepared from selected starting compounds and could also be characterized by multiple spectroscopic methods and X-ray diffraction. The final product of a one-electron reduction in THF is a diamagnetic metal-metal-bonded dimer after loss of the chlorido ligand. Bond lengths in and NMR chemical shifts of the PMI ligand backbone indicate partial electron transfer to the ligand. Two-electron reduction in THF also leads to the loss of the chlorido ligand and a pentacoordinate complex is obtained. The comparison with reported bond lengths and (13) C NMR chemical shifts of doubly reduced free pyridine monoaldimine ligands indicates that both redox equivalents in the doubly reduced rhenium complex investigated here are located in the PMI ligand. With diamagnetic complexes varying over three formal reduction stages at the PMI ligand we were, for the first time, able to establish correlations of the (13) C NMR chemical shifts with the relevant bond lengths in redox-active ligands over a full redox series.

  12. Castanospermine inhibits alpha-glucosidase activities and alters glycogen distribution in animals.

    PubMed

    Saul, R; Ghidoni, J J; Molyneux, R J; Elbein, A D

    1985-01-01

    Castanospermine, an inhibitor of alpha-glucosidase activity, was injected into rats to determine its effects in vivo. Daily injections of alkaloid, at levels of 0.5 mg/g of body weight, or higher, for 3 days decreased hepatic alpha-glucosidase to 40% of control values, whereas alpha-glucosidase in brain was reduced to 25% of control values and that in spleen and kidney was reduced to about 40%. In liver, both the neutral (pH 6.5) and the acidic (pH 4.5) alpha-glucosidase activities were inhibited, but the former was more susceptible. On the other hand, beta-N-acetylhexosaminidase activity was elevated in the livers of treated animals, whereas beta-galactosidase activity was unchanged and alpha-mannosidase activity was somewhat inhibited. Livers of treated animals were examined by light and electron microscopy and compared to control animals to determine whether changes in morphology had occurred. In treated animals fed normal rat chow, the hepatocytes were smaller in size and simplified in structure, whereas the high-glucose diet lessened these alterations. Furthermore, in those animals receiving castanospermine at 1.0 mg or higher per g of body weight for 3 days, there was a marked decrease in the amount of glycogen in the cytoplasm, while a large number of lysosomes were observed that were full of dense, granular material. That this dense material was indeed glycogen was shown by the fact that it disappeared when blocks of fixed tissue were pretreated with alpha-amylase. Glycogen levels in liver, as measured either colorimetrically or enzymatically, were somewhat depressed at the higher levels of castanospermine.

  13. Activation of phospholipase C by the alpha subunits of the Gq and G11 proteins in transfected Cos-7 cells.

    PubMed

    Wu, D Q; Lee, C H; Rhee, S G; Simon, M I

    1992-01-25

    High efficiency transient transfection was used to introduce cDNA corresponding to various G protein alpha subunits into Cos-7 cells. The proteins that were subsequently synthesized were detected with specific G protein alpha subunit antipeptide antiserum and were localized in the membrane fraction of the cell. Cells that were prelabeled with the [3H]inositol and transfected with G alpha q and G alpha 11 cDNA showed marked increases in formation of [3H]inositol phosphates after stimulation with aluminum fluoride. Co-transfection with cDNAs corresponding to phosphoinositide specific phospholipase C beta 1 (PI-PLC beta 1) and to G alpha q or G alpha 11 resulted in even higher levels of inositol phosphate formation. The introduction of mutations that convert residue glutamine 209 to leucine in G alpha q and G alpha 11 resulted in persistent activation of PI-PLC and high steady state levels of inositol phosphates. On the other hand, transfection with a variety of other G alpha subunit cDNAs, i.e. G alpha Z, G alpha OA, G alpha OB, transducin, and the glutamine 205 to leucine mutants of G alpha Z and of G alpha OA did not increase inositol phosphate formation. To further test the specificity of G protein activation of PI-PLC, a cell-free system was prepared by using washed membranes of transiently transfected cells and purified PI-PLC beta 1. Membranes derived from G alpha q and G alpha 11, but not G alpha OA transfected cells, showed guanosine 5-O-thiotriphosphate (GTP gamma S)-stimulated PIP2 hydrolysis. The activity seen in the system reconstituted with membranes derived from G alpha 11-transfected cells was blocked by preincubation with specific G alpha 11 antipeptide antibodies. All of these results are consistent with the conclusion that G alpha q and G alpha 11 cDNA encode proteins that in the presence of GTP gamma S specifically activate PI-PLC.

  14. Generation of Soluble Receptor Activator of NF-KappaB Ligand Is Critical for Osteolytic Bone Metastasis

    DTIC Science & Technology

    2010-10-01

    14-23 Kakonen SM, Mundy GR (2003) Mechanisms of osteolytic bone metastases in breast carcinoma. Cancer 97 (3 Suppl): 834-839 Kitazawa S ... Kitazawa R (2002) RANK ligand is a prerequisite for cancer-associated osteolytic lesions. J Pathol 198 (2): 228-236 Kondratiev S , Gnepp DR, Yakirevich E...et al (1998) Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 93(2):165–176 12. Kitazawa S

  15. Generation of Soluble Receptor Activator of NF-kappa B Ligand is Critical for Osteolytic Bone Metastasis

    DTIC Science & Technology

    2009-10-01

    2003) Mechanisms of osteolytic bone metastases in breast carcinoma. Cancer 97 (3 Suppl): 834-839 Kitazawa S , Kitazawa R (2002) RANK ligand is a...osteoclast differentiation and activation. Cell 93(2):165–176 12. Kitazawa S , Kitazawa R (2002) RANK ligand is a prerequisite for cancer-associated...views, opinions and/or findings contained in this report are those of the author( s ) and should not be construed as an official Department of the Army

  16. Structure- and ligand-based drug design of novel p38-alpha MAPK inhibitors in the fight against the Alzheimer's disease.

    PubMed

    Pinsetta, Flávio Roberto; Taft, Carlton Anthony; de Paula da Silva, Carlos Henrique Tomich

    2014-01-01

    Alzheimer's disease (AD) is characterized microscopically by the presence of amyloid plaques, which are accumulations of beta-amyloid protein inter-neurons, and neurofibrillary tangles formed predominantly by highly phosphorylated forms of the microtubule-associated protein, tau, which form tangled masses that consume neuronal cell body, possibly leading to neuronal dysfunction and ultimately death. p38α mitogen-activated protein kinase (MAPK) has been implicated in both events associated with AD, tau phosphorylation and inflammation. p38α MAPK pathway is activated by a dual phosphorylation at Thr180 and Tyr182 residues. Drug design of p38α MAPK inhibitors is mainly focused on small molecules that compete for Adenosine triphosphate in the catalytic site. Here, we used different approaches of structure- and ligand-based drug design and medicinal chemistry strategies based on a selected p38α MAPK structure deposited in the Protein Data Bank in complex with inhibitor, as well as others reported in literature. As a result of the virtual screening experiments performed here, as well as molecular dynamics, molecular interaction fields studies, shape and electrostatic similarities, activity and toxicity predictions, and pharmacokinetic and physicochemical properties, we have selected 13 compounds that meet the criteria of low or no toxicity potential, good pharmacotherapeutic profile, predicted activities, and calculated values ​​comparable with those obtained for the reference compounds, while maintaining the main interactions observed for the most potent inhibitors.

  17. Nitrated alpha-synuclein and microglial neuroregulatory activities

    PubMed Central

    Reynolds, Ashley D.; Kadiu, Irena; Garg, Sanjay K.; Glanzer, Jason G.; Nordgen, Tara; Ciborowski, Pawel; Banerjee, Ruma; Gendelman, Howard E.

    2008-01-01

    Microglial neuroinflammatory responses affect the onset and progression of Parkinson’s disease (PD). We posit that such neuroinflammatory responses are, in part, mediated by microglial interactions with nitrated and aggregated α-synuclein (α-syn) released from Lewy bodies as a consequence of dopaminergic neuronal degeneration. As disease progresses, secretions from α-syn activated microglia can engage neighboring glial cells in a cycle of autocrine and paracrine amplification of neurotoxic immune products. Such pathogenic processes affect the balance between a microglial neurotrophic and neurotoxic signature. We now report that microglia secrete both neurotoxic and neuroprotective factors following exposure to nitrated α-syn (N-α-syn). Proteomic [surface enhanced laser desorption-time of flight (SELDI-TOF), 1D SDS electrophoresis, and liquid chromatography-tandem mass spectrometry] and limited metabolomic profiling demonstrated that N-α-syn activated microglia secrete inflammatory, regulatory, redox-active, enzymes, and cytoskeletal proteins. Increased extracellular glutamate and cysteine, dimininshed intracellular glutathione and secreted exosomal proteins were also demonstrated. Increased redox active proteins suggest regulatory microglial responses to N-α-syn. These were linked to discontinuous cystatin expression, cathepsin activity, and NF-κB activation. Inhibition of cathepsin B attenuated, in part, N-α-syn-microglial neurotoxicity. These data support multifaceted microglia functions in PD-associated neurodegeneration. PMID:18202920

  18. Synthesis, structural elucidation, biological, antioxidant and nuclease activities of some 5-Fluorouracil-amino acid mixed ligand complexes

    NASA Astrophysics Data System (ADS)

    Shobana, Sutha; Subramaniam, Perumal; Mitu, Liviu; Dharmaraja, Jeyaprakash; Arvind Narayan, Sundaram

    2015-01-01

    Some biologically active mixed ligand complexes (1-9) have been synthesized from 5-Fluorouracil (5-FU; A) and amino acids (B) such as glycine (gly), L-alanine (ala) and L-valine (val) with Ni(II), Cu(II) and Zn(II) ions. The synthesized mixed ligand complexes (1-9) were characterized by various physico-chemical, spectral, thermal and morphological studies. 5-Fluorouracil and its mixed ligand complexes have been tested for their in vitro biological activities against some pathogenic bacterial and fungal species by the agar well diffusion method. The in vitro antioxidant activities of 5-Fluorouracil and its complexes have also been investigated by using the DPPH assay method. The results demonstrate that Cu(II) mixed ligand complexes (4-6) exhibit potent biological as well as antioxidant activities compared to 5-Fluorouracil and Ni(II) (1-3) and Zn(II) (7-9) mixed ligand complexes. Further, the cleaving activities of CT DNA under aerobic conditions show moderate activity with the synthesized Cu(II) and Ni(II) mixed ligand complexes (1-6) while no activity is seen with Zn(II) complexes (7-9). Binding studies of CT DNA with these complexes show a decrease in intensity of the charge transfer band to the extent of 5-15% along with a minor red shift. The free energy change values (Δ‡G) calculated from intrinsic binding constants indicate that the interaction between mixed ligand complex and DNA is spontaneous.

  19. Synthesis, structural elucidation, biological, antioxidant and nuclease activities of some 5-Fluorouracil-amino acid mixed ligand complexes.

    PubMed

    Shobana, Sutha; Subramaniam, Perumal; Mitu, Liviu; Dharmaraja, Jeyaprakash; Arvind Narayan, Sundaram

    2015-01-05

    Some biologically active mixed ligand complexes (1-9) have been synthesized from 5-Fluorouracil (5-FU; A) and amino acids (B) such as glycine (gly), L-alanine (ala) and L-valine (val) with Ni(II), Cu(II) and Zn(II) ions. The synthesized mixed ligand complexes (1-9) were characterized by various physico-chemical, spectral, thermal and morphological studies. 5-Fluorouracil and its mixed ligand complexes have been tested for their in vitro biological activities against some pathogenic bacterial and fungal species by the agar well diffusion method. The in vitro antioxidant activities of 5-Fluorouracil and its complexes have also been investigated by using the DPPH assay method. The results demonstrate that Cu(II) mixed ligand complexes (4-6) exhibit potent biological as well as antioxidant activities compared to 5-Fluorouracil and Ni(II) (1-3) and Zn(II) (7-9) mixed ligand complexes. Further, the cleaving activities of CT DNA under aerobic conditions show moderate activity with the synthesized Cu(II) and Ni(II) mixed ligand complexes (1-6) while no activity is seen with Zn(II) complexes (7-9). Binding studies of CT DNA with these complexes show a decrease in intensity of the charge transfer band to the extent of 5-15% along with a minor red shift. The free energy change values (Δ(‡)G) calculated from intrinsic binding constants indicate that the interaction between mixed ligand complex and DNA is spontaneous.

  20. A molecular orbital rationalization of ligand effects in N2 activation.

    PubMed

    Ariafard, Alireza; Brookes, Nigel J; Stranger, Robert; Yates, Brian F

    2008-01-01

    Molecular orbital theory has been used to study a series of [(micro-N2){ML3}2] complexes as models for dinitrogen activation, with M=Mo, Ta, W, Re and L=NH2, PH2, AsH2, SbH2 and N(BH2)2. The main aims of this study have been to provide a thorough electronic analysis of the complexes and to extend previous work involving molecular orbital analyses. Molecular orbital diagrams have been used to rationalize why for L=NH2 ligand rotation is important for the singlet state but not the triplet, to confirm the effect of ligand pi donation, and to rationalize the importance of the metal d-electron configuration. The outcomes of this study will assist with a more in-depth understanding of the electronic basis for N2 activation and allow clearer predictions to be made about the structure and multiplicity of systems involved in transition-metal catalysis.

  1. Functional Selectivity and Antidepressant Activity of Serotonin 1A Receptor Ligands.

    PubMed

    Chilmonczyk, Zdzisław; Bojarski, Andrzej Jacek; Pilc, Andrzej; Sylte, Ingebrigt

    2015-08-07

    Serotonin (5-HT) is a monoamine neurotransmitter that plays an important role in physiological functions. 5-HT has been implicated in sleep, feeding, sexual behavior, temperature regulation, pain, and cognition as well as in pathological states including disorders connected to mood, anxiety, psychosis and pain. 5-HT1A receptors have for a long time been considered as an interesting target for the action of antidepressant drugs. It was postulated that postsynaptic 5-HT1A agonists could form a new class of antidepressant drugs, and mixed 5-HT1A receptor ligands/serotonin transporter (SERT) inhibitors seem to possess an interesting pharmacological profile. It should, however, be noted that 5-HT1A receptors can activate several different biochemical pathways and signal through both G protein-dependent and G protein-independent pathways. The variables that affect the multiplicity of 5-HT1A receptor signaling pathways would thus result from the summation of effects specific to the host cell milieu. Moreover, receptor trafficking appears different at pre- and postsynaptic sites. It should also be noted that the 5-HT1A receptor cooperates with other signal transduction systems (like the 5-HT1B or 5-HT2A/2B/2C receptors, the GABAergic and the glutaminergic systems), which also contribute to its antidepressant and/or anxiolytic activity. Thus identifying brain specific molecular targets for 5-HT1A receptor ligands may result in a better targeting, raising a hope for more effective medicines for various pathologies.

  2. Functional Selectivity and Antidepressant Activity of Serotonin 1A Receptor Ligands

    PubMed Central

    Chilmonczyk, Zdzisław; Bojarski, Andrzej Jacek; Pilc, Andrzej; Sylte, Ingebrigt

    2015-01-01

    Serotonin (5-HT) is a monoamine neurotransmitter that plays an important role in physiological functions. 5-HT has been implicated in sleep, feeding, sexual behavior, temperature regulation, pain, and cognition as well as in pathological states including disorders connected to mood, anxiety, psychosis and pain. 5-HT1A receptors have for a long time been considered as an interesting target for the action of antidepressant drugs. It was postulated that postsynaptic 5-HT1A agonists could form a new class of antidepressant drugs, and mixed 5-HT1A receptor ligands/serotonin transporter (SERT) inhibitors seem to possess an interesting pharmacological profile. It should, however, be noted that 5-HT1A receptors can activate several different biochemical pathways and signal through both G protein-dependent and G protein-independent pathways. The variables that affect the multiplicity of 5-HT1A receptor signaling pathways would thus result from the summation of effects specific to the host cell milieu. Moreover, receptor trafficking appears different at pre- and postsynaptic sites. It should also be noted that the 5-HT1A receptor cooperates with other signal transduction systems (like the 5-HT1B or 5-HT2A/2B/2C receptors, the GABAergic and the glutaminergic systems), which also contribute to its antidepressant and/or anxiolytic activity. Thus identifying brain specific molecular targets for 5-HT1A receptor ligands may result in a better targeting, raising a hope for more effective medicines for various pathologies. PMID:26262615

  3. Studies of anti-fibrillogenic activity of phthalocyanines of zirconium containing out-of-plane ligands.

    PubMed

    Kovalska, Vladyslava; Losytskyy, Mykhaylo; Chernii, Viktor; Volkova, Kateryna; Tretyakova, Iryna; Cherepanov, Vsevolod; Yarmoluk, Sergiy; Volkov, Sergiy

    2012-01-01

    Series of phthalocyanines of zirconium containing lysine, citric, nonanoic acid residues and dibenzolylmethane groups as out-of-plane ligands are firstly studied as inhibitors of fibrillogenesis using cyanine-based fluorescent inhibitory assay. It was shown that studied phthalocyanines at concentration of 20μM inhibited aggregation reaction on 38.5-57.6% and inhibitory activity of phthalocyanines depended on the chemical nature of out-of-plane ligand. For the most active compound PcZrLys(2) (zirconium phthalocyanine containing lysine fragment) the efficient inhibitor concentration was estimated to be 37μM. AFM studies have shown that in the presence of PcZrLys(2) the inhibition of fibrils formation and formation of spherical oligomeric aggregates took place. Due to the ability of phthalocyanines to decrease efficiently protein aggregation into the amyloid fibrils, modification of phthalocyanine molecules via out-of-plane substitutions was proposed as approach for design of anti-fibrillogenic agents with required properties.

  4. Selective Sirt2 inhibition by ligand-induced rearrangement of the active site.

    PubMed

    Rumpf, Tobias; Schiedel, Matthias; Karaman, Berin; Roessler, Claudia; North, Brian J; Lehotzky, Attila; Oláh, Judit; Ladwein, Kathrin I; Schmidtkunz, Karin; Gajer, Markus; Pannek, Martin; Steegborn, Clemens; Sinclair, David A; Gerhardt, Stefan; Ovádi, Judit; Schutkowski, Mike; Sippl, Wolfgang; Einsle, Oliver; Jung, Manfred

    2015-02-12

    Sirtuins are a highly conserved class of NAD(+)-dependent lysine deacylases. The human isotype Sirt2 has been implicated in the pathogenesis of cancer, inflammation and neurodegeneration, which makes the modulation of Sirt2 activity a promising strategy for pharmaceutical intervention. A rational basis for the development of optimized Sirt2 inhibitors is lacking so far. Here we present high-resolution structures of human Sirt2 in complex with highly selective drug-like inhibitors that show a unique inhibitory mechanism. Potency and the unprecedented Sirt2 selectivity are based on a ligand-induced structural rearrangement of the active site unveiling a yet-unexploited binding pocket. Application of the most potent Sirtuin-rearranging ligand, termed SirReal2, leads to tubulin hyperacetylation in HeLa cells and induces destabilization of the checkpoint protein BubR1, consistent with Sirt2 inhibition in vivo. Our structural insights into this unique mechanism of selective sirtuin inhibition provide the basis for further inhibitor development and selective tools for sirtuin biology.

  5. Human glucocorticoid-induced TNF receptor ligand regulates its signaling activity through multiple oligomerization states

    PubMed Central

    Zhou, Zhaocai; Song, Xiaomin; Berezov, Alan; Zhang, Geng; Li, Yanjing; Zhang, Hongtao; Murali, Ramachandran; Li, Bin; Greene, Mark I.

    2008-01-01

    Ligation between glucocorticoid-induced tumor necrosis factor receptor (GITR) and its ligand (GITRL) provides an undefined signal that renders CD4+CD25− effector T cells resistant to the inhibitory effects of CD4+CD25+ regulatory T cells. To understand the structural basis of GITRL function, we have expressed and purified the extracellular domain of human GITR ligand in Escherichia coli. Chromotography and cross-linking studies indicate that human GITRL (hGITRL) exists as dimers and trimers in solution and also can form a supercluster. To gain insight into the nature of GITRL oligomerization, we determined the crystallographic structures of hGITRL, which revealed a loosely associated open trimer with a deep cavity at the molecular center and a flexible C-terminal tail bent for trimerization. Moreover, a tetramer of trimers (i.e., supercluster) has also been observed in the crystal, consistent with the cross-linking analysis. Deletion of the C-terminal distal three residues disrupts the loosely assembled trimer and favors the formation of a dimer that has compromised receptor binding and signaling activity. Collectively, our studies identify multiple oligomeric species of hGITRL that possess distinct kinetics of ERK activation. The studies address the functional implications and structural models for a process by which hGITRL utilizes multiple oligomerization states to regulate GITR-mediated signaling during T cell costimulation. PMID:18378892

  6. A unique alpha dosimetry technique using Gafchromic EBT3® film and feasibility study for an activity calibrator for alpha-emitting radiopharmaceuticals

    PubMed Central

    Gholami, Yaser H; Bhonsle, Uday; Hentschel, Reinhard; Khachan, Joseph

    2015-01-01

    Objective: To develop an alpha dosimetry technique for activity calibration of alpha-emitting radiopharmaceuticals using the Gafchromic® EBT3 (Gaf-EBT3) radiochromic film (International Speciality product, Wayne, NJ). Methods: The Gaf-EBT3 has a tissue equivalent radiosensitive layer (approximately 28 μm) sandwiched between two 100-μm thick polyester sheaths, thereby making it insensitive to alpha particles. We have split a Gaf-EBT3 sheet using a surgical scalpel to remove one of the polyester protective layers and covered the radiosensitive layer with thin Mylar® foil (Goodfellow Cambridge Limited, Huntingdon, UK) (2.5 μm). Small pieces of modified film were exposed at contact with a 560-Bq thin 241Am source for 5, 10, 24 and 94 h. The optical density of the films was evaluated using an optical densitometer. The alpha energy spectra of the 241Am source were recorded using a Si(Li) surface barrier detector. Results: Time-integrated specific alpha surface activity (kBq cm−2 h) was represented as a function of optical density. Conclusion: By removing one of the 100 μm thick polyester protective layers, the authors have modified the Gaf-EBT3 film to a sensitive alpha dosemeter. The calibration function relevant to a 241Am reference source was evaluated from the optical densities of the dosemeter foils. Furthermore, calibration functions for important alpha emitters such as 223Ra, 225Ac or 210Bi were parameterized from the 241Am reference data. Advances in knowledge: The authors have developed and tested the principle of a clinical alpha dosemeter using Gaf-EBT3 radiochromic films originally developed for photon dosimetry. This novel, user-friendly technique could be implemented in quality assurance and calibration procedures of important alpha-emitting radiopharmaceuticals prior to their clinical applications. PMID:26440547

  7. Digallane with redox-active diimine ligand: dualism of electron-transfer reactions.

    PubMed

    Fedushkin, Igor L; Skatova, Alexandra A; Dodonov, Vladimir A; Chudakova, Valentina A; Bazyakina, Natalia L; Piskunov, Alexander V; Demeshko, Serhiy V; Fukin, Georgy K

    2014-05-19

    The reactivity of digallane (dpp-Bian)Ga-Ga(dpp-Bian) (1), which consists of redox-active ligand 1,2-bis[(2,6-diisopropylphenyl)imino]acenaphthene (dpp-Bian), has been studied. The reaction of 1 with I2 proceeds via one-electron oxidation of each of two dpp-Bian ligands to a radical-anionic state and affords complex (dpp-Bian)IGa-GaI(dpp-Bian) (2). Dissolution of complex 2 in pyridine (Py) gives monomeric compound (dpp-Bian)GaI(Py) (3) as a result of a solvent-induced intramolecular electron transfer from the metal-metal bond to the dpp-Bian ligands. Treatment of compound 3 with B(C6F5)3 leads to removal of pyridine and restores compound 2. The reaction of compound 1 with 3,6-di-tert-butyl-ortho-benzoquinone (3,6-Q) proceeds with oxidation of all the redox-active centers in 1 (the Ga-Ga bond and two dpp-Bian dianions) and results in mononuclear catecholate (dpp-Bian)Ga(Cat) (4) (Cat = [3,6-Q](2-)). Treatment of 4 with AgBF4 gives a mixture of [(dpp-Bian)2Ag][BF4] (5) and (dpp-Bian)GaF(Cat) (6), which both consist of neutral dpp-Bian ligands. The reduction of benzylideneacetone (BA) with 1 generates the BA radical-anions, which dimerize, affording (dpp-Bian)Ga-(BA-BA)-Ga(dpp-Bian) (7). In this case the Ga-Ga bond remains unchanged. Within 10 min at 95 °C in solution compound 7 undergoes transformation to paramagnetic complex (dpp-Bian)Ga(BA-BA) (8) and metal-free compound C36H40N2 (9). The latter is a product of intramolecular addition of the C-H bond of one of the iPr groups to the C═N bond in dpp-Bian. Diamagnetic compounds 3, 5, 6, and 9 have been characterized by NMR spectroscopy, and paramagnetic complexes 2, 4, 7, and 8 by ESR spectroscopy. Molecular structures of 2-7 and 9 have been established by single-crystal X-ray analysis.

  8. Synthesis, structure and spectral and redox properties of new mixed ligand monomeric and dimeric Ru(II) complexes: predominant formation of the "cis-alpha" diastereoisomer and unusual 3MC emission by dimeric complexes.

    PubMed

    Murali, Mariappan; Palaniandavar, Mallayan

    2006-02-07

    The tetradentate ligands 1,8-bis(pyrid-2-yl)-3,6-dithiaoctane (pdto) and 1,8-bis(benzimidazol-2-yl)-3,6-dithiaoctane (bbdo) form the complexes [Ru(pdto)(mu-Cl)](2)(ClO(4))(2) 1 and [Ru(bbdo)(mu-Cl)](2)(ClO(4))(2) 2 respectively. The new di-mu-chloro dimers 1 and 2 undergo facile symmetrical bridge cleavage reactions with the diimine ligands 2,2'-bipyridine (bpy) and dipyridylamine (dpa) to form the six-coordinate complexes [Ru(pdto)(bpy)](ClO(4))(2) 3, [Ru(bbdo)(bpy)](ClO(4))(2) 4, [Ru(pdto)(dpa)](ClO(4))(2) 5 and [Ru(bbdo)(dpa)](ClO(4))(2) 6 and with the triimine ligand 2,2':6,2''-terpyridine (terpy) to form the unusual seven-coordinate complexes [Ru(pdto)(terpy)](ClO(4))(2) 7 and [Ru(bbdo)(terpy)](ClO(4))(2) 8. In 1 the dimeric cation [Ru(pdto)(mu-Cl)](2)(2+) is made up of two approximately octahedrally coordinated Ru(II) centers bridged by two chloride ions, which constitute a common edge between the two Ru(II) octahedra. Each ruthenium is coordinated also to two pyridine nitrogen and two thioether sulfur atoms of the tetradentate ligand. The ligand pdto is folded around Ru(II) as a result of the cis-dichloro coordination, which corresponds to a "cis-alpha" configuration [DeltaDelta/LambdaLambda(rac) diastereoisomer] supporting the possibility of some attractive pi-stacking interactions between the parallel py rings at each ruthenium atom. The ruthenium atom in the complex cations 3a and 4 exhibit a distorted octahedral coordination geometry composed of two nitrogen atoms of the bpy and the two thioether sulfur and two py/bzim nitrogen atoms of the pdto/bbdo ligand, which is actually folded around Ru(II) to give a "cis-alpha" isomer. The molecule of complex 5 contains a six-coordinated ruthenium atom chelated by pdto and dpa ligands in the expected distorted octahedral fashion. The (1)H and (13)C NMR spectral data of the complexes throw light on the nature of metal-ligand bonding and the conformations of the chelate rings, which indicates that the dithioether

  9. rTMS Induced Tinnitus Relief Is Related to an Increase in Auditory Cortical Alpha Activity

    PubMed Central

    Müller, Nadia; Lorenz, Isabel; Langguth, Berthold; Weisz, Nathan

    2013-01-01

    Chronic tinnitus, the continuous perception of a phantom sound, is a highly prevalent audiological symptom. A promising approach for the treatment of tinnitus is repetitive transcranial magnetic stimulation (rTMS) as this directly affects tinnitus-related brain activity. Several studies indeed show tinnitus relief after rTMS, however effects are moderate and vary strongly across patients. This may be due to a lack of knowledge regarding how rTMS affects oscillatory activity in tinnitus sufferers and which modulations are associated with tinnitus relief. In the present study we examined the effects of five different stimulation protocols (including sham) by measuring tinnitus loudness and tinnitus-related brain activity with Magnetoencephalography before and after rTMS. Changes in oscillatory activity were analysed for the stimulated auditory cortex as well as for the entire brain regarding certain frequency bands of interest (delta, theta, alpha, gamma). In line with the literature the effects of rTMS on tinnitus loudness varied strongly across patients. This variability was also reflected in the rTMS effects on oscillatory activity. Importantly, strong reductions in tinnitus loudness were associated with increases in alpha power in the stimulated auditory cortex, while an unspecific decrease in gamma and alpha power, particularly in left frontal regions, was linked to an increase in tinnitus loudness. The identification of alpha power increase as main correlate for tinnitus reduction sheds further light on the pathophysiology of tinnitus. This will hopefully stimulate the development of more effective therapy approaches. PMID:23390539

  10. Regulation of factor XIa activity by platelets and alpha 1-protease inhibitor.

    PubMed Central

    Walsh, P N; Sinha, D; Kueppers, F; Seaman, F S; Blankstein, K B

    1987-01-01

    We have studied the complex interrelationships between platelets, Factor XIa, alpha 1-protease inhibitor and Factor IX activation. Platelets were shown to secrete an inhibitor of Factor XIa, and to protect Factor XIa from inactivation in the presence of alpha 1-protease inhibitor and the secreted platelet inhibitor. This protection of Factor XIa did not arise from the binding of Factor XIa to platelets, the presence of high molecular weight kininogen, or the inactivation of alpha 1-protease inhibitor by platelets. The formation of a complex between alpha 1-protease inhibitor and the active-site-containing light chain of Factor XIa was inhibited by activated platelets and by platelet releasates, but not by high molecular weight kininogen. These results support the hypothesis that platelets can regulate Factor XIa-catalyzed Factor IX activation by secreting an inhibitor of Factor XIa that may act primarily outside the platelet microenvironment and by protecting Factor XIa from inhibition, thereby localizing Factor IX activation to the platelet plug. Images PMID:3500185

  11. The Escherichia coli RNA polymerase alpha subunit and transcriptional activation by bacteriophage lambda CII protein.

    PubMed

    Gabig, M; Obuchowski, M; Ciesielska, A; Latała, B; Wegrzyn, A; Thomas, M S; Wegrzyn, G

    1998-01-01

    Bacteriophage lambda is not able to lysogenise the Escherichia coli rpoA341 mutant. This mutation causes a single amino acid substitution Lys271Glu in the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD). Our previous studies indicated that the impaired lysogenisation of the rpoA341 host is due to a defect in transcriptional activation by the phage CII protein and suggested a role for alphaCTD in this process. Here we used a series of truncation and point mutants in the rpoA gene placed on a plasmid to investigate the process of transcriptional activation by the cII gene product. Our results indicate that amino-acid residues 265, 268 and 271 in the a subunit may play an important role in the CII-mediated activation of the pE promoter (most probably residue 271) or may be involved in putative interactions between alphaCTD and an UP-like element near pE (most probably residues 265 and 268). Measurement of the activity of pE-lacZ, pI-lacZ and p(aQ)-lacZ fusions in the rpoA+ and rpoA341 hosts demonstrated that the mechanism of activation of these CII-dependent promoters may be in each case different.

  12. Mixed Ligand Complexes of N-Methyl-N-phenyl Dithiocarbamate: Synthesis, Characterisation, Antifungal Activity, and Solvent Extraction Studies of the Ligand

    PubMed Central

    Ekennia, Anthony C.; Onwudiwe, Damian C.; Ume, Cyril; Ebenso, Eno E.

    2015-01-01

    A series of mixed ligand dithiocarbamate complexes with a general formula [ML2(py)2], where M = Mn(II), Co(II), Ni(II), and Cu(II), py = pyridine, and L = N-methyl-N-phenyl dithiocarbamate have been prepared and characterised by elemental analysis, FTIR and Uv spectroscopy, magnetic moment, and thermogravimetric and conductance analysis. The infrared spectra showed that symmetrical bidentate coordination occurred with the dithiocarbamate moiety through the sulfur atoms, while neutral monodentate coordination occurred through the nitrogen atom for the pyridine molecule in the complexes. The electronic spectra, elemental analysis, and magnetic moment results proved that the complexes adopted octahedral geometry. The conductance measurement showed that the complexes are nonelectrolytes proving their nonionic nature. The compounds were screened for three human pathogenic fungi: Aspergillus flavus, Aspergillus niger, and Candida albicans. The cobalt complex showed the best antifungal activity among the test compounds. Liquid-liquid extractive abilities of the ligand towards copper and nickel ions in different solvent media were investigated. The ligand showed a strong binding affinity towards the metals ions with an extractive efficiency of about 99%. PMID:26543441

  13. Fibroblast activation protein-alpha and dipeptidyl peptidase IV (CD26): cell-surface proteases that activate cell signaling and are potential targets for cancer therapy.

    PubMed

    Kelly, Thomas

    2005-01-01

    Fibroblast activation protein-alpha (FAP-alpha) and dipeptidyl peptidase IV (DPPIV) are serine proteases with post-prolyl peptidase activities that can modify tumor cell behavior. FAP-alpha and DPPIV can form heteromeric complexes with each other and may function coordinately to modulate the growth, differentiation, adhesion, and metastasis of tumor cells. This review is focused on FAP-alpha and summarizes a series of studies showing that elevated expression of FAP-alpha results in profound changes in growth and malignant behavior of tumor cells. Depending on the model system investigated, FAP-alpha expression causes dramatic promotion or suppression of tumor growth. In the case of tumor promotion, FAP-alpha expression can drive tumor growth by increasing angiogenesis and by decreasing the anti-tumor response of the immune system. In the case of tumor suppression, FAP-alpha can decrease tumorigenicity of mouse melanoma cells and restore contact inhibition and growth factor dependence even when it is catalytically inactive, implying that protein-protein interactions mediate these effects. Understanding how FAP-alpha activates cell signaling is critical to determining how FAP-alpha mediates growth promotion versus growth suppression in the different model systems and ultimately in human cancer patients. In particular, the roles of FAP-alpha protease activity and FAP-alpha complex formation with DPPIV and other surface molecules in activating cell signaling need to be elucidated since these represent potential targets for therapeutic intervention.

  14. AP-2{alpha} suppresses skeletal myoblast proliferation and represses fibroblast growth factor receptor 1 promoter activity

    SciTech Connect

    Mitchell, Darrion L.; DiMario, Joseph X.

    2010-01-15

    Skeletal muscle development is partly characterized by myoblast proliferation and subsequent differentiation into postmitotic muscle fibers. Developmental regulation of expression of the fibroblast growth factor receptor 1 (FGFR1) gene is required for normal myoblast proliferation and muscle formation. As a result, FGFR1 promoter activity is controlled by multiple transcriptional regulatory proteins during both proliferation and differentiation of myogenic cells. The transcription factor AP-2{alpha} is present in nuclei of skeletal muscle cells and suppresses myoblast proliferation in vitro. Since FGFR1 gene expression is tightly linked to myoblast proliferation versus differentiation, the FGFR1 promoter was examined for candidate AP-2{alpha} binding sites. Mutagenesis studies indicated that a candidate binding site located at - 1035 bp functioned as a repressor cis-regulatory element. Furthermore, mutation of this site alleviated AP-2{alpha}-mediated repression of FGFR1 promoter activity. Chromatin immunoprecipitation studies demonstrated that AP-2{alpha} interacted with the FGFR1 promoter in both proliferating myoblasts and differentiated myotubes. In total, these results indicate that AP-2{alpha} is a transcriptional repressor of FGFR1 gene expression during skeletal myogenesis.

  15. Resolution and purification of free primase activity from the DNA primase-polymerase alpha complex of HeLa cells.

    PubMed Central

    Vishwanatha, J K; Baril, E F

    1986-01-01

    DNA primase activity has been resolved from a purified DNA primase-polymerase alpha complex of HeLa cells by hydrophobic affinity chromatography on phenylSepharose followed by chromatography on hexylagarose. This procedure provides a good yield (55%) of DNA primase that is free from polymerase alpha. The free DNA primase activity was purified to near homogeneity and its properties characterized. Sodium dodecyl sulfate polyacrylamide gel electrophoretic analysis of the purified free DNA primase showed a major protein staining band of Mr 70,000. The native enzyme in velocity sedimentation has an S20'W of 5. DNA primase synthesizes RNA oligomers with single-stranded M-13 DNA, poly(dT) and poly(dC) templates that are elongated by the DNA polymerase alpha in a manner that has already been described for several purified eukaryotic DNA primase-polymerase alpha complexes. The purified free DNA primase activity is resistant to neutralizing anti-human DNA polymerase alpha antibodies, BuPdGTP and aphidicolin that specifically inhibit the free DNA polymerase alpha and also DNA polymerase alpha complexed with the primase. The free primase activity is more sensitive to monovalent salt concentrations and is more labile than polymerase alpha. Taken together these results indicate that the DNA primase and polymerase alpha activities of the DNA primase-polymerase alpha complex reside on separate polypeptides that associate tightly through hydrophobic interactions. Images PMID:3786132

  16. New metal complexes of N3 tridentate ligand: Synthesis, spectral studies and biological activity

    NASA Astrophysics Data System (ADS)

    Al-Hamdani, Abbas Ali Salih; Al Zoubi, Wail

    2015-02-01

    New tridentate ligand 3-amino-4-{1,5-dimethyl-3-[2-(5-methyl-1H-indol-3-yl)-ethylimino]-2phenyl-2,3-dihydro-1H-pyrazol-4-ylazo}-phenol L was synthesized from the reaction of 1,5-dimethyl-3-[2-(5-methyl-1H-indol-3-yl)-ethylimino]-2-phenyl-2,3-dihydro-1H-pyrazol-4-ylamine and 3.4-amino phenol. A complexes of these ligand [Ni(II)(L)(H2O)2 Cl]Cl, [pt(IV)(L)Cl3]Cl and [M(II)(L)Cl]Cl (M = Pd (II), Zn (II), Cd (II) and Hg (II) were synthesized. The complexes were characterized by spectroscopic methods and magnetic moment measurements, elemental analysis, metal content, Chloride containing and conductance. These studies revealed octahedral geometries for the Ni (II), pt (IV) complexes, square planar for Pd (II) complex and tetrahedral for the Zn (II), Cd(II) and Hg (II) complexes. The study of complexes formation via molar ratio and job method in DMF solution has been investigated and results were consistent to those found in the solid complexes with a ratio of (M:L) as (1:1). The thermodynamic parameters, such as ΔE*, ΔH*, ΔS* ΔG* and K are calculated from the TGA curve using Coats-Redfern method. Hyper Chem-8 program has been used to predict structural geometries of compounds in gas phase. The synthesized ligand and its metal complexes were screened for their biological activity against bacterial species, two Gram positive bacteria (Bacillus subtilis and Staphylococcus aureus) and two Gram negative bacteria (Escherichia coli and Pseudomonasaeruginosa).

  17. New RuII Complex for Dual Activity: Photoinduced Ligand Release and 1O2 Production

    PubMed Central

    Loftus, Lauren M.; White, Jessica K.; Albani, Bryan A.; Kohler, Lars; Kodanko, Jeremy J.; Thummel, Randolph P.

    2016-01-01

    The new complex [Ru(pydppn)(biq)(py)]2+ (1) undergoes both py photodissociation in CH3CN with Φ500=0.0070(4) and 1O2 production with ΦΔ=0.75(7) in CH3OH from a long-lived 3ππ* state centered on the pydppn ligand (pydppn=3-(pyrid-2-yl)benzo[i]dipyrido[3,2-a:2′,3′-c]phenazine; biq = 2,2′-biquinoline; py= pyridine). This represents an order of magnitude decrease in the Φ500 compared to the previously reported model compound [Ru(tpy)(biq)(py)]2+ (3) (tpy=2,2′:6′,2″-terpyridine) that undergoes only ligand exchange. The effect on the quantum yields by the addition of a second deactivation pathway through the low-lying 3ππ* state necessary for dual reactivity was investigated using ultrafast and nanosecond transient absorption spectroscopy, revealing a significantly shorter 3MLCT lifetime in 1 relative to that of the model complex 3. Due to the structural similarities between the two compounds, the lower values of Φ500 and ΦΔ compared to that of [Ru(pydppn)(bpy)(py)]2+ (2) (bpy=2,2′-bipyridine) are attributed to a competitive excited state population between the 3LF states involved in ligand dissociation and the long-lived 3ππ* state in 1. Complex 1 represents a model compound for dual activity that may be applied to photochemotherapy. PMID:26715085

  18. Catalytic Mechanism of Human Alpha-galactosidase

    SciTech Connect

    Guce, A.; Clark, N; Salgado, E; Ivanen, D; Kulinskaya, A; Brumer, H; Garman, S

    2010-01-01

    The enzyme {alpha}-galactosidase ({alpha}-GAL, also known as {alpha}-GAL A; E.C. 3.2.1.22) is responsible for the breakdown of {alpha}-galactosides in the lysosome. Defects in human {alpha}-GAL lead to the development of Fabry disease, a lysosomal storage disorder characterized by the buildup of {alpha}-galactosylated substrates in the tissues. {alpha}-GAL is an active target of clinical research: there are currently two treatment options for Fabry disease, recombinant enzyme replacement therapy (approved in the United States in 2003) and pharmacological chaperone therapy (currently in clinical trials). Previously, we have reported the structure of human {alpha}-GAL, which revealed the overall structure of the enzyme and established the locations of hundreds of mutations that lead to the development of Fabry disease. Here, we describe the catalytic mechanism of the enzyme derived from x-ray crystal structures of each of the four stages of the double displacement reaction mechanism. Use of a difluoro-{alpha}-galactopyranoside allowed trapping of a covalent intermediate. The ensemble of structures reveals distortion of the ligand into a {sup 1}S{sub 3} skew (or twist) boat conformation in the middle of the reaction cycle. The high resolution structures of each step in the catalytic cycle will allow for improved drug design efforts on {alpha}-GAL and other glycoside hydrolase family 27 enzymes by developing ligands that specifically target different states of the catalytic cycle. Additionally, the structures revealed a second ligand-binding site suitable for targeting by novel pharmacological chaperones.

  19. Phenolics from Glycyrrhiza glabra roots and their PPAR-gamma ligand-binding activity.

    PubMed

    Kuroda, Minpei; Mimaki, Yoshihiro; Honda, Shinichi; Tanaka, Hozumi; Yokota, Shinichi; Mae, Tatsumasa

    2010-01-15

    Bioassay-guided fractionation of the EtOH extract of licorice (Glycyrrhiza glabra roots), using a GAL-4-PPAR-gamma chimera assay method, resulted in the isolation of 39 phenolics, including 10 new compounds (1-10). The structures of the new compounds were determined by analysis of their spectroscopic data. Among the isolated compounds, 5'-formylglabridin (5), (2R,3R)-3,4',7-trihydroxy-3'-prenylflavane (7), echinatin, (3R)-2',3',7-trihydroxy-4'-methoxyisoflavan, kanzonol X, kanzonol W, shinpterocarpin, licoflavanone A, glabrol, shinflavanone, gancaonin L, and glabrone all exhibited significant PPAR-gamma ligand-binding activity. The activity of these compounds at a sample concentration of 10microg/mL was three times more potent than that of 0.5microM troglitazone.

  20. Sequential and selective hydrogenation of the C(alpha)-C(beta) and M-C(alpha) double bonds of an allenylidene ligand coordinated to osmium: new reaction patterns between an allenylidene complex and alcohols.

    PubMed

    Bolaño, Tamara; Castarlenas, Ricardo; Esteruelas, Miguel A; Oñate, Enrique

    2007-07-18

    Complex [OsH(=C=C=CPh2)(CH3CN)2(PiPr3)2]BF4 (1) reacts with primary and secondary alcohols to give the corresponding dehydrogenated alcohols and the hydride-carbene derivative [OsH(=CHCH=CPh2)(CH3CN)2(PiPr3)2]BF4 (2), as a result of hydrogen transfer reactions from the alcohols to the Calpha-Cbeta double bond of the allenylidene ligand of 1. The reactions with phenol and t-butanol, which do not contain any beta-hydrogen, afford the alkoxy-hydride-carbyne complexes [OsH(OR)(CCH=CPh2)(CH3CN)(PiPr3)2]BF4 (R = Ph (3), tBu (4)), as a consequence of the 1,3-addition of the O-H bond of the alcohols to the metallic center and the Cbeta atom of the allenylidene of 1. On the basis of the reactions of 1 with these tertiary alcohols, deuterium labeling experiments, and DFT calculations, the mechanism of the hydrogenation is proposed. In acetonitrile under reflux, the Os-C double bond of 2 undergoes hydrogenation to give 1,1-diphenylpropene and [Os{CH2CH(CH3)PiPr2(CH3CN)3(PiPr3)]BF4 (11), containing a metalated phosphine ligand. This reaction is a first-order process with activation parameters of DeltaH = 89.0 +/- 6.3 kJ mol-1 and DeltaS = -43.5 +/- 9.6 J mol-1 K-1. The X-ray structures of 2 and 3 are also reported.

  1. Quantitative Conformationally Sampled Pharmacophore (CSP) for δ Opioid Ligands: Reevaluation of hydrophobic moieties essential for biological activity

    PubMed Central

    Bernard, Denzil; Coop, Andrew; MacKerell, Alexander D.

    2008-01-01

    Recent studies have indicated several therapeutic applications for δ opioid agonists and antagonists. To exploit the therapeutic potential of δ opioids developing a structural basis for the activity of ligands at the δ opioid receptor is essential. The conformationally sampled pharmacophore (CSP) method (Bernard et al., JACS, 125: 3103–3107, 2003) is extended here to obtain quantitative models of δ opioid ligand efficacy and affinity. Quantification is performed via overlap integrals of the conformational space sampled by ligands with respect to a reference compound. Iterative refinement of the CSP model identified hydrophobic groups other than the traditional phenylalanine residues as important for efficacy and affinity in DSLET and ICI 174,864. The obtained models for a structurally diverse set of peptidic and non-peptidic δ opioid ligands offer good predictions with R2 values > 0.9 and the predicted efficacy for a set of test compounds was consistent with the experimental value. PMID:17367120

  2. Influence of PGF2 alpha on gastrointestinal activity in the conscious piglet.

    PubMed

    De Saedeleer, V; Wechsung, E; Houvenaghel, A

    1992-01-01

    In 5 conscious piglets with electrodes implanted on the antrum pylori, duodenum, jejunum and ileum, the effect of intravenous infusion of PGF2 alpha, 1 and 10 micrograms/kg/min during 2 h, on gastrointestinal electrical activity was studied. The influence of the PG, 10(-8) to 10(-4) M, on longitudinal tissue strips from the same segments was also examined. The in vitro results demonstrate that PGF2 alpha has only a weak contractile effect on duodenal and jejunal strips. This effect was enhanced in the presence of atropine and indomethacin. In the in vivo part of the study PGF2 alpha induced an inhibition of antral electrical activity as evidenced by a prolongation of the inhibitory phases and a reduction of the frequency of the fast oscillations. In the small intestine only ileal activity was changed significantly. PGF2 alpha provoked an increase in the phase II or irregular spiking activity and an increase in the interval of the migrating myoelectrical complexes in this segment.

  3. Tandem C-H activation/arylation catalyzed by low-valent iron complexes with bisiminopyridine ligands.

    PubMed

    Salanouve, Elise; Bouzemame, Ghania; Blanchard, Sébastien; Derat, Etienne; Desage-El Murr, Marine; Fensterbank, Louis

    2014-04-14

    Tandem C-H activation/arylation between unactivated arenes and aryl halides catalyzed by iron complexes that bear redox-active non-innocent bisiminopyridine ligands is reported. Similar reactions catalyzed by first-row transition metals have been shown to involve substrate-based aryl radicals, whereas our catalytic system likely involves ligand-centered radicals. Preliminary mechanistic investigations based on spectroscopic and reactivity studies, in conjunction with DFT calculations, led us to propose that the reaction could proceed through an inner-sphere C-H activation pathway, which is rarely observed in the case of iron complexes. This bielectronic noble-metal-like behavior could be sustained by the redox-active non-innocent bisiminopyridine ligands.

  4. Design and one-pot synthesis of alpha-aminophosphonates and bis(alpha-aminophosphonates) by iron(III) chloride and cytotoxic activity.

    PubMed

    Rezaei, Zahra; Firouzabadi, Habib; Iranpoor, Nasser; Ghaderi, Abbas; Jafari, Mohammad Reza; Jafari, Abbas Ali; Zare, Hamid Reza

    2009-11-01

    In this study, we used a solution of FeCl(3) in THF to facilitate the Mannich-type reaction of aldehyde, amine and phosphite compounds to form corresponding alpha-aminophosphonates in a one-pot, three-component reaction. Selected alpha-aminophosphonates were entered into a biological assay test and were studied by docking methods, using Autodock 3.0. The results showed that the reactions were carried out mildly and eco-friendly to form alpha-aminophosphonates in high yields. Some were found to have cytotoxic activity on the cell lines RAJI, JURKAT and MCF-7. An indole derived bis(alpha-aminophosphonates) showed maximum cytotoxic effect comparable to doxorubicin. Although the FDE (Final Docking Energy) for the most cytotoxic compound was of the most negative value, there is no correlation between FDE and cytotoxicity.

  5. Activation of hypoxia-inducible factor-1; definition of regulatory domains within the alpha subunit.

    PubMed

    Pugh, C W; O'Rourke, J F; Nagao, M; Gleadle, J M; Ratcliffe, P J

    1997-04-25

    Hypoxia-inducible factor-1 (HIF-1), a heterodimeric DNA binding complex composed of two basic-helix-loop-helix Per-AHR-ARNT-Sim proteins (HIF-1alpha and -1beta), is a key component of a widely operative transcriptional response activated by hypoxia, cobaltous ions, and iron chelation. To identify regions of HIF-1 subunits responsible for oxygen-regulated activity, we constructed chimeric genes in which portions of coding sequence from HIF-1 genes were either linked to a heterologous DNA binding domain or encoded between such a DNA binding domain and a constitutive activation domain. Sequences from HIF-1alpha but not HIF-1beta conferred oxygen-regulated activity. Two minimal domains within HIF-1alpha (amino acids 549-582 and amino acids 775-826) were defined by deletional analysis, each of which could act independently to convey inducible responses. Both these regions confer transcriptional activation, and in both cases adjacent sequences appeared functionally repressive in transactivation assays. The inducible operation of the first domain, but not the second, involved major changes in the level of the activator fusion protein in transfected cells, inclusion of this sequence being associated with a marked reduction of expressed protein level in normoxic cells, which was relieved by stimulation with hypoxia, cobaltous ions, or iron chelation. These results lead us to propose a dual mechanism of activation in which the operation of an inducible activation domain is amplified by regulation of transcription factor abundance, most likely occurring through changes in protein stability.

  6. Antifertility activity and toxicity of alpha-chlorohydrin aromatic ketal analogues in male rats.

    PubMed

    Brown-Woodman, P D; White, I G; Ridley, D D

    1986-01-01

    The antifertility activity and toxicity of alpha-chlorohydrin and seven aromatic ketal derivatives were investigated in male rats. At a dose of 5 mg/kg injected intraperitoneally each day for 14 days, alpha-chlorohydrin and the methoxy benzaldehyde derivative (compound 2) produced complete infertility. The benzaldehyde derivative (compound 1) was 89% effective and the other five compounds 71-25% effective. All compounds except the least effective antifertility agent, the methylbenzaldehyde derivative (compound 3), reduced the motility of sperm recovered from the epididymis. None of the compounds caused a decrease in body or testes weight but some increased adrenal weight.

  7. Communications: Comparison of activation barriers for the Johari-Goldstein and alpha relaxations and its implications.

    PubMed

    Goldstein, Martin

    2010-01-28

    The range of activation barrier heights for the Johari-Goldstein (JG) relaxation in glasses is shown to overlap the range for the main (alpha) relaxation, but to be on the average somewhat lower. This suggests the JG relaxation, like the alpha, involve transitions between megabasins in the energy landscape, and that the original conjecture by Johari and this author that the JG relaxation is an intrabasin one cannot be correct. A further possibility is that there is a closer connection of the JG relaxation to the phenomenon of dynamic heterogeneity in supercooled liquids than so far assumed.

  8. A Comparison of the Anorectic Effect and Safety of the Alpha2-Adrenoceptor Ligands Guanfacine and Yohimbine in Rats with Diet-Induced Obesity

    PubMed Central

    Dudek, Magdalena; Knutelska, Joanna; Bednarski, Marek; Nowiński, Leszek; Zygmunt, Małgorzata; Mordyl, Barbara; Głuch-Lutwin, Monika; Kazek, Grzegorz; Sapa, Jacek; Pytka, Karolina

    2015-01-01

    The search for drugs with anorectic activity, acting within the adrenergic system has attracted the interest of researchers. Partial α2-adrenoceptor agonists might offer the potential for effective and safe treatment of obesity. We compared the effectiveness and safety of α2-adrenoceptor ligands in reducing body mass. We also analyzed if antagonist and partial agonists of α2-adrenoceptor––yohimbine and guanfacine––act similarly, and determined which course of action is connected with anorectic activity. We tested intrinsic activity and effect on the lipolysis of these compounds in cell cultures, evaluated their effect on meal size, body weight in Wistar rats with high-fat diet-induced obesity, and determined their effect on blood pressure, heart rate, lipid profile, spontaneous locomotor activity, core temperature and glucose, as well as glycerol and cortisol levels. Both guanfacine and yohimbine showed anorectic activity. Guanfacine was much more effective than yohimbine. Both significantly reduced the amount of intraperitoneal adipose tissue and had a beneficial effect on lipid profiles. Decreased response of α2A-adrenoceptors and partial stimulation of α2B-receptors seem to be responsible for the anorectic action of guanfacine. The stimulation of α1-adrenoceptors by guanfacine is responsible for cardiovascular side effects but may also be linked with improved anorexic effect. α1-adrenoceptor blockade is connected with the side effects of yohimbine, but it is also associated with the improvement of lipid profiles. Guanfacine has been approved by the Food and Drug Administration (FDA) to treat hypertension and conduct disorder, but as it reduces body weight, it is worth examining its effectiveness and safety in models of obesity. PMID:26506439

  9. Discrete divalent rare-earth cationic ROP catalysts: ligand-dependent redox behavior and discrepancies with alkaline-earth analogues in a ligand-assisted activated monomer mechanism.

    PubMed

    Liu, Bo; Roisnel, Thierry; Maron, Laurent; Carpentier, Jean-François; Sarazin, Yann

    2013-03-18

    The first solvent-free cationic complexes of the divalent rare-earth metals, [{RO}RE(II) ](+) [A](-) (RE(II) =Yb(II) , 1; Eu(II) , 2) and [{LO}RE(II) ](+) [A](-) ([A](-) =[H2 N{B(C6 F5 )3 }2 ](-) ; RE(II) =Yb(II) , 3; Eu(II) , 4), have been prepared by using highly chelating monoanionic aminoether-fluoroalkoxide ({RO}(-) ) and aminoether-phenolate ({LO}(-) ) ligands. Complexes 1 and 2 are structurally related to their alkaline-earth analogues [{RO}AE](+) [A](-) (AE=Ca, 5; Sr, 6). Yet, the two families behave very differently during catalysis of the ring-opening polymerization (ROP) of L-lactide (L-LA) and trimethylene carbonate (TMC) performed under immortal conditions with excess BnOH as an exogenous chain-transfer agent. The ligand was found to strongly influence the behavior of the RE(II) complexes during ROP catalysis. The fluoroalkoxide RE(II) catalysts 1 and 2 are not oxidized under ROP conditions, and compare very favorably with their Ca and Sr congeners 5 and 6 in terms of activity (turnover frequency (TOF) in the range 200-400 molL-LA (molEu  h(-1) )) and control over the parameters during the immortal ROP of L-LA (Mn,theor ≈Mn,SEC , Mw /Mn <1.05). The Eu(II) -phenolate 4 provided one of the most effective ROP cationic systems known to date for L-LA polymerization, exhibiting high activity (TOF up to 1 880 molL-LA ⋅(molEu  h)(-1) ) and good control (Mw /Mn =1.05). By contrast, upon addition of L-LA the Yb(II) -phenolate 3 immediately oxidizes to inactive RE(III) species. Yet, the cyclic carbonate TMC was rapidly polymerized by combinations of 3 (or even 1) and BnOH, revealing excellent activities (TOF=5000-7000 molTMC ⋅(molEu  h)(-1) ) and unusually high control (Mn,theor ≈Mn,SEC , Mw /Mn <1.09); under identical conditions, the calcium derivative 5 was entirely inert toward TMC. Based on experimental and kinetic data, a new ligand-assisted activated monomer ROP mechanism is suggested, in which the so-called ancillary ligand plays a

  10. Renovation of Optically Active Phenanthrolines as Powerful Chiral Ligands for Versatile Asymmetric Metal Catalysis.

    PubMed

    Naganawa, Yuki; Nishiyama, Hisao

    2016-12-01

    In the field of asymmetric synthesis, the development of new chiral ligands has been regarded as an attractive challenge for decades. Novel chiral ligands can often have a great impact on synthetic protocols. In this context, we are currently interested in the application of 1,10-phenanthroline (phen) as an entirely new class of chiral ligand. To handle this issue, we designed a chiral phen ligand that provides the N,N,O-tridentate coordination of the phen moiety and an additional phenolic hydroxyl group. As phen possesses greater coordination ability with various ions, our chiral phen ligand would be valuable as one of the "privileged" chiral ligands applied to a broad range of metal catalysts and new reactions. This account summarizes the results of the application of the chiral phen ligand to various kinds of metal catalysis.

  11. Enzymatic metabolism of ergosterol by cytochrome p450scc to biologically active 17alpha,24-dihydroxyergosterol.

    PubMed

    Slominski, Andrzej; Semak, Igor; Zjawiony, Jordan; Wortsman, Jacobo; Gandy, Michael N; Li, Jinghu; Zbytek, Blazej; Li, Wei; Tuckey, Robert C

    2005-08-01

    We demonstrate the metabolism of ergosterol by cytochrome P450scc in either a reconstituted system or isolated adrenal mitochondria. The major reaction product was identified as 17alpha,24-dihydroxyergosterol. Purified P450scc also generated hydroxyergosterol as a minor product, which is probably an intermediate in the synthesis of 17alpha,24-dihydroxyergosterol. In contrast to cholesterol and 7-dehydrocholesterol, cleavage of the ergosterol side chain was not observed. NMR analysis clearly located one hydroxyl group to C24, with evidence that the second hydroxyl group is at C17. 17alpha,24-Dihydroxyergosterol inhibited cell proliferation of HaCaT keratinocytes and melanoma cells. Thus, in comparison with cholesterol and 7-dehydrocholesterol, the 24-methyl group and the C22-C23 double bond of ergosterol prevent side chain cleavage by P450scc and change the enzyme's hydroxylase activity from C22 and C20, to C24 and C17, generating bioactive product.

  12. A study of excess H-alpha emission in chromospherically active M dwarf

    NASA Technical Reports Server (NTRS)

    Young, Arthur; Skumanich, Andrew; Stauffer, John R.; Harlan, Eugene; Bopp, Bernard W.

    1989-01-01

    Spectroscopic observations from three observatories are combined to study the properties of the excess H-alpha emission which characterizes the most chromospherically active subset of the M dwarf stars, known as the dMe stars. It is demonstrated that the excess H-alpha luminosity from these stars is a monotonically decreasing function of their (R-I) color, and evidence is presented which suggests that the product of the mean surface brightness and the mean filling factor of the emissive regions is essentially constant with color. Another significant result of the study is a linear correlation between the excess luminosity in H-alpha and the coronal X-ray luminosity.

  13. Multiple ligand-binding properties of the lipocalin member chicken alpha1-acid glycoprotein studied by circular dichroism and electronic absorption spectroscopy: the essential role of the conserved tryptophan residue.

    PubMed

    Zsila, Ferenc; Matsunaga, Hisami; Bikádi, Zsolt; Haginaka, Jun

    2006-08-01

    Multiple ligand-binding properties of the 30-kDa chicken alpha(1)-acid glycoprotein (cAGP), a member of the lipocalin protein family, were investigated for the first time by using circular dichroism (CD) and UV/Vis absorption spectroscopy methods. By measuring induced CD (ICD) spectra, high-affinity binding (K(a) approximately 10(5)-10(6) M(-1)) of several drugs, dyes and natural compounds to cAGP was demonstrated including antimalarial agents (quinacrine, primaquine), phenotiazines (chlorpromazine, methylene blue), propranolol, non-steroidal antiinflammatory drugs (ketoprofen, diclofenac), tamoxifen, diazepam, tacrine, dicoumarol, cationic dyes (auramine O, thioflavine T, ethidium bromide), benzo[a]pyrene, L-thyroxine, bile pigments (bilirubin, biliverdin), alkaloids (piperine, aristolochic acid), saturated and unsaturated fatty acids. Analysis of the extrinsic CD spectra with the study of the covalently modified protein and CD displacement experiments revealed that a single Trp26 residue of cAGP conserved in the whole lipocalin family is part of the binding site, and it is essentially involved in the ligand-binding process via pi-pi stacking interaction resulting in the appearance of strong induced CD bands due to the non-degenerate intermolecular exciton coupling between the pi-pi* transitions of the stacked indole ring-ligand chromophore. The finding that cAGP is able to accommodate a broad spectrum of ligands belonging to different chemical classes suggests that its core beta-barrel cavity is unusually wide containing overlapping sub-sites. Significance of these new data in understanding of the ligand-binding properties of other lipocalins, especially that of human AGP, and potential practical applications are briefly discussed. Overall, cAGP serves as a simple, ultimate model to extend our knowledge on ligand-binding properties of lipocalins and to study the role of tryptophan residues in molecular recognition processes.

  14. Peptide insertions in domain 4 of hbeta(c), the shared signalling receptor subunit for GM-CSF, IL3 and IL5, induce ligand-independent activation.

    PubMed

    Jones, K L; Bagley, C J; Butcher, C; Barry, S C; Vadas, M A; D'Andrea, R J

    2001-06-21

    A mutant form of the common beta-subunit of the GM-CSF, interleukin-3 (IL3) and IL5 receptors is activated by a 37 residue duplicated segment which includes the WSXWS motif and an adjacent, highly conserved, aliphatic/basic element. Haemopoietic expression of this mutant, hbeta(c)FIDelta, in mice leads to myeloproliferative disease. To examine the mechanism of activation of this mutant we targetted the two conserved motifs in each repeat for mutagenesis. Here we show that this mutant exhibits constitutive activity in BaF-B03 cells in the presence of mouse or human GM-CSF receptor alpha-subunit (GMRalpha) and this activity is disrupted by mutations of the conserved motifs in the first repeat. In the presence of these mutations the receptor reverts to an alternative conformation which retains responsiveness to human IL3 in a CTLL cell line co-expressing the human IL3 receptor alpha-subunit (hIL3Ralpha). Remarkably, the activated conformation is maintained in the presence of substitutions, deletions or replacement of the second repeat. This suggests that activation occurs due to insertion of extra sequence after the WSXWS motif and is not dependent on the length or specific sequence of the insertion. Thus hbeta(c) displays an ability to fold into functional receptor conformations given insertion of up to 37 residues in the membrane-proximal region. Constitutive activation most likely results from a specific conformational change which alters a dormant, inactive receptor complex, permitting functional association with GMRalpha and ligand-independent mitogenic signalling.

  15. Bright illumination reduces parietal EEG alpha activity during a sustained attention task.

    PubMed

    Min, Byoung-Kyong; Jung, Young-Chul; Kim, Eosu; Park, Jin Young

    2013-11-13

    The influence of the illumination condition on our cognitive-performance seems to be more critical in the modern life, wherein, most people work in an office under a specific illumination condition. However, neurophysiological changes in a specific illumination state and their cognitive interpretation still remain unclear. Thereby, in the present study, the effect of different illumination conditions on the same cognitive performance was evaluated particularly by EEG wavelet analyses. During a sustained attention task, we observed that the higher illumination condition yielded significantly lower parietal tonic electroencephalogram (EEG) alpha activity before the presentation of the probe digit and longer reaction times, than that of the other illumination conditions. Although previous studies suggest that lower prestimulus EEG alpha activity is related to higher performance in an upcoming task, the reduced prestimulus alpha activity under higher illumination was associated with delayed reaction times in the present study. Presumably, the higher background illumination condition seems to be too bright for normal attentional processing and distracted participants' attention during a sustained attention task. Such a bottom-up effect by stimulus salience seemed to overwhelm a prestimulus top-down effect reflected in prestimulus alpha power during the bright background condition. This finding might imply a dynamic competition between prestimulus top-down and poststimulus bottom-up processes. Our findings provide compelling evidence that the illumination condition substantially modulates our attentional processing. Further refinement of the illumination parameters and subsequent exploration of cognitive-modulation are necessary to facilitate our cognitive performance.

  16. 5 Alpha-reductase inhibitory and antiandrogenic activities of novel steroids in hamster seminal vesicles.

    PubMed

    Cabeza, Marisa; Bratoeff, Eugene; Flores, Eugenio; Ramírez, Elena; Calleros, Jorge; Montes, Diana; Quiroz, Alexandra; Heuze, Ivonne

    2002-11-01

    The pharmacological activity of several 16-bromosubstituted trienediones 4 and 5, 16-methyl substituted dienediones 6 and 7 and the 16-methyl substituted trienedione 8 was determined on gonadectomized hamster seminal vesicles by measuring the in vitro conversion of testosterone (T) to dihydrotestosterone (DHT) as 5alpha-reductase inhibitors and also the ability of these steroids to bind to the androgen receptor. Steroids 6 and 7 when injected together with T decreased the weight of the seminal vesicles thus showing an antiandrogenic effect. Compounds 5 and 6 reduced substantially the conversion of T to DHT and therefore can be considered good inhibitors for the enzyme 5alpha-reductase; however both steroids failed to form a complex with the androgen receptor. On the other hand compound 7 which showed a very small inhibitory activity for the enzyme 5alpha-reductase, exhibited a very high affinity for the androgen receptor and thus can be considered an effective antiandrogen. This compound also reduced substantially the weight of the seminal vesicles. Steroids 4 and 8 did not reduce the weight of the seminal vesicles and exhibited a low affinity for the androgen receptor; 8 showed a weak 5alpha-reductase inhibitory activity, whereas 4 exhibited a weak androgenic effect.

  17. Role of oxidants in NF-kappa B activation and TNF-alpha gene transcription induced by hypoxia and endotoxin.

    PubMed

    Chandel, N S; Trzyna, W C; McClintock, D S; Schumacker, P T

    2000-07-15

    The transcription factor NF-kappa B stimulates the transcription of proinflammatory cytokines including TNF-alpha. LPS (endotoxin) and hypoxia both induce NF-kappa B activation and TNF-alpha gene transcription. Furthermore, hypoxia augments LPS induction of TNF-alpha mRNA. Previous reports have indicated that antioxidants abolish NF-kappa B activation in response to LPS or hypoxia, which suggests that reactive oxygen species (ROS) are involved in NF-kappa B activation. This study tested whether mitochondrial ROS are required for both NF-kappaB activation and the increase in TNF-alpha mRNA levels during hypoxia and LPS. Our results indicate that hypoxia (1.5% O2) stimulates NF-kappa B and TNF-alpha gene transcription and increases ROS generation as measured by the oxidant sensitive dye 2',7'-dichlorofluorescein diacetate in murine macrophage J774.1 cells. The antioxidants N-acetylcysteine and pyrrolidinedithiocarbamic acid abolished the hypoxic activation of NF-kappa B, TNF-alpha gene transcription, and increases in ROS levels. Rotenone, an inhibitor of mitochondrial complex I, abolished the increase in ROS signal, the activation of NF-kappa B, and TNF-alpha gene transcription during hypoxia. LPS stimulated NF-kappa B and TNF-alpha gene transcription but not ROS generation in J774.1 cells. Rotenone, pyrrolidinedithiocarbamic acid, and N-acetylcysteine had no effect on the LPS stimulation of NF-kappa B and TNF-alpha gene transcription, indicating that LPS activates NF-kappa B and TNF-alpha gene transcription through a ROS-independent mechanism. These results indicate that mitochondrial ROS are required for the hypoxic activation of NF-kappa B and TNF-alpha gene transcription, but not for the LPS activation of NF-kappa B.

  18. Recombinant human tumor necrosis factor-alpha: evidence of an indirect mode of antitumor activity.

    PubMed

    Manda, T; Shimomura, K; Mukumoto, S; Kobayashi, K; Mizota, T; Hirai, O; Matsumoto, S; Oku, T; Nishigaki, F; Mori, J

    1987-07-15

    The antitumor activity of recombinant human tumor necrosis factor (rTNF-alpha) was examined on murine tumors in mice and in cultured cells in vitro. Mice were implanted intradermally with Meth A fibrosarcoma (Meth A) on day 0. rTNF-alpha caused tumor necrosis and inhibited the tumor growth when given i.v. on day 7 or 10, but not when given on day 3. When rTNF-alpha was given i.v. in doses of 0.1-3.2 micrograms/mouse twice a week for 3 weeks beginning on day 7 or 11, the growth of solid Meth A, Colon 26 adenocarcinoma, Colon 38 carcinoma, Sarcoma-180, and M5076 reticulum cell sarcoma tumors implanted s.c. or intradermally was markedly inhibited, and the life of the mice bearing these tumors, except M5076 reticulum cell sarcoma, was prolonged. The growth of Meth A implanted i.m. was also markedly inhibited by rTNF-alpha given i.v. However, the life of mice bearing i.p. Colon 26 adenocarcinoma, MH134 hepatoma, Sarcoma-180, and Ehrlich carcinoma was not prolonged by rTNF-alpha given i.p. nine times (days 1-9) in doses up to 1.0 or 3.2 micrograms/mouse. Only in the case of mice bearing i.p. Meth A, the life was slightly prolonged by i.p. treatment with rTNF-alpha but not by i.v. treatment. In experiments against in vitro cultured cells, rTNF-alpha did not show any direct cytotoxicity against mouse tumor cells: Meth A, Colon 26 adenocarcinoma, Colon 38 carcinoma, and Sarcoma-180, but had a cytotoxic effect against L929 mouse fibroblast. The results suggest that rTNF-alpha is a unique antitumor drug with potent necrotizing activity against solid tumors in mice, and that this activity may derive from indirect mechanisms related to the growth of tumors and not to the direct cytotoxicity of the drug.

  19. General Subject 1. Report to ICUMSA on the determination of commercial alpha-amylase activity by a spectrophotometric method

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A report is given on a new industrial method for the determination of the activity or strength of commercial alpha-amylase at a sugarcane factory or refinery, as well as a recommendation. At the present time, the activities or strengths of commercial alpha-amylases cannot be directly compared becau...

  20. Top-down controlled alpha band activity in somatosensory areas determines behavioral performance in a discrimination task.

    PubMed

    Haegens, Saskia; Händel, Barbara F; Jensen, Ole

    2011-04-06

    The brain receives a rich flow of information which must be processed according to behavioral relevance. How is the state of the sensory system adjusted to up- or downregulate processing according to anticipation? We used magnetoencephalography to investigate whether prestimulus alpha band activity (8-14 Hz) reflects allocation of attentional resources in the human somatosensory system. Subjects performed a tactile discrimination task where a visual cue directed attention to their right or left hand. The strength of attentional modulation was controlled by varying the reliability of the cue in three experimental blocks (100%, 75%, or 50% valid cueing). While somatosensory prestimulus alpha power lateralized strongly with a fully predictive cue (100%), lateralization was decreased with lower cue reliability (75%) and virtually absent if the cue had no predictive value at all (50%). Importantly, alpha lateralization influenced the subjects' behavioral performance positively: both accuracy and speed of response improved with the degree of alpha lateralization. This study demonstrates that prestimulus alpha lateralization in the somatosensory system behaves similarly to posterior alpha activity observed in visual attention tasks. Our findings extend the notion that alpha band activity is involved in shaping the functional architecture of the working brain by determining both the engagement and disengagement of specific regions: the degree of anticipation modulates the alpha activity in sensory regions in a graded manner. Thus, the alpha activity is under top-down control and seems to play an important role for setting the state of sensory regions to optimize processing.

  1. Synthesis and catalytic activity of heterogeneous rare-earth metal catalysts coordinated with multitopic Schiff-base ligands.

    PubMed

    Sun, Yilin; Wu, Guangming; Cen, Dinghai; Chen, Yaofeng; Wang, Limin

    2012-08-28

    Four multitopic Schiff-base ligand precursors were synthesized via condensation of 4,4'-diol-3,3'-diformyl-1,1'-diphenyl or 1,3,5-tris(4-hydroxy-5-formylphenyl)benzene with 2,6-diisopropylaniline or 2,6-dimethylaniline. Amine elimination reactions of Ln[N(SiMe(3))(2)](3) (Ln = La, Nd, Sm or Y) with these multitopic ligand precursors gave ten heterogeneous rare-earth metal catalysts. These heterogeneous rare-earth metal catalysts are active for intramolecular hydroalkoxylation of alkynols, and the catalytic activities are influenced by the ligand and metal ion. The recycling experiment on the most active heterogeneous catalyst showed the catalyst has a good reusability.

  2. Ruthenium-based olefin metathesis catalysts bearing pH-responsive ligands: External control of catalyst solubility and activity

    NASA Astrophysics Data System (ADS)

    Balof, Shawna Lynn

    2011-12-01

    Sixteen novel, Ru-based olefin metathesis catalysts bearing pH responsive ligands were synthesized. The pH-responsive groups employed with these catalysts included dimethylamino (NMe2) modified NHC ligands as well as N-donor dimethylaminopyridine (DMAP) and 3-(o-pyridyl)propylidene ligands. These pH-responsive ligands provided the means by which the solubility and/or activity profiles of the catalysts produced could be controlled via acid addition. The main goal of this dissertation was to design catalyst systems capable of performing ring opening metathesis (ROMP) and ring closing metathesis (RCM) reactions in both organic and aqueous media. In an effort to quickly gain access to new catalyst structures, a template synthesis for functionalized NHC ligand precursors was designed, in addition to other strategies, to obtain ligand precursors with ancillary NMe2 groups. Kinetic studies for the catalysts produced from these precursors showed external control of catalyst solubility was afforded via protonation of the NMe2 groups of their NHC ligands. Additionally, this protonation afforded external control of catalyst propagation rates for several catalysts. This is the first known independent external control for the propagation rates of ROMP catalysts. The incorporation of pH-responsive N-donor ligands into catalyst structures also provided the means for the external control of metathesis activity, as the protonation of these ligands resulted in an increased initiation rate based on their fast and irreversible dissociation from the metal center. The enhanced external control makes these catalysts applicable to a wide range of applications, some of which have been explored by us and/or through collaboration. Three of the catalysts designed showed remarkable metathesis activity in aqueous media. These catalysts displayed comparable RCM activity in aqueous media to a class of water-soluble catalysts reported by Grubbs et al., considered to be the most active catalyst for

  3. Selectin ligands and tumor-associated carbohydrate structures: specificities of alpha 2,3-sialyltransferases in the assembly of 3'-sialyl-6-sialyl/sulfo Lewis a and x, 3'-sialyl-6'-sulfo Lewis x, and 3'-sialyl-6-sialyl/sulfo blood group T-hapten.

    PubMed

    Chandrasekaran, E V; Jain, R K; Larsen, R D; Wlasichuk, K; Matta, K L

    1995-03-07

    The sequence in the assembly of the functional unit of selectin ligands containing sulfate, sialic acid, and fucose and also tumor-associated O-glycan structures was studied by examining the specificities of alpha 2,3-sialyltransferases (ST). The first enzyme, porcine liver ST, was 57, 37, and 79% active (Km: 0.105, 0.420, and 0.200 mM), respectively, toward 6-sulfo, 6-sialyl, or 6-O-methyl derivatives of the Gal beta 1,3GalNAc alpha- unit; C-3 or C-6 substitution on Gal abolished sialylation. An acrylamide copolymer (MW approximately 40,000) containing approximately 40 T-haptens and asialo Cowper's gland mucin (MW approximately 200,000) containing approximately 48 T-haptens was 5-fold more active as an acceptor as compared to Gal beta 1, 3GalNAc alpha-O-Al on a molecular weight basis. The second enzyme, a cloned alpha-2,3-ST specific for lactose-based structure, was 70, 102, and 108% active (Km: 0.500, 0.210, and 0.330 mM), respectively, toward 6-sialyl, 6-sulfo, or 6-O-methyl derivatives of the Gal beta 1,3GlcNAc beta- unit; C-3 and C-6 substitution on Gal abolished sialylation. Gal beta 1,4GlcNAc beta- and its 6-sulfo derivative were approximately 20% active; the Lewis a structure, Gal beta 1,3- (Fuc alpha 1,4)GlcNAc beta-, was not an acceptor. The acrylamide copolymers containing approximately 40 units of Gal beta 1,3GlcNAc beta-, Gal beta 1,3(6-sulfo)GlcNAc beta-, or fetuin triantennary asialo or bovine IgG diantennary glycopeptides were respectively 5.9-, 5.4-, 0.7-, and 0.1-fold as active. A transfer of 7-9 mol of NeuAc per mole of the above copolymers was catalyzed by this ST, the sialyl linkage being susceptible to alpha 2,3-specific sialidase. A partially purified Colo 205 Lewis type (alpha 1, 3/4) fucosyltransferase catalyzed the formation of 3'-sialyl-6-sulfo Lewis a from [9-3H]NeuAc alpha 2, 3Gal beta 1, 3(6-sulfo)GlcNAc beta-O-Allyl and copolymer containing [9-3H]NeuAc alpha 2, 3Gal beta 1, 3(6-sulfo)GlcNAc beta- units, using GDP[14C]Fuc as fucosyl

  4. Bacillus thermoamyloliquefaciens KP1071 alpha-glucosidase II is a thermostable M(r) 540,000 homohexameric alpha-glucosidase with both exo-alpha-1,4-glucosidase and oligo-1,6-glucosidase activities.

    PubMed

    Suzuki, Y; Nobiki, M; Matsuda, M; Sawai, T

    1997-04-01

    alpha-Glucosidase II of the facultative thermophile Bacillus thermoamyloliquefaciens KP1071 (FERM-P8477; growth over 30-66 degrees C) was purified to a homogeneous state. Its M(r) was estimated as 90000 by SDS/PAGE. However, the enzyme behaved as an active Mr 540000 protein on gel filtration with each of two gels of different matrices as well as on gel electrophoresis under native conditions. The enzyme was not glycosylated. Its isoelectric point was estimated as 5.7. The N-terminal sequence of 20 residues was determined asAla1-Ile-Gln-Pro-Glu-Gln-Asp-Asp-Lys-Thr-Gln-Glu-Asp-Gly- Tyr-Ile-Asp-Ile-Gly-Asn20. The sequence did not resemble those of procaryotic and eucaryotic proteins hitherto reported including the monomeric exo-alpha-1,4-glucosidase and the monomeric oligo-1,6-glucosidase from the same microorganism. The alpha-glucosidase II had no antigenic group shared with the latter two enzymes. Analysis of substrate specificity showed that the alpha-glucosidase II has dual activity towards oligo-1,6-glucosidases and exo-alpha-1,4-glucosidases, but its preference is for non-reducing terminal alpha-1,4 glucosidic bonds in substrates. Kinetic studies proved that both activities are attributed to the same catalytic site. The enzyme was most active at 81 degrees C and pH 7.0. Its half-life at pH 6.8 was 10 min at 81 degrees C, and 5 h at 55 degrees C in 6.4 M urea, 26% ethanol or 2.5% SDS. We suggest that the alpha-glucosidase II is a thermostable, homohexameric enzyme of origin distinct from the exo-alpha-1,4-glucosidase and the oligo-1,6-glucosidase present in the same strain.

  5. [Synthesis of thiophene and alpha-terthiophene derivatives with antiproliferative activity].

    PubMed

    Székelyhidi, Zsolt; Pató, János; Hegymegi-Barakonyi, Bálint; Bánhegyi, Péter; Mészáros, György; Kéri, György; Orfi, Lászlo

    2005-01-01

    We have synthesised a series of known alpha-terthiophene lead molecules with PKC (protein kinase C) inhibitory activity and the compounds were tested in cell proliferation assay on EGF-RTK (epidermal growth factor receptor protein tyrosine kinase) over-expressing tumour cell line (A431). We found that two of them had excellent antiproliferative activity. We prepared a focused molecule library around the thiophene and the terthiophene scaffold and examined these compounds in cell proliferation assay on A431.

  6. Release of alpha 2-plasmin inhibitor from plasma fibrin clots by activated coagulation factor XIII. Its effect on fibrinolysis.

    PubMed Central

    Mimuro, J; Kimura, S; Aoki, N

    1986-01-01

    When blood coagulation takes place in the presence of calcium ions, alpha 2-plasmin inhibitor (alpha 2PI) is cross-linked to fibrin by activated coagulation Factor XIII (XIIIa) and thereby contributes to the resistance of fibrin to fibrinolysis. It was previously shown that the cross-linking reaction is a reversible one, since the alpha 2PI-fibrinogen cross-linked complex could be dissociated. In the present study we have shown that the alpha 2PI-fibrin cross-linking reaction is also a reversible reaction and alpha 2PI which had been cross-linked to fibrin can be released from fibrin by disrupting the equilibrium, resulting in a decrease of its resistance to fibrinolysis. When the fibrin clot formed from normal plasma in the presence of calcium ions was suspended in alpha 2PI-deficient plasma of buffered saline, alpha 2PI was gradually released from fibrin on incubation. When alpha 2PI was present in the suspending milieu, the release was decreased inversely to the concentrations of alpha 2PI in the suspending milieu. The release was accelerated by supplementing XIIIa or the presence of a high concentration of the NH2-terminal 12-residue peptide of alpha 2PI (N-peptide) which is cross-linked to fibrin in exchange for the release of alpha 2PI. When the release of alpha 2PI from fibrin was accelerated by XIIIa or N-peptide, the fibrin became less resistant to the fibrinolytic process, resulting in an acceleration of fibrinolysis which was proportional to the degree of the release of alpha 2PI. These results suggest the possibility that alpha 2PI could be released from fibrin in vivo by disrupting the equilibrium of the alpha 2PI-fibrin cross-linking reaction, and that the release would result in accelerated thrombolysis. Images PMID:2419360

  7. Molecular mechanisms of human IRE1 activation through dimerization and ligand binding

    PubMed Central

    Joshi, Amar; Newbatt, Yvette; McAndrew, P. Craig; Stubbs, Mark; Burke, Rosemary; Richards, Mark W.; Bhatia, Chitra; Caldwell, John J.; McHardy, Tatiana; Collins, Ian; Bayliss, Richard

    2015-01-01

    IRE1 transduces the unfolded protein response by splicing XBP1 through its C-terminal cytoplasmic kinase-RNase region. IRE1 autophosphorylation is coupled to RNase activity through formation of a back-to-back dimer, although the conservation of the underlying molecular mechanism is not clear from existing structures. We have crystallized human IRE1 in a back-to-back conformation only previously seen for the yeast homologue. In our structure the kinase domain appears primed for catalysis but the RNase domains are disengaged. Structure-function analysis reveals that IRE1 is autoinhibited through a Tyr-down mechanism related to that found in the unrelated Ser/Thr protein kinase Nek7. We have developed a compound that potently inhibits human IRE1 kinase activity while stimulating XBP1 splicing. A crystal structure of the inhibitor bound to IRE1 shows an increased ordering of the kinase activation loop. The structures of hIRE in apo and ligand-bound forms are consistent with a previously proposed model of IRE1 regulation in which formation of a back-to-back dimer coupled to adoption of a kinase-active conformation drive RNase activation. The structures provide opportunities for structure-guided design of IRE1 inhibitors. PMID:25968568

  8. Effective virtual screening strategy toward covalent ligands: identification of novel NEDD8-activating enzyme inhibitors.

    PubMed

    Zhang, Shengping; Tan, Jiani; Lai, Zhonghui; Li, Ying; Pang, Junxia; Xiao, Jianhu; Huang, Zhangjian; Zhang, Yihua; Ji, Hui; Lai, Yisheng

    2014-06-23

    The NEDD8-activating enzyme (NAE) is an emerging target for cancer therapy, which regulates the degradation and turnover of a variety of cancer-related proteins by activating the cullin-RING E3 ubiquitin ligases. Among a limited number of known NAE inhibitors, the covalent inhibitors have demonstrated the most potent efficacy through their covalently linked adducts with NEDD8. Inspired by this unique mechanism, in this study, a novel combined strategy of virtual screening (VS) was adopted with the aim to identify diverse covalent inhibitors of NAE. To be specific, a docking-enabled pharmacophore model was first built from the possible active conformations of chosen covalent inhibitors. Meanwhile, a dynamic structure-based phamacophore was also established based on the snapshots derived from molecular dynamic simulation. Subsequent screening of a focused ZINC database using these pharmacophore models combined with covalent docking discovered three novel active compounds. Among them, compound LZ3 exhibited the most potent NAE inhibitory activity with an IC50 value of 1.06 ± 0.18 μM. Furthermore, a cell-based washout experiment proved the proposed covalent binding mechanism for compound LZ3, which confirmed the successful application of our combined VS strategy, indicating it may provide a viable solution to systematically discover novel covalent ligands.

  9. LIGAND STRUCTURE-DEPENDENT ACTIVATION OF ESTROGEN RECEPTOR α/Sp BY ESTROGENS AND XENOESTROGENS

    PubMed Central

    Wu, Fei; Khan, Shaheen; Wu, Qian; Barhoumi, Rola; Burghardt, Robert; Safe, Stephen

    2008-01-01

    This study investigated the effects of E2, diethylstilbestrol (DES), antiestrogens, the phytoestrogen resveratrol, and the xenoestrogens octylphenol (OP), nonylphenol (NP), endosulfan, kepone, 2,3,4,5-tetrachlorobiphenyl-4-ol (HO-PCB-Cl4), bisphenol-A(BPA), and 2,2-bis-(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE) on induction of luciferase activity in breast cancer cells transfected with a construct (pSp13) containing three tandem GC-rich Sp binding sites linked to luciferase and wild-type or variant ERα. The results showed that induction of luciferase activity was highly structure-dependent in both MCF-7 and MDA-MB-231 cells. Moreover, RNA interference assays using small inhibitory RNAs for Sp1, Sp3 and Sp4 also demonstrated structure-dependent differences in activation of ERα/Sp1, ERα/Sp3 and ERα/Sp4. These results demonstrate for the first time that various structural classes of ER ligands differentially activate wild-type and variant ERα/Sp-dependent transactivation, selectively use different Sp proteins, and exhibit selective ER modulator (SERM)-like activity. PMID:18400491

  10. C-H functionalization: thoroughly tuning ligands at a metal ion, a chemist can greatly enhance catalyst's activity and selectivity.

    PubMed

    Shul'pin, Georgiy B

    2013-09-28

    This brief essay consists of a few "exciting stories" devoted to relations within a metal-complex catalyst between a metal ion and a coordinated ligand. When, as in the case of a human couple, the rapport of the partners is cordial and a love cements these relations, a chemist finds an ideal married couple, in other words he obtains a catalyst of choice which allows him to functionalize C-H bonds very efficiently and selectively. Examples of such lucky marriages in the catalytic world of ions and ligands are discussed here. Activity of the catalyst is characterized by turnover number (TON) or turnover frequency (TOF) as well as by yield of a target product. Introducing a chelating N,N- or N,O-ligand to the catalyst molecule (this can be an iron or manganese derivative) sharply enhances its activity. However, the activity of vanadium derivatives (with additionally added to the solution pyrazinecarboxylic acid, PCA) as well as of various osmium complexes does not dramatically depend on the nature of ligands surrounding metal ions. Complexes of these metals are very efficient catalysts in oxidations with H2O2. Osmium derivatives are record-holders exhibiting extremely high TONs whereas vanadium complexes are on the second position. Finally, elegant examples of alkane functionalization on the ions of non-transition metals (aluminium, gallium etc.) are described when one ligand within the metal complex (namely, hydroperoxyl ligand HOO(-)) helps other ligand of this complex (H2O2 molecule coordinated to the metal) to disintegrate into two species, generating very reactive hydroxyl radical. Hydrogen peroxide molecule, even ligated to the metal ion, is perfectly stable without the assistance of the neighboring HOO(-) ligand. This ligand can be easily oxidized donating an electron to its partner ligand (H2O2). In an analogous case, when the central ion in the catalyst is a transition metal, this ion changing its oxidation state can donate an electron to the coordinated H2O2

  11. Modulation of Retinoic Acid Receptor-related Orphan Receptor α and γ Activity by 7-Oxygenated Sterol Ligands*

    PubMed Central

    Wang, Yongjun; Kumar, Naresh; Solt, Laura A.; Richardson, Timothy I.; Helvering, Leah M.; Crumbley, Christine; Garcia-Ordonez, Ruben D.; Stayrook, Keith R.; Zhang, Xi; Novick, Scott; Chalmers, Michael J.; Griffin, Patrick R.; Burris, Thomas P.

    2010-01-01

    The retinoic acid receptor-related orphan receptors α and γ (RORα (NR1F1) and RORγ (NR1F3)) are orphan nuclear receptors and perform critical roles in regulation of development, metabolism, and immune function. Cholesterol and cholesterol sulfate have been suggested to be RORα ligands, but the physiological significance is unclear. To date, no endogenous RORγ ligands have been described. Here, we demonstrate that 7-oxygenated sterols function as high affinity ligands for both RORα and RORγ by directly binding to their ligand-binding domains (Ki ∼20 nm), modulating coactivator binding, and suppressing the transcriptional activity of the receptors. One of the 7-oxygenated sterols, 7α-hydroxycholesterol (7α-OHC), serves as a key intermediate in bile acid metabolism, and we show that 7α-OHC modulates the expression of ROR target genes, including Glc-6-Pase and phosphoenolpyruvate carboxykinase, in an ROR-dependent manner. Furthermore, glucose output from hepatocytes is suppressed by 7α-OHC functioning as an RORα/γ ligand. Thus, RORα and RORγ are ligand-regulated members of the NR superfamily and may serve as sensors for 7-oxygenated sterols. PMID:19965867

  12. Synthesis, Characterization, DNA Interaction, and Antitumor Activities of La (III) Complex with Schiff Base Ligand Derived from Kaempferol and Diethylenetriamine

    PubMed Central

    Wang, Qin; Huang, Yu; Zhang, Jin-Sheng; Yang, Xin-Bin

    2014-01-01

    A novel La (III) complex, [LaL(H2O)3]NO3·3H2O, with Schiff base ligand L derived from kaempferol and diethylenetriamine, has been synthesized and characterized by elemental analysis, IR, UV-visible, 1H NMR, thermogravimetric analysis, and molar conductance measurements. The fluorescence spectra, circular dichroism spectra, and viscosity measurements and gel electrophoresis experiments indicated that the ligand L and La (III) complex could bind to CT-DNA presumably via intercalative mode and the La (III) complex showed a stronger ability to bind and cleave DNA than the ligand L alone. The binding constants (Kb) were evaluated from fluorescence data and the values ranged from 0.454 to 0.659 × 105 L mol−1 and 1.71 to 17.3 × 105 L mol−1 for the ligand L and La (III) complex, respectively, in the temperature range of 298–310 K. It was also found that the fluorescence quenching mechanism of EB-DNA by ligand L and La (III) complex was a static quenching process. In comparison to free ligand L, La (III) complex exhibited enhanced cytotoxic activities against tested tumor cell lines HL-60 and HepG-2, which may correlate with the enhanced DNA binding and cleaving abilities of the La (III) complex. PMID:25371657

  13. Antibacterial activity of Pd(II) complexes with salicylaldehyde-amino acids Schiff bases ligands.

    PubMed

    Rîmbu, Cristina; Danac, Ramona; Pui, Aurel

    2014-01-01

    Palladium(II) complexes with Schiff bases ligands derived from salicylaldehyde and amino acids (Ala, Gly, Met, Ser, Val) have been synthesized and characterized by Fourier transform (FT)-IR, UV-Vis and (1)H-NMR spectroscopy. The electrospray mass spectrometry (ES-MS) spectrometry confirms the formation of palladium(II) complexes in 1/2 (M/L) molar ratio. All the Pd(II) complexes 1, [Pd(SalAla)2]Cl2; 2, [Pd(SalGly)2]Cl2; 3, [Pd(SalMet)2]Cl2; 4, [Pd(SalSer)2]Cl2; 5, [Pd(SalVal)2]Cl2; have shown antibacterial activity against Gram-positive bacteria Staphylococcus aureus and Gram-negative bacteria Escherichia coli.

  14. Cannabinoid CB2 receptor ligand profiling reveals biased signalling and off-target activity

    PubMed Central

    Soethoudt, Marjolein; Grether, Uwe; Fingerle, Jürgen; Grim, Travis W.; Fezza, Filomena; de Petrocellis, Luciano; Ullmer, Christoph; Rothenhäusler, Benno; Perret, Camille; van Gils, Noortje; Finlay, David; MacDonald, Christa; Chicca, Andrea; Gens, Marianela Dalghi; Stuart, Jordyn; de Vries, Henk; Mastrangelo, Nicolina; Xia, Lizi; Alachouzos, Georgios; Baggelaar, Marc P.; Martella, Andrea; Mock, Elliot D.; Deng, Hui; Heitman, Laura H.; Connor, Mark; Di Marzo, Vincenzo; Gertsch, Jürg; Lichtman, Aron H.; Maccarrone, Mauro; Pacher, Pal; Glass, Michelle; van der Stelt, Mario

    2017-01-01

    The cannabinoid CB2 receptor (CB2R) represents a promising therapeutic target for various forms of tissue injury and inflammatory diseases. Although numerous compounds have been developed and widely used to target CB2R, their selectivity, molecular mode of action and pharmacokinetic properties have been poorly characterized. Here we report the most extensive characterization of the molecular pharmacology of the most widely used CB2R ligands to date. In a collaborative effort between multiple academic and industry laboratories, we identify marked differences in the ability of certain agonists to activate distinct signalling pathways and to cause off-target effects. We reach a consensus that HU910, HU308 and JWH133 are the recommended selective CB2R agonists to study the role of CB2R in biological and disease processes. We believe that our unique approach would be highly suitable for the characterization of other therapeutic targets in drug discovery research. PMID:28045021

  15. Soluble NKG2D ligand promotes MDSC expansion and skews macrophage to the alternatively activated phenotype.

    PubMed

    Xiao, Gang; Wang, Xuanjun; Sheng, Jun; Lu, Shengjun; Yu, Xuezhong; Wu, Jennifer D

    2015-02-20

    Expression of surface NKG2D ligand MIC on tumor cells is deemed to stimulate NK and co-stimulate CD8 T cell anti-tumor immunity. Human cancer cells however frequently adopt a proteinase-mediated shedding strategy to generate soluble MIC (sMIC) to circumvent host immunity. High levels of sMIC have been shown to correlate with advanced disease stages in cancer patients. The underlying mechanism is currently understood as systemic downregulation of NKG2D expression on CD8 T and NK cells and perturbing NK cell periphery maintenance. Herein we report a novel mechanism by which sMIC poses immune suppressive effect on host immunity and tumor microenvironment. We demonstrate that sMIC facilitates expansion of myeloid-derived suppressor cells (MDSCs) and skews macrophages to the more immune suppressive alternative phenotype through activation of STAT3. These findings further endorse that sMIC is an important therapeutic target for cancer immunotherapy.

  16. Mapping transmembrane residues of proteinase activated receptor 2 (PAR2) that influence ligand-modulated calcium signaling.

    PubMed

    Suen, J Y; Adams, M N; Lim, J; Madala, P K; Xu, W; Cotterell, A J; He, Y; Yau, M K; Hooper, J D; Fairlie, D P

    2017-03-01

    Proteinase-activated receptor 2 (PAR2) is a G protein-coupled receptor involved in metabolism, inflammation, and cancers. It is activated by proteolysis, which exposes a nascent N-terminal sequence that becomes a tethered agonist. Short synthetic peptides corresponding to this sequence also activate PAR2, while small organic molecules show promising PAR2 antagonism. Developing PAR2 ligands into pharmaceuticals is hindered by a lack of knowledge of how synthetic ligands interact with and differentially modulate PAR2. Guided by PAR2 homology modeling and ligand docking based on bovine rhodopsin, followed by cross-checking with newer PAR2 models based on ORL-1 and PAR1, site-directed mutagenesis of PAR2 was used to investigate the pharmacology of three agonists (two synthetic agonists and trypsin-exposed tethered ligand) and one antagonist for modulation of PAR2 signaling. Effects of 28 PAR2 mutations were examined for PAR2-mediated calcium mobilization and key mutants were selected for measuring ligand binding. Nineteen of twenty-eight PAR2 mutations reduced the potency of at least one ligand by >10-fold. Key residues mapped predominantly to a cluster in the transmembrane (TM) domains of PAR2, differentially influence intracellular Ca(2+) induced by synthetic agonists versus a native agonist, and highlight subtly different TM residues involved in receptor activation. This is the first evidence highlighting the importance of the PAR2 TM regions for receptor activation by synthetic PAR2 agonists and antagonists. The trypsin-cleaved N-terminus that activates PAR2 was unaffected by residues that affected synthetic peptides, challenging the widespread practice of substituting peptides for proteases to characterize PAR2 physiology.

  17. Inhibition of cell surface mediated plasminogen activation by a monoclonal antibody against alpha-Enolase.

    PubMed

    López-Alemany, Roser; Longstaff, Colin; Hawley, Stephen; Mirshahi, Massoud; Fábregas, Pere; Jardí, Merce; Merton, Elizabeth; Miles, Lindsey A; Félez, Jordi

    2003-04-01

    Localization of plasmin activity on leukocyte surfaces plays a critical role in fibrinolysis as well as in pathological and physiological processes in which cells must degrade the extracellular matrix in order to migrate. The binding of plasminogen to leukocytic cell lines induces a 30- to 80-fold increase in the rate of plasminogen activation by tissue-type (tPA) and urokinase-type (uPA) plasminogen activators. In the present study we have examined the role of alpha-enolase in plasminogen activation on the cell surface. We produced and characterized a monoclonal antibody (MAb) 11G1 against purified alpha-enolase, which abrogated about 90% of cell-dependent plasminogen activation by either uPA or tPA on leukocytoid cell lines of different lineages: B-lymphocytic, T-lymphocytic, granulocytic, and monocytic cells. In addition, MAb 11G1 also blocked enhancement of plasmin formation by peripheral blood neutrophils and monocytes. In contrast, MAb 11G1 did not affect plasmin generation in the presence of fibrin, indicating that this antibody did not interact with fibrinolytic components in the absence of cells. These data suggest that, although leukocytic cells display several molecules that bind plasminogen, alpha-enolase is responsible for the majority of the promotion of plasminogen activation on the surfaces of leukocytic cells.

  18. Impaired ergosterol biosynthesis mediated fungicidal activity of Co(II) complex with ligand derived from cinnamaldehyde.

    PubMed

    Shreaz, Sheikh; Shiekh, Rayees A; Raja, Vaseem; Wani, Waseem A; Behbehani, Jawad M

    2016-03-05

    In this study, we have used aldehyde function of cinnamaldehyde to synthesize N, N'-Bis (cinnamaldehyde) ethylenediimine [C20H20N2] and Co(II) complex of the type [Co(C40H40N4)Cl2]. The structures of the synthesized compounds were determined on the basis of physiochemical analysis and spectroscopic data ((1)H NMR, FTIR, UV-visible and mass spectra) along with molar conductivity measurements. Anticandidal activity of cinnamaldehyde its ligand [L] and Co(II) complex was investigated by determining MIC80, time-kill kinetics, disc diffusion assay and ergosterol extraction and estimation assay. Ligand [L] and Co(II) complex are found to be 4.55 and 21.0 folds more efficient than cinnamaldehyde in a liquid medium. MIC80 of Co(II) complex correlated well with ergosterol inhibition suggesting ergosterol biosynthesis to be the primary site of action. In comparison to fluconazole, the test compounds showed limited toxicity against H9c2 rat cardiac myoblasts. In confocal microscopy propidium iodide (PI) penetrates the yeast cells when treated with MIC of metal complex, indicating a disruption of cell membrane that results in imbibition of dye. TEM analysis of metal complex treated cells exhibited notable alterations or damage to the cell membrane and the cell wall. The structural disorganization within the cell cytoplasm was noted. It was concluded that fungicidal activity of Co(II) complex originated from loss of membrane integrity and a decrease in ergosterol content is only one consequence of this.

  19. Characterization of peroxisome proliferator-activiated receptor alpha (PPARalpha)-independent effects of PPARalpha activators in the rodent liver: Di(2-ethylehexyl) phthalate activates the constitutive activated receptor

    EPA Science Inventory

    Peroxisome proliferator chemicals (PPC) are thought to mediate their effects in rodents on hepatocyte growth and liver cancer through the nuclear receptor peroxisome proliferator-activated receptor alpha (PPARalpha). Recent studies indicate that the plasticizer di-2-ethylhexyl ph...

  20. Polymer masked-unmasked protein therapy. 1. Bioresponsive dextrin-trypsin and -melanocyte stimulating hormone conjugates designed for alpha-amylase activation.

    PubMed

    Duncan, Ruth; Gilbert, Helena R P; Carbajo, Rodrigo J; Vicent, María J

    2008-04-01

    Polymer-protein conjugation, particularly PEGylation, is well-established as a means of increasing circulation time, reducing antigenicity, and improving the stability of protein therapeutics. However, PEG has limitations including lack of polymer biodegradability, and conjugation can diminish or modify protein activity. The aim of this study was to explore a novel approach for polymer-protein modification called polymer-masking-unmasking-protein therapy (PUMPT), the hypothesis being that conjugation of a biodegradable polymer to a protein would protect it and mask activity in transit, while enabling controlled reinstatement of activity at the target site by triggered degradation of the polymeric component. To test this hypothesis, dextrin (alpha-1,4 polyglucose, a natural polymer degraded by alpha-amylase) was conjugated to trypsin as a model enzyme or to melanocyte stimulating hormone (MSH) as a model receptor-binding ligand. The effect of dextrin molecular weight (7700, and 47200 g/mol) and degree of succinoylation (9-32 mol %) on its ability to mask/unmask trypsin activity was assessed using N-benzoyl-L-arginine-p-nitroanilide (L-BAPNA). Dextrin conjugation reduced enzyme activity by 34-69% depending on the molecular weight and degree of succinoylation of dextrin. However, incubation with alpha-amylase led to reinstatement of activity to a maximum of 92-115%. The highest molecular dextrin (26 mol % succinoylation) gave optimum trypsin masking-unmasking. This intermediate was used to synthesize a dextrin-MSH conjugate (dextrin Mw = 47200 g/mol; MSH content 37 wt %), and its biological activity (+/-alpha-amylase) was assessed by measuring melanin production by murine melanoma (B16F10) cells. Conjugation reduced melanin production to 11%, but addition of alpha-amylase was able to restore activity to 33% of the control value. These were the first studies to confirm the potential of PUMPT for further application to clinically important protein therapeutics. The

  1. Structure-function relationships and conformational properties of alpha-MSH(6-13) analogues with candidacidal activity.

    PubMed

    Carotenuto, Alfonso; Saviello, Maria Rosaria; Auriemma, Luigia; Campiglia, Pietro; Catania, Anna; Novellino, Ettore; Grieco, Paolo

    2007-01-01

    Alpha-melanocyte-stimulating hormone (alpha-MSH) is an endogenous linear tridecapeptide with potent anti-inflammatory effects. We firstly demonstrated that alpha-MSH and its C-terminal sequence Lys-Pro-Val [alpha-MSH(11-13)] have antimicrobial effects against two major and representative pathogens: Staphylococcus aureus and Candida albicans. Successively, in an attempt to improve the candidacidal activity of alpha-MSH and to better understand the peptide structure-antifungal activity relations, we have recently designed and synthesized novel peptide analogues. We focused on the sequence alpha-MSH(6-13), which contains the invariant melanocortin core sequence His-Phe-Arg-Trp (6-9) and also contains the sequence Lys-Pro-Val (11-13) important for antimicrobial activity. In that structure-activity study, we discovered several compounds that have greater candidacidal activity than alpha-MSH, among which the peptide [d-Nal-7,Phe-12]-alpha-MSH(6-13) was the most potent. Here, we report a detailed conformational analysis by spectroscopic and computational methods of three peptides, alpha-MSH(6-13) (1), [d-Nal-7,Phe-12]-alpha-MSH(6-13) (2) and [d-Nal-7,Asp-12]-alpha-MSH(6-13) (3). Peptides were chosen on the basis of their candidacidal activities and were studied in membrane mimetic environment (SDS micelles). Different turn structures were observed for the three peptides and a conformation-activity model was developed based on these results. This study offers a structural basis for the design of novel peptide and non-peptide analogues to be used as new antimicrobial agents.

  2. In situ autoradiography and ligand-dependent tyrosine kinase activity reveal insulin receptors and insulin-like growth factor I receptors in prepancreatic chicken embryos.

    PubMed Central

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

    1989-01-01

    We previously reported specific cross-linking of 125I-labeled insulin and 125I-labeled insulin-like growth factor I (IGF-I) to the alpha subunit of their respective receptors in chicken embryos of 20 somites and older. To achieve adequate sensitivity and localize spatially the receptors in younger embryos, we adapted an autoradiographic technique using whole-mounted chicken blastoderms. Insulin receptors and IGF-I receptors were expressed and could be localized as early as gastrulation, before the first somite is formed. Relative density was analyzed by a computer-assisted image system, revealing overall slightly higher binding of IGF-I than of insulin. Structures rich in both types of receptors were predominantly of ectodermal origin: Hensen's node in gastrulating embryos and neural folds, neural tube and optic vesicles during neurulation. The signal transduction capability of the receptors in early organogenesis was assessed by their ability to phosphorylate the exogenous substrate poly(Glu80Tyr20). Ligand-dependent tyrosine phosphorylation was demonstrable with both insulin and IGF-I in glycoprotein-enriched preparations from embryos at days 2 through 6 of embryogenesis. There was a developmentally regulated change in ligand-dependent tyrosine kinase activity, with a sharp increase from day 2 to day 4, in contrast with a small increase in the ligand binding. Binding of 125I-labeled IGF-I was, with the solubilized receptors, severalfold higher than binding of 125I-labeled insulin. However, the insulin-dependent phosphorylation was as high as the IGF-I-dependent phosphorylation at each developmental stage. Images PMID:2548191

  3. Structure-activity relationships for the inhibition of DNA polymerase alpha by aphidicolin derivatives.

    PubMed Central

    Prasad, G; Edelson, R A; Gorycki, P D; Macdonald, T L

    1989-01-01

    Aphidicolin and 17 derivatives that have been structurally modified in the A- and D-rings were assessed for their ability to inhibit DNA polymerase alpha. No derivative surpassed the activity of aphidicolin; derivatives with structural alterations in the A-ring exhibited significantly greater loss of activity relative to derivatives with structural alterations in the D-ring. The conclusions of these studies indicate a critical role for the C-18 function in the interaction of aphidicolin with polymerase alpha. Molecular modelling studies could not identify structural features of the aphidicolin-dCTP "overlap" that is unique to dCTP, relative to the remaining dNTPs, and that is consistent with the extant structure-activity data. PMID:2505232

  4. The muscle-specific laminin receptor alpha7 beta1 integrin negatively regulates alpha5 beta1 fibronectin receptor function.

    PubMed

    Tomatis, D; Echtermayer, F; Schöber, S; Balzac, F; Retta, S F; Silengo, L; Tarone, G

    1999-02-01

    alpha7 beta1 is the major integrin complex expressed in differentiated muscle cells where it functions as a laminin receptor. In this work we have expressed the alpha7 integrin subunit in CHO cells to investigate the functional properties of this receptor. After transfection with alpha7 CHO cells acquired the ability to adhere and spread on laminin 1 consistent with the laminin receptor activity of the alpha7 beta1. alpha7 transfectants, however, showed a 70% reduction in the ability to adhere to fibronectin and were unable to assemble a fibronectin matrix. The degree of reduction was inversely related to the level of alpha7 expression. To define the mechanisms underlying this adhesive defect we analyzed surface expression and functional properties of the alpha5 beta1 fibronectin receptor. Although cell surface expression of alpha5 beta1 was reduced by a factor of 20-25% in alpha7 transfectants compared to control untransfected cells, this slight reduction was not sufficient to explain the dramatic reduction in cell adhesion (70%) and matrix assembly (close to 100%). Binding studies showed that the affinity of 125I-fibronectin for its surface receptor was decreased by 50% in alpha7 transfectants, indicating that the alpha5 beta1 integrin is partially inactivated in these cells. Inactivation can be reversed by Mn2+, a cation known to increase integrin affinity for their ligands. In fact, incubation of cells with Mn2+ restored fibronectin binding affinity, adhesion to fibronectin, and assembly of fibronectin matrix in alpha7 transfectants. These data indicate that alpha7 expression leads to the functional down regulation of alpha5beta1 integrin by decreasing ligand binding affinity and surface expression. In conclusion, the data reported establish the existence of a negative cooperativity between alpha7 and alpha5 integrins that may be important in determining functional regulation of integrins during myogenic differentiation.

  5. Depressive symptoms and baseline prefrontal EEG alpha activity: a study utilizing Ecological Momentary Assessment.

    PubMed

    Putnam, Katherine M; McSweeney, Lauren B

    2008-02-01

    Prefrontal cortex (PFC) electroencephalography (EEG) alpha asymmetry has been found in individuals with major depression. However, EEG activity has never been examined in regard to specific depressive symptoms. We examine the relationship between resting baseline PFC alpha activity and both rumination and self-esteem in a depressed outpatient group (N=6) and a healthy control group (N=7) using high-density EEG sampling and multiple longitudinal self report measures, i.e. Ecological Momentary Assessment (EMA). Symptom measures were collected five times daily for 7 days, i.e. 35 assessments. Using a mixed-level analysis, significant Group x Hemisphere interactions for PFC sites and both rumination and self-esteem were found. Within the depressed group, lower bilateral PFC activity predicted higher levels of rumination, and lower right PFC activity predicted higher levels of self-esteem. There were no significant effects for the control group. Results indicate that specific symptoms of depression are uniquely associated with patterns of PFC EEG alpha activity.

  6. Structure of a potentially open state of a proton-activated pentameric ligand-gated ion channel.

    PubMed

    Hilf, Ricarda J C; Dutzler, Raimund

    2009-01-01

    The X-ray structure of a pentameric ligand-gated ion channel from Erwinia chrysanthemi (ELIC) has recently provided structural insight into this family of ion channels at high resolution. The structure shows a homo-pentameric protein with a barrel-stave architecture that defines an ion-conduction pore located on the fivefold axis of symmetry. In this structure, the wide aqueous vestibule that is encircled by the extracellular ligand-binding domains of the five subunits narrows to a discontinuous pore that spans the lipid bilayer. The pore is constricted by bulky hydrophobic residues towards the extracellular side, which probably serve as barriers that prevent the diffusion of ions. This interrupted pore architec