HSP90 inhibitors potentiate PGF2α-induced IL-6 synthesis via p38 MAP kinase in osteoblasts.

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Kazuhiko Fujita

Full Text Available Heat shock protein 90 (HSP90 that is ubiquitously expressed in various tissues, is recognized to be a major molecular chaperone. We have previously reported that prostaglandin F2α (PGF2α, a potent bone remodeling mediator, stimulates the synthesis of interleukin-6 (IL-6 through p44/p42 mitogen-activated protein (MAP kinase and p38 MAP kinase in osteoblast-like MC3T3-E1 cells, and that Rho-kinase acts at a point upstream of p38 MAP kinase. In the present study, we investigated the involvement of HSP90 in the PGF2α-stimulated IL-6 synthesis and the underlying mechanism in MC3T3-E1 cells. Geldanamycin, an inhibitor of HSP90, significantly amplified both the PGF2α-stimulated IL-6 release and the mRNA expression levels. In addition, other HSP90 inhibitors, 17-allylamino-17demethoxy-geldanamycin (17-AAG and 17-dimethylamino-ethylamino-17-demethoxy-geldanamycin (17-DMAG and onalespib, enhanced the PGF2α-stimulated IL-6 release. Geldanamycin, 17-AAG and onalespib markedly strengthened the PGF2α-induced phosphorylation of p38 MAP kinase. Geldanamycin and 17-AAG did not affect the PGF2α-induced phosphorylation of p44/p42 MAP kinase and myosin phosphatase targeting subunit (MYPT-1, a substrate of Rho-kinase, and the protein levels of RhoA and Rho-kinase. In addition, HSP90-siRNA enhanced the PGF2α-induced phosphorylation of p38 MAP kinase. Furthermore, SB203580, an inhibitor of p38 MAP kinase, significantly suppressed the amplification by geldanamycin, 17-AAG or 17-DMAG of the PGF2α-stimulated IL-6 release. Our results strongly suggest that HSP90 negatively regulates the PGF2α-stimulated IL-6 synthesis in osteoblasts, and that the effect of HSP90 is exerted through regulating p38 MAP kinase activation.

Inhibitors of Fatty Acid Synthesis Induce PPAR α -Regulated Fatty Acid β -Oxidative Genes: Synergistic Roles of L-FABP and Glucose.

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Huang, Huan; McIntosh, Avery L; Martin, Gregory G; Petrescu, Anca D; Landrock, Kerstin K; Landrock, Danilo; Kier, Ann B; Schroeder, Friedhelm

2013-01-01

While TOFA (acetyl CoA carboxylase inhibitor) and C75 (fatty acid synthase inhibitor) prevent lipid accumulation by inhibiting fatty acid synthesis, the mechanism of action is not simply accounted for by inhibition of the enzymes alone. Liver fatty acid binding protein (L-FABP), a mediator of long chain fatty acid signaling to peroxisome proliferator-activated receptor- α (PPAR α ) in the nucleus, was found to bind TOFA and its activated CoA thioester, TOFyl-CoA, with high affinity while binding C75 and C75-CoA with lower affinity. Binding of TOFA and C75-CoA significantly altered L-FABP secondary structure. High (20 mM) but not physiological (6 mM) glucose conferred on both TOFA and C75 the ability to induce PPAR α transcription of the fatty acid β -oxidative enzymes CPT1A, CPT2, and ACOX1 in cultured primary hepatocytes from wild-type (WT) mice. However, L-FABP gene ablation abolished the effects of TOFA and C75 in the context of high glucose. These effects were not associated with an increased cellular level of unesterified fatty acids but rather by increased intracellular glucose. These findings suggested that L-FABP may function as an intracellular fatty acid synthesis inhibitor binding protein facilitating TOFA and C75-mediated induction of PPAR α in the context of high glucose at levels similar to those in uncontrolled diabetes.

The TosMIC approach to 3-(oxazol-5-yl) indoles: application to the synthesis of indole-based IMPDH inhibitors.

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Dhar, T G Murali; Shen, Zhongqi; Fleener, Catherine A; Rouleau, Katherine A; Barrish, Joel C; Hollenbaugh, Diane L; Iwanowicz, Edwin J

2002-11-18

A modified approach to the synthesis of 3-(oxazolyl-5-yl) indoles is reported. This method was applied to the synthesis of series of novel indole based inhibitors of inosine monophosphate dehydrogenase (IMPDH). The synthesis and the structure-activity relationships (SARs), derived from in vitro studies, for this new series of inhibitors is given.

Design, Synthesis and Evaluation of 2,5-Diketopiperazines as Inhibitors of the MDM2-p53 Interaction.

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Mariell Pettersson

Full Text Available The transcription factor p53 is the main tumour suppressor in cells and many cancer types have p53 mutations resulting in a loss of its function. In tumours that retain wild-type p53 function, p53 activity is down-regulated by MDM2 (human murine double minute 2 via a direct protein-protein interaction. We have designed and synthesised two series of 2,5-diketopiperazines as inhibitors of the MDM2-p53 interaction. The first set was designed to directly mimic the α-helical region of the p53 peptide, containing key residues in the i, i+4 and i+7 positions of a natural α-helix. Conformational analysis indicated that 1,3,6-trisubstituted 2,5-diketopiperazines were able to place substituents in the same spatial orientation as an α-helix template. The key step of the synthesis involved the cyclisation of substituted dipeptides. The other set of tetrasubstituted 2,5-diketopiperazines were designed based on structure-based docking studies and the Ugi multicomponent reaction was used for the synthesis. This latter set comprised the most potent inhibitors which displayed micromolar IC50-values in a biochemical fluorescence polarisation assay.

A practical synthesis of a gamma-secretase inhibitor.

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Scott, Jeremy P; Lieberman, David R; Beureux, Olivier M; Brands, Karel M J; Davies, Antony J; Gibson, Andrew W; Hammond, Deborah C; McWilliams, Chris J; Stewart, Gavin W; Wilson, Robert D; Dolling, Ulf-H

2007-05-25

A practical and scaleable synthesis of the gamma-secretase inhibitor 1 is reported. The inhibitor consists of a central trisubstituted cyclohexane core with appended propionic acid, 2,5-difluorophenyl, and 4-chlorophenylsulfonyl moieties. Two alternative synthetic strategies, proceeding by way of a common disubstituted cyclohexanone derivative 5, were studied. In the preferred route, conjugate reduction of acrylonitrile derivative 4 with L-Selectride configures the desired relative stereochemistry of the cyclohexane core with >99.9:0.1 dr. A second strategy, based on catalyst-controlled hydrogenation of racemic cyclohexene derivative 2, is more convergent but less diastereoselective (up to 75:25 dr). The common cyclohexanone intermediate 5 was constructed by a regioselective Diels-Alder condensation of a 1,1-disubstituted vinyl sulfone 6 with 2-trimethylsiloxybutadiene.

Metabolic approaches to enhance transdermal drug delivery. 1. Effect of lipid synthesis inhibitors.

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Tsai, J C; Guy, R H; Thornfeldt, C R; Gao, W N; Feingold, K R; Elias, P M

1996-06-01

The intercellular domains of the stratum corneum, which contain a mixture of cholesterol, free fatty acids, and ceramides, mediate both the epidermal permeability barrier and the transdermal delivery of both lipophilic and hydrophilic molecules. Prior studies have shown that each of the three key lipid classes is required for normal barrier function. For example, selective inhibition of either cholesterol, fatty acid, or ceramide synthesis in the epidermis delays barrier recovery rates after barrier perturbation of hairless mouse skin in vivo. In this study, we investigated the potential of certain inhibitors of lipid synthesis to enhance the transdermal delivery of lidocaine or caffeine as a result of their capacity to perturb barrier homeostasis. After acetone disruption of the barrier, the extent of lidocaine delivery and the degree of altered barrier function paralleled each other. Moreover, the further alteration in barrier function produced by either the fatty acid synthesis inhibitor 5-(tetradecyloxy)-2-furancarboxylic acid (TOFA), the cholesterol synthesis inhibitor fluvastatin (FLU), or cholesterol sulfate (CS) resulted in a further increase in lidocaine absorption. Furthermore, coapplications of TOFA and CS together caused an additive increase in lidocaine uptake. Finally, a comparable increase in drug delivery occurred when the barrier was disrupted initially with DMSO instead of acetone; coapplications of TOFA and FLU together again delayed barrier recovery and increased drug delivery by about 8-fold vs delivery from a standard enhancing vehicle. Whereas these metabolic inhibitors also variably increased the octanol/water partitioning of the drugs studied (perhaps via complexion or pH alterations), physicochemical effects of the inhibitors alone did not alter drug uptake in intact skin; i.e., passive mechanisms alone cannot account for the net increase in drug delivery. Our results show that modulations of epidermal lipid biosynthesis, following

The regulation of protein synthesis and translation factors by CD3 and CD28 in human primary T lymphocytes

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Proud Christopher G

2002-05-01

Full Text Available Abstract Background Activation of human resting T lymphocytes results in an immediate increase in protein synthesis. The increase in protein synthesis after 16–24 h has been linked to the increased protein levels of translation initiation factors. However, the regulation of protein synthesis during the early onset of T cell activation has not been studied in great detail. We studied the regulation of protein synthesis after 1 h of activation using αCD3 antibody to stimulate the T cell receptor and αCD28 antibody to provide the co-stimulus. Results Activation of the T cells with both antibodies led to a sustained increase in the rate of protein synthesis. The activities and/or phosphorylation states of several translation factors were studied during the first hour of stimulation with αCD3 and αCD28 to explore the mechanism underlying the activation of protein synthesis. The initial increase in protein synthesis was accompanied by activation of the guanine nucleotide exchange factor, eukaryotic initiation factor (eIF 2B, and of p70 S6 kinase and by dephosphorylation of eukaryotic elongation factor (eEF 2. Similar signal transduction pathways, as assessed using signal transduction inhibitors, are involved in the regulation of protein synthesis, eIF2B activity and p70 S6 kinase activity. A new finding was that the p38 MAPK α/β pathway was involved in the regulation of overall protein synthesis in primary T cells. Unexpectedly, no changes were detected in the phosphorylation state of the cap-binding protein eIF4E and the eIF4E-binding protein 4E-BP1, or the formation of the cap-binding complex eIF4F. Conclusions Both eIF2B and p70 S6 kinase play important roles in the regulation of protein synthesis during the early onset of T cell activation.

Synergistic apoptosis induction in leukemic cells by the phosphatase inhibitor salubrinal and proteasome inhibitors.

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Hannes C A Drexler

Full Text Available Cells adapt to endoplasmic reticulum (ER-stress by arresting global protein synthesis while simultaneously activating specific transcription factors and their downstream targets. These processes are mediated in part by the phosphorylation-dependent inactivation of the translation initiation factor eIF2alpha. Following restoration of homeostasis protein synthesis is resumed when the serine/threonine-protein phosphatase PP1 dephosphorylates and reactivates eIF2alpha. Proteasome inhibitors, used to treat multiple myeloma patients evoke ER-stress and apoptosis by blocking the ER-associated degradation of misfolded proteins (ERAD, however, the role of eIF2alpha phosphorylation in leukemic cells under conditions of proteasome inhibitor-mediated ER stress is currently unclear.Bcr-Abl-positive and negative leukemic cell lines were used to investigate the functional implications of PP1-related phosphatase activities on eIF2alpha phosphorylation in proteasome inhibitor-mediated ER stress and apoptosis. Rather unexpectedly, salubrinal, a recently identified PP1 inhibitor capable to protect against ER stress in various model systems, strongly synergized with proteasome inhibitors to augment apoptotic death of different leukemic cell lines. Salubrinal treatment did not affect the phosphorlyation status of eIF2alpha. Furthermore, the proapoptotic effect of salubrinal occurred independently from the chemical nature of the proteasome inhibitor, was recapitulated by a second unrelated phosphatase inhibitor and was unaffected by overexpression of a dominant negative eIF2alpha S51A variant that can not be phosphorylated. Salubrinal further aggravated ER-stress and proteotoxicity inflicted by the proteasome inhibitors on the leukemic cells since characteristic ER stress responses, such as ATF4 and CHOP synthesis, XBP1 splicing, activation of MAP kinases and eventually apoptosis were efficiently abrogated by the translational inhibitor cycloheximide.Although PP1

Structure-Based Search for New Inhibitors of Cholinesterases

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Barbara Malawska

2013-03-01

Full Text Available Cholinesterases are important biological targets responsible for regulation of cholinergic transmission, and their inhibitors are used for the treatment of Alzheimer’s disease. To design new cholinesterase inhibitors, of different structure-based design strategies was followed, including the modification of compounds from a previously developed library and a fragment-based design approach. This led to the selection of heterodimeric structures as potential inhibitors. Synthesis and biological evaluation of selected candidates confirmed that the designed compounds were acetylcholinesterase inhibitors with IC50 values in the mid-nanomolar to low micromolar range, and some of them were also butyrylcholinesterase inhibitors.

Design and Synthesis of New Peptidomimetics as Potential Inhibitors of MurE.

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Zivec, Matej; Turk, Samo; Blanot, Didier; Gobec, Stanislav

2011-03-01

With the continuing emergence and spread of multidrug-resistant bacteria, there is an urgent need for the development of new antimicrobial agents. One possible source of new antibacterial targets is the biosynthesis of the bacterial cell-wall peptidoglycan. The assembly of the peptide stem is carried out by four essential enzymes, known as the Mur ligases (MurC, D, E and F). We have designed and synthesised a focused library of compounds as potential inhibitors of UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:L-lysine ligase (MurE) from Staphylococcus aureus. This was achieved using two approaches: (i) synthesis of transition-state analogues based on the methyleneamino core; and (ii) synthesis of MurE reaction product analogues. Two methyleneamino-based compounds are identified as initial hits for inhibitors of MurE.

Enantioselective synthesis of the novel chiral sulfoxide derivative as a glycogen synthase kinase 3beta inhibitor.

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Saitoh, Morihisa; Kunitomo, Jun; Kimura, Eiji; Yamano, Toru; Itoh, Fumio; Kori, Masakuni

2010-09-01

Glycogen synthase kinase 3beta (GSK-3beta) inhibitors are expected to be attractive therapeutic agents for the treatment of Alzheimer's disease (AD). Recently we discovered sulfoxides (S)-1 as a novel GSK-3beta inhibitor having in vivo efficacy. We investigated practical asymmetric preparation methods for the scale-up synthesis of (S)-1. The highly enantioselective synthesis of (S)-1 (94% ee) was achieved by titanium-mediated oxidation with D-(-)-diethyl tartrate on gram scale.

Heat Shock Protein 70 Negatively Regulates TGF-β-Stimulated VEGF Synthesis via p38 MAP Kinase in Osteoblasts

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Go Sakai

2017-11-01

Full Text Available Background/Aims: We previously demonstrated that transforming growth factor-β (TGF-β stimulates the synthesis of vascular endothelial growth factor (VEGF through the activation of p38 mitogen-activated protein (MAP kinase in osteoblast-like MC3T3-E1 cells. Heat shock protein70 (HSP70 is a ubiquitously expressed molecular chaperone. In the present study, we investigated the involvement of HSP70 in the TGF-β-stimulated VEGF synthesis and the underlying mechanism in these cells. Methods: Culture MC3T3-E1 cells were stimulated by TGF-β. Released VEGF was measured using an ELISA assay. VEGF mRNA level was quantified by RT-PCR. Phosphorylation of each protein kinase was analyzed by Western blotting. Results: VER-155008 and YM-08, both of HSP70 inhibitors, significantly amplified the TGF-β-stimulated VEGF release. In addition, the expression level of VEGF mRNA induced by TGF-β was enhanced by VER-155008. These inhibitors markedly strengthened the TGF-β-induced phosphorylation of p38 MAP kinase. The TGF-β-induced phosphorylation of p38 MAP kinase was amplified in HSP70-knockdown cells. SB203580, an inhibitor of p38 MAP kinase, significantly suppressed the amplification by these inhibitors of the TGF-β-induced VEGF release. Conclusion: These results strongly suggest that HSP70 acts as a negative regulator in the TGF-β-stimulated VEGF synthesis in osteoblasts, and that the inhibitory effect of HSP70 is exerted at a point upstream of p38 MAP kinase.

Down-Regulation by Resveratrol of Basic Fibroblast Growth Factor-Stimulated Osteoprotegerin Synthesis through Suppression of Akt in Osteoblasts

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Gen Kuroyanagi

2014-10-01

Full Text Available It is firmly established that resveratrol, a natural food compound abundantly found in grape skins and red wine, has beneficial properties for human health. In the present study, we investigated the effect of basic fibroblast growth factor (FGF-2 on osteoprotegerin (OPG synthesis in osteoblast-like MC3T3-E1 cells and whether resveratrol affects the OPG synthesis. FGF-2 stimulated both the OPG release and the expression of OPG mRNA. Resveratrol significantly suppressed the FGF-2-stimulated OPG release and the mRNA levels of OPG. SRT1720, an activator of SIRT1, reduced the FGF-2-induced OPG release and the OPG mRNA expression. PD98059, an inhibitor of upstream kinase activating p44/p42 mitogen-activated protein (MAP kinase, had little effect on the FGF-2-stimulated OPG release. On the other hand, SB203580, an inhibitor of p38 MAP kinase, SP600125, an inhibitor of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK, and Akt inhibitor suppressed the OPG release induced by FGF-2. Resveratrol failed to affect the FGF-2-induced phosphorylation of p44/p42 MAP kinase, p38 MAP kinase or SAPK/JNK. The phosphorylation of Akt induced by FGF-2 was significantly suppressed by resveratrol or SRT1720. These findings strongly suggest that resveratrol down-regulates FGF-2-stimulated OPG synthesis through the suppression of the Akt pathway in osteoblasts and that the inhibitory effect of resveratrol is mediated at least in part by SIRT1 activation.

Synthesis and Structure-Activity Relationship of Griseofulvin Analogues as Inhibitors of Centrosomal Clustering in Cancer Cells

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Rønnest, Mads Holger; Rebacz, Blanka; Markworth, Lene

2009-01-01

Griseofulvin was identified as an inhibitor of centrosomal clustering in a recently developed assay. Centrosomal clustering is an important cellular event that enables bipolar mitosis for cancer cell lines harboring supernumerary centrosomes. We report herein the synthesis and SAR of 34 griseoful......Griseofulvin was identified as an inhibitor of centrosomal clustering in a recently developed assay. Centrosomal clustering is an important cellular event that enables bipolar mitosis for cancer cell lines harboring supernumerary centrosomes. We report herein the synthesis and SAR of 34...

Abscisic acid negatively regulates elicitor-induced synthesis of capsidiol in wild tobacco.

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Mialoundama, Alexis Samba; Heintz, Dimitri; Debayle, Delphine; Rahier, Alain; Camara, Bilal; Bouvier, Florence

2009-07-01

In the Solanaceae, biotic and abiotic elicitors induce de novo synthesis of sesquiterpenoid stress metabolites known as phytoalexins. Because plant hormones play critical roles in the induction of defense-responsive genes, we have explored the effect of abscisic acid (ABA) on the synthesis of capsidiol, the major wild tobacco (Nicotiana plumbaginifolia) sesquiterpenoid phytoalexin, using wild-type plants versus nonallelic mutants Npaba2 and Npaba1 that are deficient in ABA synthesis. Npaba2 and Npaba1 mutants exhibited a 2-fold higher synthesis of capsidiol than wild-type plants when elicited with either cellulase or arachidonic acid or when infected by Botrytis cinerea. The same trend was observed for the expression of the capsidiol biosynthetic genes 5-epi-aristolochene synthase and 5-epi-aristolochene hydroxylase. Treatment of wild-type plants with fluridone, an inhibitor of the upstream ABA pathway, recapitulated the behavior of Npaba2 and Npaba1 mutants, while the application of exogenous ABA reversed the enhanced synthesis of capsidiol in Npaba2 and Npaba1 mutants. Concomitant with the production of capsidiol, we observed the induction of ABA 8'-hydroxylase in elicited plants. In wild-type plants, the induction of ABA 8'-hydroxylase coincided with a decrease in ABA content and with the accumulation of ABA catabolic products such as phaseic acid and dihydrophaseic acid, suggesting a negative regulation exerted by ABA on capsidiol synthesis. Collectively, our data indicate that ABA is not required per se for the induction of capsidiol synthesis but is essentially implicated in a stress-response checkpoint to fine-tune the amplification of capsidiol synthesis in challenged plants.

Subtype-specific suppression of Shiga toxin 2 released from Escherichia coli upon exposure to protein synthesis inhibitors

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Pedersen, Malene Gantzhorn; Hansen, Claus; Riise, Erik

2008-01-01

Shiga toxins (Stx) are important virulence factors in the pathogenesis of severe disease including hemolytic-uremic syndrome, caused by Stx-producing Escherichia coli (STEC). STEC strains increase the release of Stx in vitro following the addition of fluoroquinolones, whereas protein synthesis...... inhibitors previously have been reported to suppress the release of Stx. The amount of Stx released from wild-type STEC strains incubated with protein synthesis inhibitors was examined by a Vero cell cytotoxicity assay. The amounts released were compared to the Stx type (Stx1 or Stx2) and additionally...... to the individual subtypes and toxin variants of Stx2. In general, Stx2 release was suppressed significantly upon exposure to protein synthesis inhibitors at MICs, which was not observed in the case of Stx1. Also, the average amount of different Stx2 toxin variants released was suppressed to various levels ranging...

3-cyanoindole-based inhibitors of inosine monophosphate dehydrogenase: synthesis and initial structure-activity relationships.

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Dhar, T G Murali; Shen, Zhongqi; Gu, Henry H; Chen, Ping; Norris, Derek; Watterson, Scott H; Ballentine, Shelley K; Fleener, Catherine A; Rouleau, Katherine A; Barrish, Joel C; Townsend, Robert; Hollenbaugh, Diane L; Iwanowicz, Edwin J

2003-10-20

A series of novel small molecule inhibitors of inosine monophosphate dehydrogenase (IMPDH), based upon a 3-cyanoindole core, were explored. IMPDH catalyzes the rate determining step in guanine nucleotide biosynthesis and is a target for anticancer, immunosuppressive and antiviral therapy. The synthesis and the structure-activity relationships (SAR), derived from in vitro studies, for this new series of inhibitors is given.

Discovery of novel selenium derivatives as Pin1 inhibitors by high-throughput screening

International Nuclear Information System (INIS)

Subedi, Amit; Shimizu, Takeshi; Ryo, Akihide; Sanada, Emiko; Watanabe, Nobumoto; Osada, Hiroyuki

2016-01-01

Peptidyl prolyl cis/trans isomerization by Pin1 regulates various oncogenic signals during cancer progression, and its inhibition through multiple approaches has established Pin1 as a therapeutic target. However, lack of simplified screening systems has limited the discovery of potent Pin1 inhibitors. We utilized phosphorylation-dependent binding of Pin1 to its specific substrate to develop a screening system for Pin1 inhibitors. Using this system, we screened a chemical library, and identified a novel selenium derivative as Pin1 inhibitor. Based on structure-activity guided chemical synthesis, we developed more potent Pin1 inhibitors that inhibited cancer cell proliferation. -- Highlights: •Novel screening for Pin1 inhibitors based on Pin1 binding is developed. •A novel selenium compound is discovered as Pin1 inhibitor. •Activity guided chemical synthesis of selenium derivatives resulted potent Pin1 inhibitors.

Molecular Regulation of Histamine Synthesis

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Hua Huang

2018-06-01

Full Text Available Histamine is a critical mediator of IgE/mast cell-mediated anaphylaxis, a neurotransmitter and a regulator of gastric acid secretion. Histamine is a monoamine synthesized from the amino acid histidine through a reaction catalyzed by the enzyme histidine decarboxylase (HDC, which removes carboxyl group from histidine. Despite the importance of histamine, transcriptional regulation of HDC gene expression in mammals is still poorly understood. In this review, we focus on discussing advances in the understanding of molecular regulation of mammalian histamine synthesis.

Design, synthesis and biological activity of novel peptidyl benzyl ketone FVIIa inhibitors

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Storgaard, Morten; Henriksen, Signe Teuber; Zaragoza, Florencio

2011-01-01

Herein is described the synthesis of a novel class of peptidyl FVIIa inhibitors having a C-terminal benzyl ketone group. This class is designed to be potentially suitable as stabilization agents of liquid formulations of rFVIIa, which is a serine protease used for the treatment of hemophilia...

Angiotensin converting enzyme inhibitors mitigate collagen synthesis induced by a single dose of radiation to the whole thorax

International Nuclear Information System (INIS)

Kma, L.; Gao, F.; Fish, B.L.; Moulder, J.E.; Jacobs, E.R.; Medhora, M.

2012-01-01

Our long-term goal is to use angiotensin converting enzyme (ACE) inhibitors to mitigate the increase in lung collagen synthesis that is induced by irradiation to the lung, which could result from accidental exposure or radiological terrorism. Rats (WAG/RijCmcr) were given a single dose of 13 Gy (dose rate of 1.43 Gy/min) of X-irradiation to the thorax. Three structurally-different ACE inhibitors, captopril, enalapril and fosinopril were provided in drinking water beginning 1 week after irradiation. Rats that survived acute pneumonitis (at 6-12 weeks) were evaluated monthly for synthesis of lung collagen. Other endpoints included breathing rate, wet to dry lung weight ratio, and analysis of lung structure. Treatment with captopril (145-207 mg/m 2 /day) or enalapril (19-28 mg/m 2 /day), but not fosinopril (19-28 mg/m 2 /day), decreased morbidity from acute pneumonitis. Lung collagen in the surviving irradiated rats was increased over that of controls by 7 months after irradiation. This increase in collagen synthesis was not observed in rats treated with any of the three ACE inhibitors. Analysis of the lung morphology at 7 months supports the efficacy of ACE inhibitors against radiation-induced fibrosis. The effectiveness of fosinopril against fibrosis, but not against acute pneumonitis, suggests that pulmonary fibrosis may not be a simple consequence of injury during acute pneumonitis. In summary, three structurally-different ACE inhibitors mitigate the increase in collagen synthesis 7 months following irradiation of the whole thorax and do so, even when therapy is started one week after irradiation. (author)

L-Cysteine/D,L-homocysteine-regulated ileum motility via system L and B°(,+) transporter: Modification by inhibitors of hydrogen sulfide synthesis and dietary treatments.

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Yamane, Satoshi; Nomura, Ryouya; Yanagihara, Madoka; Nakamura, Hiroyuki; Fujino, Hiromichi; Matsumoto, Kenjiro; Horie, Syunji; Murayama, Toshihiko

2015-10-05

Previous studies including ours demonstrated that L-cysteine treatments decreased motility in gastrointestinal tissues including the ileum via hydrogen sulfide (H2S), which is formed from sulfur-containing amino acids such as L-cysteine and L-homocysteine. However, the amino acid transport systems involved in L-cysteine/L-homocysteine-induced responses have not yet been elucidated in detail; therefore, we investigated these systems pharmacologically by measuring electrical stimulation (ES)-induced contractions with amino acids in mouse ileum preparations. The treatments with L-cysteine and D,L-homocysteine inhibited ES-induced contractions in ileum preparations from fasted mice, and these responses were decreased by the treatment with 2-aminobicyclo[2.2.1]heptane-2-carboxylate (BCH), an inhibitor of systems L and B°(,+). The results obtained using ileum preparations and a model cell line (PC12 cells) with various amino acids and BCH showed that not only L-cysteine, but also aminooxyacetic acid and D,L-propargylglycine, which act as H2S synthesis inhibitors, appeared to be taken up by these preparations/cells in L and B°(,+) system-dependent manners. The L-cysteine and D,L-homocysteine responses were delayed and abolished, respectively, in ileum preparations from fed mice. Our results suggested that the regulation of ileum motility by L-cysteine and D,L-homocysteine was dependent on BCH-sensitive systems, and varied depending on feeding in mice. Therefore, the effects of aminooxyacetic acid and D,L-propargylglycine on transport systems need to be considered in pharmacological analyses. Copyright © 2015 Elsevier B.V. All rights reserved.

Effects of inhibitors of DNA synthesis and protein synthesis on the rate of DNA synthesis after exposure of mammalian cells to ultraviolet light

International Nuclear Information System (INIS)

Griffiths, T.D.; Dahle, D.B.; Meechan, P.J.; Carpenter, J.G.

1981-01-01

Chinese hamster V-79 cells were treated with metabolic inhibitors of DNA or protein synthesis for various intervals of time after exposure of 3.0 or 5.0 J m -2 . After removal of the metabolic block(s) the rate of DNA synthesis was followed by measuring the incorporation of [ 14 C]thymidine into acid-insoluble material. A 2.5 or 5.0h incubation with cycloheximide or hydroxyurea was effective in delaying the onset of the recovery in the rate of DNA synthesis that normally becomes evident several hours after exposure to ultraviolet light. By using concentrations of cycloheximide or hydroxyurea that inhibit DNA synthesis by a similar amount (70%), but protein synthesis by vastly different amounts (95% for cycloheximide; 0% for hydroxyurea), it was apparent that the delay in recovery caused by the treatment of the cells with cycloheximide could be accounted for entirely by its inhibitory effect on DNA synthesis. This suggests that the recovery in DNA synthetic rates following exposure of V-79 cells to ultraviolet light does not appear to require de novo protein synthesis, and therefore does not appear to require the involvement of an inducible DNA repair process. (Auth.)

Effects of DNA polymerase inhibitors on replicative and repair DNA synthesis in ultraviolet-irradiated HeLa cells

International Nuclear Information System (INIS)

Morita, T.; Nakamura, H.; Tsutsui, Y.; Nishiyama, Y.; Yoshida, S.

1982-01-01

Aphidicolin specifically inhibits eukaryotic DNA polymerase α, while 2',3'-dideoxythymidine 5'-triphosphate (d 2 TTP) inhibits DNA polymerase ν and ν but not α. 1-ν-D-Arabinofuranosylcytosine 5'-triphosphate (araCTP) inhibits both DNA polymerase α and ν although to a different extent. Here we measured the effects of these inhibitors on repair DNA synthesis of U.V.-irradiated HeLa cells by two different methods. Firstly, aphidicolin, 1-ν-D-arabinofuranosylcytosine (araC, a precursor of araCTP) and 2',3'-dideoxythimidine (d 2 Thd, a precursor of d 2 TTP) were added directly to the culture medium. In this case, aphidicolin and araC strongly inhibited replicative DNA synthesis of HeLa cells, and they also inhibited repair synthesis after U.V.-irradiation but to a much lesser extent. In contrast, high concentrations of d 2 Thd inhibited repair DNA synthesis to a higher extent than replicative DNA synthesis. Secondly, the active form of inhibitor, d 2 TTP, was microinjected directly into cytoplasm or nuclei or U.V.-irradiated HeLa cells. Microinjection of d 2 TTP effectively inhibited repair synthesis. The microinjection of d 2 TTP, into either cytoplasm or nucleus, strongly inhibited replicative synthesis. These results might indicate that multiple DNA polymerases are involved in repair synthesis as well as in replicative synthesis

Abscisic Acid Negatively Regulates Elicitor-Induced Synthesis of Capsidiol in Wild Tobacco1[W

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Mialoundama, Alexis Samba; Heintz, Dimitri; Debayle, Delphine; Rahier, Alain; Camara, Bilal; Bouvier, Florence

2009-01-01

In the Solanaceae, biotic and abiotic elicitors induce de novo synthesis of sesquiterpenoid stress metabolites known as phytoalexins. Because plant hormones play critical roles in the induction of defense-responsive genes, we have explored the effect of abscisic acid (ABA) on the synthesis of capsidiol, the major wild tobacco (Nicotiana plumbaginifolia) sesquiterpenoid phytoalexin, using wild-type plants versus nonallelic mutants Npaba2 and Npaba1 that are deficient in ABA synthesis. Npaba2 and Npaba1 mutants exhibited a 2-fold higher synthesis of capsidiol than wild-type plants when elicited with either cellulase or arachidonic acid or when infected by Botrytis cinerea. The same trend was observed for the expression of the capsidiol biosynthetic genes 5-epi-aristolochene synthase and 5-epi-aristolochene hydroxylase. Treatment of wild-type plants with fluridone, an inhibitor of the upstream ABA pathway, recapitulated the behavior of Npaba2 and Npaba1 mutants, while the application of exogenous ABA reversed the enhanced synthesis of capsidiol in Npaba2 and Npaba1 mutants. Concomitant with the production of capsidiol, we observed the induction of ABA 8′-hydroxylase in elicited plants. In wild-type plants, the induction of ABA 8′-hydroxylase coincided with a decrease in ABA content and with the accumulation of ABA catabolic products such as phaseic acid and dihydrophaseic acid, suggesting a negative regulation exerted by ABA on capsidiol synthesis. Collectively, our data indicate that ABA is not required per se for the induction of capsidiol synthesis but is essentially implicated in a stress-response checkpoint to fine-tune the amplification of capsidiol synthesis in challenged plants. PMID:19420326

Design, Synthesis and Biological Evaluation of Histone Deacetylase (HDAC) Inhibitors: Saha (Vorinostat) Analogs and Biaryl Indolyl Benzamide Inhibitors Display Isoform Selectivity

Science.gov (United States)

Negmeldin, Ahmed Thabet

HDAC proteins have emerged as interesting targets for anti-cancer drugs due to their involvement in cancers, as well as several other diseases. Several HDAC inhibitors have been approved by the FDA as anti-cancer drugs, including SAHA (suberoylanilide hydroxamic acid, Vorinostat). Unfortunately, SAHA inhibits most HDAC isoforms, which limit its use as a pharmacological tool and may lead to side effects in the clinic. In this work we were interested in developing isoform selective HDAC inhibitors, which may decrease or eliminate the side effects associated with non-selective inhibitors treatment. In addition, isoform selective HDAC inhibitors can be used as biological tools to help understand the HDAC-related cancer biology. Our strategy was based on synthesis and screening of several derivatives of the non-selective FDA approved drug SAHA substituted at different positions of the linker region. Several SAHA analogs modified at the C4 and C5 positions of the linker were synthesized. The new C4- and C5-modified SAHA libraries, along with the previously synthesized C2-modified SAHA analogs were screened in vitro and in cellulo for HDAC isoform selectivity. Interestingly, several analogs exhibited dual HDAC6/HDAC8 selectivity. Enantioselective syntheses of the pure enantiomers of some of the interesting analogs were performed and the enantiomers were screened in vitro. Among the most interesting analogs, ( R)-C4-benzyl SAHA displayed 520- to 1300-fold selectivity for HDAC6 and HDAC8 over HDAC1, 2, and 3, with IC50 values of 48 and 27 nM with HDAC6 and 8, respectively. Docking studies were performed to provide structural rationale for the observed selectivity of the new analogs. In addition, rational design, synthesis, and screening of several other biaryl indolyl benzamide HDAC inhibitors is discussed, and some showed modest HDAC1 selectivity. The new biaryl indolyl benzamides can be useful to further develop HDAC1 selective inhibitors. The dual HDAC6/8 selective

Effect of decoyinine on the regulation of alpha-amylase synthesis in Bacillus subtilis.

OpenAIRE

Nicholson, W L; Chambliss, G H

1987-01-01

Decoyinine, an inhibitor of GMP synthetase, allows sporulation in Bacillus subtilis to initiate and proceed under otherwise catabolite-repressing conditions. The effect of decoyinine on alpha-amylase synthesis in B. subtilis, an event which exhibits regulatory features resembling sporulation initiation, was examined. Decoyinine did not overcome catabolite repression of alpha-amylase synthesis in a wild-type strain of B. subtilis but did cause premature and enhanced synthesis in a mutant strai...

Synthesis and study on biological activity of nitrogen-containing heterocyclic compounds – regulators of enzymes of nucleic acid biosynthesis

Directory of Open Access Journals (Sweden)

Alexeeva I. V.

2013-07-01

Full Text Available Results of investigations on the development of new regulators of functional activity of nucleic acid biosynthesis enzymes based on polycyclic nitrogen-containing heterosystems are summarized. Computer design and molecular docking in the catalytic site of target enzyme (T7pol allowed to perform the directed optimization of basic structures. Several series of compounds were obtained and efficient inhibitors of herpes family (simple herpes virus type 2, Epstein-Barr virus, influenza A and hepatitis C viruses were identified, as well as compounds with potent antitumor, antibacterial and antifungal activity. It was established that the use of model test systems based on enzymes participating in nucleic acids synthesis is a promising approach to the primary screening of potential inhibitors in vitro.

Synthesis and Characterization of Novel Acyl-Glycine Inhibitors of GlyT2.

Science.gov (United States)

Mostyn, Shannon N; Carland, Jane E; Shimmon, Susan; Ryan, Renae M; Rawling, Tristan; Vandenberg, Robert J

2017-09-20

It has been demonstrated previously that the endogenous compound N-arachidonyl-glycine inhibits the glycine transporter GlyT2, stimulates glycinergic neurotransmission, and provides analgesia in animal models of neuropathic and inflammatory pain. However, it is a relatively weak inhibitor with an IC 50 of 9 μM and is subject to oxidation via cyclooxygenase, limiting its therapeutic value. In this paper we describe the synthesis and testing of a novel series of monounsaturated C18 and C16 acyl-glycine molecules as inhibitors of the glycine transporter GlyT2. We demonstrate that they are up to 28 fold more potent that N-arachidonyl-glycine with no activity at the closely related GlyT1 transporter at concentrations up to 30 μM. This novel class of compounds show considerable promise as a first generation of GlyT2 transport inhibitors.

Thromboxane synthesis inhibitors and postprandial jejunal capillary exchange capacity.

Science.gov (United States)

Mangino, M J; Chou, C C

1988-05-01

The effects of thromboxane synthesis inhibitors (imidazole and U 63557A; Upjohn) and the cyclooxygenase inhibitor, mefenamic acid, on jejunal capillary filtration coefficients (Kfc) were determined in dogs before and during the presence of predigested food in the jejunal lumen. The jejunal Kfc increased significantly soon after the placement of a predigested test food containing all major constituents of diet. The Kfc remained elevated as long as the food was present in the lumen (15 min). Mefenamic acid (10 mg/kg iv) did not significantly alter resting jejunal Kfc or alter the food-induced increase in Kfc. Imidazole (5.0 mg/min ia) or U 63557A (5.0 mg/kg iv) per se significantly increased jejunal Kfc. Placement of digested food further increased the Kfc to levels significantly higher than those observed before administration of the two thromboxane synthase inhibitors. Production of thromboxane B2 by jejunal tissue was significantly reduced and 6-ketoprostaglandin F1 alpha (the stable hydrolysis product of prostacyclin) production was significantly increased after administration of U 63557A. Our study indicates that the relative production of endogenous thromboxanes and other prostanoids modulates jejunal capillary exchange capacity in the absence or presence of digested food in the jejunal lumen.

Sugar-mediated semidian oscillation of gene expression in the cassava storage root regulates starch synthesis

Energy Technology Data Exchange (ETDEWEB)

Jansson, Christer; Baguma, Yona; Sun, Chuanxin; Boren, Mats; Olsson, Helena; Rosenqvist, Sara; Mutisya, Joel; Rubaihayo, Patrick R.; Jansson, Christer

2008-01-15

Starch branching enzyme (SBE) activity in the cassava storage root exhibited a diurnal fluctuation, dictated by a transcriptional oscillation of the corresponding SBE genes. The peak of SBE activity coincided with the onset of sucrose accumulation in the storage, and we conclude that the oscillatory mechanism keeps the starch synthetic apparatus in the storage root sink in tune with the flux of sucrose from the photosynthetic source. When storage roots were uncoupled from the source, SBE expression could be effectively induced by exogenous sucrose. Turanose, a sucrose isomer that cannot be metabolized by plants, mimicked the effect of sucrose, demonstrating that downstream metabolism of sucrose was not necessary for signal transmission. Also glucose and glucose-1-P induced SBE expression. Interestingly, induction by sucrose, turanose and glucose but not glucose-1-P sustained an overt semidian (12-h) oscillation in SBE expression and was sensitive to the hexokinase (HXK) inhibitor glucosamine. These results suggest a pivotal regulatory role for HXK during starch synthesis. Abscisic acid (ABA) was another potent inducer of SBE expression. Induction by ABA was similar to that of glucose-1-P in that it bypassed the semidian oscillator. Both the sugar and ABA signaling cascades were disrupted by okadaic acid, a protein phosphatase inhibitor. Based on these findings, we propose a model for sugar signaling in regulation of starch synthesis in the cassava storage root.

Design and synthesis of potent, orally-active DGAT-1 inhibitors containing a dioxino[2,3-d]pyrimidine core.

Science.gov (United States)

Dow, Robert L; Andrews, Melissa; Aspnes, Gary E; Balan, Gayatri; Michael Gibbs, E; Guzman-Perez, Angel; Karki, Kapil; Laperle, Jennifer L; Li, Jian-Cheng; Litchfield, John; Munchhof, Michael J; Perreault, Christian; Patel, Leena

2011-10-15

A novel series of potent DGAT-1 inhibitors was developed originating from the lactam-based clinical candidate PF-04620110. Incorporation of a dioxino[2,3-d]pyrimidine-based core afforded good alignment of pharmacophore features and resulted in improved passive permeability. Development of an efficient, homochiral synthesis of these targets facilitated confirmation of predictions regarding the stereochemical-dependence of DGAT-1 inhibition for this series. Compound 10 was shown to be a potent inhibitor of human DGAT-1 (10 nM) and to suppress triglyceride synthesis at oral doses of <3mg/kg. Copyright © 2011 Elsevier Ltd. All rights reserved.

Direct modulation of T-box riboswitch-controlled transcription by protein synthesis inhibitors.

Science.gov (United States)

Stamatopoulou, Vassiliki; Apostolidi, Maria; Li, Shuang; Lamprinou, Katerina; Papakyriakou, Athanasios; Zhang, Jinwei; Stathopoulos, Constantinos

2017-09-29

Recently, it was discovered that exposure to mainstream antibiotics activate numerous bacterial riboregulators that control antibiotic resistance genes including metabolite-binding riboswitches and other transcription attenuators. However, the effects of commonly used antibiotics, many of which exhibit RNA-binding properties, on the widespread T-box riboswitches, remain unknown. In Staphylococcus aureus, a species-specific glyS T-box controls the supply of glycine for both ribosomal translation and cell wall synthesis, making it a promising target for next-generation antimicrobials. Here, we report that specific protein synthesis inhibitors could either significantly increase T-box-mediated transcription antitermination, while other compounds could suppress it, both in vitro and in vivo. In-line probing of the full-length T-box combined with molecular modelling and docking analyses suggest that the antibiotics that promote transcription antitermination stabilize the T-box:tRNA complex through binding specific positions on stem I and the Staphylococcal-specific stem Sa. By contrast, the antibiotics that attenuate T-box transcription bind to other positions on stem I and do not interact with stem Sa. Taken together, our results reveal that the transcription of essential genes controlled by T-box riboswitches can be directly modulated by commonly used protein synthesis inhibitors. These findings accentuate the regulatory complexities of bacterial response to antimicrobials that involve multiple riboregulators. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

A phase I study of a new polyamine biosynthesis inhibitor, SAM486A, in cancer patients with solid tumours

NARCIS (Netherlands)

Paridaens, R; Uges, DRA; Barbet, N; Choi, L; Seeghers, M; van der Graaf, WTA; Groen, HJM; Dumez, H; Van Buuren, [No Value; Muskiet, F; Capdeville, R; van Oosterom, AT; de Vries, EGE

Because tumour cell proliferation is highly dependent upon up-regulation of de-novo polyamine synthesis, inhibition of the polyamine synthesis pathway represents a potential target for anticancer therapy. SAM486A (CGP 48664) is a new inhibitor of the polyamine biosynthetic enzyme

The CYP2E1 inhibitor DDC up-regulates MMP-1 expression in hepatic stellate cells via an ERK1/2- and Akt-dependent mechanism.

Science.gov (United States)

Liu, Tianhui; Wang, Ping; Cong, Min; Xu, Youqing; Jia, Jidong; You, Hong

2013-06-05

DDC (diethyldithiocarbamate) could block collagen synthesis in HSC (hepatic stellate cells) through the inhibition of ROS (reactive oxygen species) derived from hepatocyte CYP2E1 (cytochrome P450 2E1). However, the effect of DDC on MMP-1 (matrix metalloproteinase-1), which is the main collagen degrading matrix metalloproteinase, has not been reported. In co-culture experiments, we found that DDC significantly enhanced MMP-1 expression in human HSC (LX-2) that were cultured with hepatocyte C3A cells either expressing or not expressing CYP2E1. The levels of both proenzyme and active MMP-1 enzyme were up-regulated in LX-2 cells, accompanied by elevated enzyme activity of MMP-1 and decreased collagen I, in both LX-2 cells and the culture medium. H2O2 treatment abrogated DDC-induced MMP-1 up-regulation and collagen I decrease, while catalase treatment slightly up-regulated MMP-1 expression. These data suggested that the decrease in ROS by DDC was partially responsible for the MMP-1 up-regulation. ERK1/2 (extracellular signal-regulated kinase 1/2), Akt (protein kinase B) and p38 were significantly activated by DDC. The ERK1/2 inhibitor (U0126) and Akt inhibitor (T3830) abrogated the DDC-induced MMP-1 up-regulation. In addition, a p38 inhibitor (SB203580) improved MMP-1 up-regulation through the stimulation of ERK1/2. Our data indicate that DDC significantly up-regulates the expression of MMP-1 in LX-2 cells which results in greater MMP-1 enzyme activity and decreased collagen I. The enhancement of MMP-1 expression by DDC was associated with H2O2 inhibition and coordinated regulation by the ERK1/2 and Akt pathways. These data provide some new insights into treatment strategies for hepatic fibrosis.

Effects of inhibitors of protein synthesis and intracellular transport on the gamma-aminobutyric acid agonist-induced functional differentiation of cultured cerebellar granule cells

DEFF Research Database (Denmark)

Belhage, B; Hansen, Gert Helge; Meier, E

1990-01-01

The effect of inhibitors of protein synthesis (actinomycin D, cycloheximide), proteases (leupeptin), and intracellular transport (colchicine, monensin) on the gamma-aminobutyric acid (GABA) agonist [4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP)]-induced changes in morphological...... an intracellular and a plasma membrane localization of the receptors. In all experiments cultures treated with THIP alone served as controls. The inhibitors of protein synthesis totally abolished the ability of THIP to induce low-affinity GABA receptors. In contrast, the inhibitors of intracellular transport...

ERalpha and ERbeta expression and transcriptional activity are differentially regulated by HDAC inhibitors

Science.gov (United States)

Duong, Vanessa; Licznar, Anne; Margueron, Raphaël; Boulle, Nathalie; Busson, Muriel; Lacroix, Matthieu; Katzenellenbogen, Benita S.; Cavaillès, Vincent; Lazennec, Gwendal

2006-01-01

The proliferative action of ERα largely accounts for the carcinogenic activity of estrogens. By contrast, recent data show that ERβ displays tumor-suppressor properties, thus supporting the interest to identify compounds which could increase its activity. Here, we show that histone deacetylase inhibitors (HDI) up-regulated ERβ protein levels, whereas it decreased ERα expression. Part of this regulation took place at the mRNA level through a mechanism independent of de novo protein synthesis. In addition, we found that, in various cancer cells, the treatment with different HDI enhanced the ligand-dependent activity of ERβ more strongly than that of ERα. On the other hand, in MDA-MB231 and HeLa cells, the expression of ERs modified the transcriptional response to HDI. The use of deletion mutants of both receptors demonstrated that AF1 domain of the receptors was required. Finally, we show that ERβ expression led to a dramatic increased in the antiproliferative activity of HDI, which correlated with a modification of the transcription of genes involved in cell cycle control by HDI. Altogether, these data demonstrate that the interference of ERβ and HDAC on the control of transcription and cell proliferation constitute a promising approach for cancer therapy. PMID:16158045

Cloning and regulation of rat tissue inhibitor of metalloproteinases-2 in osteoblastic cells

Science.gov (United States)

Cook, T. F.; Burke, J. S.; Bergman, K. D.; Quinn, C. O.; Jeffrey, J. J.; Partridge, N. C.

1994-01-01

Rat tissue inhibitor of metalloproteinases-2 (TIMP-2) was cloned from a UMR 106-01 rat osteoblastic osteosarcoma cDNA library. The 969-bp full-length clone demonstrates 98 and 86% sequence identity to human TIMP-2 at the amino acid and nucleic acid levels, respectively. Parathyroid hormone (PTH), at 10(-8) M, stimulates an approximately twofold increase in both the 4.2- and 1.0-kb transcripts over basal levels in UMR cells after 24 h of exposure. The PTH stimulation of TIMP-2 transcripts was not affected by the inhibitor of protein synthesis, cycloheximide (10(-5) M), suggesting a primary effect of the hormone. This is in contradistinction to regulation of interstitial collagenase (matrix metalloproteinase-1) by PTH in these same cells. Nuclear run-on assays demonstrate that PTH causes an increase in TIMP-2 transcription that parallels the increase in message levels. Parathyroid hormone, in its stimulation of TIMP-2 mRNA, appears to act through a signal transduction pathway involving protein kinase A (PKA) since the increase in TIMP-2 mRNA is reproduced by treatment with the cAMP analogue, 8-bromo-cAMP (5 x 10(-3) M). The protein kinase C and calcium pathways do not appear to be involved due to the lack of effect of phorbol 12-myristate 13-acetate (2.6 x 10(-6) M) and the calcium ionophore, ionomycin (10(-7) M), on TIMP-2 transcript abundance. In this respect, regulation of TIMP-2 and collagenase in osteoblastic cells by PTH are similar. However, we conclude that since stimulation of TIMP-2 transcription is a primary event, the PKA pathway must be responsible for a direct increase in transcription of this gene.

The host-encoded Heme Regulated Inhibitor (HRI facilitates virulence-associated activities of bacterial pathogens.

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Niraj Shrestha

Full Text Available Here we show that cells lacking the heme-regulated inhibitor (HRI are highly resistant to infection by bacterial pathogens. By examining the infection process in wild-type and HRI null cells, we found that HRI is required for pathogens to execute their virulence-associated cellular activities. Specifically, unlike wild-type cells, HRI null cells infected with the gram-negative bacterial pathogen Yersinia are essentially impervious to the cytoskeleton-damaging effects of the Yop virulence factors. This effect is due to reduced functioning of the Yersinia type 3 secretion (T3S system which injects virulence factors directly into the host cell cytosol. Reduced T3S activity is also observed in HRI null cells infected with the bacterial pathogen Chlamydia which results in a dramatic reduction in its intracellular proliferation. We go on to show that a HRI-mediated process plays a central role in the cellular infection cycle of the Gram-positive pathogen Listeria. For this pathogen, HRI is required for the post-invasion trafficking of the bacterium to the infected host cytosol. Thus by depriving Listeria of its intracellular niche, there is a highly reduced proliferation of Listeria in HRI null cells. We provide evidence that these infection-associated functions of HRI (an eIF2α kinase are independent of its activity as a regulator of protein synthesis. This is the first report of a host factor whose absence interferes with the function of T3S secretion and cytosolic access by pathogens and makes HRI an excellent target for inhibitors due to its broad virulence-associated activities.

Design, Synthesis, and Biological Activity of 1,2,3-Triazolobenzodiazepine BET Bromodomain Inhibitors.

Science.gov (United States)

Sharp, Phillip P; Garnier, Jean-Marc; Hatfaludi, Tamas; Xu, Zhen; Segal, David; Jarman, Kate E; Jousset, Hélène; Garnham, Alexandra; Feutrill, John T; Cuzzupe, Anthony; Hall, Peter; Taylor, Scott; Walkley, Carl R; Tyler, Dean; Dawson, Mark A; Czabotar, Peter; Wilks, Andrew F; Glaser, Stefan; Huang, David C S; Burns, Christopher J

2017-12-14

A number of diazepines are known to inhibit bromo- and extra-terminal domain (BET) proteins. Their BET inhibitory activity derives from the fusion of an acetyl-lysine mimetic heterocycle onto the diazepine framework. Herein we describe a straightforward, modular synthesis of novel 1,2,3-triazolobenzodiazepines and show that the 1,2,3-triazole acts as an effective acetyl-lysine mimetic heterocycle. Structure-based optimization of this series of compounds led to the development of potent BET bromodomain inhibitors with excellent activity against leukemic cells, concomitant with a reduction in c- MYC expression. These novel benzodiazepines therefore represent a promising class of therapeutic BET inhibitors.

Structure-Based Design and Synthesis of Potent and Selective Matrix Metalloproteinase 13 Inhibitors.

Science.gov (United States)

Choi, Jun Yong; Fuerst, Rita; Knapinska, Anna M; Taylor, Alexander B; Smith, Lyndsay; Cao, Xiaohang; Hart, P John; Fields, Gregg B; Roush, William R

2017-07-13

We describe the use of comparative structural analysis and structure-guided molecular design to develop potent and selective inhibitors (10d and (S)-17b) of matrix metalloproteinase 13 (MMP-13). We applied a three-step process, starting with a comparative analysis of the X-ray crystallographic structure of compound 5 in complex with MMP-13 with published structures of known MMP-13·inhibitor complexes followed by molecular design and synthesis of potent but nonselective zinc-chelating MMP inhibitors (e.g., 10a and 10b). After demonstrating that the pharmacophores of the chelating inhibitors (S)-10a, (R)-10a, and 10b were binding within the MMP-13 active site, the Zn 2+ chelating unit was replaced with nonchelating polar residues that bridged over the Zn 2+ binding site and reached into a solvent accessible area. After two rounds of structural optimization, these design approaches led to small molecule MMP-13 inhibitors 10d and (S)-17b, which bind within the substrate-binding site of MMP-13 and surround the catalytically active Zn 2+ ion without chelating to the metal. These compounds exhibit at least 500-fold selectivity versus other MMPs.

Design, Synthesis, and Evaluation of Novel Tyrosine-Based DNA Gyrase B Inhibitors.

Science.gov (United States)

Cotman, Andrej E; Trampuž, Marko; Brvar, Matjaž; Kikelj, Danijel; Ilaš, Janez; Peterlin-Mašič, Lucija; Montalvão, Sofia; Tammela, Päivi; Frlan, Rok

2017-08-01

The discovery and synthesis of new tyrosine-based inhibitors of DNA gyrase B (GyrB), which target its ATPase subunit, is reported. Twenty-four compounds were synthesized and evaluated for activity against DNA gyrase and DNA topoisomerase IV. The antibacterial properties of selected GyrB inhibitors were demonstrated by their activity against Staphylococcus aureus and Enterococcus faecalis in the low micromolar range. The most promising compounds, 8a and 13e, inhibited Escherichia coli and S. aureus GyrB with IC 50 values of 40 and 30 µM. The same compound also inhibited the growth of S. aureus and E. faecalis with minimal inhibitory concentrations (MIC 90 ) of 14 and 28 µg/mL, respectively. © 2017 Deutsche Pharmazeutische Gesellschaft.

Inhibition of Estradiol Synthesis Impairs Fear Extinction in Male Rats

Science.gov (United States)

Graham, Bronwyn M.; Milad, Mohammed R.

2014-01-01

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

Synthesis of suicide inhibitors of monoamine oxidase: carbon-11 labeled clorgyline, L-deprenyl and D-deprenyl

International Nuclear Information System (INIS)

MacGregor, R.R.; Fowler, J.S.; Wolf, A.P.; Halldin, C.; Langstroem, B.

1988-01-01

The suicide inhibitors of monoamine oxidase type A and B, clorgyline and L-deprenyl have been labeled with carbon-11 by [ 11 C]methylation of the norbases with [ 11 C]H 3 I. The less active enantiomer of deprenyl (D-deprenyl) was also labeled using this procedure. The synthesis time was 35 minutes, the radiochemical yield was 25-40% and the specific activity was 0.8-2.0 Ci/μmol (calculated to EOB). Procedures for synthesis of the precursor norbases as well as the synthesis of unlabeled clorgyline, L-deprenyl and D-deprenyl are given. (author)

Purification and properties of a mitochondrial lipoprotein inhibitor of sterol synthesis

International Nuclear Information System (INIS)

Madhosingh, C.; Migicovsky, B.B.; Starratt, A.N.

1976-01-01

A lipoprotein inhibitor of hydroxymethylglutaryl CoA reductase (EC 1.1.1.34) and of cholesterol synthesis by rat liver homogenates, was isolated from the mitochondria of starved rats' livers. The isolated lipoprotein complex contained a low molecular weight protein and fatty acids. The fatty acids identified were arachidonic, linoleic, oleic, stearic and palmitic. The saturated fatty acids and oleic acid did not inhibit. Inhibition of the enzyme was to a large extent related to the degree of fatty acid unsaturation. (auth.)

The use of transcription inhibitors in the study of the mechanism of abscisic acid action in germinating triticale caryopses

Directory of Open Access Journals (Sweden)

Stanisław Weidner

2014-01-01

Full Text Available The study was conducted on germinating triticale (var. Grado caryopses. The purpose of the experiments was to compare the effect of selected inhibitors of transcription with the action of abscisic acid during germination of caryopses. The following inhibitors were used: α-amanitin, cordycepin, cycloheximide and 5-fluorouracil. Studied were the synthesis of total and polyribosomal RNA, the process of polyribosome formation and the synthesis of ribosomal proteins. The effect of exogenous ABA, especially in the early stages of germination, was not similar to any of the four above inhibitors of transcription. After 12 h of imbibition at a lowered temperature and 3 h of germination, ABA caused a relatively low level of inhibition of RNA synthesis, whereas all of the inhibitors used halted RNA synthesis in embryos by about 50-60%. After 6 h of germination, the same proportion of polyribosomes in the total ribosome fraction (46% was found in both the embryos from the control sample and treated with ABA. The use of inhibitors brought this figure down to below 40%. The conclusion is drawn that in the early stages of germination, regulation of protein synthesis by ABA in triticale caryopses must occur on a level other than transcription.

Direct observation of the effects of cellulose synthesis inhibitors using live cell imaging of Cellulose Synthase (CESA) in Physcomitrella patens.

Science.gov (United States)

Tran, Mai L; McCarthy, Thomas W; Sun, Hao; Wu, Shu-Zon; Norris, Joanna H; Bezanilla, Magdalena; Vidali, Luis; Anderson, Charles T; Roberts, Alison W

2018-01-15

Results from live cell imaging of fluorescently tagged Cellulose Synthase (CESA) proteins in Cellulose Synthesis Complexes (CSCs) have enhanced our understanding of cellulose biosynthesis, including the mechanisms of action of cellulose synthesis inhibitors. However, this method has been applied only in Arabidopsis thaliana and Brachypodium distachyon thus far. Results from freeze fracture electron microscopy of protonemal filaments of the moss Funaria hygrometrica indicate that a cellulose synthesis inhibitor, 2,6-dichlorobenzonitrile (DCB), fragments CSCs and clears them from the plasma membrane. This differs from Arabidopsis, in which DCB causes CSC accumulation in the plasma membrane and a different cellulose synthesis inhibitor, isoxaben, clears CSCs from the plasma membrane. In this study, live cell imaging of the moss Physcomitrella patens indicated that DCB and isoxaben have little effect on protonemal growth rates, and that only DCB causes tip rupture. Live cell imaging of mEGFP-PpCESA5 and mEGFP-PpCESA8 showed that DCB and isoxaben substantially reduced CSC movement, but had no measureable effect on CSC density in the plasma membrane. These results suggest that DCB and isoxaben have similar effects on CSC movement in P. patens and Arabidopsis, but have different effects on CSC intracellular trafficking, cell growth and cell integrity in these divergent plant lineages.

The synthesis of a tritium, carbon-14, and stable isotope-labeled cathepsin C inhibitors.

Science.gov (United States)

Allen, Paul; Bragg, Ryan A; Caffrey, Moya; Ericsson, Cecilia; Hickey, Michael J; Kingston, Lee P; Elmore, Charles S

2017-02-01

As part of a medicinal chemistry program aimed at developing a highly potent and selective cathepsin C inhibitor, tritium, carbon-14, and stable isotope-labeled materials were required. The synthesis of tritium-labeled methanesulfonate 5 was achieved via catalytic tritiolysis of a chloro precursor, albeit at a low radiochemical purity of 67%. Tritium-labeled AZD5248 was prepared via a 3-stage synthesis, utilizing amide-directed hydrogen isotope exchange. Carbon-14 and stable isotope-labeled AZD5248 were successfully prepared through modifications of the medicinal chemistry synthetic route, enabling the use of available labeled intermediates. Copyright © 2016 John Wiley & Sons, Ltd.

Regulation of cellulose synthesis in response to stress.

Science.gov (United States)

Kesten, Christopher; Menna, Alexandra; Sánchez-Rodríguez, Clara

2017-12-01

The cell wall is a complex polysaccharide network that provides stability and protection to the plant and is one of the first layers of biotic and abiotic stimuli perception. A controlled remodeling of the primary cell wall is essential for the plant to adapt its growth to environmental stresses. Cellulose, the main component of plant cell walls is synthesized by plasma membrane-localized cellulose synthases moving along cortical microtubule tracks. Recent advancements demonstrate a tight regulation of cellulose synthesis at the primary cell wall by phytohormone networks. Stress-induced perturbations at the cell wall that modify cellulose synthesis and microtubule arrangement activate similar phytohormone-based stress response pathways. The integration of stress perception at the primary cell wall and downstream responses are likely to be tightly regulated by phytohormone signaling pathways in the context of cellulose synthesis and microtubule arrangement. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Potent New Small-Molecule Inhibitor of Botulinum Neurotoxin Serotype A Endopeptidase Developed by Synthesis-Based Computer-Aided Molecular Design

Science.gov (United States)

2009-11-01

simulations; (4) synthesis and evaluation of the molecules from Step 2 or 3 (e.g., synthesizing and testing AHP). From synthetic chemistry point of view...2000) Synthesis of 6H-indolo [2,3-b][1,6]naphthyridines and related compounds as the 5-Aza analogues of ellipticine alkaloids . J Org Chem 65: 7977–7983...Potent New Small-Molecule Inhibitor of Botulinum Neurotoxin Serotype A Endopeptidase Developed by Synthesis -Based Computer-Aided Molecular Design

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

DEFF Research Database (Denmark)

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

2016-01-01

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

Design and synthesis of 4-heteroaryl 1,2,3,4-tetrahydroisoquinolines as triple reuptake inhibitors.

Science.gov (United States)

Liu, Shuang; Zha, Congxiang; Nacro, Kassoum; Hu, Min; Cui, Wenge; Yang, Yuh-Lin; Bhatt, Ulhas; Sambandam, Aruna; Isherwood, Matthew; Yet, Larry; Herr, Michael T; Ebeltoft, Sarah; Hassler, Carla; Fleming, Linda; Pechulis, Anthony D; Payen-Fornicola, Anne; Holman, Nicholas; Milanowski, Dennis; Cotterill, Ian; Mozhaev, Vadim; Khmelnitsky, Yuri; Guzzo, Peter R; Sargent, Bruce J; Molino, Bruce F; Olson, Richard; King, Dalton; Lelas, Snjezana; Li, Yu-Wen; Johnson, Kim; Molski, Thaddeus; Orie, Anitra; Ng, Alicia; Haskell, Roy; Clarke, Wendy; Bertekap, Robert; O'Connell, Jonathan; Lodge, Nicholas; Sinz, Michael; Adams, Stephen; Zaczek, Robert; Macor, John E

2014-07-10

A series of 4-bicyclic heteroaryl 1,2,3,4-tetrahydroisoquinoline inhibitors of the serotonin transporter (SERT), norepinephrine transporter (NET), and dopamine transporter (DAT) was discovered. The synthesis and structure-activity relationship (SAR) of these triple reuptake inhibitors (TRIs) will be discussed. Compound 10i (AMR-2), a very potent inhibitor of SERT, NET, and DAT, showed efficacy in the rat forced-swim and mouse tail suspension models with minimum effective doses of 0.3 and 1 mg/kg (po), respectively. At efficacious doses in these assays, 10i exhibited substantial occupancy levels at the three transporters in both rat and mouse brain. The study of the metabolism of 10i revealed the formation of a significant active metabolite, compound 13.

Synthesis of the proteinase inhibitor LEKTI domain 6 by the fragment condensation method and regioselective disulfide bond formation.

Science.gov (United States)

Vasileiou, Zoe; Barlos, Kostas K; Gatos, Dimitrios; Adermann, Knut; Deraison, Celine; Barlos, Kleomenis

2010-01-01

Proteinase inhibitors are of high pharmaceutical interest and are drug candidates for a variety of indications. Specific kallikrein inhibitors are important for their antitumor activity and their potential application to the treatment of skin diseases. In this study we describe the synthesis of domain 6 of the kallikrein inhibitor Lympho-Epithilial Kazal-Type Inhibitor (LEKTI) by the fragment condensation method and site-directed cystine bridge formation. To obtain the linear LEKTI precursor, the condensation was best performed in solution, coupling the protected fragment 1-22 to 23-68. This method yielded LEKTI domain 6 of high purity and equipotent to the recombinantly produced peptide. (c) 2010 Wiley Periodicals, Inc.

Protein synthesis inhibitors attenuate water flow in vasopressin-stimulated toad urinary bladder

International Nuclear Information System (INIS)

Hoch, B.S.; Ast, M.B.; Fusco, M.J.; Jacoby, M.; Levine, S.D.

1988-01-01

Vasopressin stimulates the introduction of aggregated particles, which may represent pathways for water flow, into the luminal membrane of toad urinary bladder. It is not known whether water transport pathways are degraded on removal from membrane or whether they are recycled. The authors examined the effect of the protein synthesis inhibitors cycloheximide and puromycin using repeated 30-min cycles of vasopressin followed by washout of vasopressin, all in the presence of an osmotic gradient, a protocol that maximizes aggregate turnover. High dose cycloheximide inhibited flow immediately. Low dose cycloheximide did not affect initial flow. In the absence of vasopressin, inhibition did not develop. Despite the inhibition of flow in vasopressin-treated tissues, the cAMP-dependent protein kinase ratio was elevated in cycloheximide-treated tissues, suggesting modulation at a distal site in the stimulatory cascade. [ 14 C]urea permeability was not inhibited by cycloheximide. Puromycin also inhibited water flow by the fourth challenge with vasopressin. The data suggest that protein synthesis inhibitors attenuate flow at a site that is distal to cAMP-dependent protein kinase. However, the reversal of inhibition in MIX-treated tissues suggests that the water pathway can be fully manifested given suitable stimulation. They conclude that either large stores of the transport system are available or that the transport system is extensively recycled on retrieval from the membrane

Asymmetric synthesis of a potent, aminopiperidine-fused imidazopyridine dipeptidyl peptidase IV inhibitor.

Science.gov (United States)

Xu, Feng; Corley, Edward; Zacuto, Michael; Conlon, David A; Pipik, Brenda; Humphrey, Guy; Murry, Jerry; Tschaen, David

2010-03-05

A practical asymmetric synthesis of a novel aminopiperidine-fused imidazopyridine dipeptidyl peptidase IV (DPP-4) inhibitor 1 has been developed. Application of a unique three-component cascade coupling with chiral nitro diester 7, which is easily accessed via a highly enantioselective Michael addition of dimethyl malonate to a nitrostyrene, allows for the assembly of the functionalized piperidinone skeleton in one pot. Through a base-catalyzed, dynamic crystallization-driven process, the cis-piperidionone 16a is epimerized to the desired trans isomer 16b, which is directly crystallized from the crude reaction stream in high yield and purity. Isomerization of the allylamide 16b in the presence of RhCl(3) is achieved without any epimerization of the acid/base labile stereogenic center adjacent to the nitro group on the piperidinone ring, while the undesired enamine intermediate is consumed to <0.5% by utilizing a trace amount of HCl generated from RhCl(3). The amino lactam 4, obtained through hydrogenation and hydrolysis, is isolated as its crystalline pTSA salt from the reaction solution directly, as such intramolecular transamidation has been dramatically suppressed via kinetic control. Finally, a Cu(I) catalyzed coupling-cyclization allows for the formation of the tricyclic structure of the potent DPP-4 inhibitor 1. The synthesis, which is suitable for large scale preparation, is accomplished in 23% overall yield.

A modified approach to 2-(N-aryl)-1,3-oxazoles: application to the synthesis of the IMPDH inhibitor BMS-337197 and analogues.

Science.gov (United States)

Dhar, T G Murali; Guo, Junqing; Shen, Zhongqi; Pitts, William J; Gu, Henry H; Chen, Bang-Chi; Zhao, Rulin; Bednarz, Mark S; Iwanowicz, Edwin J

2002-06-13

[structure: see text] A modified approach to the synthesis of 2-(N-aryl)-1,3-oxazoles, employing an optimized iminophosphorane/heterocumulene-mediated methodology, and its application to the synthesis of BMS-337197, a potent inhibitor of IMPDH, are described.

X-linked inhibitor of apoptosis regulates T cell effector function

DEFF Research Database (Denmark)

Zehntner, Simone P; Bourbonnière, Lyne; Moore, Craig S

2007-01-01

To understand how the balance between pro- and anti-apoptotic signals influences effector function in the immune system, we studied the X-linked inhibitor of apoptosis (XIAP), an endogenous regulator of cellular apoptosis. Real-time PCR showed increased XIAP expression in blood of mice with exper......To understand how the balance between pro- and anti-apoptotic signals influences effector function in the immune system, we studied the X-linked inhibitor of apoptosis (XIAP), an endogenous regulator of cellular apoptosis. Real-time PCR showed increased XIAP expression in blood of mice...... dramatically reduced within the CNS. Flow cytometry showed an 88-93% reduction in T cells. The proportion of TUNEL(+) apoptotic CD4(+) T cells in the CNS was increased from Neurons...... and oligodendrocytes were not affected; neither did apoptosis increase in liver, where XIAP knockdown also occurred. ASO-XIAP increased susceptibility of T cells to activation-induced apoptosis in vitro. Our results identify XIAP as a critical controller of apoptotic susceptibility of effector T cell function...

Targeting tumor-initiating cells: Eliminating anabolic cancer stem cells with inhibitors of protein synthesis or by mimicking caloric restriction

Science.gov (United States)

Lamb, Rebecca; Harrison, Hannah; Smith, Duncan L.; Townsend, Paul A.; Jackson, Thomas; Ozsvari, Bela; Martinez-Outschoorn, Ubaldo E.; Pestell, Richard G.; Howell, Anthony; Lisanti, Michael P.; Sotgia, Federica

2015-01-01

We have used an unbiased proteomic profiling strategy to identify new potential therapeutic targets in tumor-initiating cells (TICs), a.k.a., cancer stem cells (CSCs). Towards this end, the proteomes of mammospheres from two breast cancer cell lines were directly compared to attached monolayer cells. This allowed us to identify proteins that were highly over-expressed in CSCs and/or progenitor cells. We focused on ribosomal proteins and protein folding chaperones, since they were markedly over-expressed in mammospheres. Overall, we identified >80 molecules specifically associated with protein synthesis that were commonly upregulated in mammospheres. Most of these proteins were also transcriptionally upregulated in human breast cancer cells in vivo, providing evidence for their potential clinical relevance. As such, increased mRNA translation could provide a novel mechanism for enhancing the proliferative clonal expansion of TICs. The proteomic findings were functionally validated using known inhibitors of protein synthesis, via three independent approaches. For example, puromycin (which mimics the structure of tRNAs and competitively inhibits protein synthesis) preferentially targeted CSCs in both mammospheres and monolayer cultures, and was ~10-fold more potent for eradicating TICs, than “bulk” cancer cells. In addition, rapamycin, which inhibits mTOR and hence protein synthesis, was very effective at reducing mammosphere formation, at nanomolar concentrations. Finally, mammosphere formation was also markedly inhibited by methionine restriction, which mimics the positive effects of caloric restriction in cultured cells. Remarkably, mammosphere formation was >18-fold more sensitive to methionine restriction and replacement, as directly compared to monolayer cell proliferation. Methionine is absolutely required for protein synthesis, since every protein sequence starts with a methionine residue. Thus, the proliferation and survival of CSCs is very sensitive to

Functional Dynamics within the Human Ribosome Regulate the Rate of Active Protein Synthesis.

Science.gov (United States)

Ferguson, Angelica; Wang, Leyi; Altman, Roger B; Terry, Daniel S; Juette, Manuel F; Burnett, Benjamin J; Alejo, Jose L; Dass, Randall A; Parks, Matthew M; Vincent, C Theresa; Blanchard, Scott C

2015-11-05

The regulation of protein synthesis contributes to gene expression in both normal physiology and disease, yet kinetic investigations of the human translation mechanism are currently lacking. Using single-molecule fluorescence imaging methods, we have quantified the nature and timing of structural processes in human ribosomes during single-turnover and processive translation reactions. These measurements reveal that functional complexes exhibit dynamic behaviors and thermodynamic stabilities distinct from those observed for bacterial systems. Structurally defined sub-states of pre- and post-translocation complexes were sensitive to specific inhibitors of the eukaryotic ribosome, demonstrating the utility of this platform to probe drug mechanism. The application of three-color single-molecule fluorescence resonance energy transfer (smFRET) methods further revealed a long-distance allosteric coupling between distal tRNA binding sites within ribosomes bearing three tRNAs, which contributed to the rate of processive translation. Copyright © 2015 Elsevier Inc. All rights reserved.

Dihydroquinazolines as a novel class of Trypanosoma brucei trypanothione reductase inhibitors: discovery, synthesis, and characterization of their binding mode by protein crystallography.

Science.gov (United States)

Patterson, Stephen; Alphey, Magnus S; Jones, Deuan C; Shanks, Emma J; Street, Ian P; Frearson, Julie A; Wyatt, Paul G; Gilbert, Ian H; Fairlamb, Alan H

2011-10-13

Trypanothione reductase (TryR) is a genetically validated drug target in the parasite Trypanosoma brucei , the causative agent of human African trypanosomiasis. Here we report the discovery, synthesis, and development of a novel series of TryR inhibitors based on a 3,4-dihydroquinazoline scaffold. In addition, a high resolution crystal structure of TryR, alone and in complex with substrates and inhibitors from this series, is presented. This represents the first report of a high resolution complex between a noncovalent ligand and this enzyme. Structural studies revealed that upon ligand binding the enzyme undergoes a conformational change to create a new subpocket which is occupied by an aryl group on the ligand. Therefore, the inhibitor, in effect, creates its own small binding pocket within the otherwise large, solvent exposed active site. The TryR-ligand structure was subsequently used to guide the synthesis of inhibitors, including analogues that challenged the induced subpocket. This resulted in the development of inhibitors with improved potency against both TryR and T. brucei parasites in a whole cell assay.

JTP-103237, a monoacylglycerol acyltransferase inhibitor, prevents fatty liver and suppresses both triglyceride synthesis and de novo lipogenesis

Directory of Open Access Journals (Sweden)

Chihiro Okuma

2015-07-01

Conclusion: In the present study, JTP-103237 prevented carbohydrate-induced fatty liver and suppressed both TG synthesis and de novo lipogenesis, suggesting MGAT inhibitor may prevent carbohydrate-induced metabolic disorders, including NAFLD, obesity and diabetes.

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

Science.gov (United States)

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

2017-12-09

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

Akt Inhibitor A-443654 Interferes with Mitotic Progression by Regulating Aurora A Kinase Expression

Directory of Open Access Journals (Sweden)

Xuesong Liu

2008-08-01

Full Text Available Both Akt and Aurora A kinase have been shown to be important targets for intervention for cancer therapy. We report here that Compound A (A-443654, a specific Akt inhibitor, interferes with mitotic progression and bipolar spindle formation. Compound A induces G2/M accumulation, defects in centrosome separation, and formation of either monopolar arrays or disorganized spindles. On the basis of gene expression array studies, we identified Aurora A as one of the genes regulated transcriptionally by Akt inhibitors including Compound A. Inhibition of the phosphatidylinositol 3-kinase (PI3K/Akt pathway, either by PI3K inhibitor LY294002 or by Compound A, dramatically inhibits the promoter activity of Aurora A, whereas the mammalian target of rapamycin inhibitor has little effect, suggesting that Akt might be responsible for up-regulating Aurora A for mitotic progression. Further analysis of the Aurora A promoter region indicates that the Ets element but not the Sp1 element is required for Compound A-sensitive transcriptional control of Aurora A. Overexpression of Aurora A in cells treated with Compound A attenuates the mitotic arrest and the defects in bipolar spindle formation induced by Akt inhibition. Our studies suggest that that Akt may promote mitotic progression through the transcriptional regulation of Aurora A.

Regulation of ribonucleic acid synthesis by polyamines. Reversal by spermine of inhibition by methylglyoxal bis(guanylhydrazone) of ribonucleic acid synthesis and histone acetylation in rabbit heart.

Science.gov (United States)

Caldarera, C M; Casti, A; Guarnier, C; Moruzzi, G

1975-10-01

The relationship between polyamines and RNA synthesis was studied by considering the action of spermine on histone acetylation in perfused heart. In addition, the effect of methylglyoxal bis(guanylhydrazone), inhibitor of putrescine-activated S-adenosylmethionine decarboxylase activity, on RNA and polyamine specific radioactivity and on acetylation of histone fractions was also investigated in perfused heart. Different concentrations of spermine and/or methylglyoxas bis(guanylhydrazone) were injected into the heart, 15 min after beginning the perfusion. The results demonstrate that spermine stimulates the specific radioactivity of RNA of subcellular fractions. Acetylation of the arginine-rich histone fractions, involved in the regulation of RNA transcription, is enhanced by spermine. The perfusion with methylglyoxal bis(guanylhydrazone) causes a decrease in the specific radioactivity of polyamines and RNA, and in acetylation of histone fractions. However, spermine is able to reverse the methylglyoxal bis(guanylhydrazone) inhibition when injected simultaneously. From these results we may assume a possible role for spermine in the regulation of RNA transcription.

Synthesis and Biological Evaluation of Glycosidase Inhibitors: gem-Difluoromethylenated Nojirimycin Analogues

DEFF Research Database (Denmark)

Bols, Mikael; Wang, Ruo-Wen; Qiu, Xiao-Long

2006-01-01

In our ongoing program aimed at the design, synthesis, and biological evaluation of novel gem-difluoromethylenated glycosidase inhibitors, gem-4,4-difluoromethylenated iminosugars (5-9) were synthesized. The biological evaluation of these synthetic iminosugars showed that the gem....... It is proposed that the unprotonated iminosugar is the species preferably bound by beta-glucosidase, due to the lower pK(a) value of iminosugar 6 than of 1 or 36, leaving iminosugars 1 and 36 mostly protonated at pH 5.0, while iminosugar 6 is not. Iminosugar 6 also displayed good and selective inhibition of beta...

Effects of ionizing radiations and of metabolic inhibitors on the synthesis of RNA in lymphocites bred in culture

Energy Technology Data Exchange (ETDEWEB)

Farulla, A; Alimena, G; Castagna, R; Naro, G; Percoco, V D [Rome Univ. (Italy)

1954-01-01

Studies are conducted of the activation kinetics of the RNA synthesis and of proteins, during their blasto transformation stage of human circulating lymphocytes in culture breeding and of the effects caused by some metabolic inhibitors and by ionizing radiations on RNA synthesis. The results obtained lead to assume that the initial activation concern RNA nuclear linkages with fast turnover and scarce sensibility to actinomycin and that the exposure to ionizing radiations causes a significant reduction of the RNA synthesis rate at the initial phases of the activation process while it hasn't any effect on the later stage.

Proteomic and functional analyses reveal MAPK1 regulates milk protein synthesis.

Science.gov (United States)

Lu, Li-Min; Li, Qing-Zhang; Huang, Jian-Guo; Gao, Xue-Jun

2012-12-27

L-Lysine (L-Lys) is an essential amino acid that plays fundamental roles in protein synthesis. Many nuclear phosphorylated proteins such as Stat5 and mTOR regulate milk protein synthesis. However, the details of milk protein synthesis control at the transcript and translational levels are not well known. In this current study, a two-dimensional gel electrophoresis (2-DE)/MS-based proteomic technology was used to identify phosphoproteins responsible for milk protein synthesis in dairy cow mammary epithelial cells (DCMECs). The effect of L-Lys on DCMECs was analyzed by CASY technology and reversed phase high performance liquid chromatography (RP-HPLC). The results showed that cell proliferation ability and β-casein expression were enhanced in DCMECs treated with L-Lys. By phosphoproteomics analysis, six proteins, including MAPK1, were identified up-expressed in DCMECs treated with 1.2 mM L-Lys for 24 h, and were verified by quantitative real-time PCR (qRT-PCR) and western blot. Overexpression and siRNA inhibition of MAPK1 experiments showed that MAPK1 upregulated milk protein synthesis through Stat5 and mTOR pathway. These findings that MAPK1 involves in regulation of milk synthesis shed new insights for understanding the mechanisms of milk protein synthesis.

DMPD: Mechanism of age-associated up-regulation in macrophage PGE2 synthesis. [Dynamic Macrophage Pathway CSML Database

Lifescience Database Archive (English)

Full Text Available 15331118 Mechanism of age-associated up-regulation in macrophage PGE2 synthesis. Wu...e-associated up-regulation in macrophage PGE2 synthesis. PubmedID 15331118 Title Mechanism of age-associated... up-regulation in macrophage PGE2 synthesis. Authors Wu D, Meydani SN. Publicatio

Design and synthesis of imidazopyridine analogues as inhibitors of phosphoinositide 3-kinase signaling and angiogenesis.

Science.gov (United States)

Kim, Okseon; Jeong, Yujeong; Lee, Hyunseung; Hong, Sun-Sun; Hong, Sungwoo

2011-04-14

Phosphatidylinositol 3-kinase α (PI3Kα) is an important regulator of intracellular signaling pathways, controlling remarkably diverse arrays of physiological processes. Because the PI3K pathway is frequently up-regulated in human cancers, the inhibition of PI3Kα can be a promising approach to cancer therapy. In this study, we have designed and synthesized a new series of imidazo[1,2-a]pyridine derivatives as PI3Kα inhibitors through the fragment-growing strategy. By varying groups at the 3- and 6-positions of imidazo[1,2-a]pyridines, we studied the structure-activity relationships (SAR) profiles and identified a series of potent PI3Kα inhibitors. Representative derivatives showed good activity in cellular proliferation and apoptosis assays. Moreover, these inhibitors exhibited noteworthy antiangiogenic activity.

A Gibberellin-Mediated DELLA-NAC Signaling Cascade Regulates Cellulose Synthesis in Rice.

Science.gov (United States)

Huang, Debao; Wang, Shaogan; Zhang, Baocai; Shang-Guan, Keke; Shi, Yanyun; Zhang, Dongmei; Liu, Xiangling; Wu, Kun; Xu, Zuopeng; Fu, Xiangdong; Zhou, Yihua

2015-06-01

Cellulose, which can be converted into numerous industrial products, has important impacts on the global economy. It has long been known that cellulose synthesis in plants is tightly regulated by various phytohormones. However, the underlying mechanism of cellulose synthesis regulation remains elusive. Here, we show that in rice (Oryza sativa), gibberellin (GA) signals promote cellulose synthesis by relieving the interaction between SLENDER RICE1 (SLR1), a DELLA repressor of GA signaling, and NACs, the top-layer transcription factors for secondary wall formation. Mutations in GA-related genes and physiological treatments altered the transcription of CELLULOSE SYNTHASE genes (CESAs) and the cellulose level. Multiple experiments demonstrated that transcription factors NAC29/31 and MYB61 are CESA regulators in rice; NAC29/31 directly regulates MYB61, which in turn activates CESA expression. This hierarchical regulation pathway is blocked by SLR1-NAC29/31 interactions. Based on the results of anatomical analysis and GA content examination in developing rice internodes, this signaling cascade was found to be modulated by varied endogenous GA levels and to be required for internode development. Genetic and gene expression analyses were further performed in Arabidopsis thaliana GA-related mutants. Altogether, our findings reveal a conserved mechanism by which GA regulates secondary wall cellulose synthesis in land plants and provide a strategy for manipulating cellulose production and plant growth. © 2015 American Society of Plant Biologists. All rights reserved.

Sensitization of human cells by inhibitors of DNA synthesis following the action of DNA-damaging agents

Energy Technology Data Exchange (ETDEWEB)

Filatov, M.V.; Noskin, L.A. (Leningrad Inst. of Nuclear Physics, Gatchina (USSR))

1983-08-01

Inhibitors of DNA synthesis 1-..beta..-arabinofuranosylcytosine (Ac) and hydroxyurea (Hu) taken together drastically sensitized human cells to the killing effect of DNA-damaging agents. For UV-irradiation this sensitization depended on the cells' ability for excision repair. By using viscoelastometric methods of measurement of double-strand breaks (DSB) in the genome, it was established that the first DSB were generated after incubation of the damaged cells in the mixture of inhibitors at about the same dose when sensitization appeared. A scheme is proposed to describe molecular events associated with the phenomenon studied. 35 refs.

Sensitization of multidrug-resistant human cancer cells to Hsp90 inhibitors by down-regulation of SIRT1

Science.gov (United States)

Kim, Hak-Bong; Lee, Su-Hoon; Um, Jee-Hyun; Oh, Won Keun; Kim, Dong-Wan; Kang, Chi-Dug; Kim, Sun-Hee

2015-01-01

The effectiveness of Hsp90 inhibitors as anticancer agents was limited in multidrug-resistant (MDR) human cancer cells due to induction of heat shock proteins (Hsps) such as Hsp70/Hsp27 and P-glycoprotein (P-gp)-mediated efflux. In the present study, we showed that resistance to Hsp90 inhibitors of MDR human cancer cells could be overcome with SIRT1 inhibition. SIRT1 knock-down or SIRT1 inhibitors (amurensin G and EX527) effectively suppressed the resistance to Hsp90 inhibitors (17-AAG and AUY922) in several MDR variants of human lymphoblastic leukemia and human breast cancer cell lines. SIRT1 inhibition down-regulated the expression of heat shock factor 1 (HSF1) and subsequently Hsps and facilitated Hsp90 multichaperone complex disruption via hyperacetylation of Hsp90/Hsp70. These findings were followed by acceleration of ubiquitin ligase CHIP-mediated mutant p53 (mut p53) degradation and subsequent down-regulation of P-gp in 17-AAG-treated MDR cancer cells expressing P-gp and mut p53 after inhibition of SIRT1. Therefore, combined treatment with Hsp90 inhibitor and SIRT1 inhibitor could be a more effective therapeutic approach for Hsp90 inhibitor-resistant MDR cells via down-regulation of HSF1/Hsps, mut p53 and P-gp. PMID:26416354

Regulation of urea synthesis during the acute phase response in rats

DEFF Research Database (Denmark)

Thomsen, Karen Louise; Jessen, Niels; Buch Møller, Andreas

2013-01-01

The acute-phase response is a catabolic event involving increased waste of amino-nitrogen (N) via hepatic urea synthesis, despite an increased need for amino-N incorporation into acute-phase proteins. This study aimed to clarify the regulation of N elimination via urea during different phases...... of the tumor necrosis factor-α (TNF-α)-induced acute-phase response in rats. We used four methods to study the regulation of urea synthesis: We examined urea cycle enzyme mRNA levels in liver tissue, the hepatocyte urea cycle enzyme proteins, the in vivo capacity of urea-N synthesis (CUNS), and known humoral...... regulators of CUNS at 1, 3, 24, and 72 h after TNF-α injection (25 μg/kg iv rrTNF-α) in rats. Serum acute-phase proteins and their liver mRNA levels were also measured. The urea cycle enzyme mRNA levels acutely decreased and then gradually normalized, whereas the urea cycle enzyme proteins remained...

Smad signaling pathway is a pivotal component of tissue inhibitor of metalloproteinases-3 regulation by transforming growth factor beta in human chondrocytes.

Science.gov (United States)

Qureshi, Hamid Yaqoob; Ricci, Gemma; Zafarullah, Muhammad

2008-09-01

Transforming growth factor beta (TGF-beta1) promotes cartilage matrix synthesis and induces tissue inhibitor of metalloproteinases-3 (TIMP-3), which inhibits matrix metalloproteinases, aggrecanases and TNF-alpha-converting enzyme implicated in articular cartilage degradation and joint inflammation. TGF-beta1 activates Akt, ERK and Smad2 pathways in chondrocytes. Here we investigated previously unexplored roles of specific Smads in TGF-beta1 induction of TIMP-3 gene by pharmacological and genetic knockdown approaches. TGF-beta1-induced Smad2 phosphorylation and TIMP-3 protein expression could be inhibited by the Smad2/3 phosphorylation inhibitors, PD169316 and SB203580 and by Smad2-specific siRNA. Specific inhibitor of Smad3 (SIS3) and Smad3 siRNA abolished TGF-beta induction of TIMP-3. Smad2/3 siRNAs also down regulated TIMP-3 promoter-driven luciferase activities, suggesting transcriptional regulation. SiRNA-driven co-Smad4 knockdown abrogated TIMP-3 augmentation by TGF-beta. TIMP-3 promoter deletion analysis revealed that -828 deletion retains the original promoter activity while -333 and -167 deletions display somewhat reduced activity suggesting that most of the TGF-beta-responsive, cis-acting elements are found in the -333 fragment. Chromatin Immunoprecipitation (ChIP) analysis confirmed binding of Smad2 and Smad4 with the -940 and -333 promoter sequences. These results suggest that receptor-activated Smad2 and Smad3 and co-Smad4 critically mediate TGF-beta-stimulated TIMP-3 expression in human chondrocytes and TIMP-3 gene is a target of Smad signaling pathway.

Probing the Influence of Linker Length and Flexibility in the Design and Synthesis of New Trehalase Inhibitors

Directory of Open Access Journals (Sweden)

Giampiero D’Adamio

2018-02-01

Full Text Available This work aims to synthesize new trehalase inhibitors selective towards the insect trehalase versus the porcine trehalase, in view of their application as potentially non-toxic insecticides and fungicides. The synthesis of a new pseudodisaccharide mimetic 8, by means of a stereoselective α-glucosylation of the key pyrrolizidine intermediate 13, was accomplished. The activity of compound 8 as trehalase inhibitor towards C. riparius trehalase was evaluated and the results showed that 8 was active in the μM range and showed a good selectivity towards the insect trehalase. To reduce the overall number of synthetic steps, simpler and more flexible disaccharide mimetics 9–11 bearing a pyrrolidine nucleus instead of the pyrrolizidine core were synthesized. The biological data showed the key role of the linker chain’s length in inducing inhibitory properties, since only compounds 9 (α,β-mixture, bearing a two-carbon atom linker chain, maintained activity as trehalase inhibitors. A proper change in the glucosyl donor-protecting groups allowed the stereoselective synthesis of the β-glucoside 9β, which was active in the low micromolar range (IC50 = 0.78 μM and 12-fold more potent (and more selective than 9α towards the insect trehalase.

A Gibberellin-Mediated DELLA-NAC Signaling Cascade Regulates Cellulose Synthesis in Rice[OPEN

Science.gov (United States)

Huang, Debao; Wang, Shaogan; Zhang, Baocai; Shang-Guan, Keke; Shi, Yanyun; Zhang, Dongmei; Liu, Xiangling; Wu, Kun; Xu, Zuopeng; Fu, Xiangdong; Zhou, Yihua

2015-01-01

Cellulose, which can be converted into numerous industrial products, has important impacts on the global economy. It has long been known that cellulose synthesis in plants is tightly regulated by various phytohormones. However, the underlying mechanism of cellulose synthesis regulation remains elusive. Here, we show that in rice (Oryza sativa), gibberellin (GA) signals promote cellulose synthesis by relieving the interaction between SLENDER RICE1 (SLR1), a DELLA repressor of GA signaling, and NACs, the top-layer transcription factors for secondary wall formation. Mutations in GA-related genes and physiological treatments altered the transcription of CELLULOSE SYNTHASE genes (CESAs) and the cellulose level. Multiple experiments demonstrated that transcription factors NAC29/31 and MYB61 are CESA regulators in rice; NAC29/31 directly regulates MYB61, which in turn activates CESA expression. This hierarchical regulation pathway is blocked by SLR1-NAC29/31 interactions. Based on the results of anatomical analysis and GA content examination in developing rice internodes, this signaling cascade was found to be modulated by varied endogenous GA levels and to be required for internode development. Genetic and gene expression analyses were further performed in Arabidopsis thaliana GA-related mutants. Altogether, our findings reveal a conserved mechanism by which GA regulates secondary wall cellulose synthesis in land plants and provide a strategy for manipulating cellulose production and plant growth. PMID:26002868

Synthesis and application of labelled growth regulators

International Nuclear Information System (INIS)

Shyutte, G.R.

1982-01-01

For the investigation of the metabolism both of phytoeffectors like herbicides and plant growth regulators such compounds are needed in radioactive labelled form. The synthesis of radioactive labelled fluorodifen, nitrofen, ethephon, diphenylic acetic acid, 2,4-dichlorophenoxyisobutyric acid, abscisic acid, hydroxybenzoic acids and different conjugates are described. Some examples of these compounds metabolism in plants are discussed [ru

Synthesis and characterization of a novel organic corrosion inhibitor for mild steel in 1 M hydrochloric acid

Science.gov (United States)

Ahmed, Mohammed H. Othman; Al-Amiery, Ahmed A.; Al-Majedy, Yasmin K.; Kadhum, Abdul Amir H.; Mohamad, Abu Bakar; Gaaz, Tayser Sumer

2018-03-01

The synthesis and characterization of a novel organic corrosion inhibitor (4-(3-mercapto-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-b][1,2,4,5]tetrazin-6-yl)phenol), for mild steel in 1 M hydrochloric acid (HCl) has been successfully reported for the first time. The inhibitor evaluated as corrosion inhibitor for mild steel in 1 M of Hydrochloric acid solution using electrochemical impedance spectroscopy (EIS), and electrochemical frequency modulation (EFM) measurement techniques. Changes in the impedance parameters suggested an adsorption of the inhibitor onto the mild steel surface, leading to the formation of protective films. The results show that the inhibition efficiencies increased with increasing the concentrations of the inhibitors and decreased with increasing temperature. The maximum inhibition efficiency up to 67% at the maximum concentration 0.5 mM. This shows that those inhibitors are effective in helping to reduce and slowing down the corrosion process that occurs to mild steel with a hydrochloric acid solution by providing an organic inhibitor for the mild steel that can be weakened by increasing the temperature. The adsorption process of the synthesized organic inhibitor depends on its electronic characteristics in addition to steric effects and the nature of metal surface, temperature degree and the varying degrees of surface-site activity. The synthesized inhibitor molecules were absorbed by metal surface and follow Langmuir isotherms.

Syndecan-2 regulates melanin synthesis via protein kinase C βII-mediated tyrosinase activation.

Science.gov (United States)

Jung, Hyejung; Chung, Heesung; Chang, Sung Eun; Choi, Sora; Han, Inn-Oc; Kang, Duk-Hee; Oh, Eok-Soo

2014-05-01

Syndecan-2, a transmembrane heparan sulfate proteoglycan that is highly expressed in melanoma cells, regulates melanoma cell functions (e.g. migration). Since melanoma is a malignant tumor of melanocytes, which largely function to synthesize melanin, we investigated the possible involvement of syndecan-2 in melanogenesis. Syndecan-2 expression was increased in human skin melanoma tissues compared with normal skin. In both mouse and human melanoma cells, siRNA-mediated knockdown of syndecan-2 was associated with reduced melanin synthesis, whereas overexpression of syndecan-2 increased melanin synthesis. Similar effects were also detected in human primary epidermal melanocytes. Syndecan-2 expression did not affect the expression of tyrosinase, a key enzyme in melanin synthesis, but instead enhanced the enzymatic activity of tyrosinase by increasing the membrane and melanosome localization of its regulator, protein kinase CβII. Furthermore, UVB caused increased syndecan-2 expression, and this up-regulation of syndecan-2 was required for UVB-induced melanin synthesis. Taken together, these data suggest that syndecan-2 regulates melanin synthesis and could be a potential therapeutic target for treating melanin-associated diseases. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Microwave-Assisted Synthesis of a MK2 Inhibitor by Suzuki-Miyaura Coupling for Study in Werner Syndrome Cells

Directory of Open Access Journals (Sweden)

Mark C. Bagley

2015-06-01

Full Text Available Microwave-assisted Suzuki-Miyaura cross-coupling reactions have been employed towards the synthesis of three different MAPKAPK2 (MK2 inhibitors to study accelerated aging in Werner syndrome (WS cells, including the cross-coupling of a 2-chloroquinoline with a 3-pyridinylboronic acid, the coupling of an aryl bromide with an indolylboronic acid and the reaction of a 3-amino-4-bromopyrazole with 4-carbamoylphenylboronic acid. In all of these processes, the Suzuki-Miyaura reaction was fast and relatively efficient using a palladium catalyst under microwave irradiation. The process was incorporated into a rapid 3-step microwave-assisted method for the synthesis of a MK2 inhibitor involving 3-aminopyrazole formation, pyrazole C-4 bromination using N-bromosuccinimide (NBS, and Suzuki-Miyaura cross-coupling of the pyrazolyl bromide with 4-carbamoylphenylboronic acid to give the target 4-arylpyrazole in 35% overall yield, suitable for study in WS cells.

INVITED REVIEW: Inhibitors of myostatin as methods of enhancing muscle growth and development.

Science.gov (United States)

Chen, P R; Lee, K

2016-08-01

With the increasing demand for affordable, high-quality meat, livestock and poultry producers must continually find ways to maximize muscle growth in their animals without compromising palatability of the meat products. Muscle mass relies on myoblast proliferation during prenatal or prehatch stages and fiber hypertrophy through protein synthesis and nuclei donation by satellite cells after birth or hatch. Therefore, understanding the cellular and molecular mechanisms of myogenesis and muscle development is of great interest. Myostatin is a well-known negative regulator of muscle growth and development that inhibits proliferation and differentiation in myogenic cells as well as protein synthesis in existing muscle fibers. In this review, various inhibitors of myostatin activity or signaling are examined that may be used in animal agriculture for enhancing muscle growth. Myostatin inhibitors are relevant as potential therapies for muscle-wasting diseases and muscle weakness in humans and animals. Currently, there are no commercial myostatin inhibitors for agriculture or biomedical purposes because the safest and most effective option has yet to be identified. Further investigation of myostatin inhibitors and administration strategies may revolutionize animal production and the medical field.

Insect P450 inhibitors and insecticides: challenges and opportunities.

Science.gov (United States)

Feyereisen, René

2015-06-01

P450 enzymes are encoded by a large number of genes in insects, often over a hundred. They play important roles in insecticide metabolism and resistance, and growing numbers of P450 enzymes are now known to catalyse important physiological reactions, such as hormone metabolism or cuticular hydrocarbon synthesis. Ways to inhibit P450 enzymes specifically or less specifically are well understood, as P450 inhibitors are found as drugs, as fungicides, as plant growth regulators and as insecticide synergists. Yet there are no P450 inhibitors as insecticides on the market. As new modes of action are constantly needed to support insecticide resistance management, P450 inhibitors should be considered because of their high potential for insect selectivity, their well-known mechanisms of action and the increasing ease of rational design and testing. © 2014 Society of Chemical Industry.

A chiral synthesis of dapoxetine hydrochloride, a serotonin re-uptake inhibitor, and its 14C isotopomer

International Nuclear Information System (INIS)

Wheeler, W.J.; O'Bannon, D.D.

1992-01-01

The 14 C-isotopmer of dapoxetine-[ 14 C] HCl (S (+) -N,N-dimethyl-α[2-(1-naphthalenyloxy)ethyl-2- 14 C]benzenemeth a-n amine hydrochloride, 1a), a potent serotonin re-uptake inhibitor has been prepared by a chiral synthesis, starting with tert. -butyloxyphenylglycine (3). Borane reduction, followed by activation of the resulting alcohol 4 as its mesylate 5b, provided the chiral starting material. The radiolabel was introduced by reaction of 5b with sodium cyanide-[ 14 C]. The desired product (1) was then elaborated from nitrile 6a,b via a five step synthesis in an overall 19.5% radiochemical yield. (Author)

Endogenous Natural Complement Inhibitor Regulates Cardiac Development

DEFF Research Database (Denmark)

Mortensen, Simon A; Skov, Louise L; Kjaer-Sorensen, Kasper

2017-01-01

mechanisms during fetal development and adult homeostasis. In this article, we describe the function of an endogenous complement inhibitor, mannan-binding lectin (MBL)-associated protein (MAp)44, in regulating the composition of a serine protease-pattern recognition receptor complex, MBL-associated serine...... of MAp44 caused impaired cardiogenesis, lowered heart rate, and decreased cardiac output. These defects were associated with aberrant neural crest cell behavior. We found that MAp44 competed with MASP-3 for pattern recognition molecule interaction, and knockdown of endogenous MAp44 expression could...... be rescued by overexpression of wild-type MAp44. Our observations provide evidence that immune molecules are centrally involved in the orchestration of cardiac tissue development....

Roles of Transcriptional and Translational Control Mechanisms in Regulation of Ribosomal Protein Synthesis in Escherichia coli.

Science.gov (United States)

Burgos, Hector L; O'Connor, Kevin; Sanchez-Vazquez, Patricia; Gourse, Richard L

2017-11-01

Bacterial ribosome biogenesis is tightly regulated to match nutritional conditions and to prevent formation of defective ribosomal particles. In Escherichia coli , most ribosomal protein (r-protein) synthesis is coordinated with rRNA synthesis by a translational feedback mechanism: when r-proteins exceed rRNAs, specific r-proteins bind to their own mRNAs and inhibit expression of the operon. It was recently discovered that the second messenger nucleotide guanosine tetra and pentaphosphate (ppGpp), which directly regulates rRNA promoters, is also capable of regulating many r-protein promoters. To examine the relative contributions of the translational and transcriptional control mechanisms to the regulation of r-protein synthesis, we devised a reporter system that enabled us to genetically separate the cis -acting sequences responsible for the two mechanisms and to quantify their relative contributions to regulation under the same conditions. We show that the synthesis of r-proteins from the S20 and S10 operons is regulated by ppGpp following shifts in nutritional conditions, but most of the effect of ppGpp required the 5' region of the r-protein mRNA containing the target site for translational feedback regulation and not the promoter. These results suggest that most regulation of the S20 and S10 operons by ppGpp following nutritional shifts is indirect and occurs in response to changes in rRNA synthesis. In contrast, we found that the promoters for the S20 operon were regulated during outgrowth, likely in response to increasing nucleoside triphosphate (NTP) levels. Thus, r-protein synthesis is dynamic, with different mechanisms acting at different times. IMPORTANCE Bacterial cells have evolved complex and seemingly redundant strategies to regulate many high-energy-consuming processes. In E. coli , synthesis of ribosomal components is tightly regulated with respect to nutritional conditions by mechanisms that act at both the transcription and translation steps. In

Epitope-Targeting of Tertiary Protein Structure Enables Target-Guided Synthesis of a Potent in Cell Inhibitor of Botulinum Neurotoxin**

OpenAIRE

Farrow, Blake; Wong, Michelle; Malette, Jacquie; Lai, Bert; Deyle, Kaycie M.; Das, Samir; Nag, Arundhati; Agnew, Heather D.; Heath, James R.

2015-01-01

Botulinum neurotoxin (BoNT) serotype A is the most lethal known toxin and has an occluded structure, which prevents direct inhibition of its active site before it enters the cytosol. Target-guided synthesis by in situ click chemistry is combined with synthetic epitope targeting to exploit the tertiary structure of the BoNT protein as a landscape for assembling a competitive inhibitor. A substrate-mimicking peptide macrocycle is used as a direct inhibitor of BoNT. An epitope-targeting in situ ...

Plasminogen activator inhibitor-I-related regulation of procollagen I (α1 and α2) by antitransforming growth factor-β1 treatment during radiation-impaired wound healing

International Nuclear Information System (INIS)

Schultze-Mosgau, Stefan; Kopp, Juergen; Thorwarth, Michael; Roedel, Franz; Melnychenko, Ivan; Grabenbauer, Gerhard G.; Amann, Kerstin; Wehrhan, Falk

2006-01-01

Purpose: Plasminogen activator inhibitor (PAI)-1 mediates transforming growth factor-β 1 (TGF-β 1 )-related signaling by stimulating collagen Type I synthesis in radiation-impaired wound healing. The regulation of α(I)-procollagen is contradictory in fibroblasts of different fibrotic lesions. It is not known whether anti-TGF-β 1 treatment specifically inhibits α(I)-procollagen synthesis. We used an experimental wound healing study to address anti-TGF-β 1 -associated influence on α(I)-procollagen synthesis. Methods and Materials: A free flap was transplanted into the preirradiated (40 Gy) or nonirradiated neck region of Wistar rats: Group 1 (n = 8) surgery alone; Group 2 (n = 14) irradiation and surgery; Group 3 (n = 8) irradiation and surgery and anti-TGF-β 1 treatment. On the 14th postoperative day, skin samples were processed for fibroblast culture, in situ hybridization for TGF-β 1 , immunohistochemistry, and immunoblotting for PAI-1, α 1 /α 2 (I)-procollagen. Results: Anti-TGF-β 1 significantly reduced TGF-β 1 mRNA (p 1 treatment in vivo significantly reduced α 1 (I)-procollagen protein (p 2 (I)-procollagen expression. Conclusion: These results emphasize anti-TGF-β 1 treatment to reduce radiation-induced fibrosis by decreasing α 1 (I)-procollagen synthesis in vivo. α 1 (I)-procollagen and α 2 (I)-procollagen might be differentially regulated by anti-TGF-β 1 treatment. Increased TGF-β signaling in irradiated skin fibroblasts seemed to be reversible, as shown by a reduction in PAI-1 expression after anti-TGF-β 1 treatment

Potent new small-molecule inhibitor of botulinum neurotoxin serotype A endopeptidase developed by synthesis-based computer-aided molecular design.

Directory of Open Access Journals (Sweden)

Yuan-Ping Pang

2009-11-01

Full Text Available Botulinum neurotoxin serotype A (BoNTA causes a life-threatening neuroparalytic disease known as botulism. Current treatment for post exposure of BoNTA uses antibodies that are effective in neutralizing the extracellular toxin to prevent further intoxication but generally cannot rescue already intoxicated neurons. Effective small-molecule inhibitors of BoNTA endopeptidase (BoNTAe are desirable because such inhibitors potentially can neutralize the intracellular BoNTA and offer complementary treatment for botulism. Previously we reported a serotype-selective, small-molecule BoNTAe inhibitor with a K(i (app value of 3.8+/-0.8 microM. This inhibitor was developed by lead identification using virtual screening followed by computer-aided optimization of a lead with an IC(50 value of 100 microM. However, it was difficult to further improve the lead from micromolar to even high nanomolar potency due to the unusually large enzyme-substrate interface of BoNTAe. The enzyme-substrate interface area of 4,840 A(2 for BoNTAe is about four times larger than the typical protein-protein interface area of 750-1,500 A(2. Inhibitors must carry several functional groups to block the unusually large interface of BoNTAe, and syntheses of such inhibitors are therefore time-consuming and expensive. Herein we report the development of a serotype-selective, small-molecule, and competitive inhibitor of BoNTAe with a K(i value of 760+/-170 nM using synthesis-based computer-aided molecular design (SBCAMD. This new approach accounts the practicality and efficiency of inhibitor synthesis in addition to binding affinity and selectivity. We also report a three-dimensional model of BoNTAe in complex with the new inhibitor and the dynamics of the complex predicted by multiple molecular dynamics simulations, and discuss further structural optimization to achieve better in vivo efficacy in neutralizing BoNTA than those of our early micromolar leads. This work provides new insight

Acquisition of iron from transferrin regulates reticulocyte heme synthesis

International Nuclear Information System (INIS)

Ponka, P.; Schulman, H.M.

1985-01-01

Fe-salicylaldehyde isonicotinoylhydrazone (SIH), which can donate iron to reticulocytes without transferrin as a mediator, has been utilized to test the hypothesis that the rate of iron uptake from transferrin limits the rate of heme synthesis in erythroid cells. Reticulocytes take up 59 Fe from [ 59 Fe]SIH and incorporate it into heme to a much greater extent than from saturating concentrations of [ 59 Fe]transferrin. Also, Fe-SIH stimulates [2- 14 C]glycine into heme when compared to the incorporation observed with saturating levels of Fe-transferrin. In addition, delta-aminolevulinic acid does not stimulate 59 Fe incorporation into heme from either [ 59 Fe]transferrin or [ 59 Fe]SIH but does reverse the inhibition of 59 Fe incorporation into heme caused by isoniazid, an inhibitor of delta-aminolevulinic acid synthase. Taken together, these results suggest the hypothesis that some step(s) in the pathway of iron from extracellular transferrin to intracellular protoporphyrin limits the overall rate of heme synthesis in reticulocytes

Protein degradation and protein synthesis in long-term memory formation

Directory of Open Access Journals (Sweden)

Timothy J Jarome

2014-06-01

Full Text Available Long-term memory (LTM formation requires transient changes in the activity of intracellular signaling cascades that are thought to regulate new gene transcription and de novo protein synthesis in the brain. Consistent with this, protein synthesis inhibitors impair LTM for a variety of behavioral tasks when infused into the brain around the time of training or following memory retrieval, suggesting that protein synthesis is a critical step in LTM storage in the brain. However, evidence suggests that protein degradation mediated by the ubiquitin-proteasome system may also be a critical regulator of LTM formation and stability following retrieval. This requirement for increased protein degradation has been shown in the same brain regions in which protein synthesis is required for LTM storage. Additionally, increases in the phosphorylation of proteins involved in translational control parallel increases in protein polyubiquitination and the increased demand for protein degradation is regulated by intracellular signaling molecules thought to regulate protein synthesis during LTM formation. In some cases inhibiting proteasome activity can rescue memory impairments that result from pharmacological blockade of protein synthesis, suggesting that protein degradation may control the requirement for protein synthesis during the memory storage process. Results such as these suggest that protein degradation and synthesis are both critical for LTM formation and may interact to properly consolidate and store memories in the brain. Here, we review the evidence implicating protein synthesis and degradation in LTM storage and highlight the areas of overlap between these two opposing processes. We also discuss evidence suggesting these two processes may interact to properly form and store memories. LTM storage likely requires a coordinated regulation between protein degradation and synthesis at multiple sites in the mammalian brain.

Protein targeting to glycogen is a master regulator of glycogen synthesis in astrocytes

KAUST Repository

Ruchti, E.

2016-10-08

The storage and use of glycogen, the main energy reserve in the brain, is a metabolic feature of astrocytes. Glycogen synthesis is regulated by Protein Targeting to Glycogen (PTG), a member of specific glycogen-binding subunits of protein phosphatase-1 (PPP1). It positively regulates glycogen synthesis through de-phosphorylation of both glycogen synthase (activation) and glycogen phosphorylase (inactivation). In cultured astrocytes, PTG mRNA levels were previously shown to be enhanced by the neurotransmitter noradrenaline. To achieve further insight into the role of PTG in the regulation of astrocytic glycogen, its levels of expression were manipulated in primary cultures of mouse cortical astrocytes using adenovirus-mediated overexpression of tagged-PTG or siRNA to downregulate its expression. Infection of astrocytes with adenovirus led to a strong increase in PTG expression and was associated with massive glycogen accumulation (>100 fold), demonstrating that increased PTG expression is sufficient to induce glycogen synthesis and accumulation. In contrast, siRNA-mediated downregulation of PTG resulted in a 2-fold decrease in glycogen levels. Interestingly, PTG downregulation strongly impaired long-term astrocytic glycogen synthesis induced by insulin or noradrenaline. Finally, these effects of PTG downregulation on glycogen metabolism could also be observed in cultured astrocytes isolated from PTG-KO mice. Collectively, these observations point to a major role of PTG in the regulation of glycogen synthesis in astrocytes and indicate that conditions leading to changes in PTG expression will directly impact glycogen levels in this cell type.

Role of c-Src inhibitor in the regulation of hepatocarcinoma cell ...

African Journals Online (AJOL)

SAM

2014-03-19

Mar 19, 2014 ... BEL-7402 cell line was used as HCC cell model for investigating the regulation of cell migration upon c-. Src inhibitors (PP2 and .... PDGF-BB were purchased from Enzo Life Sciences International,. USA; SU6656 Sigma (USA). .... Statistical analysis was done with Student's t-test for comparison of two ...

Skeletal muscle protein synthesis and the abundance of the mRNA translation initiation repressor PDCD4 are inversely regulated by fasting and refeeding in rats.

Science.gov (United States)

Zargar, Sana; Moreira, Tracy S; Samimi-Seisan, Helena; Jeganathan, Senthure; Kakade, Dhanshri; Islam, Nushaba; Campbell, Jonathan; Adegoke, Olasunkanmi A J

2011-06-01

Optimal skeletal muscle mass is vital to human health, because defects in muscle protein metabolism underlie or exacerbate human diseases. The mammalian target of rapamycin complex 1 is critical in the regulation of mRNA translation and protein synthesis. These functions are mediated in part by the ribosomal protein S6 kinase 1 (S6K1) through mechanisms that are poorly understood. The tumor suppressor programmed cell death 4 (PDCD4) has been identified as a novel substrate of S6K1. Here, we examined 1) the expression of PDCD4 in skeletal muscle and 2) its regulation by feed deprivation (FD) and refeeding. Male rats (~100 g; n = 6) were subjected to FD for 48 h; some rats were refed for 2 h. FD suppressed muscle fractional rates of protein synthesis and Ser(67) phosphorylation of PDCD4 (-50%) but increased PDCD4 abundance (P muscle fractional rates of protein synthesis and reduced PDCD4 abundance relative to FD. Finally, when myoblasts were grown in amino acid- and serum-free medium, phenylalanine incorporation into proteins in cells depleted of PDCD4 more than doubled the values in cells with a normal level of PDCD4 (P skeletal muscle in parallel with the reduction of the abundance of this mRNA translation inhibitor.

Synthesis, cytotoxicity and molecular modelling studies of new phenylcinnamide derivatives as potent inhibitors of cholinesterases.

Science.gov (United States)

Saeed, Aamer; Mahesar, Parvez Ali; Zaib, Sumera; Khan, Muhammad Siraj; Matin, Abdul; Shahid, Mohammad; Iqbal, Jamshed

2014-05-06

The present study reports the synthesis of cinnamide derivatives and their biological activity as inhibitors of both cholinesterases and anticancer agents. Controlled inhibition of brain acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) may slow neurodegeneration in Alzheimer's diseases (AD). The anticholinesterase activity of phenylcinnamide derivatives was determined against Electric Eel acetylcholinesterase (EeAChE) and horse serum butyrylcholinesterase (hBChE) and some of the compounds appeared as moderately potent inhibitors of EeAChE and hBChE. The compound 3-(2-(Benzyloxy)phenyl)-N-(3,4,5-trimethoxyphenyl)acrylamide (3i) showed maximum activity against EeAChE with an IC50 0.29 ± 0.21 μM whereas 3-(2-chloro-6-nitrophenyl)-N-(3,4,5-trimethoxyphenyl)acrylamide (3k) was proved to be the most potent inhibitor of hBChE having IC50 1.18 ± 1.31 μM. To better understand the enzyme-inhibitor interaction of the most active compounds toward cholinesterases, molecular modelling studies were carried out on high-resolution crystallographic structures. The anticancer effects of synthesized compounds were also evaluated against cancer cell line (lung carcinoma). The compounds may be useful leads for the design of a new class of anticancer drugs for the treatment of cancer and cholinesterase inhibitors for Alzheimer's disease (AD). Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Regulation of protein synthesis during sea urchin early development

International Nuclear Information System (INIS)

Kelso, L.C.

1989-01-01

Fertilization of the sea urchin egg results in a 20-40 fold increase in the rate of protein synthesis. The masked message hypothesis proposes that mRNAs are masked or unavailable for translation in the egg. We devised an in vivo assay to test this hypothesis. Our results show that masked mRNAs limit protein synthesis in the unfertilized egg. In addition, we show that protein synthesis is also regulated at the level of translational machinery. Following fertilization is a period of rapid cell divisions. This period, known as the rapid cleavage stage, is characterized by the transient synthesis of a novel set of proteins. The synthesis of these proteins is programmed by maternal mRNAs stored in the unfertilized egg. To study the behavior of these mRNAs, we prepared a cDNA library from polysomal poly (A+) RNA from 2-hour embryos. [ 32 P] labeled probes, prepared from the cDNA library, were used to monitor the levels of individual mRNAs in polysomes at fertilization and during early development

Endogenous secreted phospholipase A2 group X regulates cysteinyl leukotrienes synthesis by human eosinophils.

Science.gov (United States)

Hallstrand, Teal S; Lai, Ying; Hooper, Kathryn A; Oslund, Rob C; Altemeier, William A; Matute-Bello, Gustavo; Gelb, Michael H

2016-01-01

Phospholipase A2s mediate the rate-limiting step in the formation of eicosanoids such as cysteinyl leukotrienes (CysLTs). Group IVA cytosolic PLA2α (cPLA2α) is thought to be the dominant PLA2 in eosinophils; however, eosinophils also have secreted PLA2 (sPLA2) activity that has not been fully defined. To examine the expression of sPLA2 group X (sPLA2-X) in eosinophils, the participation of sPLA2-X in the formation of CysLTs, and the mechanism by which sPLA2-X initiates the synthesis of CysLTs in eosinophils. Peripheral blood eosinophils were obtained from volunteers with asthma and/or allergy. A rabbit polyclonal anti-sPLA2-X antibody identified sPLA2-X by Western blot. We used confocal microscopy to colocalize the sPLA2-X to intracellular structures. An inhibitor of sPLA2-X (ROC-0929) that does not inhibit other mammalian sPLA2s, as well as inhibitors of the mitogen-activated kinase cascade (MAPK) and cPLA2α, was used to examine the mechanism of N-formyl-methionyl-leucyl-phenylalanine (fMLP)-mediated formation of CysLT. Eosinophils express the mammalian sPLA2-X gene (PLA2G10). The sPLA2-X protein is located in the endoplasmic reticulum, golgi, and granules of eosinophils and moves to the granules and lipid bodies during fMLP-mediated activation. Selective sPLA2-X inhibition attenuated the fMLP-mediated release of arachidonic acid and CysLT formation by eosinophils. Inhibitors of p38, extracellular-signal-regulated kinases 1/2 (p44/42 MAPK), c-Jun N-terminal kinase, and cPLA2α also attenuated the fMLP-mediated formation of CysLT. The sPLA2-X inhibitor reduced the phosphorylation of p38 and extracellular-signal-regulated kinases 1/2 (p44/42 MAPK) as well as cPLA2α during cellular activation, indicating that sPLA2-X is involved in activating the MAPK cascade leading to the formation of CysLT via cPLA2α. We further demonstrate that sPLA2-X is activated before secretion from the cell during activation. Short-term priming with IL-13 and TNF/IL-1β increased the

Uncoupled regulation of fibronectin and collagen synthesis in Rous sarcoma virus transformed avian tendon cells

International Nuclear Information System (INIS)

Parry, G.; Soo, W.J.; Bissell, M.J.

1979-01-01

The regulation of fibronectin and procollagen synthesis has been investigated in normal and Rous sarcoma virus transformed primary avian tendon cells. These two proteins interact at the cell periphery and both are reportedly lost upon transformation. Whether their synthesis was coordinately regulated in Rous sarcoma virus-infected cells was thus examined. It was found that while the synthesis of both pro α 1 and pro α 2 peptides was reduced upon transformation, the synthesis of fibronectin was not altered. Nevertheless, long term radiolabeling demonstrated that fibronectin levels were reduced in transformed cells. It is concluded that the reduction in levels of these components at the surface is brought about by different mechanisms; collagen levels being regulated by procollagen synthesis and fibronectin levels by degradation and/or release into the culture medium. The possibility is discussed that fibronectin is lost from the cell periphery of primary avian tendon cells as a consequence of decreased levels of anchoring collagen molecules

Protein targeting to glycogen is a master regulator of glycogen synthesis in astrocytes

OpenAIRE

E. Ruchti; P.J. Roach; A.A. DePaoli-Roach; P.J. Magistretti; I. Allaman

2016-01-01

The storage and use of glycogen, the main energy reserve in the brain, is a metabolic feature of astrocytes. Glycogen synthesis is regulated by Protein Targeting to Glycogen (PTG), a member of specific glycogen-binding subunits of protein phosphatase-1 (PPP1). It positively regulates glycogen synthesis through de-phosphorylation of both glycogen synthase (activation) and glycogen phosphorylase (inactivation). In cultured astrocytes, PTG mRNA levels were previously shown to be enhanced by the ...

A SARS-coronovirus 3CL protease inhibitor isolated from the marine sponge Axinella cf. corrugata: structure elucidation and synthesis

International Nuclear Information System (INIS)

Lira, Simone P. de; Seleghim, Mirna H.R.; Berlinck, Roberto G.S.

2007-01-01

Two coumarin derivatives, esculetin-4-carboxylic acid methyl ester (1) and esculetin-4- carboxylic acid ethyl ester (2), have been isolated from the marine sponge Axinella cf. corrugata. Structure determination included analysis of spectroscopic data and total synthesis of compound 2. These are the first coumarin derivatives isolated from a marine sponge. The ethyl ester 2 was found to be an in vitro inhibitor of SARS 3CL-protease and an effective inhibitor of SARS-CoV replication in Vero cells at non-cytotoxic concentrations. (author)

Potential inhibitors of dapE and argE enzymes as the new antimicrobial agents: Synthesis and characterization

Czech Academy of Sciences Publication Activity Database

Hlaváček, Jan; Pícha, Jan; Vaněk, Václav; Jiráček, Jiří; Slaninová, Jiřina; Fučík, Vladimír; Holz, R. C.

2008-01-01

Roč. 14, č. 8 (2008), s. 83-84 ISSN 1075-2617. [European Peptide Symposium /30./. 31.08.2008-05.09.2008, Helsinki] R&D Projects: GA AV ČR IAA400550614 Institutional research plan: CEZ:AV0Z40550506 Keywords : antimicrobial agents * dapE and argE inhibitors * synthesis and activity Subject RIV: CC - Organic Chemistry

Synthesis, Pharmacological Profile and Docking Studies of New Sulfonamides Designed as Phosphodiesterase-4 Inhibitors.

Directory of Open Access Journals (Sweden)

Isabelle Karine da Costa Nunes

Full Text Available Prior investigations showed that increased levels of cyclic AMP down-regulate lung inflammatory changes, stimulating the interest in phosphodiesterase (PDE4 as therapeutic target. Here, we described the synthesis, pharmacological profile and docking properties of a novel sulfonamide series (5 and 6a-k designed as PDE4 inhibitors. Compounds were screened for their selectivity against the four isoforms of human PDE4 using an IMAP fluorescence polarized protocol. The effect on allergen- or LPS-induced lung inflammation and airway hyper-reactivity (AHR was studied in A/J mice, while the xylazine/ketamine-induced anesthesia test was employed as a behavioral correlate of emesis in rodents. As compared to rolipram, the most promising screened compound, 6a (LASSBio-448 presented a better inhibitory index concerning PDE4D/PDE4A or PDE4D/PDE4B. Accordingly, docking analyses of the putative interactions of LASSBio-448 revealed similar poses in the active site of PDE4A and PDE4C, but slight unlike orientations in PDE4B and PDE4D. LASSBio-448 (100 mg/kg, oral, 1 h before provocation, inhibited allergen-induced eosinophil accumulation in BAL fluid and lung tissue samples. Under an interventional approach, LASSBio-448 reversed ongoing lung eosinophilic infiltration, mucus exacerbation, peribronchiolar fibrosis and AHR by allergen provocation, in a mechanism clearly associated with blockade of pro-inflammatory mediators such as IL-4, IL-5, IL-13 and eotaxin-2. LASSBio-448 (2.5 and 10 mg/kg also prevented inflammation and AHR induced by LPS. Finally, the sulfonamide derivative was shown to be less pro-emetic than rolipram and cilomilast in the assay employed. These findings suggest that LASSBio-448 is a new PDE4 inhibitor with marked potential to prevent and reverse pivotal pathological features of diseases characterized by lung inflammation, such as asthma.

Radioprotection of intestinal crypt cells by cox-inhibitors

International Nuclear Information System (INIS)

Bisnar, Paul O.; Dones, Rosa Angela S.A.; Serna, Paulene-Ver A.; Deocaris, Chester C.; Guttierez, Kalangitan V.; Deocaris, Custer C.

2006-01-01

The regulation of tissue homeostasis in the gastrointestinal epithelium after epithelial injury focuses on the prostaglandins(PGs) as its major mediators. The two cyclooxygenase isoforms, cox-1 and cox-2, catalyze synthesis of PGs. Cox-1 is the predominant cyclooxygenase isoform found in the normal intestine. In contrast, cox-2 is present at low levels in normal intestine but is elevated at sites of inflammation, and in adenomas and carcinomas. To study the effects of various commercially-available cox-inhibitors (Ketorolac: cox-1 selective; Celecoxib: cox-2 selective; and Indocid: cox-1/2 non-selective), we determine mouse crypt epithelial cell fate after genotoxic injury with whole-body gamma-ray exposure at 15 Gy. Intestinal tissues of mice treated with cox-2 inhibitors that showed invariable apoptotic event, however, have increased occurrence of regenerating cells. Our results suggest a potential application of cox-2 selective inhibitors as radioprotective agent for normal cells after radiotherapy. (Author)

Nitric oxide, prostaglandins and angiotensin II in the regulation of renal medullary blood flow during volume expansion.

Science.gov (United States)

Moreno, Carol; Llinás, María T; Rodriguez, Francisca; Moreno, Juan M; Salazar, F Javier

2016-03-01

Regulation of medullary blood flow (MBF) is essential in maintaining renal function and blood pressure. However, it is unknown whether outer MBF (OMBF) and papillary blood flow (PBF) are regulated independently when extracellular volume (ECV) is enhanced. The aim of this study was to determine whether OMBF and PBF are differently regulated and whether there is an interaction between nitric oxide (NO), prostaglandins (PGs) and angiotensin II (Ang II) in regulating OMBF and PBF when ECV is enhanced. To achieve these goals, OMBF and PBF were measured by laser-Doppler in volume-expanded rats treated with a cyclooxygenase inhibitor (meclofenamate, 3 mg/kg) and/or a NO synthesis inhibitor (L-nitro-arginine methyl ester (L-NAME), 3 μg/kg/min) and/or Ang II (10 ng/kg/min). OMBF was unchanged by NO or PGs synthesis inhibition but decreased by 36 % (P blood flows to the outer medulla and renal papilla are differently regulated and showing that there is a complex interaction between NO, PGs and Ang II in regulating OMBF and PBF when ECV is enhanced.

Synthesis and characterization of a novel organic corrosion inhibitor for mild steel in 1 M hydrochloric acid

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Mohammed H. Othman Ahmed

2018-03-01

Full Text Available The synthesis and characterization of a novel organic corrosion inhibitor (4-(3-mercapto-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-b][1,2,4,5]tetrazin-6-ylphenol, for mild steel in 1 M hydrochloric acid (HCl has been successfully reported for the first time. The inhibitor evaluated as corrosion inhibitor for mild steel in 1 M of Hydrochloric acid solution using electrochemical impedance spectroscopy (EIS, and electrochemical frequency modulation (EFM measurement techniques. Changes in the impedance parameters suggested an adsorption of the inhibitor onto the mild steel surface, leading to the formation of protective films. The results show that the inhibition efficiencies increased with increasing the concentrations of the inhibitors and decreased with increasing temperature. The maximum inhibition efficiency up to 67% at the maximum concentration 0.5 mM. This shows that those inhibitors are effective in helping to reduce and slowing down the corrosion process that occurs to mild steel with a hydrochloric acid solution by providing an organic inhibitor for the mild steel that can be weakened by increasing the temperature. The adsorption process of the synthesized organic inhibitor depends on its electronic characteristics in addition to steric effects and the nature of metal surface, temperature degree and the varying degrees of surface-site activity. The synthesized inhibitor molecules were absorbed by metal surface and follow Langmuir isotherms. Keywords: Corrosion, Inhibitor, Mild steel, EIS spectroscopy

Effects of indomethacin, NS-398 (a selective prostaglandin H synthase-2 inhibitor) and protein synthesis inhibitors on prostaglandin production by the guinea-pig placenta.

Science.gov (United States)

Aitken, H; Poyser, N L

2001-01-01

The outputs of PGF(2 alpha), PGE2 and 6-keto-PGF(1 alpha)were similar from the day 22 guinea-pig placenta and sub-placenta in culture, except for PGE2 output from the sub-placenta which was lower. Between days 22 and 29 of pregnancy, the outputs of PGF(2 alpha), PGE2 and 6-keto-PGF(1 alpha)during the initial 2 h culture period increased 6.9-, 1.1- and 3.2-fold, respectively, from the placenta, and 2.1-, 1.4- and 2.2-fold, respectively, from the sub-placenta. Therefore, there was a relatively specific increase in PGF(2 alpha)production by the guinea-pig placenta between days 22 and 29 of pregnancy. The output of PGFM from the cultured placenta also increased between days 22 and 29, indicating that the increase in PGF(2 alpha)output was due to increased synthesis rather than to decreased metabolism. By comparing the amounts of prostaglandins produced by tissue homogenates during a 1 h incubation period, it appears that there is approximately a 2-fold increase in the amount of prostaglandin H synthase (PGHS) present in the guinea-pig placenta between days 22 and 29. NS-398 (a specific inhibitor of PGHS-2) and indomethacin (an inhibitor of both PGHS-1 and PGHS-2) both inhibited prostaglandin production by homogenates of day 22 and day 29 placenta. Indomethacin was more effective than NS-398, except for their actions on PGF(2 alpha)production by the day 29 placenta where indomethacin and NS-398 were equiactive. Indomethacin and NS-398 were both very effective at inhibiting the outputs of PGF(2 alpha), PGE2 and 6-keto-PGF(1 alpha)from the day 22 and day 29 placenta and sub-placenta in culture, indicating that prostaglandin production by the guinea-pig placenta and sub-placenta in culture is largely dependent upon the activity of PGHS-2. The high production of PGF(2 alpha)by the day 29 placenta is not dependent on the continual synthesis of fresh protein(s), as inhibitors of protein synthesis did not reduce PGF(2 alpha)output from the day 29 guinea-pig placenta in culture

Adverse Effects of COX-2 Inhibitors

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Jagdish N. Sharma

2005-01-01

Full Text Available Cyclooxygenase-2 selective inhibitors (COXIBs were developed with the prime object of minimizing gastrointestinal adverse effects, which are seen with the use of traditional nonsteroidal anti-inflammatory drugs (NSAIDs. Their long-term use is limited by the development of hypertension, edema, and congestive heart failure in a significant proportion of patients. NSAIDs block the activity of both COX isozymes, COX-1 and COX-2, which mediate the enzymatic conversion of arachidonate to prostaglandin H2 (PGH2 and other prostaglandin (PG metabolites. It is well established that the cardiovascular profile of COX-2 inhibitors can be accounted for by inhibition of COX-dependent PG synthesis. Following the COX-mediated synthesis of PGH2 from arachidonate, PGH2 is metabolized to one of at least five bioactive PGs, including PGE2, PGI2, PGF2, PGD2, or thromboxane A2 (TXA2. These prostanoids have pleiotropic cardiovascular effects, altering platelet function and renal function, and they are acting either as vasodilators or vasoconstrictors. Although COX-1 and COX-2 exhibit similar biochemical activity in converting arachidonate to PGH2in vitro, the ultimate prostanoids they produce in vivo may be different due to differential regulation of COX-1 and COX-2, tissue distribution, and availability of the prostanoid synthases. PGs have been established as being critically involved in mitigating hypertension, helping to maintain medullary blood flow (MBF, promoting urinary salt excretion, and preserving the normal homeostasis of thrombosis, and the researchers found that the use of COX-2 inhibitors caused many serious complications in altering the normal body homeostasis. The purpose of the present research is to explain briefly the side effects of COX-2 inhibitors on the renal and cardiovascular system.

Histone deacetylase inhibitors up-regulate LL-37 expression independent of toll-like receptor mediated signalling in airway epithelial cells.

Science.gov (United States)

Liu, Quan; Liu, Juan; Roschmann, Kristina Irene Lisolette; van Egmond, Danielle; Golebski, Korneliusz; Fokkens, Wytske Johanna; Wang, Dehui; van Drunen, Cornelis Maria

2013-04-11

HDAC inhibitors have been proposed as anticancer agents. However, their roles in innate genes expression remain not well known. Cathelicidin LL-37 is one of the few human bactericidal peptides, but the regulation of histone acetylation on LL-37 expression in airway epithelium remains largely unknown. Therefore, we investigated the effects of two non-selective HDACi, trichostatin A (TSA) and sodium butyrate (SB), on the expression of the cathelicidin LL-37 in human airway epithelial cells. LL37 in human NCI-H292 airway epithelial cells and the primary cultures of normal nasal epithelial cells(PNEC) in response to HDAC inhibitors with or without poly (I:C) stimulation was assessed using real-time PCR and western blot. In parallel, IL-6 expression was evaluated by ELISA. Our results showed that HDAC inhibitors up-regulated LL-37 gene expression independent of poly (I:C) stimulation in PNEC as well as in NCI-H292 cells. HDAC inhibitors increased LL37 protein expression in NCI-H292 cells but not in PNEC. In addition, HDAC inhibitors significantly inhibited poly (I:C)-induced IL-6 production in both of the epithelial cells. In conclusion, HDAC inhibitors directly up-regulated LL-37 gene expression in human airway epithelial cells.

Different effects of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors on sterol synthesis in various human cell types

NARCIS (Netherlands)

Vliet, A.K. van; Thiel, G.C.F. van; Huisman, R.H.; Moshage, H.; Yap, S.H.; Cohen, L.H.

1995-01-01

The three vastatins examined, lovastatin, simvastatin and pravastatin, are equally strong inhibitors of the sterol synthesis in human hepatocytes in culture with IC50-values of 4.1, 8.0 and 2.0 nM, respectively. However, in the human extrahepatic cells: umbilical vascular endothelial cells, retinal

Chiral gold(I vs chiral silver complexes as catalysts for the enantioselective synthesis of the second generation GSK-hepatitis C virus inhibitor

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María Martín-Rodríguez

2011-07-01

Full Text Available The synthesis of a GSK 2nd generation inhibitor of the hepatitis C virus, by enantioselective 1,3-dipolar cycloaddition between a leucine derived iminoester and tert-butyl acrylate, was studied. The comparison between silver(I and gold(I catalysts in this reaction was established by working with chiral phosphoramidites or with chiral BINAP. The best reaction conditions were used for the total synthesis of the hepatitis C virus inhibitor by a four step procedure affording this product in 99% ee and in 63% overall yield. The origin of the enantioselectivity of the chiral gold(I catalyst was justified according to DFT calculations, the stabilizing coulombic interaction between the nitrogen atom of the thiazole moiety and one of the gold atoms being crucial.

Safe taste memory consolidation is disrupted by a protein synthesis inhibitor in the nucleus accumbens shell.

Science.gov (United States)

Pedroza-Llinás, R; Ramírez-Lugo, L; Guzmán-Ramos, K; Zavala-Vega, S; Bermúdez-Rattoni, F

2009-07-01

Consolidation is the process by which a new memory is stabilized over time, and is dependent on de novo protein synthesis. A useful model for studying memory formation is gustatory memory, a type of memory in which a novel taste may become either safe by not being followed by negative consequences (attenuation of neophobia, AN), or aversive by being followed by post-digestive malaise (conditioned taste aversion, CTA). Here we evaluated the effects of the administration of a protein synthesis inhibitor in the nucleus accumbens (NAc) shell for either safe or aversive taste memory trace consolidation. To test the effects on CTA and AN of protein synthesis inhibition, anisomycin (100microg/microl) was bilaterally infused into the NAc shell of Wistar rats' brains. We found that post-trial protein synthesis blockade impaired the long-term safe taste memory. However, protein synthesis inhibition failed to disrupt the long-term memory of CTA. In addition, we infused anisomycin in the NAc shell after the pre-exposure to saccharin in a latent inhibition of aversive taste. We found that the protein synthesis inhibition impaired the consolidation of safe taste memory, allowing the aversive taste memory to form and consolidate. Our results suggest that protein synthesis is required in the NAc shell for consolidation of safe but not aversive taste memories, supporting the notion that consolidation of taste memory is processed in several brain regions in parallel, and implying that inhibitory interactions between both taste memory traces do occur.

Genetic regulation of phenazine-1-carboxamide synthesis by Pseudomonas chlororaphis strain PCL1391

NARCIS (Netherlands)

Girard, Genevieve

2006-01-01

A general overview of regulation of secondary metabolism in Pseudomonas species is given in Chapter 1. Several approaches were combined to identify novel genes involved in the regulation of PCN synthesis and to study their interactions with other regulators. Site-directed mutagenesis was used to

Inhibitors of apoptosis (IAPs) regulate intestinal immunity and inflammatory bowel disease (IBD) inflammation

DEFF Research Database (Denmark)

Pedersen, Jannie; LaCasse, Eric C; Seidelin, Jakob B

2014-01-01

The inhibitor of apoptosis (IAP) family members, notably cIAP1, cIAP2, and XIAP, are critical and universal regulators of tumor necrosis factor (TNF) mediated survival, inflammatory, and death signaling pathways. Furthermore, IAPs mediate the signaling of nucleotide-binding oligomerization domain...

Lewis lung carcinoma regulation of mechanical stretch-induced protein synthesis in cultured myotubes.

Science.gov (United States)

Gao, Song; Carson, James A

2016-01-01

Mechanical stretch can activate muscle and myotube protein synthesis through mammalian target of rapamycin complex 1 (mTORC1) signaling. While it has been established that tumor-derived cachectic factors can induce myotube wasting, the effect of this catabolic environment on myotube mechanical signaling has not been determined. We investigated whether media containing cachectic factors derived from Lewis lung carcinoma (LLC) can regulate the stretch induction of myotube protein synthesis. C2C12 myotubes preincubated in control or LLC-derived media were chronically stretched. Protein synthesis regulation by anabolic and catabolic signaling was then examined. In the control condition, stretch increased mTORC1 activity and protein synthesis. The LLC treatment decreased basal mTORC1 activity and protein synthesis and attenuated the stretch induction of protein synthesis. LLC media increased STAT3 and AMP-activated protein kinase phosphorylation in myotubes, independent of stretch. Both stretch and LLC independently increased ERK1/2, p38, and NF-κB phosphorylation. In LLC-treated myotubes, the inhibition of ERK1/2 and p38 rescued the stretch induction of protein synthesis. Interestingly, either leukemia inhibitory factor or glycoprotein 130 antibody administration caused further inhibition of mTORC1 signaling and protein synthesis in stretched myotubes. AMP-activated protein kinase inhibition increased basal mTORC1 signaling activity and protein synthesis in LLC-treated myotubes, but did not restore the stretch induction of protein synthesis. These results demonstrate that LLC-derived cachectic factors can dissociate stretch-induced signaling from protein synthesis through ERK1/2 and p38 signaling, and that glycoprotein 130 signaling is associated with the basal stretch response in myotubes. Copyright © 2016 the American Physiological Society.

Discovery of pyrazolo[1,5-a]pyrimidine-based CHK1 inhibitors: A template-based approach-Part 2

Energy Technology Data Exchange (ETDEWEB)

Labroli, Marc; Paruch, Kamil; Dwyer, Michael P.; Alvarez, Carmen; Keertikar, Kartik; Poker, Cory; Rossman, Randall; Duca, Jose S.; Fischmann, Thierry O.; Madison, Vincent; Parry, David; Davis, Nicole; Seghezzi, Wolfgang; Wiswell, Derek; Guzi, Timothy J. [Merck

2013-11-20

Previous efforts by our group have established pyrazolo[1,5-a]pyrimidine as a viable core for the development of potent and selective CDK inhibitors. As part of an effort to utilize the pyrazolo[1,5-a]pyrimidine core as a template for the design and synthesis of potent and selective kinase inhibitors, we focused on a key regulator in the cell cycle progression, CHK1. Continued SAR development of the pyrazolo[1,5-a]pyrimidine core at the C5 and C6 positions, in conjunction with previously disclosed SAR at the C3 and C7 positions, led to the discovery of potent and selective CHK1 inhibitors.

DMPD: Regulation of nitric oxide synthesis and apoptosis by arginase and argininerecycling. [Dynamic Macrophage Pathway CSML Database

Lifescience Database Archive (English)

Full Text Available 17513437 Regulation of nitric oxide synthesis and apoptosis by arginase and arginin...tion of nitric oxide synthesis and apoptosis by arginase and argininerecycling. A...erecycling. Mori M. J Nutr. 2007 Jun;137(6 Suppl 2):1616S-1620S. (.png) (.svg) (.html) (.csml) Show Regulation of nitric oxide synthe...sis and apoptosis by arginase and argininerecycling. PubmedID 17513437 Title Regula

Regulation of hemopoiesis: inhibitors and stimulators produced by a murine bone marrow stromal cell line (H-1)

International Nuclear Information System (INIS)

Cronkite, E.P.; Miller, M.E.; Garnett, H.; Harigaya, K.

1982-01-01

A murine cell line (H-1) probably derived from the adventitial reticular cell has been isolated and preserved. This line produces: (1) CSF; (2) a labile inhibitor of CFU-c proliferation with preferential action on granulopoiesis; (3) PGE; (4) proliferation inhibitors of BFU-E and GEMM; and (5) suppression of entry of CFU-S into DNA synthesis in vitro. It is postulated that the adventitial reticular cell (ARC) may play a major regulatory role in hemopoiesis through intramedullary production of the factors described. The nature of the signals that activate the genes in the ARC which are coded for the factors described is not known

Design, Synthesis and Evaluation of Ribose-modified Anilinopyrimidine Derivatives as EGFR Tyrosine Kinase Inhibitors

Science.gov (United States)

Hu, Xiuqin; Wang, Disha; Tong, Yi; Tong, Linjiang; Wang, Xia; Zhu, Lili; Xie, Hua; Li, Shiliang; Yang, You; Xu, Yufang

2017-11-01

The synthesis of a series of ribose-modified anilinopyrimidine derivatives was efficiently achieved by utilizing DBU or tBuOLi-promoted coupling of ribosyl alcohols with 2,4,5-trichloropyrimidine as key step. Preliminary biological evaluation of this type of compounds as new EGFR tyrosine kinase inhibitors for combating EGFR L858R/T790M mutant associated with drug resistance in the treatment of non-small cell lung cancer revealed that 3-N-acryloyl-5-O-anilinopyrimidine ribose derivative 1a possessed potent and specific inhibitory activity against EGFR L858R/T790M over WT EGFR. Based upon molecular docking studies of the binding mode between compound 1a and EGFR, the distance between the Michael receptor and the pyrimidine scaffold is considered as an important factor for the inhibitory potency and future design of selective EGFR tyrosine kinase inhibitors against EGFR L858R/T790M mutants.

Manipulating chromosome structure and metaphase status with ultraviolet light and repair synthesis inhibitors

Energy Technology Data Exchange (ETDEWEB)

Mullinger, A.M.; Johnson, R.T. (Cambridge Univ. (UK). Dept. of Zoology)

1985-02-01

DNA repair occurs in metaphase-arrested cells in response to ultraviolet irradiation. In the presence of the repair synthesis inhibitors, hydroxyurea and 1-..beta..-D-arabinofuranosylcytosine, the chromosomes of such cells are decondensed. The dose response of chromosome decondensation varies between different cell types. In human cells defective in excision repair there is much less chromosome decondensation in response to the same ultraviolet dose and time of repair inhibition. However, a simian virus 40-transformed muntjac cell displays pronounced chromosome decondensation but has limited incision ability. Both chromosome decondensation and single-strand break accumulation in the presence of inhibitors are reversed when DNA precursors are provided, but reversal after higher ultraviolet doses and longer period of incubation leads to recondensed chromosomes that are fragmented. Although the chromosomes of repair-inhibited metaphase cells are decondensed in fixed preparations, their morphology appears normal in intact cells. The cells also retain a capacity to induce prematurely condensed chromosomes (PCC) when fused with interphase cells: compared with control mitotic cells, the speed of induction is sometimes reduced but the final amount of PCC produced is similar.

Synthesis and Application of Phenyl Nitrone Derivatives as Acidic and Microbial Corrosion Inhibitors

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Shijun Chen

2015-01-01

Full Text Available Nitrone has drawn great attention due to its wide applications as a 1,3-dipole in heterocyclic compounds synthesis and the bioactivities. With the special structure, nitrone can also be used as ligand in inorganic chemistry. Based on the current research, the nitrones are anticipated to be effective inhibitors against acidic and microbial corrosion. The aim of this work is to investigate the inhibitory action of nitrones. In this work, a series of phenyl nitrone derivatives (PN was synthesized and used as acidic and microbial corrosion inhibitors. The results indicate that several compounds show moderate to high inhibition efficiency (IE in 3% HCl. Accompanied with HMTA or BOZ, the IEs greatly increase, and the highest efficiency of 98.5% was obtained by using PN4 + BOZ. Investigation of the antibacterial activity against oilfield microorganism shows that the nitrone derivatives can inhibit SRB, IB, and TGB with moderate to high efficiency under 1,000 mg/L, which makes them potential to be used as bifunctional oilfield chemicals.

Human keratinocytes produce the complement inhibitor factor H: synthesis is regulated by interferon-gamma

NARCIS (Netherlands)

Timár, Krisztina K.; Pasch, Marcel C.; van den Bosch, Norbert H. A.; Jarva, Hanna; Junnikkala, Sami; Meri, Seppo; Bos, Jan D.; Asghar, Syed S.

2006-01-01

Locally synthesized complement is believed to play an important role in host defense and inflammation at organ level. In the epidermis, keratinocytes have so far been shown to synthesize two complement components, C3 and factor B. Here, we studied the synthesis of factor H by human keratinocytes. We

Synthesis and characterisation of 5-acyl-6,7-dihydrothieno[3,2-c]pyridine inhibitors of Hedgehog acyltransferase

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Thomas Lanyon-Hogg

2016-06-01

Full Text Available In this data article we describe synthetic and characterisation data for four members of the 5-acyl-6,7-dihydrothieno[3,2-c]pyridine (termed “RU-SKI” class of inhibitors of Hedgehog acyltransferase, including associated NMR spectra for final compounds. RU-SKI compounds were selected for synthesis based on their published high potencies against the enzyme target. RU-SKI 41 (9a, RU-SKI 43 (9b, RU-SKI 101 (9c, and RU-SKI 201 (9d were profiled for activity in the related article “Click chemistry armed enzyme linked immunosorbent assay to measure palmitoylation by Hedgehog acyltransferase” (Lanyon-Hogg et al., 2015 [1]. 1H NMR spectral data indicate different amide conformational ratios between the RU-SKI inhibitors, as has been observed in other 5-acyl-6,7-dihydrothieno[3,2-c]pyridines. The synthetic and characterisation data supplied in the current article provide validated access to the class of RU-SKI inhibitors.

Plasminogen Activator Inhibitor-1 Controls Vascular Integrity by Regulating VE-Cadherin Trafficking.

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Anna E Daniel

Full Text Available Plasminogen activator inhibitor-1 (PAI-1, a serine protease inhibitor, is expressed and secreted by endothelial cells. Patients with PAI-1 deficiency show a mild to moderate bleeding diathesis, which has been exclusively ascribed to the function of PAI-1 in down-regulating fibrinolysis. We tested the hypothesis that PAI-1 function plays a direct role in controlling vascular integrity and permeability by keeping endothelial cell-cell junctions intact.We utilized PAI-039, a specific small molecule inhibitor of PAI-1, to investigate the role of PAI-1 in protecting endothelial integrity. In vivo inhibition of PAI-1 resulted in vascular leakage from intersegmental vessels and in the hindbrain of zebrafish embryos. In addition PAI-1 inhibition in human umbilical vein endothelial cell (HUVEC monolayers leads to a marked decrease of transendothelial resistance and disrupted endothelial junctions. The total level of the endothelial junction regulator VE-cadherin was reduced, whereas surface VE-cadherin expression was unaltered. Moreover, PAI-1 inhibition reduced the shedding of VE-cadherin. Finally, we detected an accumulation of VE-cadherin at the Golgi apparatus.Our findings indicate that PAI-1 function is important for the maintenance of endothelial monolayer and vascular integrity by controlling VE-cadherin trafficking to and from the plasma membrane. Our data further suggest that therapies using PAI-1 antagonists like PAI-039 ought to be used with caution to avoid disruption of the vessel wall.

The Design, Synthesis and Structure-Activity Relationship of Mixed Serotonin, Norepinephrine and Dopamine Uptake Inhibitors

Science.gov (United States)

Chen, Zhengming; Yang, Ji; Skolnick, Phil

The evolution of antidepressants over the past four decades has involved the replacement of drugs with a multiplicity of effects (e.g., TCAs) by those with selective actions (i.e., SSRIs). This strategy was employed to reduce the adverse effects of TCAs, largely by eliminating interactions with certain neurotransmitters or receptors. Although these more selective compounds may be better tolerated by patients, selective drugs, specifically SSRIs, are not superior to older drugs in treating depressed patients as measured by response and remission rates. It may be an advantage to increase synaptic levels of both serotonin and norepinephrine, as in the case of dual uptake inhibitors like duloxetine and venlafaxine. An important recent development has been the emergence of the triple-uptake inhibitors (TUIs/SNDRIs), which inhibit the uptake of the three neurotransmitters most closely linked to depression: serotonin, norepinephrine, and dopamine. Preclinical studies and clinical trials indicate that a drug inhibiting the reuptake of all three of these neurotransmitters could produce more rapid onset of action and greater efficacy than traditional antidepressants. This review will detail the medicinal chemistry involved in the design, synthesis and discovery of mixed serotonin, norepinephrine and dopamine transporter uptake inhibitors.

Type 1 plaminogen activator inhibitor gene: Functional analysis and glucocorticoid regulation of its promoter

International Nuclear Information System (INIS)

Van Zonneveld, A.J.; Curriden, S.A.; Loskutoff, D.J.

1988-01-01

Plasminogen activator inhibitor type 1 is an important component of the fibrinolytic system and its biosynthesis is subject to complex regulation. To study this regulation at the level of transcription, the authors have identified and sequenced the promoter of the human plasminogen activator inhibitor type 1 gene. Nuclease protection experiments were performed by using endothelial cell mRNA and the transcription initiation (cap) site was established. Sequence analysis of the 5' flanking region of the gene revealed a perfect TATA box at position -28 to position -23, the conserved distance from the cap site. Comparative functional studies with the firefly luciferase gene as a reporter gene showed that fragments derived from this 5' flanking region exhibited high promoter activity when transfected into bovine aortic endothelial cells and mouse Ltk - fibroblasts but were inactive when introduced into HeLa cells. These studies indicate that the fragments contain the plasminogen activator inhibitor type 1 promoter and that it is expressed in a tissue-specific manner. Although the fragments were also silent in rat FTO2B hepatoma cells, their promoter activity could be induced up to 40-fold with the synthetic glucocorticoid dexamethasone. Promoter deletion mapping experiments and studies involving the fusion of promoter fragments to a heterologous gene indicated that dexamethasone induction is mediated by a glucocorticoid responsive element with enhancer-like properties located within the region between nucleotides -305 and +75 of the plasminogen activator inhibitor type 1 gene

Laminin peptide YIGSR induces collagen synthesis in Hs27 human dermal fibroblasts

International Nuclear Information System (INIS)

Yoon, Jong Hyuk; Kim, Jaeyoon; Lee, Hyeongjoo; Kim, So Young; Jang, Hwan-Hee; Ryu, Sung Ho; Kim, Beom Joon; Lee, Taehoon G.

2012-01-01

Highlights: ► We identify a function of the YIGSR peptide to enhance collagen synthesis in Hs27. ► YIGSR peptide enhanced collagen type 1 synthesis both of gene and protein levels. ► There were no changes in cell proliferation and MMP-1 level in YIGSR treatment. ► The YIGSR effect on collagen synthesis mediated activation of FAK, pyk2 and ERK. ► The YIGSR-induced FAK and ERK activation was modulated by FAK and MEK inhibitors. -- Abstract: The dermal ECM is synthesized from fibroblasts and is primarily compromised of fibrillar collagen and elastic fibers, which support the mechanical strength and resiliency of skin, respectively. Laminin, a major glycoprotein located in the basement membrane, promotes cell adhesion, cell growth, differentiation, and migration. The laminin tyrosine-isoleucine-glycine-serine-arginine (YIGSR) peptide, corresponding to the 929–933 sequence of the β1 chain, is known to be a functional motif with effects on the inhibition of tumor metastasis, the regulation of sensory axonal response and the inhibition of angiogenesis through high affinity to the 67 kDa laminin receptor. In this study, we identified a novel function of the YIGSR peptide to enhance collagen synthesis in human dermal fibroblasts. To elucidate this novel function regarding collagen synthesis, we treated human dermal fibroblasts with YIGSR peptide in both a time- and dose-dependent manner. According to subsequent experiments, we found that the YIGSR peptide strongly enhanced collagen type 1 synthesis without changing cell proliferation or cellular MMP-1 level. This YIGSR peptide-mediated collagen type 1 synthesis was modulated by FAK inhibitor and MEK inhibitor. This study clearly reveals that YIGSR peptide plays a novel function on the collagen type 1 synthesis of dermal fibroblasts and also suggests that YIGSR is a strong candidate peptide for the treatment of skin aging and wrinkles.

Radiation metagenesis and inhibition of DNA synthesis

International Nuclear Information System (INIS)

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

1983-01-01

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

Regulation and inhibition of collagenase expression by long-wavelength ultraviolet radiation in cultured human skin fibroblasts

International Nuclear Information System (INIS)

Petersen, Marta; Hamilton, Tiffani; Haili Li

1995-01-01

The cellular mechanisms responsible for the connective tissue changes produced by chronic exposure to UV light are poorly understood. collagenase, a metalloproteinase, initiates degradation of types I and III collagen and thus plays a key role in the remodeling of dermal collagen. Collagenase synthesis by fibroblasts and keratinocytes involves the protein kinase C (PKC) second messenger system, and corticosteroids have been shown to suppress its synthesis at the level of gene transcription. Long-wavelength UV light (UVA, 320-400 nm) stimulates the synthesis of interstitial collagenase, as well as increasing PKC activity, in human skin fibroblasts in vitro. This study explores the regulation of collagenase expression by UVA in cultured human skin fibroblasts. Specifically, the time course, the effect of actinomycin D, an inhibitor of RNA synthesis, as well as the effect of PKC inhibitors and dexamethansone on expression of collagenase following UVA irradiation were examined. (Author)

Pollination in Nicotiana alata stimulates synthesis and transfer to the stigmatic surface of NaStEP, a vacuolar Kunitz proteinase inhibitor homologue.

Science.gov (United States)

Busot, Grethel Yanet; McClure, Bruce; Ibarra-Sánchez, Claudia Patricia; Jiménez-Durán, Karina; Vázquez-Santana, Sonia; Cruz-García, Felipe

2008-01-01

After landing on a wet stigma, pollen grains hydrate and germination generally occurs. However, there is no certainty of the pollen tube growth through the style to reach the ovary. The pistil is a gatekeeper that evolved in many species to recognize and reject the self-pollen, avoiding endogamy and encouraging cross-pollination. However, recognition is a complex process, and specific factors are needed. Here the isolation and characterization of a stigma-specific protein from N. alata, NaStEP (N. alata Stigma Expressed Protein), that is homologous to Kunitz-type proteinase inhibitors, are reported. Activity gel assays showed that NaStEP is not a functional serine proteinase inhibitor. Immunohistochemical and protein blot analyses revealed that NaStEP is detectable in stigmas of self-incompatible (SI) species N. alata, N. forgetiana, and N. bonariensis, but not in self-compatible (SC) species N. tabacum, N. plumbaginifolia, N. benthamiana, N. longiflora, and N. glauca. NaStEP contains the vacuolar targeting sequence NPIVL, and immunocytochemistry experiments showed vacuolar localization in unpollinated stigmas. After self-pollination or pollination with pollen from the SC species N. tabacum or N. plumbaginifolia, NaStEP was also found in the stigmatic exudate. The synthesis and presence in the stigmatic exudate of this protein was strongly induced in N. alata following incompatible pollination with N. tabacum pollen. The transfer of NaStEP to the stigmatic exudate was accompanied by perforation of the stigmatic cell wall, which appeared to release the vacuolar contents to the apoplastic space. The increase in NaStEP synthesis after pollination and its presence in the stigmatic exudates suggest that this protein may play a role in the early pollen-stigma interactions that regulate pollen tube growth in Nicotiana.

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

Science.gov (United States)

Igarashi, K; Morris, D R

1984-11-01

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

Three pectin methylesterase inhibitors protect cell wall integrity for arabidopsis immunity to Botrytis

DEFF Research Database (Denmark)

Lionetti, Vincenzo; Fabri, Eleonora; Caroli, Monica De

2017-01-01

to the control plants. A higher stimulation of the fungal oxalic acid biosynthetic pathway also can contribute to the higher susceptibility of pmei mutants. The lack of PMEI expression does not affect hemicellulose strengthening, callose deposition, and the synthesis of structural defense proteins, proposed...... is posttranscriptionally regulated by endogenous protein inhibitors (PMEIs). Here, AtPMEI10, AtPMEI11, and AtPMEI12 are identified as functional PMEIs induced in Arabidopsis (Arabidopsis thaliana) during B. cinerea infection. AtPMEI expression is strictly regulated by jasmonic acid and ethylene signaling, while only At...

Synthesis, structure-activity relationship, and pharmacological profile of analogs of the ASIC-3 inhibitor A-317567.

Science.gov (United States)

Kuduk, Scott D; Di Marco, Christina N; Bodmer-Narkevitch, Vera; Cook, Sean P; Cato, Matthew J; Jovanovska, Aneta; Urban, Mark O; Leitl, Michael; Sain, Nova; Liang, Annie; Spencer, Robert H; Kane, Stefanie A; Hartman, George D; Bilodeau, Mark T

2010-01-20

The synthesis, structure-activity relationship (SAR), and pharmacological evaluation of analogs of the acid-sensing ion channel (ASIC) inhibitor A-317567 are reported. It was found that the compound with an acetylenic linkage was the most potent ASIC-3 channel blocker. This compound reversed mechanical hypersensitivity in the rat iodoacetate model of osteoarthritis pain, although sedation was noted. Sedation was also observed in ASIC-3 knockout mice, questioning whether sedation and antinociception are mediated via a non-ASIC-3 specific mechanism.

Identification of Small Molecule Translesion Synthesis Inhibitors That Target the Rev1-CT/RIR Protein-Protein Interaction.

Science.gov (United States)

Sail, Vibhavari; Rizzo, Alessandro A; Chatterjee, Nimrat; Dash, Radha C; Ozen, Zuleyha; Walker, Graham C; Korzhnev, Dmitry M; Hadden, M Kyle

2017-07-21

Translesion synthesis (TLS) is an important mechanism through which proliferating cells tolerate DNA damage during replication. The mutagenic Rev1/Polζ-dependent branch of TLS helps cancer cells survive first-line genotoxic chemotherapy and introduces mutations that can contribute to the acquired resistance so often observed with standard anticancer regimens. As such, inhibition of Rev1/Polζ-dependent TLS has recently emerged as a strategy to enhance the efficacy of first-line chemotherapy and reduce the acquisition of chemoresistance by decreasing tumor mutation rate. The TLS DNA polymerase Rev1 serves as an integral scaffolding protein that mediates the assembly of the active multiprotein TLS complexes. Protein-protein interactions (PPIs) between the C-terminal domain of Rev1 (Rev1-CT) and the Rev1-interacting region (RIR) of other TLS DNA polymerases play an essential role in regulating TLS activity. To probe whether disrupting the Rev1-CT/RIR PPI is a valid approach for developing a new class of targeted anticancer agents, we designed a fluorescence polarization-based assay that was utilized in a pilot screen for small molecule inhibitors of this PPI. Two small molecule scaffolds that disrupt this interaction were identified, and secondary validation assays confirmed that compound 5 binds to Rev1-CT at the RIR interface. Finally, survival and mutagenesis assays in mouse embryonic fibroblasts and human fibrosarcoma HT1080 cells treated with cisplatin and ultraviolet light indicate that these compounds inhibit mutagenic Rev1/Polζ-dependent TLS in cells, validating the Rev1-CT/RIR PPI for future anticancer drug discovery and identifying the first small molecule inhibitors of TLS that target Rev1-CT.

Synthesis and biological evaluation of several dephosphonated analogues of CMP-Neu5Ac as inhibitors of GM3-synthase.

Science.gov (United States)

Rota, Paola; Cirillo, Federica; Piccoli, Marco; Gregorio, Antonio; Tettamanti, Guido; Allevi, Pietro; Anastasia, Luigi

2015-10-05

Previous studies demonstrated that reducing the GM3 content in myoblasts increased the cell resistance to hypoxic stress, suggesting that a pharmacological inhibition of the GM3 synthesis could be instrumental for the development of new treatments for ischemic diseases. Herein, the synthesis of several dephosphonated CMP-Neu5Ac congeners and their anti-GM3-synthase activity is reported. Biological activity testes revealed that some inhibitors almost completely blocked the GM3-synthase activity in vitro and reduced the GM3 content in living embryonic kidney 293A cells, eventually activating the epidermal growth factor receptor (EGFR) signaling cascade. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Design, synthesis, in vitro Evaluation and docking studies on dihydropyrimidine-based urease inhibitors.

Science.gov (United States)

Iftikhar, Fatima; Ali, Yousaf; Ahmad Kiani, Farooq; Fahad Hassan, Syed; Fatima, Tabeer; Khan, Ajmal; Niaz, Basit; Hassan, Abbas; Latif Ansari, Farzana; Rashid, Umer

2017-10-01

In our previous report, we have identified 3,4-dihydropyrimidine scaffold as promising class of urease inhibitor in a structure based virtual screen (SBVS) experiment. In present study, we attempted to optimize the scaffold by varying C-5 substituent. The elongation of the C-5 chain was achieved by the reaction of C-5 ester with hydrazine leading to C-5 carbohydrazides which were further used as building blocks for the synthesis of fifteen new compounds having diverse moieties. A significantly higher in vitro urease inhibitory activity with IC 50 values in submicromolar range was observed for semithiocarbazide derivatives (4a-c, 0.58-0.79µM) and isatin Schiff base derivative 5a (0.23µM). Docking analysis suggests that the synthesized compounds were anchored well in the catalytic site and extending to the entrance of binding pocket and thus restrict the mobility of the flap by interacting with its key amino acid residues. The overall results of urease inhibition have shown that these compounds can be further optimized and developed as lead urease inhibitors. Copyright © 2017 Elsevier Inc. All rights reserved.

Design, synthesis, X-ray studies, and biological evaluation of novel macrocyclic HIV-1 protease inhibitors involving the P1'-P2' ligands

Energy Technology Data Exchange (ETDEWEB)

Ghosh, Arun K.; Sean Fyvie, W.; Brindisi, Margherita; Steffey, Melinda; Agniswamy, Johnson; Wang, Yuan-Fang; Aoki, Manabu; Amano, Masayuki; Weber, Irene T.; Mitsuya, Hiroaki

2017-11-01

Design, synthesis, and evaluation of a new class of HIV-1 protease inhibitors containing diverse flexible macrocyclic P1'-P2' tethers are reported. Inhibitor 5a with a pyrrolidinone-derived macrocycle exhibited favorable enzyme inhibitory and antiviral activity (Ki = 13.2 nM, IC50 = 22 nM). Further incorporation of heteroatoms in the macrocyclic skeleton provided macrocyclic inhibitors 5m and 5o. These compounds showed excellent HIV-1 protease inhibitory (Ki = 62 pM and 14 pM, respectively) and antiviral activity (IC50 = 5.3 nM and 2.0 nM, respectively). Inhibitor 5o also remained highly potent against a DRV-resistant HIV-1 variant.

The NADPH oxidase inhibitor apocynin induces nitric oxide synthesis via oxidative stress

International Nuclear Information System (INIS)

Riganti, Chiara; Costamagna, Costanzo; Doublier, Sophie; Miraglia, Erica; Polimeni, Manuela; Bosia, Amalia; Ghigo, Dario

2008-01-01

We have recently shown that apocynin elicits an oxidative stress in N11 mouse glial cells and other cell types. Here we report that apocynin increased the accumulation of nitrite, the stable derivative of nitric oxide (NO), in the extracellular medium of N11 cell cultures, and the NO synthase (NOS) activity in cell lysates. The increased synthesis of NO was associated with increased expression of inducible NOS (iNOS) mRNA, increased nuclear translocation of the redox-sensitive transcription factor NF-κB and decreased intracellular level of its inhibitor IkBα. These effects, accompanied by increased production of H 2 O 2 , were very similar to those observed after incubation with bacterial lipopolysaccharide (LPS) and were inhibited by catalase. These results suggest that apocynin, similarly to LPS, induces increased NO synthesis by eliciting a generation of reactive oxygen species (ROS), which in turn causes NF-κB activation and increased expression of iNOS. Therefore, the increased bioavailability of NO reported in the literature after in vivo or in vitro treatments with apocynin might depend, at least partly, on the drug-elicited induction of iNOS, and not only on the inhibition of NADPH oxidase and the subsequent decreased scavenging of NO by oxidase-derived ROS, as it is often supposed

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

Directory of Open Access Journals (Sweden)

Andrzej P. Herman

2014-01-01

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

Epitope targeting of tertiary protein structure enables target-guided synthesis of a potent in-cell inhibitor of botulinum neurotoxin.

Science.gov (United States)

Farrow, Blake; Wong, Michelle; Malette, Jacquie; Lai, Bert; Deyle, Kaycie M; Das, Samir; Nag, Arundhati; Agnew, Heather D; Heath, James R

2015-06-08

Botulinum neurotoxin (BoNT) serotype A is the most lethal known toxin and has an occluded structure, which prevents direct inhibition of its active site before it enters the cytosol. Target-guided synthesis by in situ click chemistry is combined with synthetic epitope targeting to exploit the tertiary structure of the BoNT protein as a landscape for assembling a competitive inhibitor. A substrate-mimicking peptide macrocycle is used as a direct inhibitor of BoNT. An epitope-targeting in situ click screen is utilized to identify a second peptide macrocycle ligand that binds to an epitope that, in the folded BoNT structure, is active-site-adjacent. A second in situ click screen identifies a molecular bridge between the two macrocycles. The resulting divalent inhibitor exhibits an in vitro inhibition constant of 165 pM against the BoNT/A catalytic chain. The inhibitor is carried into cells by the intact holotoxin, and demonstrates protection and rescue of BoNT intoxication in a human neuron model. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Trichloroacetimidates as Alkylating Reagents and Their Application in the Synthesis of Pyrroloindoline Natural Products and Synthesis of Small Molecule Inhibitors of Src Homology 2 Domain-Containing Inositol Phosphatase (SHIP)

Science.gov (United States)

Adhikari, Arijit A.

was applied towards the synthesis of natural products and their analogs. The pyrroloindoline ring system is found in many alkaloids and cyclic peptides which mainly differ in the substitution at the C3a position. To provide rapid access to these natural products a diversity-oriented strategy was established via displacement of C3a-trichloroacetimidate pyrroloindoline. Carbon, oxygen, sulfur and nitrogen nucleophiles were all shown to undergo substitution reactions with these trichloroacetimidates in the presence of a Lewis acid catalyst. In order to demonstrate the utility of this new method it was applied towards the synthesis of arundinine and a formal synthesis of psychotriasine. Current investigations involve the application of this method towards the synthesis of a complex pyrroloindoline natural product kapakahine C and the progress made therein has been discussed. The reactivity of trichloroacetimidates was also investigated for the selective C3-alkylation of 2,3-disubstituted indoles to provide indolenines. Indolenines serve as useful intermediates in the synthesis of many complex alkaloids. Different benzylic and allylic trichloroacetimidates were shown to provide 3,3'-disubstituted indolenines with high yields in the presence of catalytic amounts of Lewis acids. Various substituted indoles were evaluated under these reaction conditions. This methodology was also applied towards the synthesis of the core tetracyclic ring system found in communesin natural products. In addition to the above work, synthesis of small molecule inhibitors of Src Homology 2 Domain-Containing Inositol Phosphatase (SHIP) has also been described. Aberrations in the phosphoinositide 3-kinase (PI3K) cellular signaling pathway can lead to diseased cellular states like cancer. Herein we have reported stereoselective synthesis of two quinoline based small molecule SHIP inhibitors. The lead compounds and their analogs were tested for their activities against SHIP by Malachite green assay

Nε-Acryloyllysine Piperazides as Irreversible Inhibitors of Transglutaminase 2: Synthesis, Structure-Activity Relationships, and Pharmacokinetic Profiling.

Science.gov (United States)

Wodtke, Robert; Hauser, Christoph; Ruiz-Gómez, Gloria; Jäckel, Elisabeth; Bauer, David; Lohse, Martin; Wong, Alan; Pufe, Johanna; Ludwig, Friedrich-Alexander; Fischer, Steffen; Hauser, Sandra; Greif, Dieter; Pisabarro, M Teresa; Pietzsch, Jens; Pietsch, Markus; Löser, Reik

2018-05-24

Transglutaminase 2 (TGase 2)-catalyzed transamidation represents an important post-translational mechanism for protein modification with implications in physiological and pathophysiological conditions, including fibrotic and neoplastic processes. Consequently, this enzyme is considered a promising target for the diagnosis of and therapy for these diseases. In this study, we report on the synthesis and kinetic characterization of N ε -acryloyllysine piperazides as irreversible inhibitors of TGase 2. Systematic structural modifications on 54 new compounds were performed with a major focus on fluorine-bearing substituents due to the potential of such compounds to serve as radiotracer candidates for positron emission tomography. The determined inhibitory activities ranged from 100 to 10 000 M -1 s -1 , which resulted in comprehensive structure-activity relationships. Structure-activity correlations using various substituent parameters accompanied by covalent docking studies provide an advanced understanding of the molecular recognition for this inhibitor class within the active site of TGase 2. Selectivity profiling of selected compounds for other transglutaminases demonstrated an excellent selectivity toward transglutaminase 2. Furthermore, an initial pharmacokinetic profiling of selected inhibitors was performed, including the assessment of potential membrane permeability and liver microsomal stability.

ApoB-100 secretion by HepG2 cells is regulated by the rate of triglyceride biosynthesis but not by intracellular lipid pools.

Science.gov (United States)

Benoist, F; Grand-Perret, T

1996-10-01

Triglycerides (TGs), cholesteryl esters (CEs), cholesterol, and phosphatidylcholine have been independently proposed as playing regulatory roles in apoB-100 secretion; the results depended on the cellular model used. In this study, we reinvestigate the role of lipids in apoB-100 production in HepG2 cells and in particular, we clarify the respective roles of intracellular mass and the biosynthesis of lipids in the regulation of apoB-100 production. In a first set of experiments, the pool size of cholesterol, CEs, and TGs was modulated by a 3-day treatment with either lipid precursors or inhibitors of enzymes involved in lipid synthesis. We used simvastatin (a hydroxymethylglutaryl coenzyme A reductase inhibitor), 58-035 (an acyl coenzyme A cholesterol acyltransferase inhibitor), 5-tetradecyloxy-2-furancarboxylic acid (TOFA, an inhibitor of fatty acid synthesis), and oleic acid. The secretion rate of apoB-100 was not affected by the large modulation of lipid mass induced by these various pre-treatments. In a second set of experiments, the same lipid modulators were added during a 4-hour labeling period. Simvastatin and 58-035 inhibited cholesterol and CE synthesis without affecting apoB-100 secretion. By contrast, treatment of HepG2 cells with TOFA resulted in the inhibition of TG synthesis and apoB-100 secretion. This effect was highly specific for apoB-100 and was reversed by adding oleic acid, which stimulated both TG synthesis and apoB-100 secretion. Moreover, a combination of oleic acid and 58-035 inhibited CE biosynthesis and increased both TG synthesis and apoB-100 secretion. These results show that in HepG2 cells TG biosynthesis regulates apoB-100 secretion, whereas the rate of cholesterol or CE biosynthesis has no effect.

Inhibitors of HIV-protease from computational design. A history of theory and synthesis still to be fully appreciated.

Science.gov (United States)

Berti, Federico; Frecer, Vladimir; Miertus, Stanislav

2014-01-01

Despite the fact that HIV-Protease is an over 20 years old target, computational approaches to rational design of its inhibitors still have a great potential to stimulate the synthesis of new compounds and the discovery of new, potent derivatives, ever capable to overcome the problem of drug resistance. This review deals with successful examples of inhibitors identified by computational approaches, rather than by knowledge-based design. Such methodologies include the development of energy and scoring functions, docking protocols, statistical models, virtual combinatorial chemistry. Computations addressing drug resistance, and the development of related models as the substrate envelope hypothesis are also reviewed. In some cases, the identified structures required the development of synthetic approaches in order to obtain the desired target molecules; several examples are reported.

Methanol Metabolism in Yeasts : Regulation of the Synthesis of Catabolic Enzymes

NARCIS (Netherlands)

Egli, Th.; Dijken, J.P. van; Veenhuis, M.; Harder, W.; Fiechter, A.

1980-01-01

The regulation of the synthesis of four dissimilatory enzymes involved in methanol metabolism, namely alcohol oxidase, formaldehyde dehydrogenase, formate dehydrogenase and catalase was investigated in the yeasts Hansenula polymorpha and Kloeckera sp. 2201. Enzyme profiles in cell-free extracts of

The cochaperone BAG3 coordinates protein synthesis and autophagy under mechanical strain through spatial regulation of mTORC1.

Science.gov (United States)

Kathage, Barbara; Gehlert, Sebastian; Ulbricht, Anna; Lüdecke, Laura; Tapia, Victor E; Orfanos, Zacharias; Wenzel, Daniela; Bloch, Wilhelm; Volkmer, Rudolf; Fleischmann, Bernd K; Fürst, Dieter O; Höhfeld, Jörg

2017-01-01

The cochaperone BAG3 is a central protein homeostasis factor in mechanically strained mammalian cells. It mediates the degradation of unfolded and damaged forms of the actin-crosslinker filamin through chaperone-assisted selective autophagy (CASA). In addition, BAG3 stimulates filamin transcription in order to compensate autophagic disposal and to maintain the actin cytoskeleton under strain. Here we demonstrate that BAG3 coordinates protein synthesis and autophagy through spatial regulation of the mammalian target of rapamycin complex 1 (mTORC1). The cochaperone utilizes its WW domain to contact a proline-rich motif in the tuberous sclerosis protein TSC1 that functions as an mTORC1 inhibitor in association with TSC2. Interaction with BAG3 results in a recruitment of TSC complexes to actin stress fibers, where the complexes act on a subpopulation of mTOR-positive vesicles associated with the cytoskeleton. Local inhibition of mTORC1 is essential to initiate autophagy at sites of filamin unfolding and damage. At the same time, BAG3-mediated sequestration of TSC1/TSC2 relieves mTORC1 inhibition in the remaining cytoplasm, which stimulates protein translation. In human muscle, an exercise-induced association of TSC1 with the cytoskeleton coincides with mTORC1 activation in the cytoplasm. The spatial regulation of mTORC1 exerted by BAG3 apparently provides the basis for a simultaneous induction of autophagy and protein synthesis to maintain the proteome under mechanical strain. Copyright © 2016 Elsevier B.V. All rights reserved.

Endothelin-1 Regulation of Exercise-Induced Changes in Flow: Dynamic Regulation of Vascular Tone

Directory of Open Access Journals (Sweden)

Robert M. Rapoport

2017-10-01

Full Text Available Although endothelin (ET-1 is a highly potent vasoconstrictor with considerable efficacy in numerous vascular beds, the role of endogenous ET-1 in the regulation of vascular tone remains unclear. The perspective that ET-1 plays little role in the on-going regulation of vascular tone at least under physiologic conditions is supported by findings that potential ET-1 constriction is minimized by the release of the vasodilator and ET-1 synthesis inhibitor, nitric oxide (NO. Indeed, ET-1 release and constriction is self-limited by ET-1-induced, endothelial ETB receptor-mediated release of NO. Moreover, even if the balance between ET-1 and NO were reversed as the result of lowered NO activity, as occurs in a number of pathophysiologies associated with endothelial dysfunction, the well-known resistance of ET-1 constriction to reversal (as determined with exogenous ET-1 precludes ET-1 in the dynamic, i.e., moment-to-moment, regulation of vascular tone. On the other hand, and as presently reviewed, findings of ET-1-dependent modulation of organ blood flow with exercise under physiologic conditions demonstrate the dynamic regulation of vascular tone by ET-1. We speculate that this regulation is mediated at least in part through changes in ET-1 synthesis/release caused by pulsatile flow-induced shear stress and NO.

Synthesis and Biological Evaluation of Benzochromenopyrimidinones as Cholinesterase Inhibitors and Potent Antioxidant, Non-Hepatotoxic Agents for Alzheimer’s Disease

Directory of Open Access Journals (Sweden)

Youssef Dgachi

2016-05-01

Full Text Available We report herein the straightforward two-step synthesis and biological assessment of novel racemic benzochromenopyrimidinones as non-hepatotoxic, acetylcholinesterase inhibitors with antioxidative properties. Among them, compound 3Bb displayed a mixed-type inhibition of human acetylcholinesterase (IC50 = 1.28 ± 0.03 μM, good antioxidant activity, and also proved to be non-hepatotoxic on human HepG2 cell line.

Fatty Amides from Crude Rice Bran Oil as Green Corrosion Inhibitors

Directory of Open Access Journals (Sweden)

E. Reyes-Dorantes

2017-01-01

Full Text Available Due to its high oil content, this research proposes the use of an agroindustrial byproduct (rice bran as a sustainable option for the synthesis of corrosion inhibitors. From the crude rice bran oil, the synthesis of fatty amide-type corrosion inhibitors was carried out. The corrosion inhibitory capacity of the fatty amides was evaluated on an API X-70 steel using electrochemical techniques such as real-time corrosion monitoring and potentiodynamic polarization curves. As a corrosive medium, a CO2-saturated solution (3.5% NaCl was used at three temperatures (30, 50, and 70°C and different concentrations of inhibitor (0, 5, 10, 25, 50, and 100 ppm. The results demonstrate that the sustainable use of agroindustrial byproducts is a good alternative to the synthesis of environmentally friendly inhibitors with high corrosion inhibition efficiencies.

A novel PKB/Akt inhibitor, MK-2206, effectively inhibits insulin-stimulated glucose metabolism and protein synthesis in isolated rat skeletal muscle.

Science.gov (United States)

Lai, Yu-Chiang; Liu, Yang; Jacobs, Roxane; Rider, Mark H

2012-10-01

PKB (protein kinase B), also known as Akt, is a key component of insulin signalling. Defects in PKB activation lead to insulin resistance and metabolic disorders, whereas PKB overactivation has been linked to tumour growth. Small-molecule PKB inhibitors have thus been developed for cancer treatment, but also represent useful tools to probe the roles of PKB in insulin action. In the present study, we examined the acute effects of two allosteric PKB inhibitors, MK-2206 and Akti 1/2 (Akti) on PKB signalling in incubated rat soleus muscles. We also assessed the effects of the compounds on insulin-stimulated glucose uptake, glycogen and protein synthesis. MK-2206 dose-dependently inhibited insulin-stimulated PKB phosphorylation, PKBβ activity and phosphorylation of PKB downstream targets (including glycogen synthase kinase-3α/β, proline-rich Akt substrate of 40 kDa and Akt substrate of 160 kDa). Insulin-stimulated glucose uptake, glycogen synthesis and glycogen synthase activity were also decreased by MK-2206 in a dose-dependent manner. Incubation with high doses of MK-2206 (10 μM) inhibited insulin-induced p70 ribosomal protein S6 kinase and 4E-BP1 (eukaryotic initiation factor 4E-binding protein-1) phosphorylation associated with increased eEF2 (eukaryotic elongation factor 2) phosphorylation. In contrast, Akti only modestly inhibited insulin-induced PKB and mTOR (mammalian target of rapamycin) signalling, with little or no effect on glucose uptake and protein synthesis. MK-2206, rather than Akti, would thus be the tool of choice for studying the role of PKB in insulin action in skeletal muscle. The results point to a key role for PKB in mediating insulin-stimulated glucose uptake, glycogen synthesis and protein synthesis in skeletal muscle.

Design and synthesis of novel N-benzylidenesulfonohydrazide inhibitors of MurC and MurD as potential antibacterial agents.

Science.gov (United States)

Frlan, Rok; Kovac, Andreja; Blanot, Didier; Gobec, Stanislav; Pecar, Slavko; Obreza, Ales

2008-01-11

A series of novel N-benzylidenesulfonohydrazide compounds were designed and synthesized as inhibitors of UDP-N-acetylmuramic acid: L-alanine ligase (MurC) and UDP-N-acetylmuramoyl-L-alanine: D-glutamate ligase (MurD) from E. coli, involved in the biosynthesis of bacterial cell-walls. Some compounds possessed inhibitory activity against both enzymes with IC(50) values as low as 30 microM. In addition, a new, one-pot synthesis of amidobenzaldehydes is reported.

Regulation of collagenase inhibitor production in chondrosarcoma chondrocytes

International Nuclear Information System (INIS)

Harper, J.; Harper, E.

1987-01-01

Swarm rat chondrosarcoma chondrocytes produce an inhibitor of collagenase. This inhibitor is similar to those isolated from normal cartilage tissues. These cells will synthesize proteins in the absence of serum. Since serum contains inhibitors of collagenase, it is necessary to culture cells without serum in order to obtain accurate measurements of enzyme and inhibitor levels. They examined the effect of insulin on inhibitor secretion by cultures of Swarm rat chondrosarcoma chondrocytes. They observed a 2.5 to 3.5 fold stimulation of inhibitory activity in the presence of as little as 10 ng/ml insulin as compared to controls in serum free Dulbecco's modified Eagle's medium supplemented with 4.5 g/l glucose. The units of inhibitor were determined over a 7 day culture period. Medium was harvested daily and assayed for collagenase activity and for inhibition of a known collagenase from rabbit skin or human skin, using the 14 C-glycine peptide release assay. The amount of inhibitor obtained from days 2 through 7 were: 1.4 unit (control), 3.8 units (10 ng/ml insulin), 5.2 units (1 μg/ml insulin). The addition of 1 mM dibutyryl cyclic AMP to these chondrocytes in the presence of 1 μg/ml insulin caused a decrease in the level of inhibitor, suggesting that a dephosphorylation event may be necessary for this stimulation by insulin to occur

Steroid synthesis by primary human keratinocytes; implications for skin disease

Energy Technology Data Exchange (ETDEWEB)

Hannen, Rosalind F., E-mail: r.f.hannen@qmul.ac.uk [Centre for Cutaneous Research, Institute of Cell and Molecular Science, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London E1 2AT (United Kingdom); Michael, Anthony E. [Centre for Developmental and Endocrine Signalling, Academic Section of Obstetrics and Gynaecology, Division of Clinical Developmental Sciences, 3rd Floor, Lanesborough Wing, St. George' s, University of London, Cranmer Terrace, Tooting, London SW17 0RE (United Kingdom); Jaulim, Adil [Centre for Cutaneous Research, Institute of Cell and Molecular Science, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London E1 2AT (United Kingdom); Bhogal, Ranjit [Life Science, Unilever R and D Colworth House, Sharnbrook, Bedfordshire MK44 1LQ (United Kingdom); Burrin, Jacky M. [Centre for Endocrinology, William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London EC1M 6BQ (United Kingdom); Philpott, Michael P. [Centre for Cutaneous Research, Institute of Cell and Molecular Science, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London E1 2AT (United Kingdom)

2011-01-07

Research highlights: {yields} Primary keratinocytes express the steroid enzymes required for cortisol synthesis. {yields} Normal primary human keratinocytes can synthesise cortisol. {yields} Steroidogenic regulators, StAR and MLN64, are expressed in normal epidermis. {yields} StAR expression is down regulated in eczema and psoriatic epidermis. -- Abstract: Cortisol-based therapy is one of the most potent anti-inflammatory treatments available for skin conditions including psoriasis and atopic dermatitis. Previous studies have investigated the steroidogenic capabilities of keratinocytes, though none have demonstrated that these skin cells, which form up to 90% of the epidermis are able to synthesise cortisol. Here we demonstrate that primary human keratinocytes (PHK) express all the elements required for cortisol steroidogenesis and metabolise pregnenolone through each intermediate steroid to cortisol. We show that normal epidermis and cultured PHK express each of the enzymes (CYP11A1, CYP17A1, 3{beta}HSD1, CYP21 and CYP11B1) that are required for cortisol synthesis. These enzymes were shown to be metabolically active for cortisol synthesis since radiometric conversion assays traced the metabolism of [7-{sup 3}H]-pregnenolone through each steroid intermediate to [7-{sup 3}H]-cortisol in cultured PHK. Trilostane (a 3{beta}HSD1 inhibitor) and ketoconazole (a CYP17A1 inhibitor) blocked the metabolism of both pregnenolone and progesterone. Finally, we show that normal skin expresses two cholesterol transporters, steroidogenic acute regulatory protein (StAR), regarded as the rate-determining protein for steroid synthesis, and metastatic lymph node 64 (MLN64) whose function has been linked to cholesterol transport in steroidogenesis. The expression of StAR and MLN64 was aberrant in two skin disorders, psoriasis and atopic dermatitis, that are commonly treated with cortisol, suggesting dysregulation of epidermal steroid synthesis in these patients. Collectively these data

Steroid synthesis by primary human keratinocytes; implications for skin disease

International Nuclear Information System (INIS)

Hannen, Rosalind F.; Michael, Anthony E.; Jaulim, Adil; Bhogal, Ranjit; Burrin, Jacky M.; Philpott, Michael P.

2011-01-01

Research highlights: → Primary keratinocytes express the steroid enzymes required for cortisol synthesis. → Normal primary human keratinocytes can synthesise cortisol. → Steroidogenic regulators, StAR and MLN64, are expressed in normal epidermis. → StAR expression is down regulated in eczema and psoriatic epidermis. -- Abstract: Cortisol-based therapy is one of the most potent anti-inflammatory treatments available for skin conditions including psoriasis and atopic dermatitis. Previous studies have investigated the steroidogenic capabilities of keratinocytes, though none have demonstrated that these skin cells, which form up to 90% of the epidermis are able to synthesise cortisol. Here we demonstrate that primary human keratinocytes (PHK) express all the elements required for cortisol steroidogenesis and metabolise pregnenolone through each intermediate steroid to cortisol. We show that normal epidermis and cultured PHK express each of the enzymes (CYP11A1, CYP17A1, 3βHSD1, CYP21 and CYP11B1) that are required for cortisol synthesis. These enzymes were shown to be metabolically active for cortisol synthesis since radiometric conversion assays traced the metabolism of [7- 3 H]-pregnenolone through each steroid intermediate to [7- 3 H]-cortisol in cultured PHK. Trilostane (a 3βHSD1 inhibitor) and ketoconazole (a CYP17A1 inhibitor) blocked the metabolism of both pregnenolone and progesterone. Finally, we show that normal skin expresses two cholesterol transporters, steroidogenic acute regulatory protein (StAR), regarded as the rate-determining protein for steroid synthesis, and metastatic lymph node 64 (MLN64) whose function has been linked to cholesterol transport in steroidogenesis. The expression of StAR and MLN64 was aberrant in two skin disorders, psoriasis and atopic dermatitis, that are commonly treated with cortisol, suggesting dysregulation of epidermal steroid synthesis in these patients. Collectively these data show that PHK are capable of extra

Plasminogen activator inhibitor type 1 regulates microglial motility and phagocytic activity

Directory of Open Access Journals (Sweden)

Jeon Hyejin

2012-06-01

Full Text Available Abstract Background Plasminogen activator inhibitor type 1 (PAI-1 is the primary inhibitor of urokinase type plasminogen activators (uPA and tissue type plasminogen activators (tPA, which mediate fibrinolysis. PAI-1 is also involved in the innate immunity by regulating cell migration and phagocytosis. However, little is known about the role of PAI-1 in the central nervous system. Methods In this study, we identified PAI-1 in the culture medium of mouse mixed glial cells by liquid chromatography and tandem mass spectrometry. Secretion of PAI-1 from glial cultures was detected by ELISA and western blotting analysis. Cell migration was evaluated by in vitro scratch-wound healing assay or Boyden chamber assay and an in vivo stab wound injury model. Phagocytic activity was measured by uptake of zymosan particles. Results The levels of PAI-1 mRNA and protein expression were increased by lipopolysaccharide and interferon-γ stimulation in both microglia and astrocytes. PAI-1 promoted the migration of microglial cells in culture via the low-density lipoprotein receptor-related protein (LRP 1/Janus kinase (JAK/signal transducer and activator of transcription (STAT1 axis. PAI-1 also increased microglial migration in vivo when injected into mouse brain. PAI-1-mediated microglial migration was independent of protease inhibition, because an R346A mutant of PAI-1 with impaired PA inhibitory activity also promoted microglial migration. Moreover, PAI-1 was able to modulate microglial phagocytic activity. PAI-1 inhibited microglial engulfment of zymosan particles in a vitronectin- and Toll-like receptor 2/6-dependent manner. Conclusion Our results indicate that glia-derived PAI-1 may regulate microglial migration and phagocytosis in an autocrine or paracrine manner. This may have important implications in the regulation of brain microglial activities in health and disease.

Coordinate Regulation of Yeast Sterol Regulatory Element-binding Protein (SREBP) and Mga2 Transcription Factors.

Science.gov (United States)

Burr, Risa; Stewart, Emerson V; Espenshade, Peter J

2017-03-31

The Mga2 and Sre1 transcription factors regulate oxygen-responsive lipid homeostasis in the fission yeast Schizosaccharomyces pombe in a manner analogous to the mammalian sterol regulatory element-binding protein (SREBP)-1 and SREBP-2 transcription factors. Mga2 and SREBP-1 regulate triacylglycerol and glycerophospholipid synthesis, whereas Sre1 and SREBP-2 regulate sterol synthesis. In mammals, a shared activation mechanism allows for coordinate regulation of SREBP-1 and SREBP-2. In contrast, distinct pathways activate fission yeast Mga2 and Sre1. Therefore, it is unclear whether and how these two related pathways are coordinated to maintain lipid balance in fission yeast. Previously, we showed that Sre1 cleavage is defective in the absence of mga2 Here, we report that this defect is due to deficient unsaturated fatty acid synthesis, resulting in aberrant membrane transport. This defect is recapitulated by treatment with the fatty acid synthase inhibitor cerulenin and is rescued by addition of exogenous unsaturated fatty acids. Furthermore, sterol synthesis inhibition blocks Mga2 pathway activation. Together, these data demonstrate that Sre1 and Mga2 are each regulated by the lipid product of the other transcription factor pathway, providing a source of coordination for these two branches of lipid synthesis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Laminin peptide YIGSR induces collagen synthesis in Hs27 human dermal fibroblasts

Energy Technology Data Exchange (ETDEWEB)

Yoon, Jong Hyuk; Kim, Jaeyoon; Lee, Hyeongjoo [NovaCell Technology Inc., Pohang, Kyungbuk 790-784 (Korea, Republic of); Kim, So Young [Department of Dermatology, Chung-Ang University College of Medicine, Seoul 156-756 (Korea, Republic of); Department of Convergence Medicine and Pharmaceutical Biosciences, Graduate School, Chung-Ang University, Seoul 156-756 (Korea, Republic of); Jang, Hwan-Hee [Functional Food and Nutrition Division, Department of Agrofood Resources, Rural Development Administration, Suwon 441-853 (Korea, Republic of); Ryu, Sung Ho [Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology (POSTECH), Pohang, Kyungbuk 790-784 (Korea, Republic of); Kim, Beom Joon [Department of Dermatology, Chung-Ang University College of Medicine, Seoul 156-756 (Korea, Republic of); Department of Convergence Medicine and Pharmaceutical Biosciences, Graduate School, Chung-Ang University, Seoul 156-756 (Korea, Republic of); Lee, Taehoon G., E-mail: taehoon@novacelltech.com [NovaCell Technology Inc., Pohang, Kyungbuk 790-784 (Korea, Republic of)

2012-11-23

Highlights: Black-Right-Pointing-Pointer We identify a function of the YIGSR peptide to enhance collagen synthesis in Hs27. Black-Right-Pointing-Pointer YIGSR peptide enhanced collagen type 1 synthesis both of gene and protein levels. Black-Right-Pointing-Pointer There were no changes in cell proliferation and MMP-1 level in YIGSR treatment. Black-Right-Pointing-Pointer The YIGSR effect on collagen synthesis mediated activation of FAK, pyk2 and ERK. Black-Right-Pointing-Pointer The YIGSR-induced FAK and ERK activation was modulated by FAK and MEK inhibitors. -- Abstract: The dermal ECM is synthesized from fibroblasts and is primarily compromised of fibrillar collagen and elastic fibers, which support the mechanical strength and resiliency of skin, respectively. Laminin, a major glycoprotein located in the basement membrane, promotes cell adhesion, cell growth, differentiation, and migration. The laminin tyrosine-isoleucine-glycine-serine-arginine (YIGSR) peptide, corresponding to the 929-933 sequence of the {beta}1 chain, is known to be a functional motif with effects on the inhibition of tumor metastasis, the regulation of sensory axonal response and the inhibition of angiogenesis through high affinity to the 67 kDa laminin receptor. In this study, we identified a novel function of the YIGSR peptide to enhance collagen synthesis in human dermal fibroblasts. To elucidate this novel function regarding collagen synthesis, we treated human dermal fibroblasts with YIGSR peptide in both a time- and dose-dependent manner. According to subsequent experiments, we found that the YIGSR peptide strongly enhanced collagen type 1 synthesis without changing cell proliferation or cellular MMP-1 level. This YIGSR peptide-mediated collagen type 1 synthesis was modulated by FAK inhibitor and MEK inhibitor. This study clearly reveals that YIGSR peptide plays a novel function on the collagen type 1 synthesis of dermal fibroblasts and also suggests that YIGSR is a strong candidate

Design, synthesis, and protein crystallography of biaryltriazoles as potent tautomerase inhibitors of macrophage migration inhibitory factor.

Science.gov (United States)

Dziedzic, Pawel; Cisneros, José A; Robertson, Michael J; Hare, Alissa A; Danford, Nadia E; Baxter, Richard H G; Jorgensen, William L

2015-03-04

Optimization is reported for biaryltriazoles as inhibitors of the tautomerase activity of human macrophage migration inhibitory factor (MIF), a proinflammatory cytokine associated with numerous inflammatory diseases and cancer. A combined approach was taken featuring organic synthesis, enzymatic assaying, crystallography, and modeling including free-energy perturbation (FEP) calculations. X-ray crystal structures for 3a and 3b bound to MIF are reported and provided a basis for the modeling efforts. The accommodation of the inhibitors in the binding site is striking with multiple hydrogen bonds and aryl-aryl interactions. Additional modeling encouraged pursuit of 5-phenoxyquinolinyl analogues, which led to the very potent compound 3s. Activity was further enhanced by addition of a fluorine atom adjacent to the phenolic hydroxyl group as in 3w, 3z, 3aa, and 3bb to strengthen a key hydrogen bond. It is also shown that physical properties of the compounds can be modulated by variation of solvent-exposed substituents. Several of the compounds are likely the most potent known MIF tautomerase inhibitors; the most active ones are more than 1000-fold more active than the well-studied (R)-ISO-1 and more than 200-fold more active than the chromen-4-one Orita-13.

Imidazopyridine-based inhibitors of glycogen synthase kinase 3: synthesis and evaluation of amide isostere replacements of the carboxamide scaffold.

Science.gov (United States)

Yngve, Ulrika; Söderman, Peter; Svensson, Mats; Rosqvist, Susanne; Arvidsson, Per I

2012-11-01

In this study, we explored the effect of bioisostere replacement in a series of glycogen synthase kinase 3 (GSK3) inhibitors based on the imidazopyridine core. The synthesis and biological evaluation of a number of novel sulfonamide, 1,2,4-oxadiazole, and thiazole derivates as amide bioisosteres, as well as a computational rationalization of the obtained results are reported. Copyright © 2012 Verlag Helvetica Chimica Acta AG, Zürich.

Synergism between demethylation inhibitor fungicides or gibberellin inhibitor plant growth regulators and bifenthrin in a pyrethroid-resistant population of Listronotus maculicollis (Coleoptera: Curculionidae).

Science.gov (United States)

Ramoutar, D; Cowles, R S; Requintina, E; Alm, S R

2010-10-01

In 2007-2008, the "annual bluegrass weevil," Listronotus maculicollis Kirby (Coleoptera: Curculionidae), a serious pest of Poa annua L. (Poales: Poaceae) on U.S. golf courses, was shown to be resistant to two pyrethroids, bifenthrin and lambda-cyhalothrin. In 2008, we showed that bifenthrin resistance was principally mediated by oxidase detoxification (cytochrome P450 [P450]). P450s can be inhibited by demethylation inhibitor fungicides and gibberellin inhibitor plant growth regulators, both of which are commonly used on golf courses. We tested these compounds for synergistic activity with bifenthin against a pyrethroid-resistant population of L. maculicollis. The LD50 value for bifenthrin was significantly reduced from 87 ng per insect (without synergists) to 9.6-40 ng per insect after exposure to the fungicides fenarimol, fenpropimorph, prochloraz, propiconazole, and pyrifenox and the plant growth regulators flurprimidol, paclobutrazol, and trinexapac-ethyl. Simulated field exposure with formulated products registered for use on turf revealed enhanced mortality when adult weevils were exposed to bifenthrin (25% mortality, presented alone) combined with field dosages of propiconizole, fenarimol, flurprimidol, or trinexapac-ethyl (range, 49-70% mortality).

Total synthesis and structural revision of TMG-chitotriomycin, a specific inhibitor of insect and fungal beta-N-acetylglucosaminidases.

Science.gov (United States)

Yang, You; Li, Yao; Yu, Biao

2009-09-02

TMG-chitotriomycin, a potent and selective inhibitor of the beta-N-acetylglucosaminidases that possesses an unique N,N,N-trimethyl-d-glucosamine (TMG) residue, is revised to be the TMG-beta-(1-->4)-chitotriose instead of the originally proposed alpha-anomer via its total synthesis, for which a highly convergent approach was developed in which the sterically demanding (1-->4)-glycosidic linkages are efficiently constructed by the Au(I)-catalyzed glycosylation protocol with glycosyl o-hexynylbenzoates as donors.

Optimization of microtubule affinity regulating kinase (MARK) inhibitors with improved physical properties

Energy Technology Data Exchange (ETDEWEB)

Sloman, David L.; Noucti, Njamkou; Altman, Michael D.; Chen, Dapeng; Mislak, Andrea C.; Szewczak, Alexander; Hayashi, Mansuo; Warren, Lee; Dellovade, Tammy; Wu, Zhenhua; Marcus, Jacob; Walker, Deborah; Su, Hua-Poo; Edavettal, Suzanne C.; Munshi, Sanjeev; Hutton, Michael; Nuthall, Hugh; Stanton, Matthew G. (Merck)

2016-09-01

Inhibition of microtubule affinity regulating kinase (MARK) represents a potentially attractive means of arresting neurofibrillary tangle pathology in Alzheimer’s disease. This manuscript outlines efforts to optimize a pyrazolopyrimidine series of MARK inhibitors by focusing on improvements in potency, physical properties and attributes amenable to CNS penetration. A unique cylcyclohexyldiamine scaffold was identified that led to remarkable improvements in potency, opening up opportunities to reduce MW, Pgp efflux and improve pharmacokinetic properties while also conferring improved solubility.

Dual functional cholinesterase and MAO inhibitors for the treatment of Alzheimer's disease: synthesis, pharmacological analysis and molecular modeling of homoisoflavonoid derivatives.

Science.gov (United States)

Wang, Yali; Sun, Yang; Guo, Yueyan; Wang, Zechen; Huang, Ling; Li, Xingshu

2016-01-01

Because of the complexity of Alzheimer's disease (AD), the multi-target-directed ligand (MTDL) strategy is expected to provide superior effects for the treatment of AD, instead of the classic one-drug-one-target strategy. In this context, we focused on the design, synthesis and evaluation of homoisoflavonoid derivatives as dual acetyl cholinesterase (AChE) and monoamine oxidase (MAO-B) inhibitors. Among all the synthesized compounds, compound 10 provided a desired balance of AChE and hMAO-B inhibition activities, with IC50 value of 3.94 and 3.44 μM, respectively. Further studies revealed that compound 10 was a mixed-type inhibitor of AChE and an irreversible inhibitor of hMAO-B, which was also confirmed by molecular modeling studies. Taken together, the data indicated that 10 was a promising dual functional agent for the treatment of AD.

Differential regulation of TNF-α and IL-1β production from endotoxin stimulated human monocytes by phosphodiesterase inhibitors

Directory of Open Access Journals (Sweden)

K. L. Molnar-Kimber

1992-01-01

Full Text Available The effect of selective PDE-I (vinpocetine, PDE-III (milrinone, CI-930, PDE-IV (rolipram, nitroquazone, and PDE-V (zaprinast isozyme inhibitors on TNF-α and IL-1β production from LPS stimulated human monocytes was investigated. The PDE-IV inhibitors caused a concentration dependent inhibition of TNF-α production, but only partially inhibited IL-1β at high concentrations. High concentrations of the PDE-III inhibitors weakly inhibited TNF-α, but had no effect on IL-1β production. PDE-V inhibition was associated with an augmentation of cytokine secretion. Studies with combinations of PDE isozyme inhibitors indicated that PDE-III and PDE-V inhibitors modulate rolipram's suppression of TNF production in an additive manner. These data confirm that TNF-α and IL-1β production from LPS stimulated human monocytes are differentially regulated, and suggest that PDE-IV inhibitors have the potential to suppress TNF levels in man.

Phosphorylation of mouse serine racemase regulates D-serine synthesis

DEFF Research Database (Denmark)

Foltyn, Veronika N; Zehl, Martin; Dikopoltsev, Elena

2010-01-01

Serine racemase (SR) catalyses the synthesis of the transmitter/neuromodulator D-serine, which plays a major role in synaptic plasticity and N-methyl D-aspartate receptor neurotoxicity. We now report that SR is phosphorylated at Thr71 and Thr227 as revealed by mass spectrometric analysis and in v...... with a phosphorylation-deficient SR mutant indicate that Thr71 phosphorylation increases SR activity, suggesting a novel mechanism for regulating D-serine production....

Evaluation of novel arginine based inhibitors of DDAH and investigations into radical hydroacylation of vinyl sulfonates

OpenAIRE

Khanom, N.

2010-01-01

The thesis is in two main sections. In the first section, studies on methylarginine processing enzymes are presented. Dimethyalrginine dimethylaminohydrolase (DDAH) is a class of enzymes involved in the metabolism of methylarginines ADMA and L-NMMA, which indirectly regulate physiological nitric oxide levels. It is desirable to inhibit excess NO in pathological situations, and the arginine mimetic L-257 is a DDAH inhibitor which reduces levels of NO. Synthesis of ester analo...

Computational study on the inhibitor binding mode and allosteric regulation mechanism in hepatitis C virus NS3/4A protein.

Directory of Open Access Journals (Sweden)

Weiwei Xue

Full Text Available HCV NS3/4A protein is an attractive therapeutic target responsible for harboring serine protease and RNA helicase activities during the viral replication. Small molecules binding at the interface between the protease and helicase domains can stabilize the closed conformation of the protein and thus block the catalytic function of HCV NS3/4A protein via an allosteric regulation mechanism. But the detailed mechanism remains elusive. Here, we aimed to provide some insight into the inhibitor binding mode and allosteric regulation mechanism of HCV NS3/4A protein by using computational methods. Four simulation systems were investigated. They include: apo state of HCV NS3/4A protein, HCV NS3/4A protein in complex with an allosteric inhibitor and the truncated form of the above two systems. The molecular dynamics simulation results indicate HCV NS3/4A protein in complex with the allosteric inhibitor 4VA adopts a closed conformation (inactive state, while the truncated apo protein adopts an open conformation (active state. Further residue interaction network analysis suggests the communication of the domain-domain interface play an important role in the transition from closed to open conformation of HCV NS3/4A protein. However, the inhibitor stabilizes the closed conformation through interaction with several key residues from both the protease and helicase domains, including His57, Asp79, Asp81, Asp168, Met485, Cys525 and Asp527, which blocks the information communication between the functional domains interface. Finally, a dynamic model about the allosteric regulation and conformational changes of HCV NS3/4A protein was proposed and could provide fundamental insights into the allosteric mechanism of HCV NS3/4A protein function regulation and design of new potent inhibitors.

Design, Synthesis and in vitro Biochemical Activity of Novel Amino Acid Sulfonohydrazide Inhibitors of MurC.

Science.gov (United States)

Frlan, Rok; Kovač, Andreja; Blanot, Didier; Gobec, Stanislav; Pečar, Slavko; Obreza, Aleš

2011-06-01

Mur ligases are essential enzymes involved in the cytoplasmic steps of peptidoglycan synthesis which remain attractive, yet unexploited targets. In order to develop new antibacterial agents, we have designed a series of new MurC and MurD inhibitors bearing amino acid sulfonohydrazide moiety. The L-Leu series of this class displayed the highest enzyme inhibition with IC50 in the concentration range between 100 and 500 µM, with L-Thr, L-Pro and L-Ala derivatives being inactive. The most promising compound of the series also expressed weak antibacterial activity against S. aureus with MIC = 128 µg/mL.

Small ring constrained peptidomimetics. Synthesis of epoxy peptidomimetics, inhibitors of cysteine proteases.

Science.gov (United States)

Demarcus, M; Ganadu, M L; Mura, G M; Porcheddu, A; Quaranta, L; Reginato, G; Taddei, M

2001-02-09

Different dipeptide analogues containing an oxirane ring in the place of the peptidic bond were prepared starting from naturally occurring amino acids. N-Fmoc-amino aldehydes were transformed into the corresponding methoxyvinyl derivatives through a Wittig reaction, and the addition of PhSeCl gave a series of different alpha-phenylselenyl aldehydes. Mukajiama reaction with silylketene acetals gave an intermediate product that was finally transformed into the desired oxiranyl peptidomimetics. Following this strategy we were able to control three new contiguous stereocenters starting from the enantiomerically pure amino acid. The dipeptide analogues could be used in SPPS on a SASRIN resin as the final epoxides were relatively unstable under acidic conditions. Moreover the synthesis of the single dipeptide mimetics was carried out on solid phase to generate a small library of epoxy peptidomimetics. Some of the products prepared in this work resulted as time-dependent reversible inhibitors of cysteine protease.

Fragment-based discovery of potent inhibitors of the anti-apoptotic MCL-1 protein.

Science.gov (United States)

Petros, Andrew M; Swann, Steven L; Song, Danying; Swinger, Kerren; Park, Chang; Zhang, Haichao; Wendt, Michael D; Kunzer, Aaron R; Souers, Andrew J; Sun, Chaohong

2014-03-15

Apoptosis is regulated by the BCL-2 family of proteins, which is comprised of both pro-death and pro-survival members. Evasion of apoptosis is a hallmark of malignant cells. One way in which cancer cells achieve this evasion is thru overexpression of the pro-survival members of the BCL-2 family. Overexpression of MCL-1, a pro-survival protein, has been shown to be a resistance factor for Navitoclax, a potent inhibitor of BCL-2 and BCL-XL. Here we describe the use of fragment screening methods and structural biology to drive the discovery of novel MCL-1 inhibitors from two distinct structural classes. Specifically, cores derived from a biphenyl sulfonamide and salicylic acid were uncovered in an NMR-based fragment screen and elaborated using high throughput analog synthesis. This culminated in the discovery of selective and potent inhibitors of MCL-1 that may serve as promising leads for medicinal chemistry optimization efforts. Copyright © 2014 Elsevier Ltd. All rights reserved.

Bluetongue virus non-structural protein 1 is a positive regulator of viral protein synthesis

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Boyce Mark

2012-08-01

Full Text Available Abstract Background Bluetongue virus (BTV is a double-stranded RNA (dsRNA virus of the Reoviridae family, which encodes its genes in ten linear dsRNA segments. BTV mRNAs are synthesised by the viral RNA-dependent RNA polymerase (RdRp as exact plus sense copies of the genome segments. Infection of mammalian cells with BTV rapidly replaces cellular protein synthesis with viral protein synthesis, but the regulation of viral gene expression in the Orbivirus genus has not been investigated. Results Using an mRNA reporter system based on genome segment 10 of BTV fused with GFP we identify the protein characteristic of this genus, non-structural protein 1 (NS1 as sufficient to upregulate translation. The wider applicability of this phenomenon among the viral genes is demonstrated using the untranslated regions (UTRs of BTV genome segments flanking the quantifiable Renilla luciferase ORF in chimeric mRNAs. The UTRs of viral mRNAs are shown to be determinants of the amount of protein synthesised, with the pre-expression of NS1 increasing the quantity in each case. The increased expression induced by pre-expression of NS1 is confirmed in virus infected cells by generating a replicating virus which expresses the reporter fused with genome segment 10, using reverse genetics. Moreover, NS1-mediated upregulation of expression is restricted to mRNAs which lack the cellular 3′ poly(A sequence identifying the 3′ end as a necessary determinant in specifically increasing the translation of viral mRNA in the presence of cellular mRNA. Conclusions NS1 is identified as a positive regulator of viral protein synthesis. We propose a model of translational regulation where NS1 upregulates the synthesis of viral proteins, including itself, and creates a positive feedback loop of NS1 expression, which rapidly increases the expression of all the viral proteins. The efficient translation of viral reporter mRNAs among cellular mRNAs can account for the observed

Bluetongue virus non-structural protein 1 is a positive regulator of viral protein synthesis.

Science.gov (United States)

Boyce, Mark; Celma, Cristina C P; Roy, Polly

2012-08-29

Bluetongue virus (BTV) is a double-stranded RNA (dsRNA) virus of the Reoviridae family, which encodes its genes in ten linear dsRNA segments. BTV mRNAs are synthesised by the viral RNA-dependent RNA polymerase (RdRp) as exact plus sense copies of the genome segments. Infection of mammalian cells with BTV rapidly replaces cellular protein synthesis with viral protein synthesis, but the regulation of viral gene expression in the Orbivirus genus has not been investigated. Using an mRNA reporter system based on genome segment 10 of BTV fused with GFP we identify the protein characteristic of this genus, non-structural protein 1 (NS1) as sufficient to upregulate translation. The wider applicability of this phenomenon among the viral genes is demonstrated using the untranslated regions (UTRs) of BTV genome segments flanking the quantifiable Renilla luciferase ORF in chimeric mRNAs. The UTRs of viral mRNAs are shown to be determinants of the amount of protein synthesised, with the pre-expression of NS1 increasing the quantity in each case. The increased expression induced by pre-expression of NS1 is confirmed in virus infected cells by generating a replicating virus which expresses the reporter fused with genome segment 10, using reverse genetics. Moreover, NS1-mediated upregulation of expression is restricted to mRNAs which lack the cellular 3' poly(A) sequence identifying the 3' end as a necessary determinant in specifically increasing the translation of viral mRNA in the presence of cellular mRNA. NS1 is identified as a positive regulator of viral protein synthesis. We propose a model of translational regulation where NS1 upregulates the synthesis of viral proteins, including itself, and creates a positive feedback loop of NS1 expression, which rapidly increases the expression of all the viral proteins. The efficient translation of viral reporter mRNAs among cellular mRNAs can account for the observed replacement of cellular protein synthesis with viral protein

Down-regulation of UDP-glucose dehydrogenase affects glycosaminoglycans synthesis and motility in HCT-8 colorectal carcinoma cells

Energy Technology Data Exchange (ETDEWEB)

Wang, Tsung-Pao; Pan, Yun-Ru; Fu, Chien-Yu; Chang, Hwan-You, E-mail: hychang@life.nthu.edu.tw

2010-10-15

UDP-glucose dehydrogenase (UGDH) catalyzes oxidation of UDP-glucose to yield UDP-glucuronic acid, a precursor of hyaluronic acid (HA) and other glycosaminoglycans (GAGs) in extracellular matrix. Although association of extracellular matrix with cell proliferation and migration has been well documented, the importance of UGDH in these behaviors is not clear. Using UGDH-specific small interference RNA to treat HCT-8 colorectal carcinoma cells, a decrease in both mRNA and protein levels of UGDH, as well as the cellular UDP-glucuronic acid and GAG production was observed. Treatment of HCT-8 cells with either UGDH-specific siRNA or HA synthesis inhibitor 4-methylumbelliferone effectively delayed cell aggregation into multicellular spheroids and impaired cell motility in both three-dimensional collagen gel and transwell migration assays. The reduction in cell aggregation and migration rates could be restored by addition of exogenous HA. These results indicate that UGDH can regulate cell motility through the production of GAG. The enzyme may be a potential target for therapeutic intervention of colorectal cancers.

A new class of HIV-1 protease inhibitor: the crystallographic structure, inhibition and chemical synthesis of an aminimide peptide isostere.

Science.gov (United States)

Rutenber, E E; McPhee, F; Kaplan, A P; Gallion, S L; Hogan, J C; Craik, C S; Stroud, R M

1996-09-01

The essential role of HIV-1 protease (HIV-1 PR) in the viral life cycle makes it an attractive target for the development of substrate-based inhibitors that may find efficacy as anti-AIDS drugs. However, resistance has arisen to potent peptidomimetic drugs necessitating the further development of novel chemical backbones for diversity based chemistry focused on probing the active site for inhibitor interactions and binding modes that evade protease resistance. AQ148 is a potent inhibitor of HIV-1 PR and represents a new class of transition state analogues incorporating an aminimide peptide isostere. A 3-D crystallographic structure of AQ148, a tetrapeptide isostere, has been determined in complex with its target HIV-1 PR to a resolution of 2.5 A and used to evaluate the specific structural determinants of AQ148 potency and to correlate structure-activity relationships within the class of related compounds. AQ148 is a competitive inhibitor of HIV-1 PR with a Ki value of 137 nM. Twenty-nine derivatives have been synthesized and chemical modifications have been made at the P1, P2, P1', and P2' sites. The atomic resolution structure of AQ148 bound to HIV-1 PR reveals both an inhibitor binding mode that closely resembles that of other peptidomimetic inhibitors and specific protein/inhibitor interactions that correlate with structure-activity relationships. The structure provides the basis for the design, synthesis and evaluation of the next generation of hydroxyethyl aminimide inhibitors. The aminimide peptide isostere is a scaffold with favorable biological properties well suited to both the combinatorial methods of peptidomimesis and the rational design of potent and specific substrate-based analogues.

Amino acid metabolism in dairy cows and their regulation in milk synthesis.

Science.gov (United States)

Wang, Feiran; Shi, Haitao; Wang, Shuxiang; Wang, Yajing; Cao, Zhijun; Li, Shengli

2018-06-10

Reducing dietary crude protein (CP) and supplementing with certain amino acids (AAs) has been known as a potential solution to improve nitrogen (N) efficiency in dairy production. Thus understanding how AAs are utilized in various sites along the gut is critical. AA flow from the intestine to portal-drained viscera (PDV) and liver then to the mammary gland was elaborated in this article. Recoveries in individual AA in PDV and liver seem to share similar AA pattern with input: output ratio in mammary gland, which subdivides essential AA (EAA) into two groups, lysine (Lys) and branched-chain AA (BCAA) in group 1, input: output ratio > 1; methionine (Met), histidine (His), phenylalanine (Phe) etc. in group 2, input: output ratio close to 1. AAs in the mammary gland are either utilized for milk protein synthesis or retained as body tissue, or catabolized. The fractional removal of AAs and the number and activity of AA transporters together contribute to the ability of AAs going through mammary cells. Mammalian target of rapamycin (mTOR) pathway is closely related to milk protein synthesis and provides alternatives for AA regulation of milk protein synthesis, which connects AA with lactose synthesis via α-lactalbumin (gene: LALBA) and links with milk fat synthesis via sterol regulatory element-binding transcription protein 1 (SREBP1) and peroxisome proliferator-activated receptor (PPAR). Overall, AA flow across various tissues reveal AA metabolism and utilization in dairy cows on one hand. While the function of AA in the biosynthesis of milk protein, fat and lactose at both transcriptional and posttranscriptional level from another angle provides the possibility for us to regulate them for higher efficiency. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Src regulates the activity of SIRT2

International Nuclear Information System (INIS)

Choi, You Hee; Kim, Hangun; Lee, Sung Ho; Jin, Yun-Hye; Lee, Kwang Youl

2014-01-01

Highlights: • Src decreases the protein levels of Sirt2. • Src inhibitor and knockdown of Src increase the protein levels of Sirt2. • Src interacts with and phosphorylates Sirt2. • Src regulate the activity of Sirt2. - Abstract: SIRT2 is a mammalian member of the Sirtuin family of NAD + -dependent protein deacetylases. The tyrosine kinase Src is involved in a variety of cellular signaling pathways, leading to the induction of DNA synthesis, cell proliferation, and cytoskeletal reorganization. The function of SIRT2 is modulated by post-translational modifications; however, the precise molecular signaling mechanism of SIRT2 through interactions with c-Src has not yet been established. In this study, we investigated the potential regulation of SIRT2 function by c-Src. We found that the protein levels of SIRT2 were decreased by c-Src, and subsequently rescued by the addition of a Src specific inhibitor, SU6656, or by siRNA-mediated knockdown of c-Src. The c-Src interacts with and phosphorylates SIRT2 at Tyr104. c-Src also showed the ability to regulate the deacetylation activity of SIRT2. Investigation on the phosphorylation of SIRT2 suggested that this was the method of c-Src-mediated SIRT2 regulation

Synthesis of the highly selective p38 MAPK inhibitor UR-13756 for possible therapeutic use in Werner syndrome.

Science.gov (United States)

Bagley, Mark C; Davis, Terence; Rokicki, Michal J; Widdowson, Caroline S; Kipling, David

2010-02-01

UR-13756 is a potent and selective p38 mitogen-activated protein kinase (MAPK) inhibitor, reported to have good bioavailability and pharmacokinetic properties and, thus, is of potential use in the treatment of accelerated aging in Werner syndrome. Irradiation of 2-chloroacrylonitrile and methylhydrazine in ethanol at 100 °C gives 1-methyl-3-aminopyrazole, which reacts with 4-fluorobenzaldehyde and a ketone, obtained by Claisen condensation of 4-picoline, in a Hantzsch-type 3-component hereocyclocondensation, to give the pyrazolopyridine UR-13756. UR-13756 shows p38 MAPK inhibitory activity in human telomerase reverse transcriptase-immortalized HCA2 dermal fibroblasts, with an IC(50) of 80 nm, as shown by ELISA, is 100% efficacious for up to 24 h at 1.0 μm and displays excellent kinase selectivity over the related stress-activated c-Jun kinases. In addition, UR-13756 is an effective p38 inhibitor at 1.0 μm in Werner syndrome cells, as shown by immunoblot. The convergent synthesis of UR-13756 is realized using microwave dielectric heating and provides a highly selective inhibitor that shows excellent selectivity for p38 MAPK over c-Jun N-terminal kinase.

AMPD2 Regulates GTP Synthesis and is Mutated in a Potentially-Treatable Neurodegenerative Brainstem Disorder

Science.gov (United States)

Akizu, Naiara; Cantagrel, Vincent; Schroth, Jana; Cai, Na; Vaux, Keith; McCloskey, Douglas; Naviaux, Robert K.; Vleet, Jeremy Van; Fenstermaker, Ali G.; Silhavy, Jennifer L.; Scheliga, Judith S.; Toyama, Keiko; Morisaki, Hiroko; Sonmez, Fatma Mujgan; Celep, Figen; Oraby, Azza; Zaki, Maha S.; Al-Baradie, Raidah; Faqeih, Eissa; Saleh, Mohammad; Spencer, Emily; Rosti, Rasim Ozgur; Scott, Eric; Nickerson, Elizabeth; Gabriel, Stacey; Morisaki, Takayuki; Holmes, Edward W.; Gleeson, Joseph G.

2013-01-01

Purine biosynthesis and metabolism, conserved in all living organisms, is essential for cellular energy homeostasis and nucleic acids synthesis. The de novo synthesis of purine precursors is under tight negative feedback regulation mediated by adenosine and guanine nucleotides. We describe a new distinct early-onset neurodegenerative condition resulting from mutations in the adenosine monophosphate deaminase 2 gene (AMPD2). Patients have characteristic brain imaging features of pontocerebellar hypoplasia (PCH), due to loss of brainstem and cerebellar parenchyma. We found that AMPD2 plays an evolutionary conserved role in the maintenance of cellular guanine nucleotide pools by regulating the feedback inhibition of adenosine derivatives on de novo purine synthesis. AMPD2 deficiency results in defective GTP-dependent initiation of protein translation, which can be rescued by administration of purine precursors. These data suggest AMPD2-related PCH as a new, potentially treatable early-onset neurodegenerative disease. PMID:23911318

Zonation of heme synthesis enzymes in mouse liver and their regulation by β-catenin and Ha-ras.

Science.gov (United States)

Braeuning, Albert; Schwarz, Michael

2010-11-01

Cytochrome P450 (CYP) hemoproteins play an important role in hepatic biotransformation. Recently, β-catenin and Ha-ras signaling have been identified as players controlling transcription of various CYP genes in mouse liver. The aim of the present study was to analyze the role of β-catenin and Ha-ras in the regulation of heme synthesis. Heme synthesis-related gene expression was analyzed in normal liver, in transgenic mice expressing activated β-catenin or Ha-ras, and in hepatomas. Regulation of the aminolevulinate dehydratase promoter was studied in vitro. Elevated expression of mRNAs and proteins involved in heme biosynthesis was linked to β-catenin activation in perivenous hepatocytes, in transgenic hepatocytes, and in hepatocellular tumors. Stimulation of the aminolevulinate dehydratase promoter by β-catenin was independent of the β-catenin/T-cell-specific transcription factor dimer. By contrast, activation of Ha-ras repressed heme synthesis-related gene expression. The present data suggest that β-catenin enhances the expression of both CYPs and heme synthesis-related genes, thus coordinating the availability of CYP apoprotein and its prosthetic group heme. The reciprocal regulation of heme synthesis by β-catenin and Ha-ras-dependent signaling supports our previous hypothesis that antagonistic action of these pathways plays a major role in the control of zonal gene expression in healthy mouse liver and aberrant expression patterns in hepatocellular tumors.

The multi-targeted kinase inhibitor sorafenib inhibits enterovirus 71 replication by regulating IRES-dependent translation of viral proteins.

Science.gov (United States)

Gao, Meng; Duan, Hao; Liu, Jing; Zhang, Hao; Wang, Xin; Zhu, Meng; Guo, Jitao; Zhao, Zhenlong; Meng, Lirong; Peng, Yihong

2014-06-01

The activation of ERK and p38 signal cascade in host cells has been demonstrated to be essential for picornavirus enterovirus 71 (EV71) replication and up-regulation of virus-induced cyclooxygenase-2 (COX-2)/prostaglandins E2 (PGE2) expression. The aim of this study was to examine the effects of sorafenib, a clinically approved anti-cancer multi-targeted kinase inhibitor, on the propagation and pathogenesis of EV71, with a view to its possible mechanism and potential use in the design of therapy regimes for Hand foot and mouth disease (HFMD) patients with life threatening neurological complications. In this study, non-toxic concentrations of sorafenib were shown to inhibit the yield of infectious progeny EV71 (clinical BC08 strain) by about 90% in three different cell types. A similar inhibitory effect of sorafenib was observed on the synthesis of both viral genomic RNA and the VP1 protein. Interestingly, sorafenib exerted obvious inhibition of the EV71 internal ribosomal entry site (IRES)-mediated translation, the first step in picornavirus replication, by linking it to a firefly luciferase reporter gene. Sorafenib was also able to prevent both EV71-induced CPE and the activation of ERK and p38, which contributes to up-regulation COX-2/PGE2 expression induced by the virus. Overall, this study shows that sorafenib strongly inhibits EV71 replication at least in part by regulating viral IRES-dependent translation of viral proteins, indicating a novel potential strategy for the treatment of HFMD patients with severe neurological complications. To our knowledge, this is the first report that investigates the mechanism by which sorafenib inhibits EV71 replication. Copyright © 2014 Elsevier B.V. All rights reserved.

Rapid Assessment of Resistance to Antibiotic Inhibitors of Protein Synthesis in the Gram-Positive Pathogens, Enterococcus faecalis and Streptococcus pneumoniae, Based on Evaluation of the Lytic Response.

Science.gov (United States)

Otero, Fátima; Tamayo, María; Santiso, Rebeca; Gosálvez, Jaime; Bou, Germán; Fernández, José Luis

2017-04-01

A novel assay for rapid determination of resistance to antibiotic inhibitors of protein synthesis was developed for the gram-positive pathogens, Enterococcus faecalis and Streptococcus pneumoniae. To this purpose, a lytic response was obtained by a brief incubation with lysozyme or a mixture of lysozyme, Triton X-100, and EDTA for E. faecalis (n = 82) and S. pneumoniae (n = 51), respectively. Lysis was quantified by visualizing the released nucleoids. Antibiotic-susceptible bacteria treated with Clinical and Laboratory Standards Institute (CLSI) breakpoint doses of erythromycin, azithromycin, or doxycycline that inhibited protein synthesis demonstrated a large reduction of lysed cells with respect to the control, that is, without antibiotics. However, cell lysis prevention was much lower in nonsusceptible strains, with unsuccessful inhibition of protein synthesis. ROC analysis showed that a reduction value of ≥35.6% and ≥40.4% discriminates susceptible and nonsusceptible strains for erythromycin and for doxycycline, respectively, in E. faecalis, whereas ≥20.0% is adequate for both macrolides and doxycycline in S. pneumoniae. Resistant stains were identified in 90-120 min with sensitivity and specificity between 91.7% and 100%. This is a proof of concept that evaluation of the lytic response may be a rapid and efficient test for determination of resistance to antibiotic inhibitors of protein synthesis.

Novel diamide-based inhibitors of IMPDH.

Science.gov (United States)

Gu, Henry H; Iwanowicz, Edwin J; Guo, Junqing; Watterson, Scott H; Shen, Zhongqi; Pitts, William J; Dhar, T G Murali; Fleener, Catherine A; Rouleau, Katherine; Sherbina, N Z; Witmer, Mark; Tredup, Jeffrey; Hollenbaugh, Diane

2002-05-06

A series of novel amide-based small molecule inhibitors of inosine monophosphate dehydrogenase is described. The synthesis and the structure-activity relationships (SARs) derived from in vitro studies are presented.

A Heme-responsive Regulator Controls Synthesis of Staphyloferrin B in Staphylococcus aureus.

Science.gov (United States)

Laakso, Holly A; Marolda, Cristina L; Pinter, Tyler B; Stillman, Martin J; Heinrichs, David E

2016-01-01

Staphylococcus aureus possesses a multitude of mechanisms by which it can obtain iron during growth under iron starvation conditions. It expresses an effective heme acquisition system (the iron-regulated surface determinant system), it produces two carboxylate-type siderophores staphyloferrin A and staphyloferrin B (SB), and it expresses transporters for many other siderophores that it does not synthesize. The ferric uptake regulator protein regulates expression of genes encoding all of these systems. Mechanisms of fine-tuning expression of iron-regulated genes, beyond simple iron regulation via ferric uptake regulator, have not been uncovered in this organism. Here, we identify the ninth gene of the sbn operon, sbnI, as encoding a ParB/Spo0J-like protein that is required for expression of genes in the sbn operon from sbnD onward. Expression of sbnD-I is drastically decreased in an sbnI mutant, and the mutant does not synthesize detectable SB during early phases of growth. Thus, SB-mediated iron acquisition is impaired in an sbnI mutant strain. We show that the protein forms dimers and tetramers in solution and binds to DNA within the sbnC coding region. Moreover, we show that SbnI binds heme and that heme-bound SbnI does not bind DNA. Finally, we show that providing exogenous heme to S. aureus growing in an iron-free medium results in delayed synthesis of SB. This is the first study in S. aureus that identifies a DNA-binding regulatory protein that senses heme to control gene expression for siderophore synthesis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Co-ordinate transcriptional regulation of dopamine synthesis genes by alpha-synuclein in human neuroblastoma cell lines.

Science.gov (United States)

Baptista, Melisa J; O'Farrell, Casey; Daya, Sneha; Ahmad, Rili; Miller, David W; Hardy, John; Farrer, Matthew J; Cookson, Mark R

2003-05-01

Abnormal accumulation of alpha-synuclein in Lewy bodies is a neuropathological hallmark of both sporadic and familial Parkinson's disease (PD). Although mutations in alpha-synuclein have been identified in autosomal dominant PD, the mechanism by which dopaminergic cell death occurs remains unknown. We investigated transcriptional changes in neuroblastoma cell lines transfected with either normal or mutant (A30P or A53T) alpha-synuclein using microarrays, with confirmation of selected genes by quantitative RT-PCR. Gene products whose expression was found to be significantly altered included members of diverse functional groups such as stress response, transcription regulators, apoptosis-inducing molecules, transcription factors and membrane-bound proteins. We also found evidence of altered expression of dihydropteridine reductase, which indirectly regulates the synthesis of dopamine. Because of the importance of dopamine in PD, we investigated the expression of all the known genes in dopamine synthesis. We found co-ordinated downregulation of mRNA for GTP cyclohydrolase, sepiapterin reductase (SR), tyrosine hydroxylase (TH) and aromatic acid decarboxylase by wild-type but not mutant alpha-synuclein. These were confirmed at the protein level for SR and TH. Reduced expression of the orphan nuclear receptor Nurr1 was also noted, suggesting that the co-ordinate regulation of dopamine synthesis is regulated through this transcription factor.

Glycation inhibitors extend yeast chronological lifespan by reducing advanced glycation end products and by back regulation of proteins involved in mitochondrial respiration.

Science.gov (United States)

Kazi, Rubina S; Banarjee, Reema M; Deshmukh, Arati B; Patil, Gouri V; Jagadeeshaprasad, Mashanipalya G; Kulkarni, Mahesh J

2017-03-06

Advanced Glycation End products (AGEs) are implicated in aging process. Thus, reducing AGEs by using glycation inhibitors may help in attenuating the aging process. In this study using Saccharomyces cerevisiae yeast system, we show that Aminoguanidine (AMG), a well-known glycation inhibitor, decreases the AGE modification of proteins in non-calorie restriction (NR) (2% glucose) and extends chronological lifespan (CLS) similar to that of calorie restriction (CR) condition (0.5% glucose). Proteomic analysis revealed that AMG back regulates the expression of differentially expressed proteins especially those involved in mitochondrial respiration in NR condition, suggesting that it switches metabolism from fermentation to respiration, mimicking CR. AMG induced back regulation of differentially expressed proteins could be possibly due to its chemical effect or indirectly by glycation inhibition. To delineate this, Metformin (MET), a structural analog of AMG and a mild glycation inhibitor and Hydralazine (HYD), another potent glycation inhibitor but not structural analog of AMG were used. HYD was more effective than MET in mimicking AMG suggesting that glycation inhibition was responsible for restoration of differentially expressed proteins. Thus glycation inhibitors particularly AMG, HYD and MET extend yeast CLS by reducing AGEs, modulating the expression of proteins involved in mitochondrial respiration and possibly by scavenging glucose. This study reports the role of glycation in aging process. In the non-caloric restriction condition, carbohydrates such as glucose promote protein glycation and reduce CLS. While, the inhibitors of glycation such as AMG, HYD, MET mimic the caloric restriction condition by back regulating deregulated proteins involved in mitochondrial respiration which could facilitate shift of metabolism from fermentation to respiration and extend yeast CLS. These findings suggest that glycation inhibitors can be potential molecules that can be used

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

Science.gov (United States)

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

2011-01-28

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

AMPD2 regulates GTP synthesis and is mutated in a potentially treatable neurodegenerative brainstem disorder.

Science.gov (United States)

Akizu, Naiara; Cantagrel, Vincent; Schroth, Jana; Cai, Na; Vaux, Keith; McCloskey, Douglas; Naviaux, Robert K; Van Vleet, Jeremy; Fenstermaker, Ali G; Silhavy, Jennifer L; Scheliga, Judith S; Toyama, Keiko; Morisaki, Hiroko; Sonmez, Fatma M; Celep, Figen; Oraby, Azza; Zaki, Maha S; Al-Baradie, Raidah; Faqeih, Eissa A; Saleh, Mohammed A M; Spencer, Emily; Rosti, Rasim Ozgur; Scott, Eric; Nickerson, Elizabeth; Gabriel, Stacey; Morisaki, Takayuki; Holmes, Edward W; Gleeson, Joseph G

2013-08-01

Purine biosynthesis and metabolism, conserved in all living organisms, is essential for cellular energy homeostasis and nucleic acid synthesis. The de novo synthesis of purine precursors is under tight negative feedback regulation mediated by adenosine and guanine nucleotides. We describe a distinct early-onset neurodegenerative condition resulting from mutations in the adenosine monophosphate deaminase 2 gene (AMPD2). Patients have characteristic brain imaging features of pontocerebellar hypoplasia (PCH) due to loss of brainstem and cerebellar parenchyma. We found that AMPD2 plays an evolutionary conserved role in the maintenance of cellular guanine nucleotide pools by regulating the feedback inhibition of adenosine derivatives on de novo purine synthesis. AMPD2 deficiency results in defective GTP-dependent initiation of protein translation, which can be rescued by administration of purine precursors. These data suggest AMPD2-related PCH as a potentially treatable early-onset neurodegenerative disease. Copyright © 2013 Elsevier Inc. All rights reserved.

Induction of glutathione synthesis in human hepatocytes by acute and chronic arsenic exposure: Differential roles of mitogen-activated protein kinases

International Nuclear Information System (INIS)

Hou, Yongyong; Wang, Yi; Wang, Huihui; Xu, Yuanyuan

2014-01-01

Highlights: • Arsenic exposure increased intracellular levels of glutathione. • Mitogen-activated protein kinases were involved in glutathione homeostasis. • ERK contributed to glutathione synthesis during acute arsenic exposure. • Glutathione synthesis was regulated by p38 at least in part independent of NRF2 during chronic arsenic exposure. - Abstract: Glutathione (GSH) is a vital component of antioxidant defense which protects cells from toxic insults. Previously we found intracellular GSH was involved in cell resistance against arsenic-induced cytotoxicity. However, molecular mechanisms of GSH homeostasis during arsenic exposure are largely undefined. Here, we investigated roles of mitogen-activated protein kinases (MAPKs) in GSH synthesis pathway with two arsenic exposure strategies by using Chang human hepatocytes. In one strategy, acute arsenic exposure (20 μM, 24 h) was applied, as MAPK signaling is generally considered to be transient. In the other one, chronic arsenic exposure (500 nM, 20 weeks) was applied, which mimicked the general human exposure to arsenic. We found that acute arsenic exposure activated extracellular signal-regulated 1/2 kinases (ERK1/2) and c-Jun N-terminal kinase (JNK) in parallel with increased transcription and nuclear translocation of factor-erythroid 2-related factor 2 (NRF2) and enhanced expression of γ-glutamyl cysteine ligase catalytic subunit (GCLC), resulting in elevated intracellular GSH levels. Specific ERK inhibitor abolished arsenic-induced NRF2 nuclear translocation and GSH synthesis. During chronic arsenic exposure which induced a malignant cellular phenotype, continuous p38 activation and NRF2 nuclear translocation were observed with enhanced GSH synthesis. Specific p38 inhibitor attenuated arsenic-enhanced GSH synthesis without changing NRF2 nuclear translocation. Taken together, our results indicate MAPK pathways play an important role in cellular GSH homeostasis in response to arsenic. However, the

The protein phosphatase-1/inhibitor-2 complex differentially regulates GSK3 dephosphorylation and increases sarcoplasmic/endoplasmic reticulum calcium ATPase 2 levels

International Nuclear Information System (INIS)

King, Taj D.; Gandy, Johanna C.; Bijur, Gautam N.

2006-01-01

The ubiquitously expressed protein glycogen synthase kinase-3 (GSK3) is constitutively active, however its activity is markedly diminished following phosphorylation of Ser21 of GSK3α and Ser9 of GSK3β. Although several kinases are known to phosphorylate Ser21/9 of GSK3, for example Akt, relatively much less is known about the mechanisms that cause the dephosphorylation of GSK3 at Ser21/9. In the present study KCl-induced plasma membrane depolarization of SH-SY5Y cells, which increases intracellular calcium concentrations caused a transient decrease in the phosphorylation of Akt at Thr308 and Ser473, and GSK3 at Ser21/9. Overexpression of the selective protein phosphatase-1 inhibitor protein, inhibitor-2, increased basal GSK3 phosphorylation at Ser21/9 and significantly blocked the KCl-induced dephosphorylation of GSK3β, but not GSK3α. The phosphorylation of Akt was not affected by the overexpression of inhibitor-2. GSK3 activity is known to affect sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) levels. Overexpression of inhibitor-2 or treatment of cells with the GSK3 inhibitors lithium and SB216763 increased the levels of SERCA2. These results indicate that the protein phosphatase-1/inhibitor-2 complex differentially regulates GSK3 dephosphorylation induced by KCl and that GSK3 activity regulates SERCA2 levels

A multidisciplinary study of 3-(β-d-glucopyranosyl)-5-substituted-1,2,4-triazole derivatives as glycogen phosphorylase inhibitors: Computation, synthesis, crystallography and kinetics reveal new potent inhibitors.

Science.gov (United States)

Kun, Sándor; Begum, Jaida; Kyriakis, Efthimios; Stamati, Evgenia C V; Barkas, Thomas A; Szennyes, Eszter; Bokor, Éva; Szabó, Katalin E; Stravodimos, George A; Sipos, Ádám; Docsa, Tibor; Gergely, Pál; Moffatt, Colin; Patraskaki, Myrto S; Kokolaki, Maria C; Gkerdi, Alkistis; Skamnaki, Vassiliki T; Leonidas, Demetres D; Somsák, László; Hayes, Joseph M

2018-03-10

3-(β-d-Glucopyranosyl)-5-substituted-1,2,4-triazoles have been revealed as an effective scaffold for the development of potent glycogen phosphorylase (GP) inhibitors but with the potency very sensitive to the nature of the alkyl/aryl 5-substituent (Kun et al., Eur. J. Med. Chem. 2014, 76, 567). For a training set of these ligands, quantum mechanics-polarized ligand docking (QM-PLD) demonstrated good potential to identify larger differences in potencies (predictive index PI = 0.82) and potent inhibitors with K i 's synthesis. The compounds were prepared in O-perbenzoylated forms by either ring transformation of 5-β-d-glucopyranosyl tetrazole by N-benzyl-arenecarboximidoyl chlorides, ring closure of C-(β-d-glucopyranosyl)formamidrazone with aroyl chlorides, or that of N-(β-d-glucopyranosylcarbonyl)arenethiocarboxamides by hydrazine, followed by deprotections. Kinetics experiments against rabbit muscle GPb (rmGPb) and human liver GPa (hlGPa) revealed five compounds as potent low μM inhibitors with three of these on the submicromolar range for rmGPa. X-ray crystallographic analysis sourced the potency to a combination of favorable interactions from the 1,2,4-triazole and suitable aryl substituents in the GP catalytic site. The compounds also revealed promising calculated pharmacokinetic profiles. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Novel Small Molecule Inhibitors of Choline Kinase Identified by Fragment-Based Drug Discovery.

Science.gov (United States)

Zech, Stephan G; Kohlmann, Anna; Zhou, Tianjun; Li, Feng; Squillace, Rachel M; Parillon, Lois E; Greenfield, Matthew T; Miller, David P; Qi, Jiwei; Thomas, R Mathew; Wang, Yihan; Xu, Yongjin; Miret, Juan J; Shakespeare, William C; Zhu, Xiaotian; Dalgarno, David C

2016-01-28

Choline kinase α (ChoKα) is an enzyme involved in the synthesis of phospholipids and thereby plays key roles in regulation of cell proliferation, oncogenic transformation, and human carcinogenesis. Since several inhibitors of ChoKα display antiproliferative activity in both cellular and animal models, this novel oncogene has recently gained interest as a promising small molecule target for cancer therapy. Here we summarize our efforts to further validate ChoKα as an oncogenic target and explore the activity of novel small molecule inhibitors of ChoKα. Starting from weakly binding fragments, we describe a structure based lead discovery approach, which resulted in novel highly potent inhibitors of ChoKα. In cancer cell lines, our lead compounds exhibit a dose-dependent decrease of phosphocholine, inhibition of cell growth, and induction of apoptosis at low micromolar concentrations. The druglike lead series presented here is optimizable for improvements in cellular potency, drug target residence time, and pharmacokinetic parameters. These inhibitors may be utilized not only to further validate ChoKα as antioncogenic target but also as novel chemical matter that may lead to antitumor agents that specifically interfere with cancer cell metabolism.

Methyl gallate from Acer barbinerve decreases melanin synthesis in Mel-Ab cells.

Science.gov (United States)

Kim, In Wook; jeong, Hyo-Soon; Kim, Jin Kyu; Lee, Jin-Koo; Kim, Hak Rim; Yun, Hye-Young; Baek, Kwang Jin; Kwon, Nyoun Soo; Park, Kyoung-Chan; Kim, Dong-Seok

2015-01-01

Methyl gallate (MG) was isolated from the bark of Acer barbinerve, which has traditionally been used in Oriental medicine. In the present study, we examined the effects of MG on melanin synthesis in Mel-Ab melanocyte cells. MG decreased melanin pigmentation in a concentration-dependent manner, but did not directly inhibit tyrosinase activity. Further analysis showed that MG had no effect on extracellular signal-regulated kinase (ERK) activation, but induced phosphorylation of glycogen synthase kinase (GSK)3β, which is known to increase β-catenin accumulation. Accordingly, the β-catenin level was increased by MG. However, a specific GSK3β inhibitor did not rescue the MG-induced inhibition of melanogenesis. Additionally, MG decreased the protein expression of microphthalmia-associated transcription factor (MITF) and tyrosinase, which regulate melanin synthesis. Based on these results, we conclude that MG inhibits melanogenesis by decreasing the expression of MITF and tyrosinase.

Severe Acute Respiratory Syndrome-Coronavirus Papain-Like Novel Protease Inhibitors: Design, Synthesis, Protein-Ligand X-ray Structure and Biological Evaluation

Energy Technology Data Exchange (ETDEWEB)

Ghosh, Arun K.; Takayama, Jun; Rao, Kalapala Venkateswar; Ratia, Kiira; Chaudhuri, Rima; Mulhearn, Debbie C.; Lee, Hyun; Nichols, Daniel B.; Baliji, Surendranath; Baker, Susan C.; Johnson, Michael E.; Mesecar, Andrew D. (Purdue); (UC); (UIC)

2012-02-21

The design, synthesis, X-ray crystal structure, molecular modeling, and biological evaluation of a series of new generation SARS-CoV PLpro inhibitors are described. A new lead compound 3 (6577871) was identified via high-throughput screening of a diverse chemical library. Subsequently, we carried out lead optimization and structure-activity studies to provide a series of improved inhibitors that show potent PLpro inhibition and antiviral activity against SARS-CoV infected Vero E6 cells. Interestingly, the (S)-Me inhibitor 15h (enzyme IC{sub 50} = 0.56 {mu}M; antiviral EC{sub 50} = 9.1 {mu}M) and the corresponding (R)-Me 15g (IC{sub 50} = 0.32 {mu}M; antiviral EC{sub 50} = 9.1 {mu}M) are the most potent compounds in this series, with nearly equivalent enzymatic inhibition and antiviral activity. A protein-ligand X-ray structure of 15g-bound SARS-CoV PLpro and a corresponding model of 15h docked to PLpro provide intriguing molecular insight into the ligand-binding site interactions.

Target sites for chemical regulation of strigolactone signaling

Directory of Open Access Journals (Sweden)

Hidemitsu eNakamura

2014-11-01

Full Text Available Demands for plant growth regulators (chemicals that control plant growth are increasing globally, especially in developing countries. Both positive and negative plant growth regulators are widely used to enhance crop production and to suppress unwanted shoot growth, respectively. Strigolactones (SLs are multifunctional molecules that function as phytohormones, inhibiting shoot branching and also functioning in the rhizospheric communication with symbiotic fungi and parasitic weeds. Therefore, it is anticipated that chemicals that regulate the functions of SLs will be widely used in agricultural applications. Although the SL biosynthetic pathway is not fully understood, it has been demonstrated that beta-carotene isomerases, carotenoid cleavage dioxygenases (CCDs, and a cytochrome P450 monooxygenase are involved in strigolactone biosynthesis. A CCD inhibitor, abamine, which is also an inhibitor of abscisic acid biosynthesis, reduces the levels of SL in several plant species and reduces the germination rate of Orobanche minor seeds grown with tobacco. On the basis of the structure of abamine, several chemicals have been designed to specifically inhibit CCDs during SL synthesis. Cytochrome P450 monooxygenase is another target enzyme in the development of SL biosynthesis inhibitors, and the triazole-derived TIS series of chemicals is known to include SL biosynthesis inhibitors, although their target enzyme has not been identified. Recently, DWARF14 (D14 has been shown to be a receptor for SLs, and the D-ring moiety of SL is essential for its recognition by D14. A variety of SL agonists are currently under development and most agonists commonly contain the D-ring or a D-ring-like moiety. Several research groups have also resolved the crystal structure of D14 in the last two years. It is expected that this information on the D14 structure will be invaluable not only for developing SL agonists with novel structures but also in the design of inhibitors

Regulation of antibody synthesis in the X-irradiated Mexican axolotl

International Nuclear Information System (INIS)

Charlemagne, J.

1981-01-01

The effects of X-irradiation were studied on the Mexican axolotl antibody synthesis. To reduce the anti-horse red blood cell (HRBC) antibody titers, 150 rd and smaller doses are ineffective, 200-450 rd are increasingly effective, and 700 rd are maximally effective (and lethal). A significant enhancement of the anti-HRBC titers was observed in low doses (50-150 rd X-irradiated animals). This enhancement was also observed when a low X-ray dose was applied only on the thymic areas. In whole body, but thymus area-shielded, 100 rd X-irradiated animals, the anti-HRBC titers were similar to those of the nonirradiated, HRBC-immunized control group. To explain these phenomena, it is suggested that a radiosensitive, adult thymectomy-sensitive and hydrocortisone-sensitive suppressor T cell subpopulation regulates the antibody synthesis in the axolotl. (orig.) [de

Regulation of antibody synthesis in the X-irradiated Mexican axolotl

Energy Technology Data Exchange (ETDEWEB)

Charlemagne, J.

1981-09-01

The effects of X-irradiation were studied on the Mexican axolotl antibody synthesis. To reduce the anti-horse red blood cell (HRBC) antibody titers, 150 rd and smaller doses are ineffective, 200-450 rd are increasingly effective, and 700 rd are maximally effective (and lethal). A significant enhancement of the anti-HRBC titers was observed in low doses (50-150 rd X-irradiated animals). This enhancement was also observed when a low X-ray dose was applied only on the thymic areas. In whole body, but thymus area-shielded, 100 rd X-irradiated animals, the anti-HRBC titers were similar to those of the nonirradiated, HRBC-immunized control group. To explain these phenomena, it is suggested that a radiosensitive, adult thymectomy-sensitive and hydrocortisone-sensitive suppressor T cell subpopulation regulates the antibody synthesis in the axolotl.

A novel pulse-chase SILAC strategy measures changes in protein decay and synthesis rates induced by perturbation of proteostasis with an Hsp90 inhibitor.

Directory of Open Access Journals (Sweden)

Ivo Fierro-Monti

Full Text Available Standard proteomics methods allow the relative quantitation of levels of thousands of proteins in two or more samples. While such methods are invaluable for defining the variations in protein concentrations which follow the perturbation of a biological system, they do not offer information on the mechanisms underlying such changes. Expanding on previous work [1], we developed a pulse-chase (pc variant of SILAC (stable isotope labeling by amino acids in cell culture. pcSILAC can quantitate in one experiment and for two conditions the relative levels of proteins newly synthesized in a given time as well as the relative levels of remaining preexisting proteins. We validated the method studying the drug-mediated inhibition of the Hsp90 molecular chaperone, which is known to lead to increased synthesis of stress response proteins as well as the increased decay of Hsp90 "clients". We showed that pcSILAC can give information on changes in global cellular proteostasis induced by treatment with the inhibitor, which are normally not captured by standard relative quantitation techniques. Furthermore, we have developed a mathematical model and computational framework that uses pcSILAC data to determine degradation constants kd and synthesis rates Vs for proteins in both control and drug-treated cells. The results show that Hsp90 inhibition induced a generalized slowdown of protein synthesis and an increase in protein decay. Treatment with the inhibitor also resulted in widespread protein-specific changes in relative synthesis rates, together with variations in protein decay rates. The latter were more restricted to individual proteins or protein families than the variations in synthesis. Our results establish pcSILAC as a viable workflow for the mechanistic dissection of changes in the proteome which follow perturbations. Data are available via ProteomeXchange with identifier PXD000538.

A Novel Pulse-Chase SILAC Strategy Measures Changes in Protein Decay and Synthesis Rates Induced by Perturbation of Proteostasis with an Hsp90 Inhibitor

Science.gov (United States)

Fierro-Monti, Ivo; Racle, Julien; Hernandez, Celine; Waridel, Patrice; Hatzimanikatis, Vassily; Quadroni, Manfredo

2013-01-01

Standard proteomics methods allow the relative quantitation of levels of thousands of proteins in two or more samples. While such methods are invaluable for defining the variations in protein concentrations which follow the perturbation of a biological system, they do not offer information on the mechanisms underlying such changes. Expanding on previous work [1], we developed a pulse-chase (pc) variant of SILAC (stable isotope labeling by amino acids in cell culture). pcSILAC can quantitate in one experiment and for two conditions the relative levels of proteins newly synthesized in a given time as well as the relative levels of remaining preexisting proteins. We validated the method studying the drug-mediated inhibition of the Hsp90 molecular chaperone, which is known to lead to increased synthesis of stress response proteins as well as the increased decay of Hsp90 “clients”. We showed that pcSILAC can give information on changes in global cellular proteostasis induced by treatment with the inhibitor, which are normally not captured by standard relative quantitation techniques. Furthermore, we have developed a mathematical model and computational framework that uses pcSILAC data to determine degradation constants kd and synthesis rates Vs for proteins in both control and drug-treated cells. The results show that Hsp90 inhibition induced a generalized slowdown of protein synthesis and an increase in protein decay. Treatment with the inhibitor also resulted in widespread protein-specific changes in relative synthesis rates, together with variations in protein decay rates. The latter were more restricted to individual proteins or protein families than the variations in synthesis. Our results establish pcSILAC as a viable workflow for the mechanistic dissection of changes in the proteome which follow perturbations. Data are available via ProteomeXchange with identifier PXD000538. PMID:24312217

Gram-scale synthesis of the p38α MAPK-inhibitor VX-745 for preclinical studies into Werner syndrome.

Science.gov (United States)

Bagley, Mark C; Davis, Terence; Dix, Matthew C; Fusillo, Vincenzo; Pigeaux, Morgane; Rokicki, Michal J; Kipling, David

2010-09-01

The ATP-competitive p38α MAPK inhibitor VX-745 exhibits an exquisite kinase selectivity profile, is effective in blocking p38 stress signaling in Werner syndrome dermal fibroblasts, has efficacy in clinical trials and may have therapeutic value against Werner syndrome. Previous synthetic routes, however, have only resulted in milligram quantities suitable for cell-based studies, whereas gram quantities would be required for in vivo use. Microwave irradiation using a stop-flow monomodal microwave reactor has been found to facilitate scale-up of the synthesis of VX-745. Ullmann-type C-S bond formation using thiophenol, chloropyridazine, copper(I) catalyst and diol ligand proceeds rapidly and efficiently in this apparatus for elaboration to the pyrimido[1,6-b]pyridazinone core of VX-745 on gram scale and with good overall yield. This method delivers the p38 inhibitor VX-745 in sufficient quantities for preclinical studies to rescue the aging phenotype in Werner syndrome.

A Heme-responsive Regulator Controls Synthesis of Staphyloferrin B in Staphylococcus aureus*♦

Science.gov (United States)

Laakso, Holly A.; Marolda, Cristina L.; Pinter, Tyler B.; Stillman, Martin J.; Heinrichs, David E.

2016-01-01

Staphylococcus aureus possesses a multitude of mechanisms by which it can obtain iron during growth under iron starvation conditions. It expresses an effective heme acquisition system (the iron-regulated surface determinant system), it produces two carboxylate-type siderophores staphyloferrin A and staphyloferrin B (SB), and it expresses transporters for many other siderophores that it does not synthesize. The ferric uptake regulator protein regulates expression of genes encoding all of these systems. Mechanisms of fine-tuning expression of iron-regulated genes, beyond simple iron regulation via ferric uptake regulator, have not been uncovered in this organism. Here, we identify the ninth gene of the sbn operon, sbnI, as encoding a ParB/Spo0J-like protein that is required for expression of genes in the sbn operon from sbnD onward. Expression of sbnD–I is drastically decreased in an sbnI mutant, and the mutant does not synthesize detectable SB during early phases of growth. Thus, SB-mediated iron acquisition is impaired in an sbnI mutant strain. We show that the protein forms dimers and tetramers in solution and binds to DNA within the sbnC coding region. Moreover, we show that SbnI binds heme and that heme-bound SbnI does not bind DNA. Finally, we show that providing exogenous heme to S. aureus growing in an iron-free medium results in delayed synthesis of SB. This is the first study in S. aureus that identifies a DNA-binding regulatory protein that senses heme to control gene expression for siderophore synthesis. PMID:26534960

Antitumor effects of a sirtuin inhibitor, tenovin-6, against gastric cancer cells via death receptor 5 up-regulation.

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Sachiko Hirai

Full Text Available Up-regulated sirtuin 1 (SIRT1, an NAD+-dependent class III histone deacetylase, deacetylates p53 and inhibits its transcriptional activity, leading to cell survival. SIRT1 overexpression has been reported to predict poor survival in some malignancies, including gastric cancer. However, the antitumor effect of SIRT1 inhibition remains elusive in gastric cancer. Here, we investigated the antitumor mechanisms of a sirtuin inhibitor, tenovin-6, in seven human gastric cancer cell lines (four cell lines with wild-type TP53, two with mutant-type TP53, and one with null TP53. Interestingly, tenovin-6 induced apoptosis in all cell lines, not only those with wild-type TP53, but also mutant-type and null versions, accompanied by up-regulation of death receptor 5 (DR5. In the KatoIII cell line (TP53-null, DR5 silencing markedly attenuated tenovin-6-induced apoptosis, suggesting that the pivotal mechanism behind its antitumor effects is based on activation of the death receptor signal pathway. Although endoplasmic reticulum stress caused by sirtuin inhibitors was reported to induce DR5 up-regulation in other cancer cell lines, we could not find marked activation of its related molecules, such as ATF6, PERK, and CHOP, in gastric cancer cells treated with tenovin-6. Tenovin-6 in combination with docetaxel or SN-38 exerted a slight to moderate synergistic cytotoxicity against gastric cancer cells. In conclusion, tenovin-6 has potent antitumor activity against human gastric cancer cells via DR5 up-regulation. Our results should be helpful for the future clinical development of sirtuin inhibitors.

Urotensin II contributes to collagen synthesis and up-regulates Egr-1 expression in cultured pulmonary arterial smooth muscle cells through the ERK1/2 pathway

Energy Technology Data Exchange (ETDEWEB)

Li, Wei [Biomedical Engineering Institute, School of Control Science and Engineering, Shandong University, Jinan 250061 (China); Cai, Zhifeng; Liu, Mengmeng [Department of Pediatrics, Qilu Hospital, Shandong University, Jinan 250012 (China); Zhao, Cuifen, E-mail: zhaocuifen@sdu.edu.cn [Department of Pediatrics, Qilu Hospital, Shandong University, Jinan 250012 (China); Li, Dong [Research Room of Hypothermia Medicine, Qilu Hospital, Shandong University, Jinan 250012 (China); Lv, Chenguang; Wang, Yuping; Xu, Tengfei [Biomedical Engineering Institute, School of Control Science and Engineering, Shandong University, Jinan 250061 (China)

2015-11-27

Aim: The objective of this study was to investigate the effects of urotensin II (UII) treatment on the proliferation and collagen synthesis of cultured rat pulmonary arterial smooth muscle cells (PASMCs) and to explore whether these effects are mediated by mitogen-activated protein kinase (MAPK) signaling pathways and early growth response 1 (Egr-1). Methods: The proliferation of cultured PASMCs stimulated with different doses of UII was detected by BrdU incorporation. The mRNA expression levels of procollagen I (procol I), procollagen III (procol III), extracellular regulated protein kinase 1/2 (ERK1/2), stress-stimulated protein kinase (Sapk), p38 MAPK (p38), and Egr-1 mRNA in cultured PASMCs after treatment with UII, the UII-specific antagonist urantide, and the ERK1/2 inhibitor PD98059 were detected by real-time polymerase chain reaction (PCR), and the protein expression levels of procol I, procol III, phosphorylated (p)-ERK1/2, p-Sapk, p-p38, and Egr-1 were detected by Western blotting. Results: Treatment with UII increased the proliferation of cultured PASMCs in a dose-dependent manner (P < 0.05). However, treatment with urantide and PD98059 inhibited the promoting effect of UII on PASMC proliferation (P < 0.05). Real-time PCR analysis showed that UII up-regulated the expression of procol I, procol III, ERK1/2, Sapk, and Egr-1 mRNA (P < 0.05), but not p38 mRNA. However, the up-regulating effect of UII was inhibited by PD98059 and urantide. Western blotting analysis showed that UII increased the synthesis of collagen I, collagen III, p-ERK1/2, p-Sapk, and Egr-1, and these effects also were inhibited by PD98059 and urantide (P < 0.05). Conclusions: Egr-1 participates in the UII-mediated proliferation and collagen synthesis of cultured rat PASMCs via activation of the ERK1/2 signaling pathway.

Thiocarbamate-Directed Tandem Olefination-Intramolecular Sulfuration of Two Ortho C-H Bonds: Application to Synthesis of a COX-2 Inhibitor.

Science.gov (United States)

Li, Wendong; Zhao, Yingwei; Mai, Shaoyu; Song, Qiuling

2018-02-16

A palladium-catalyzed dual ortho C-H bond activation of aryl thiocarbamates is developed. This tandem reaction initiates by thiocarbamate-directed ortho C-H palladation, which leads to favorable olefin insertion rather than reductive elimination. The oxidative Heck reaction followed by another C-H activation and sulfuration affords the dual-functionalized products. This reaction provides a concise route to the S,O,C multisubstituted benzene skeleton which could be successfully applied for the synthesis of a COX-2 inhibitor.

Extra-adrenal glucocorticoid synthesis: immune regulation and aspects on local organ homeostasis.

Science.gov (United States)

Talabér, Gergely; Jondal, Mikael; Okret, Sam

2013-11-05

Systemic glucocorticoids (GCs) mainly originate from de novo synthesis in the adrenal cortex under the control of the hypothalamus-pituitary-adrenal (HPA)-axis. However, research during the last 1-2 decades has revealed that additional organs express the necessary enzymes and have the capacity for de novo synthesis of biologically active GCs. This includes the thymus, intestine, skin and the brain. Recent research has also revealed that locally synthesized GCs most likely act in a paracrine or autocrine manner and have significant physiological roles in local homeostasis, cell development and immune cell activation. In this review, we summarize the nature, regulation and known physiological roles of extra-adrenal GC synthesis. We specifically focus on the thymus in which GC production (by both developing thymocytes and epithelial cells) has a role in the maintenance of proper immunological function. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Model for the mechanism and regulation of chitosan synthesis in Mucor rouxii

International Nuclear Information System (INIS)

Davis, L.L.; Bartnicki-Garcia, S.

1984-01-01

The cell walls of mucoraceous fungi are characterized by the joint occurrence of chitosan and chitin, the β-1,4-linked polysaccharides of G1cN and G1cNAc, respectively. It has been proposed that chitosan is made from chitin by enzymatic deacetylation, but the evidence is inconclusive since the deacetylase isolated from Mucor rouxii is effective against glycol chitin, but not against genuine chitin; consequently, chitosan synthesis in vitro was not achieved. The authors discovered that the same deacetylase can deacetylate chitin efficiently if it is allowed to act on chitin chains as they are being formed; i.e. the simultaneous presence and operation of chitin synthetase and chitin deacetylase is required for chitosan synthesis. Subsequent studies on the effect of digitonin on chitosan synthesis were the basis for a model the authors have developed for the regulation of chitosan and chitin syntheses in vivo

Mig-6 plays a critical role in the regulation of cholesterol homeostasis and bile acid synthesis.

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Bon Jeong Ku

Full Text Available The disruption of cholesterol homeostasis leads to an increase in cholesterol levels which results in the development of cardiovascular disease. Mitogen Inducible Gene 6 (Mig-6 is an immediate early response gene that can be induced by various mitogens, stresses, and hormones. To identify the metabolic role of Mig-6 in the liver, we conditionally ablated Mig-6 in the liver using the Albumin-Cre mouse model (Alb(cre/+Mig-6(f/f; Mig-6(d/d. Mig-6(d/d mice exhibit hepatomegaly and fatty liver. Serum levels of total, LDL, and HDL cholesterol and hepatic lipid were significantly increased in the Mig-6(d/d mice. The daily excretion of fecal bile acids was significantly decreased in the Mig-6(d/d mice. DNA microarray analysis of mRNA isolated from the livers of these mice showed alterations in genes that regulate lipid metabolism, bile acid, and cholesterol synthesis, while the expression of genes that regulate biliary excretion of bile acid and triglyceride synthesis showed no difference in the Mig-6(d/d mice compared to Mig-6(f/f controls. These results indicate that Mig-6 plays an important role in cholesterol homeostasis and bile acid synthesis. Mice with liver specific conditional ablation of Mig-6 develop hepatomegaly and increased intrahepatic lipid and provide a novel model system to investigate the genetic and molecular events involved in the regulation of cholesterol homeostasis and bile acid synthesis. Defining the molecular mechanisms by which Mig-6 regulates cholesterol homeostasis will provide new insights into the development of more effective ways for the treatment and prevention of cardiovascular disease.

Regulation of dopamine synthesis and release in striatal and prefrontal cortical brain slices

International Nuclear Information System (INIS)

Wolf, M.E.

1986-01-01

Brain slices were used to investigate the role of nerve terminal autoreceptors in modulating dopamine (DA) synthesis and release in striatum and prefrontal cortex. Accumulation of dihydroxyphenylalanine (DOPA) was used as an index of tyrosine hydroxylation in vitro. Nomifensine, a DA uptake blocker, inhibited DOPA synthesis in striatal but not prefrontal slices. This effect was reversed by the DA antagonist sulpiride, suggesting it involved activation of DA receptors by elevated synaptic levels of DA. The autoreceptor-selective agonist EMD-23-448 also inhibited striatal but not prefrontal DOPA synthesis. DOPA synthesis was stimulated in both brain regions by elevated K + , however only striatal synthesis could be further enhanced by sulpiride. DA release was measured by following the efflux of radioactivity from brain slices prelabeled with [ 3 H]-DA. EMD-23-448 and apomorphine inhibited, while sulpiride enhanced, the K + -evoked overflow of radioactivity from both striatal and prefrontal cortical slices. These findings suggest that striatal DA nerve terminals possess autoreceptors which modulate tyrosine hydroxylation as well as autoreceptors which modulate release. Alternatively, one site may be coupled to both functions through distinct transduction mechanisms. In contrast, autoreceptors on prefrontal cortical terminals appear to regulate DA release but not DA synthesis

Novel guanidine-based inhibitors of inosine monophosphate dehydrogenase.

Science.gov (United States)

Iwanowicz, Edwin J; Watterson, Scott H; Liu, Chunjian; Gu, Henry H; Mitt, Toomas; Leftheris, Katerina; Barrish, Joel C; Fleener, Catherine A; Rouleau, Katherine; Sherbina, N Z; Hollenbaugh, Diane L

2002-10-21

A series of novel guanidine-based small molecule inhibitors of inosine monophosphate dehydrogenase (IMPDH) was explored. IMPDH catalyzes the rate determining step in guanine nucleotide biosynthesis and is a target for anticancer, immunosuppressive and antiviral therapy. The synthesis and the structure-activity relationships (SARs), derived from in vitro studies, for this new series of inhibitors is given.

Physical change in cytoplasmic messenger ribonucleoproteins in cells treated with inhibitors of mRNA transcription

International Nuclear Information System (INIS)

Dreyfuss, G.; Adam, S.A.; Choi, Y.D.

1984-01-01

Exposure of intact cells to UV light brings about cross-linking of polyadenylated mRNA to a set of cytoplasmic proteins which are in direct contact with the mRNA in vivo. Substantial amounts of an additional protein of molecular weight 38,000 become cross-linked to the mRNA when cells are treated with inhibitors of mRNA synthesis (actinomycin D, camptothecin, and 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole) or after infection with vesicular stomatitis virus. Cordycepin, which inhibits polyadenylation but not mRNA synthesis, has no such effect. Inhibitors of protein synthesis and of rRNA synthesis are also without effect on 38K cross-linking to mRNA. The onset of the effect of inhibitors of mRNA synthesis on the UV cross-linkable interaction between mRNA and 38K is rapid and reaches a maximal level in less than 60 min, and it is completely and rapidly reversible. In cells treated with actinomycin D, the amount of 38K which becomes cross-linked to mRNA is proportional to the extent of inhibition of mRNA synthesis. The association of 38K with mRNA during transcriptional arrest does not require protein synthesis because simultaneous treatment with the protein synthesis inhibitor emetine does not interfere with it. The effectors which promote the interaction of 38K with mRNA do not affect the proteins which are in contact with polyadenylated heterogeneous nuclear RNA and do not markedly affect protein synthesis in the cell. The 38K protein can be isolated with the polyribosomal polyadenylated fraction from which it was purified, and monoclonal antibodies against it were prepared

The Zymomonas mobilis regulator hfq contributes to tolerance against multiple lignocellulosic pretreatment inhibitors

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Lu Tse-Yuan S

2010-05-01

mutant construction in Z. mobilis, and constructed a Gateway compatible expression plasmid for use in Z. mobilis for the first time. We have also used genetics to show Z. mobilis Hfq and S. cerevisiae Lsm proteins play important roles in resisting multiple, important industrially relevant inhibitors. The conserved nature of this global regulator offers the potential to apply insights from these fundamental studies for further industrial strain development.

DGAT inhibitors for obesity.

Science.gov (United States)

Matsuda, Daisuke; Tomoda, Hiroshi

2007-10-01

Obesity is characterized by the accumulation of triacylglycerol in adipocytes. Diacylglycerol acyltransferase (DGAT) catalyzes the final reaction of triacylgycerol synthesis. Two isozymes of DGAT, DGAT1 and DGAT2, have been reported. Increased DGAT2 activity has a role in steatosis, while DGAT1 plays a role in very (V)LDL synthesis; increased plasma VLDL concentrations may promote obesity and thus DGAT1 is considered a potential therapeutic target of inhibition for obesity control. Several DGAT inhibitors of natural and synthetic origin have been reported, and their future prospect as anti-obesity drugs is discussed in this review.

The design and synthesis of novel SGLT2 inhibitors: C-glycosides with benzyltriazolopyridinone and phenylhydantoin as the aglycone moieties.

Science.gov (United States)

Guo, Cheng; Hu, Min; DeOrazio, Russell J; Usyatinsky, Alexander; Fitzpatrick, Kevin; Zhang, Zhenjun; Maeng, Jun-Ho; Kitchen, Douglas B; Tom, Susan; Luche, Michele; Khmelnitsky, Yuri; Mhyre, Andrew J; Guzzo, Peter R; Liu, Shuang

2014-07-01

The sodium glucose co-transporter 2 (SGLT2) has received considerable attention in recent years as a target for the treatment of type 2 diabetes mellitus. This report describes the design, synthesis and structure-activity relationship (SAR) of C-glycosides with benzyltriazolopyridinone and phenylhydantoin as the aglycone moieties as novel SGLT2 inhibitors. Compounds 5p and 33b demonstrated high potency in inhibiting SGLT2 and high selectivity against SGLT1. The in vitro ADMET properties of these compounds will also be discussed. Copyright © 2014 Elsevier Ltd. All rights reserved.

Novel peptide-based protease inhibitors

DEFF Research Database (Denmark)

Roodbeen, Renée

of novel peptide-based protease inhibitors, efforts were made towards improved methods for peptide synthesis. The coupling of Fmoc-amino acids onto N-methylated peptidyl resins was investigated. These couplings can be low yielding and the effect of the use of microwave heating combined with the coupling...

The MMP inhibitor (R)-2-(N-benzyl-4-(2-[18F]fluoroethoxy)phenylsulphonamido) -N-hydroxy-3-methylbutanamide: Improved precursor synthesis and fully automated radiosynthesis

International Nuclear Information System (INIS)

Wagner, Stefan; Faust, Andreas; Breyholz, Hans-Joerg; Schober, Otmar; Schaefers, Michael; Kopka, Klaus

2011-01-01

Summary: The CGS 25966 derivative (R)-2-(N-Benzyl-4-(2-[ 18 F]fluoroethoxy)phenyl-sulphonamido) -N-hydroxy-3-methylbutanamide [ 18 F]9 represents a very potent radiolabelled matrix metalloproteinase inhibitor. For first human PET studies it is mandatory to have a fully automated radiosynthesis and a straightforward precursor synthesis available. The realisation of both requirements is reported herein. In particular, the corresponding precursor 8 was obtained in a reliable 7 step synthesis with an overall chemical yield of 2.3%. Furthermore, the target compound [ 18 F]9 was prepared with a radiochemical yield of 14.8±3.9% (not corrected for decay).

SYNTHESIS OF VOLTAGES OF UNIFORM PWM IN TIME REGULATION

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A. G. Stryzhniou

2014-01-01

Full Text Available The article describes a process of synthesis and qualitative assessment of the harmonic composition of voltages of multiple and single PWM pulses in time regulation, being, along with amplitude, frequency and phase method, one of control methods of an asynchronous motor. The main point of time regulation is that a pause after any two single PWM pulses with different polarity or after any two groups of multiple PWM pulses with different polarity changes during a process of regulation. Feature of time regulation is that a motor has fast response in the range of small-signal of control and good linearity of speed-torque characteristics in the whole control range. Analytical expressions of parameters of PWM pulses ai and ti are obtained which allow to simplify considerably a process of formation and implementation of time regulation using tabular or indexed-tabular methods. These expressions allow not only to define voltage amplitude of  harmonic but also to perform qualitative assessment of harmonic composition of output voltages at time regulation. It is specified that harmonic frequencies wi = w0/q change in inverse proportion to magnitude of parameter q during a process of regulation and there is a replacement of a fundamental frequency by frequencies of higher harmonics.The offered approach allows to synthesize voltage of uniform single and multiple PWM pulses and to perform their comparative and qualitative analysis and the obtained expressions can be used at modeling of AC motor work. Voltage of multiple PWM pulses which is formed using stepped reference voltage with even quantity of steps in a half period and a pause on a zero level has the best parameters by criterion of a minimum of harmonic components and a maximum of a factor of anharmonicity Kнс at time regulation.

Synthesis, conformational analysis, and biological activity of new analogues of thiazole-4-carboxamide adenine dinucleotide (TAD) as IMP dehydrogenase inhibitors.

Science.gov (United States)

Franchetti, Palmarisa; Cappellacci, Loredana; Pasqualini, Michela; Petrelli, Riccardo; Jayaprakasan, Vetrichelvan; Jayaram, Hiremagalur N; Boyd, Donald B; Jain, Manojkumar D; Grifantini, Mario

2005-03-15

Thiazole-4-carboxamide adenine dinucleotide (TAD) analogues T-2'-MeAD (1) and T-3'-MeAD (2) containing, respectively, a methyl group at the ribose 2'-C-, and 3'-C-position of the adenosine moiety, were prepared as potential selective human inosine monophosphate dehydrogenase (IMPDH) type II inhibitors. The synthesis of heterodinucleotides was carried out by CDI-catalyzed coupling reaction of unprotected 2'-C-methyl- or 3'-C-methyl-adenosine 5'-monophosphate with 2',3'-O-isopropylidene-tiazofurin 5'-monophosphate, and then deisopropylidenation. Biological evaluation of dinucleotides 1 and 2 as inhibitors of recombinant human IMPDH type I and type II resulted in a good activity. Inhibition of both isoenzymes by T-2'-MeAD and T-3'-MeAD was noncompetitive with respect to NAD substrate. Binding of T-3'-MeAD was comparable to that of parent compound TAD, while T-2'-MeAD proved to be a weaker inhibitor. However, no significant difference was found in inhibition of the IMPDH isoenzymes. T-2'-MeAD and T-3'-MeAD were found to inhibit the growth of K562 cells (IC(50) 30.7 and 65.0muM, respectively).

Ribonuclease inhibitor 1 regulates erythropoiesis by controlling GATA1 translation.

Science.gov (United States)

Chennupati, Vijaykumar; Veiga, Diogo Ft; Maslowski, Kendle M; Andina, Nicola; Tardivel, Aubry; Yu, Eric Chi-Wang; Stilinovic, Martina; Simillion, Cedric; Duchosal, Michel A; Quadroni, Manfredo; Roberts, Irene; Sankaran, Vijay G; MacDonald, H Robson; Fasel, Nicolas; Angelillo-Scherrer, Anne; Schneider, Pascal; Hoang, Trang; Allam, Ramanjaneyulu

2018-04-02

Ribosomal proteins (RP) regulate specific gene expression by selectively translating subsets of mRNAs. Indeed, in Diamond-Blackfan anemia and 5q- syndrome, mutations in RP genes lead to a specific defect in erythroid gene translation and cause anemia. Little is known about the molecular mechanisms of selective mRNA translation and involvement of ribosomal-associated factors in this process. Ribonuclease inhibitor 1 (RNH1) is a ubiquitously expressed protein that binds to and inhibits pancreatic-type ribonucleases. Here, we report that RNH1 binds to ribosomes and regulates erythropoiesis by controlling translation of the erythroid transcription factor GATA1. Rnh1-deficient mice die between embryonic days E8.5 and E10 due to impaired production of mature erythroid cells from progenitor cells. In Rnh1-deficient embryos, mRNA levels of Gata1 are normal, but GATA1 protein levels are decreased. At the molecular level, we found that RNH1 binds to the 40S subunit of ribosomes and facilitates polysome formation on Gata1 mRNA to confer transcript-specific translation. Further, RNH1 knockdown in human CD34+ progenitor cells decreased erythroid differentiation without affecting myelopoiesis. Our results reveal an unsuspected role for RNH1 in the control of GATA1 mRNA translation and erythropoiesis.

The Serine Protease Inhibitor Neuroserpin Is Required for Normal Synaptic Plasticity and Regulates Learning and Social Behavior

Science.gov (United States)

Reumann, Rebecca; Vierk, Ricardo; Zhou, Lepu; Gries, Frederice; Kraus, Vanessa; Mienert, Julia; Romswinkel, Eva; Morellini, Fabio; Ferrer, Isidre; Nicolini, Chiara; Fahnestock, Margaret; Rune, Gabriele; Glatzel, Markus; Galliciotti, Giovanna

2017-01-01

The serine protease inhibitor neuroserpin regulates the activity of tissue-type plasminogen activator (tPA) in the nervous system. Neuroserpin expression is particularly prominent at late stages of neuronal development in most regions of the central nervous system (CNS), whereas it is restricted to regions related to learning and memory in the…

Estradiol Synthesis in Gut-Associated Lymphoid Tissue: Leukocyte Regulation by a Sexually Monomorphic System.

Science.gov (United States)

Oakley, Oliver R; Kim, Kee Jun; Lin, Po-Ching; Barakat, Radwa; Cacioppo, Joseph A; Li, Zhong; Whitaker, Alexandra; Chung, Kwang Chul; Mei, Wenyan; Ko, CheMyong

2016-12-01

17β-estradiol is a potent sex hormone synthesized primarily by gonads in females and males that regulates development and function of the reproductive system. Recent studies show that 17β-estradiol is locally synthesized in nonreproductive tissues and regulates a myriad of events, including local inflammatory responses. In this study, we report that mesenteric lymph nodes (mLNs) and Peyer's patches (Pps) are novel sites of de novo synthesis of 17β-estradiol. These secondary lymphoid organs are located within or close to the gastrointestinal tract, contain leukocytes, and function at the forefront of immune surveillance. 17β-estradiol synthesis was initially identified using a transgenic mouse with red fluorescent protein coexpressed in cells that express aromatase, the enzyme responsible for 17β-estradiol synthesis. Subsequent immunohistochemistry and tissue culture experiments revealed that aromatase expression was localized to high endothelial venules of these lymphoid organs, and these high endothelial venule cells synthesized 17β-estradiol when isolated and cultured in vitro. Both mLNs and Pps contained 17β-estradiol with concentrations that were significantly higher than those of peripheral blood. Furthermore, the total amount of 17β-estradiol in these organs exceeded that of the gonads. Mice lacking either aromatase or estrogen receptor-β had hypertrophic Pps and mLNs with more leukocytes than their wild-type littermates, demonstrating a role for 17β-estradiol in leukocyte regulation. Importantly, we did not observe any sex-dependent differences in aromatase expression, 17β-estradiol content, or steroidogenic capacity in these lymphoid organs.

In Silico Screening, Synthesis and In Vitro Evaluation of Some Quinazolinone and Pyridine Derivatives as Dihydrofolate Reductase Inhibitors for Anticancer Activity

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A. G. Nerkar

2009-01-01

Full Text Available Dihydrofolate reductase (DHFR is the important target for anticancer drugs belonging to the class of antimetabolites as the enzyme plays important role in the de novo purine synthesis. We here report the in silico screening to obtain best fit molecules as DHFR inhibitors, synthesis of some ʻbest fitʼ quinazolinone from 2-phenyl-3-(substituted-benzilidine-amino quinazolinones (Quinazolinone Shiff's bases QSB1-5 and pyridine-4-carbohydrazide Shiff's bases (ISB1-5 derivatives and their in vitro anticancer assay. Synthesis of the molecules was performed using microwave assisted synthesis. The structures of these molecules were elucidated by IR and 1H-NMR. These compounds were then subjected for in vitro anticancer evaluation against five human cancer cell-lines for anticancer cyto-toxicity assay. Methotrexate (MTX was used as standard for this evaluation to give a comparable inhibition of the cell proliferation by DHFR inhibition. Placlitaxel, adriamycin and 5-fluoro-uracil were also used as standard to give a comparable activity of these compounds with other mechanism of anticancer activity. ISB3 (4-(N, N-dimethyl-amino-phenyl Schiff''s base derivative of pyridine carbohydrazide showed equipotent activity with the standards used in in vitro anticancer assay as per the NCI (National Cancer Institute guidelines.

EFFECTS OF PSYCHOTROPIC DRUGS AS BACTERIAL EFFLUX PUMP INHIBITORS ON QUORUM SENSING REGULATED BEHAVIORS

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Aynur Aybey

2014-10-01

Full Text Available Psychotropic drugs are known to have antimicrobial activity against several groups of microorganisms. The antidepressant agents such as duloxetine, paroxetine, hydroxyzine and venlafaxine are shown to act as efflux pump inhibitors in bacterial cells. In order to the investigation of the effects of psychotropic drugs were determined for clinically significant pathogens by using standart broth microdillusion method. The anti-quorum sensing (anti-QS activity of psychotropic drugs was tested against four test pathogens using the agar well diffusion method. All drugs showed strong inhibitory effect on the growth of S. typhimurium. Additionally, quorum sensing-regulated behaviors of Pseudomonas aeruginosa, including swarming, swimming and twitching motility and alkaline protease production were investigated. Most effective drugs on swarming, swimming and twitching motility and alkaline protease production, respectively, were paroxetine and duloxetine; duloxetine; hydroxyzine and venlafaxine; paroxetine and venlafaxine; venlafaxine. Accordingly, psychotropic drugs were shown strongly anti-QS activity by acting as bacterial efflux pump inhibitors and effection on motility and alkaline protease production of P. aeruginosa.

Synthesis and biological activities of the respiratory chain inhibitor aurachin D and new ring versus chain analogues

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Xu-Wen Li

2013-07-01

Full Text Available Aurachins are myxobacterial 3-farnesyl-4(1H-quinolone derived compounds initially described as respiratory chain inhibitors, more specifically as inhibitors of various cytochrome complexes. They are also known as potent antibiotic compounds. We describe herein the first synthesis of aurachin D through a key Conrad–Limpach reaction. The same strategy was used to reach some ring as opposed to chain analogues, allowing for the description of structure–activity relationships. Biological screening of the analogues showed antiparasitic, cytotoxic, antibacterial and antifungal activities, and depletion of the mitochondrial membrane potential. The strongest activity was found on Plasmodium falciparum with a selectivity index of 345, compared to Vero cells, for the natural product and its geranyl analogue. The loss of mitochondrial membrane potential induced by aurachins in human U-2 OS osteosarcoma cells was studied, showing the best activity for aurachin D and a naphthalene analogue, yet without totally explaining the observed cytotoxic activity of the compounds. Finally, a synthetic entry is given to the complete carboheterocyclic core of aurachin H through the N-oxidation/epoxidation of aurachin D and a shorter chain analogue, followed by subsequent biomimetic cyclization.

Low survival of mice following lethal gamma-irradiation after administration of inhibitors of prostaglandin synthesis

International Nuclear Information System (INIS)

Hofer, M.; Pospisil, M.; Tkadlecek, L.; Viklicka, S.; Pipalova, I.; Hola, J.

1992-01-01

An impairment was observed of the survival of mice subjected to whole-body gamma-irradiation with a lethal dose of 10 Gy and treated with a repeated postirradiation administration of the prostaglandin synthesis inhibitors (PGSIs) indomethacin or diclofenac. Morphological examination of the gastrointestinal tract and estimation of the blood loss into its lumen in animals treated with diclofenac did not show serious damage such as hemorrhages or perforation, but revealed structural injury to the intestinal mucosa indicating inflammatory processes. The lesions found are supposed to be connected with increased intestinal permeability which leads to endotoxin escape from the gut and a subsequent increased mortality rate of irradiated animals. It may be concluded that PGSIs are not suitable for the management of radiation sickness after an exposure to lethal doses of ionizing radiation. (author) 2 tabs., 4 figs., 20 refs

Identification of an inhibitor of the MurC enzyme, which catalyzes an essential step in the peptidoglycan precursor synthesis pathway.

Science.gov (United States)

Zawadzke, Laura E; Norcia, Michael; Desbonnet, Charlene R; Wang, Hong; Freeman-Cook, Kevin; Dougherty, Thomas J

2008-02-01

The pathway for synthesis of the peptidoglycan precursor UDP-N-acetylmuramyl pentapeptide is essential in Gram-positive and Gram-negative bacteria. This pathway has been exploited in the recent past to identify potential new antibiotics as inhibitors of one or more of the Mur enzymes. In the present study, a high-throughput screen was employed to identify potential inhibitors of the Escherichia coli MurC (UDP-N-acetylmuramic acid:L-alanine ligase), the first of four paralogous amino acid-adding enzymes. Inhibition of ATP consumed during the MurC reaction, using an adaptation of a kinase assay format, identified a number of potential inhibitory chemotypes. After nonspecific inhibition testing and chemical attractiveness were assessed, C-1 emerged as a compound for further characterization. The inhibition of MurC by this compound was confirmed in both a kinetic-coupled enzyme assay and a direct nuclear magnetic resonance product detection assay. C-1 was found to be a low micromolar inhibitor of the E. coli MurC reaction, with preferential inhibition by one of two enantiomeric forms. Experiments indicated that it was a competitive inhibitor of ATP binding to the MurC enzyme. Further work with MurC enzymes from several bacterial sources revealed that while the compound was equally effective at inhibiting MurC from genera (Proteus mirabilis and Klebsiella pneumoniae) closely related to E. coli, MurC enzymes from more distant Gram-negative species such as Haemophilus influenzae, Acinetobacter baylyi, and Pseudomonas aeruginosa were not inhibited.

Fluoxetine Is a Potent Inhibitor of Coxsackievirus Replication

OpenAIRE

Zuo, Jun; Quinn, Kevin K.; Kye, Steve; Cooper, Paige; Damoiseaux, Robert; Krogstad, Paul

2012-01-01

No antiviral drugs currently exist for the treatment of enterovirus infections, which are often severe and potentially life threatening. Molecular screening of small molecule libraries identified fluoxetine, a selective serotonin reuptake inhibitor, as a potent inhibitor of coxsackievirus replication. Fluoxetine did not interfere with either viral entry or translation of the viral genome. Instead, fluoxetine and its metabolite norfluoxetine markedly reduced the synthesis of viral RNA and prot...

A regulator of ubiquitin-proteasome activity, 2-hexyldecanol, suppresses melanin synthesis and the appearance of facial hyperpigmented spots.

Science.gov (United States)

Hakozaki, T; Laughlin, T; Zhao, S; Wang, J; Deng, D; Jewell-Motz, E; Elstun, L

2013-07-01

2-Hexyldecanol has long been used in skin-care products, but has not previously been reported as an active ingredient for skin benefits. To evaluate 2-hexyldecanol in in vitro and ex vivo systems and, if found to be active, progress it to topical clinical testing to determine effects on pigmentation in skin. 2-Hexyldecanol was tested in melanocyte cell culture systems (B16 mouse melanoma cells and normal human melanocytes) for its effect on proteolytic activity and melanin production, in the absence and presence of the proteasome-specific inhibitor, MG132. It was further tested in a human skin explant model for its effect on melanin production. Lastly, topically applied 2-hexyldecanol was evaluated for its effect on the appearance of facial pigmentation in an 8-week, randomized, double-blind, vehicle-controlled, split-face incomplete block design study in Chinese women. In submerged cell culture, 2-hexyldecanol upregulated proteolytic activity and decreased melanin synthesis. These effects were antagonized by the proteasome-specific inhibitor MG132. MG132, tested in the absence of 2-hexyldecanol, increased melanin production. In a human skin explant model, topical 2-hexyldecanol suppressed the production of melanin vs. a vehicle control. In a human clinical study in Chinese women (n = 110 observations per test material), a 2-hexyldecanol-containing formulation significantly reduced the appearance of facial hyperpigmented spots vs. its control. These data indicate that regulation of proteasome activity is a viable target for control of melanin production, that 2-hexyldecanol upregulates proteasomal activity in melanocytes, and that topical 2-hexyldecanol reduces the appearance of hyperpigmentation. © 2013 The Authors BJD © 2013 British Association of Dermatologists.

Potential New H1N1 Neuraminidase Inhibitors from Ferulic Acid and Vanillin: Molecular Modelling, Synthesis and in Vitro Assay

Science.gov (United States)

Hariono, Maywan; Abdullah, Nurshariza; Damodaran, K. V.; Kamarulzaman, Ezatul E.; Mohamed, Nornisah; Hassan, Sharifah Syed; Shamsuddin, Shaharum; Wahab, Habibah A.

2016-12-01

We report the computational and experimental efforts in the design and synthesis of novel neuraminidase (NA) inhibitors from ferulic acid and vanillin. Two proposed ferulic acid analogues, MY7 and MY8 were predicted to inhibit H1N1 NA using molecular docking. From these two analogues, we designed, synthesised and evaluated the biological activities of a series of ferulic acid and vanillin derivatives. The enzymatic H1N1 NA inhibition assay showed MY21 (a vanillin derivative) has the lowest IC50 of 50 μM. In contrast, the virus inhibition assay showed MY15, a ferulic acid derivative has the best activity with the EC50 of ~0.95 μM. Modelling studies further suggest that these predicted activities might be due to the interactions with conserved and essential residues of NA with ΔGbind values comparable to those of oseltamivir and zanamivir, the two commercial NA inhibitors.

Synthesis of three bromophenols from red algae as PTP1B inhibitors

Science.gov (United States)

Guo, Shuju; Li, Jing; Li, Ting; Shi, Dayong; Han, Lijun

2011-01-01

Bromophenols are a set of natural products widely distributed in seaweed, most of which exhibit interesting and useful biological activities. To develop a reliable and efficient synthetic route to these natural bromophenols, three of them, 3,4-dibromo-5-(2'-bromo-3',4'-dihydroxy-6'-methoxymethyl-benzyl)-benzene-1,2-diol (compound 9), 3,4-dibromo-5-(2'-bromo-6'-ethoxy methyl-3',4'-dihydroxybenzyl)-benzene-1,2-diol (compound 10), and 3-bromo-4-(3'-bromo-4',5'-dihydroxy benzyl)-5-(ethoxymethyl)-benzene-1,2-diol (compound 14), isolated from red marine algae, have been synthesized in eight steps with an overall yield of 14.4%, 14.4%, and 18.2% respectively, via a practical approach employing bromination, Wolff-Kishner-Huang reduction and a Friedel-Crafts reaction as key steps. The protein tyrosine phosphatase 1B (PTP1B) inhibitory activities of the synthetic compounds were evaluated by the colorimetric assay. The results show that these compounds are moderate PTP1B inhibitors. The synthesis of these bromophenol derivatives makes in vivo studies of their structure-activity relationships and inhibition activity against PTP1B possible.

The protease inhibitor HAI-2, but not HAI-1, regulates matriptase activation and shedding through prostasin

DEFF Research Database (Denmark)

Friis, Stine; Sales, Katiuchia Uzzun; Schafer, Jeffrey Martin

2014-01-01

cells. Whereas ablation of HAI-1 did not affect matriptase in epithelial cells of the small or large intestine, ablation of HAI-2 resulted in the loss of matriptase from both tissues. Gene silencing studies in intestinal Caco-2 cell monolayers revealed that this loss of cell-associated matriptase......The membrane-anchored serine proteases, matriptase and prostasin, and the membrane-anchored serine protease inhibitors, hepatocyte growth factor activator inhibitor (HAI)-1 and HAI-2, are critical effectors of epithelial development and postnatal epithelial homeostasis. Matriptase and prostasin...... form a reciprocal zymogen activation complex that results in the formation of active matriptase and prostasin that are targets for inhibition by HAI-1 and HAI-2. Conflicting data, however, have accumulated as to the existence of auxiliary functions for both HAI-1 and HAI-2 in regulating...

TOR Pathway-Mediated Juvenile Hormone Synthesis Regulates Nutrient-Dependent Female Reproduction in Nilaparvata lugens (Stål).

Science.gov (United States)

Lu, Kai; Chen, Xia; Liu, Wen-Ting; Zhou, Qiang

2016-03-28

The "target of rapamycin" (TOR) nutritional signaling pathway and juvenile hormone (JH) regulation of vitellogenesis has been known for a long time. However, the interplay between these two pathways regulating vitellogenin (Vg) expression remains obscure. Here, we first demonstrated the key role of amino acids (AAs) in activation of Vg synthesis and egg development in Nilaparvata lugens using chemically defined artificial diets. AAs induced the expression of TOR and S6K (S6 kinase), whereas RNAi-mediated silencing of these two TOR pathway genes and rapamycin application strongly inhibited the AAs-induced Vg synthesis. Furthermore, knockdown of Rheb (Ras homologue enriched in brain), TOR, S6K and application of rapamycin resulted in a dramatic reduction in the mRNA levels of jmtN (juvenile hormone acid methyltransferase, JHAMT). Application of JH III on the RNAi (Rheb and TOR) and rapamycin-treated females partially rescued the Vg expression. Conversely, knockdown of either jmtN or met (methoprene-tolerant, JH receptor) and application of JH III had no effects on mRNA levels of Rheb, TOR and S6K and phosphorylation of S6K. In summary, our results demonstrate that the TOR pathway induces JH biosynthesis that in turn regulates AAs-mediated Vg synthesis in N. lugens.

Histone deacetylase inhibitors up-regulate LL-37 expression independent of toll-like receptor mediated signalling in airway epithelial cells

NARCIS (Netherlands)

Liu, Quan; Liu, Juan; Roschmann, Kristina Irene Lisolette; van Egmond, Danielle; Golebski, Korneliusz; Fokkens, Wytske Johanna; Wang, Dehui; van Drunen, Cornelis Maria

2013-01-01

HDAC inhibitors have been proposed as anticancer agents. However, their roles in innate genes expression remain not well known. Cathelicidin LL-37 is one of the few human bactericidal peptides, but the regulation of histone acetylation on LL-37 expression in airway epithelium remains largely

New halogenated phenylcoumarins as tyrosinase inhibitors.

Science.gov (United States)

Matos, Maria João; Santana, Lourdes; Uriarte, Eugenio; Delogu, Giovanna; Corda, Marcella; Fadda, Maria Benedetta; Era, Benedetta; Fais, Antonella

2011-06-01

With the aim to find out structural features for the tyrosinase inhibitory activity, in the present communication we report the synthesis and pharmacological evaluation of a new series of phenylcoumarin derivatives with different number of hydroxyl or ether groups and bromo substituent in the scaffold. The synthesized compounds 5-12 were evaluated as mushroom tyrosinase inhibitors showing, two of them, lower IC(50) than the umbelliferone. Compound 12 (IC(50)=215 μM) is the best tyrosinase inhibitor of this series. Copyright © 2011 Elsevier Ltd. All rights reserved.

Purification, crystallization and preliminary X-ray crystallographic analysis of rice bifunctional α-amylase/subtilisin inhibitor from Oryza sativa

International Nuclear Information System (INIS)

Lin, Yi-Hung; Peng, Wen-Yan; Huang, Yen-Chieh; Guan, Hong-Hsiang; Hsieh, Ying-Cheng; Liu, Ming-Yih; Chang, Tschining; Chen, Chun-Jung

2006-01-01

The crystallization of rice α-amylase/subtilisin bifunctional inhibitor is reported. Rice bifunctional α-amylase/subtilisin inhibitor (RASI) can inhibit both α-amylase from larvae of the red flour beetle (Tribolium castaneum) and subtilisin from Bacillus subtilis. The synthesis of RASI is up-regulated during the late milky stage in developing seeds. The 8.9 kDa molecular-weight RASI from rice has been crystallized using the hanging-drop vapour-diffusion method. According to 1.81 Å resolution X-ray diffraction data from rice RASI crystals, the crystal belongs to space group P2 1 2 1 2, with unit-cell parameters a = 79.99, b = 62.95, c = 66.70 Å. Preliminary analysis indicates two RASI molecules in an asymmetric unit with a solvent content of 44%

Exploiting Chemical Libraries, Structure, and Genomics in the Search for Kinase Inhibitors

NARCIS (Netherlands)

Gray, Nathanael S.; Wodicka, Lisa; Thunnissen, Andy-Mark W.H.; Norman, Thea C.; Kwon, Soojin; Espinoza, F. Hernan; Morgan, David O.; Barnes, Georjana; LeClerc, Sophie; Meijer, Laurent; Kim, Sung-Hou; Lockhart, David J.; Schultz, Peter G.

1998-01-01

Selective protein kinase inhibitors were developed on the basis of the unexpected binding mode of 2,6,9-trisubstituted purines to the adenosine triphosphate-binding site of the human cyclin-dependent kinase 2 (CDK2). By iterating chemical library synthesis and biological screening, potent inhibitors

Design, synthesis, and biological evaluation of enantiomeric beta-N-acetylhexosaminidase inhibitors LABNAc and DABNAc as potential agents against Tay-Sachs and Sandhoff disease.

Science.gov (United States)

Rountree, J S Shane; Butters, Terry D; Wormald, Mark R; Boomkamp, Stephanie D; Dwek, Raymond A; Asano, Naoki; Ikeda, Kyoko; Evinson, Emma L; Nash, Robert J; Fleet, George W J

2009-03-01

N-Acetylhexosaminidases are of considerable importance in mammals and are involved in various significant biological processes. In humans, deficiencies of these enzymes in the lysosome, resulting from inherited genetic defects, cause the glycolipid storage disorders Tay-Sachs and Sandhoff diseases. One promising therapy for these diseases involves the use of beta-N-acetylhexosaminidase inhibitors as chemical chaperones to enhance the enzyme activity above sub-critical levels. Herein we describe the synthesis and biological evaluation of a potent inhibitor, 2-acetamido-1,4-imino-1,2,4-trideoxy-L-arabinitol (LABNAc), in a high-yielding 11-step procedure from D-lyxonolactone. The N-benzyl and N-butyl analogues were also prepared and found to be potent inhibitors. The enantiomers DABNAc and NBn-DABNAc were synthesised from L-lyxonolactone, and were also evaluated. The L-iminosugar LABNAc and its derivatives were found to be potent noncompetitive inhibitors of some beta-N-acetylhexosaminidases, while the D-iminosugar DABNAc and its derivatives were found to be weaker competitive inhibitors. These results support previous work postulating that D-iminosugar mimics inhibit D-glycohydrolases competitively, and that their corresponding L-enantiomers show noncompetitive inhibition of these enzymes. Molecular modelling studies confirm that the spatial organisation in enantiomeric inhibitors leads to a different overlay with the monosaccharide substrate. Initial cell-based studies suggest that NBn-LABNAc can act as a chemical chaperone to enhance the deficient enzyme's activity to levels that may cause a positive pharmacological effect. LABNAc, NBn-LABNAc, and NBu-LABNAc are potent and selective inhibitors of beta-N-acetylhexosaminidase and may be useful as therapeutic agents for treating adult Tay-Sachs and Sandhoff diseases.

IL-6 has no acute effect on the regulation of urea synthesis in vivo in rats

DEFF Research Database (Denmark)

Thomsen, Karen; Aagaard, Niels Kristian; Grønbæk, Henning

2011-01-01

Clinical or experimentally induced, active inflammation up-regulates the in vivo capacity of urea synthesis (CUNS), which promotes nitrogen removal from the body and metabolic catabolism. We have shown that tumor necrosis factor a (TNF-a) up-regulates CUNS and increases interleukin 6 expression (IL......-6) within hours of administration. The described effect of TNF-a on nitrogen homeostasis may, therefore, depend on IL-6....

Caspase-1 inhibitor regulates humoral responses in experimental autoimmune myasthenia gravis via IL-6- dependent inhibiton of STAT3.

Science.gov (United States)

Wang, Cong-Cong; Zhang, Min; Li, Heng; Li, Xiao-Li; Yue, Long-Tao; Zhang, Peng; Liu, Ru-Tao; Chen, Hui; Li, Yan-Bin; Duan, Rui-Sheng

2017-08-24

We have previously demonstrated that Cysteinyl aspartate-specific proteinase-1 (caspase-1) inhibitor ameliorates experimental autoimmune myasthenia gravis (EAMG) by inhibited cellular immune response, via suppressing DC IL-1 β, CD4 + T and γdT cells IL-17 pathways. In this study, we investigated the effect of caspase-1 inhibitor on humoral immune response of EAMG and further explore the underlying mechanisms. An animal model of MG was induced by region 97-116 of the rat AChR α subunit (R97-116 peptide) in Lewis rats. Rats were treated with caspase-1 inhibitor Ac-YVAD-cmk intraperitoneally (i.p.) every second day from day 13 after the first immunization. Flow cytometry, western blot, immunofluorescence, and enzyme-linked immunosorbent assay (ELISA) were performed to evaluate the neuroprotective effect of caspase-1 inhibitor on humoral immune response of EAMG. The results showed that caspase-1 inhibitor reduced the relative affinity of anti-R97-116 IgG, suppressed germinal center response, decreased follicular helper T cells, and increased follicular regulatory T cells and regulatory B cells. In addition, we found that caspase-1 inhibitor inhibited humoral immunity response in EAMG rats via suppressing IL-6-STAT3-Bcl-6 pathways. These results suggest that caspase-1 inhibitor ameliorates EAMG by regulating humoral immune response, thus providing new insights into the development of myasthenia gravis and other autoimmune diseases therapies. Copyright © 2017 Elsevier B.V. All rights reserved.

Aurintricarboxylic acid is a potent inhibitor of phosphofructokinase.

Science.gov (United States)

McCune, S A; Foe, L G; Kemp, R G; Jurin, R R

1989-01-01

Aurintricarboxylic acid (ATA) was found to be a very potent inhibitor of purified rabbit liver phosphofructokinase (PFK), giving 50% inhibition at 0.2 microM. The inhibition was in a manner consistent with interaction at the citrate-inhibitory site of the enzyme. The data suggest that inhibition of PFK by ATA was not due to denaturation of the enzyme or the irreversible binding of inhibitor, since the inhibition could be reversed by addition of allosteric activators of PFK, i.e. fructose 2,6-bisphosphate or AMP. Two other tricarboxylic acids, agaric acid and (-)-hydroxycitrate, were found to inhibit PFK. ATA at much higher concentrations (500 microM) was shown to inhibit fatty acid synthesis from endogenous glycogen in rat hepatocytes; however, protein synthesis was not altered. PMID:2525029

Fibulin-1C, C1 esterase inhibitor and glucose regulated protein 75 interact with the CREC proteins, calumenin and reticulocalbin

DEFF Research Database (Denmark)

Hansen, Gry Aune Westergaard; Ludvigsen, Maja; Jacobsen, Christian

2015-01-01

Affinity purification, immunoprecipitation, gel electrophoresis and mass spectrometry were used to identify fibulin-1C, C1 esterase inhibitor and glucose regulated protein 75, grp75, as binding partners of the CREC proteins, calumenin and reticulocalbin. Surface plasmon resonance was used to verify...... the interaction of all three proteins with each of the CREC proteins. Fibulin-1C interacts with calumenin and reticulocalbin with an estimated dissociation constant around 50-60 nM. The interaction, at least for reticulocalbin, was not dependent upon the presence of Ca2+. C1 esterase inhibitor interacted...

Potent nonnucleoside reverse transcriptase inhibitors target HIV-1 Gag-Pol.

Directory of Open Access Journals (Sweden)

Anna Figueiredo

2006-11-01

Full Text Available Nonnucleoside reverse transcriptase inhibitors (NNRTIs target HIV-1 reverse transcriptase (RT by binding to a pocket in RT that is close to, but distinct, from the DNA polymerase active site and prevent the synthesis of viral cDNA. NNRTIs, in particular, those that are potent inhibitors of RT polymerase activity, can also act as chemical enhancers of the enzyme's inter-subunit interactions. However, the consequences of this chemical enhancement effect on HIV-1 replication are not understood. Here, we show that the potent NNRTIs efavirenz, TMC120, and TMC125, but not nevirapine or delavirdine, inhibit the late stages of HIV-1 replication. These potent NNRTIs enhanced the intracellular processing of Gag and Gag-Pol polyproteins, and this was associated with a decrease in viral particle production from HIV-1-transfected cells. The increased polyprotein processing is consistent with premature activation of the HIV-1 protease by NNRTI-enhanced Gag-Pol multimerization through the embedded RT sequence. These findings support the view that Gag-Pol multimerization is an important step in viral assembly and demonstrate that regulation of Gag-Pol/Gag-Pol interactions is a novel target for small molecule inhibitors of HIV-1 production. Furthermore, these drugs can serve as useful probes to further understand processes involved in HIV-1 particle assembly and maturation.

Novel amide-based inhibitors of inosine 5'-monophosphate dehydrogenase.

Science.gov (United States)

Watterson, Scott H; Liu, Chunjian; Dhar, T G Murali; Gu, Henry H; Pitts, William J; Barrish, Joel C; Fleener, Catherine A; Rouleau, Katherine; Sherbina, N Z; Hollenbaugh, Diane L; Iwanowicz, Edwin J

2002-10-21

A series of novel amide-based small molecule inhibitors of inosine monophosphate dehydrogenase (IMPDH) was explored. The synthesis and the structure-activity relationships (SARs) derived from in vitro studies are described.

Genetic mapping of the regulator gene determining enterotoxin synthesis in Vibrio cholerae

International Nuclear Information System (INIS)

Smirnova, N.I.; Livanova, L.F.; Shaginyan, I.A.; Motin, V.L.

1986-01-01

Data on the genetic mapping of mutation tox-7 (the mutation affecting the synthesis of the cholera toxin) were obtained by conjugation crosses between the atoxigenic donor strain Vibrio cholerae Eltor and the toxigenic recipient strain V. cholera classica. The molecular and genetic analysis of the Tox - recombinants indicated that, when the synthesis of the cholera toxin is disrupted in these strains, the tox-7 mutation (which impairs the regulator gene tox) is gained. Close linkage between the tox-7 and pur-63 mutations was established (during the selection procedure there was 81.1% combined transfer with respect to marker pur-63 situated in the donor strain chromosome more proximal than mutation tox-7). The markers were localized in the following order in the region under investigation: asp-cys-nal-pur-61-trp-his-pur-63-tox-7-ile

Modifications to the translational apparatus which affect the regulation of protein synthesis in sea urchin embryos

International Nuclear Information System (INIS)

Scalise, F.W.

1988-01-01

Protein synthesis can be regulated at a number of cellular levels. I have examined how modifications to specific components of the protein synthetic machinery are involved in regulating the efficiency of initiation of translation during early sea urchin embryogenesis. It is demonstrated that Ca 2+ concentrations exceeding 500 uM cause the inhibition of protein synthesis in cell-free translation lysates prepared from sea urchin embryos. Specific changes in the state of phosphorylation of at least 8 proteins occur during this Ca 2+ -mediated repression of translation. Analysis of these proteins has indicated that, unlike mammalian systems, there is no detectable level of Ca 2+ -dependent phosphorylation of the αsubunit eIF-2. Two of the proteins which do become phosphorylated in response to Ca 2+ are calmodulin and an isoelectric form of sea urchin eIF-4D. In addition, 2 proteins which share similarities with kinases involved in the regulation of protein synthesis in mammalian cells, also become phosphorylated. I have investigated the consequences of changes in eIF-4D during sea urchin embryogenesis because it has been proposed that a polyamine-mediated conversion of lysine to hypusine in this factor may enhance translational activity. It is demonstrated that [ 3 H] spermidine-derived radioactivity is incorporated into a number of proteins when sea urchin embryos are labeled in vivo, and that the pattern of individual proteins that become labeled changes over the course of the first 30 hr of development

Aromatic inhibitors derived from ammonia-pretreated lignocellulose hinder bacterial ethanologenesis by activating regulatory circuits controlling inhibitor efflux and detoxification

Directory of Open Access Journals (Sweden)

David H. Keating

2014-08-01

Full Text Available Efficient microbial conversion of lignocellulosic hydrolysates to biofuels is a key barrier to the economically viable deployment of lignocellulosic biofuels. A chief contributor to this barrier is the impact on microbial processes and energy metabolism of lignocellulose-derived inhibitors, including phenolic carboxylates, phenolic amides (for ammonia-pretreated biomass, phenolic aldehydes, and furfurals. To understand the bacterial pathways induced by inhibitors present in ammonia-pretreated biomass hydrolysates, which are less well studied than acid-pretreated biomass hydrolysates, we developed and exploited synthetic mimics of ammonia-pretreated corn stover hydrolysate (ACSH. To determine regulatory responses to the inhibitors normally present in ACSH, we measured transcript and protein levels in an Escherichia coli ethanologen using RNA-seq and quantitative proteomics during fermentation to ethanol of synthetic hydrolysates containing or lacking the inhibitors. Our study identified four major regulators mediating these responses, the MarA/SoxS/Rob network, AaeR, FrmR, and YqhC. Induction of these regulons was correlated with a reduced rate of ethanol production, buildup of pyruvate, depletion of ATP and NAD(PH, and an inhibition of xylose conversion. The aromatic aldehyde inhibitor 5-hydroxymethylfurfural appeared to be reduced to its alcohol form by the ethanologen during fermentation, whereas phenolic acid and amide inhibitors were not metabolized. Together, our findings establish that the major regulatory responses to lignocellulose-derived inhibitors are mediated by transcriptional rather than translational regulators, suggest that energy consumed for inhibitor efflux and detoxification may limit biofuel production, and identify a network of regulators for future synthetic biology efforts.

Design and synthesis of new esters of terpenoid alcohols as 15-lipoxygenase inhibitors

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Hamid Sadeghian

2018-07-01

Full Text Available Objective(s: 15-Lipoxygenases are one of the iron-containing proteins capable of performing peroxidation of unsaturated fatty acids in animals and plants. The critical role of enzymes in the formation of inflammations, sensitivities, and some cancers has been demonstrated in mammals. The importance of enzymes has led to the development of mechanistic studies, product analysis, and synthesis of inhibitors. Materials and Methods: The inhibitory activity of all synthetic compounds against SLO (soybean 15-lipoxygenase: L1; EC 1,13,11,12 was determined using the peroxide formation method. In this method, the basis of evaluation of lipoxygenase activity is measuring the concentration of fatty acid peroxide. All measurements were compared with  4-​methyl-​2-​(4-​methylpiperazinylpyrimido[4,​5-​b]benzothiazine (4-MMPB as one of the known lipoxygenase inhibitors. The radical scavenging ability of all synthetic compounds using stable free radicals (DPPH: 2,2-diphenyl-1-picrylhydrazyl was measured for further investigation.Results: In this study, a series of esters from phenolic acids with terpenoid alcohols was synthesized and their inhibitory potency against soybean 15-lipoxygenase and their free radical scavenging properties were determined. Among the synthetic compounds, adamantyl protocatetuate 2j and bornyl protocatetuate 2o showed the most potent inhibitory activity with IC50 values of 0.95 and 0.78 μm, respectively.Conclusion: By changing the alcohol and acyl portions of stylosin, it was found that electronic properties play main role in lipoxygenase inhibition potency in contrast with steric features. Insertion of more reductive phenolic moiety such as catechuate and gallate lead to more lipoxygenase inhibition potency of the esters as observed in their radical scavenging activity.

Aberrant regulation of synthesis and degradation of viral proteins in coliphage lambda-infected UV-irradiated cells and in minicells

International Nuclear Information System (INIS)

Shaw, J.E.; Epp, C.; Pearson, M.L.; Reeve, J.N.

1987-01-01

The patterns of bacteriophage lambda proteins synthesized in UV-irradiated Escherichia coli cells and in anucleate minicells are significantly different; both systems exhibit aberrations of regulation in lambda gene expression. In unirradiated cells or cells irradiated with low UV doses (less than 600 J/m2), regulation of lambda protein synthesis is controlled by the regulatory proteins CI, N, CII, CIII, Cro, and Q. As the UV dose increases, activation of transcription of the cI, rexA, and int genes by CII and CIII proteins fails to occur and early protein synthesis, normally inhibited by the action of Cro, continues. After high UV doses (greater than 2000 J/m2), late lambda protein synthesis does not occur. Progression through the sequence of regulatory steps in lambda gene expression is slower in infected minicells. In minicells, there is no detectable cII- and cIII-dependent synthesis of CI, RexA, or Int proteins and inhibition of early protein synthesis by Cro activity is always incomplete. The synthesis of early b region proteins is not subject to control by CI, N, or Cro proteins, and evidence is presented suggesting that, in minicells, transcription of the early b region is initiated at a promoter(s) within the b region. Proteolytic cleavage of the regulatory proteins O and N and of the capsid proteins C, B, and Nu3 is much reduced in infected minicells. Exposure of minicells to very high UV doses before infection does not completely inhibit late lambda protein synthesis

Apolipoprotein B synthesis in rat small intestine: regulation by dietary triglyceride and biliary lipid

International Nuclear Information System (INIS)

Davidson, N.O.; Kollmer, M.E.; Glickman, R.M.

1986-01-01

Apolipoprotein B (apoB) synthesis rates have been determined, in vivo, in rat enterocytes. Following intralumenal administration of a pulse of [ 3 H]leucine, newly synthesized apoB was quantitated by specific immunoprecipitation and compared to [ 3 H]leucine incorporation into total, trichloroacetic acid-insoluble protein. ApoB synthesis rates were determined after acute administration of either 0.1 or 1 g of triglyceride to fasting animals. No differences were found at any time from 90 min to 6 hr after challenge and values were not different from the basal values established in fasted controls. Animals rechallenged with triglyceride after 8 days' intake of fat-free chow also failed to demonstrate a change in intestinal apoB synthesis rate. By contrast, enterocyte content of apoB appeared to fall, temporarily, with the onset of active triglyceride flux. Groups of animals were then subjected to external bile diversion for 48 hr, a maneuver designed to remove all lumenal sources of lipid. Jejunal apoB synthesis rates fell by 43% (from 0.76% +/- 0.14 to 0.43% +/- 0.12, P less than 0.001), a change that was completely prevented by continuous replacement with 10 mM Na taurocholate. The suppression of jejunal apoB synthesis, induced by prolonged bile diversion, was reversed after 14 hr, but not 8 hr, of intralumenal perfusion with 10 mM Na taurocholate. The addition of micellar fatty acid-monoolein to the perfusate for 4 hr produced no further change in apoB synthesis. Ileal apoB synthesis rates fell by 70% (from 0.61% +/- 0.15 to 0.18% +/- 0.10, P less than 0.001) following 48 hr external bile diversion, a change that was only partially prevented by continuous bile salt replacement. These results suggest that jejunal apoB synthesis demonstrates bile salt dependence but not regulation by acute triglyceride flux

A novel selective prostaglandin E2 synthesis inhibitor relieves pyrexia and arthritis in Guinea pigs inflammatory models

Directory of Open Access Journals (Sweden)

Ryusuke Sugita

2016-02-01

Full Text Available Prostaglandin E2 (PGE2, one of the terminal products in the cyclooxygenase pathway, plays an important role in various inflammatory responses. To determine whether selective inhibition of PGE2 may relieve these inflammatory symptoms, we synthesized a selective PGE2 synthesis inhibitor, compound A [1-(6-fluoro-5,7-dimethyl-1,3-benzothiazol-2-yl-N-[(1S,2R-2-(hydroxymethylcyclohexyl]piperidine-4-carboxamide], then investigated the effects on pyrexia, arthritis and inflammatory pain in guinea pigs. In LPS-stimulated guinea pig macrophages, compound A selectively inhibited inducible PGE2 biosynthesis in a dose-dependent manner whereas enhanced the formation of thromboxane B2 (TXB2. Compound A suppressed yeast-evoked PGE2 production selectively and enhanced the production of TXB2 and 6-keto PGF1α in vivo. In addition, compound A relieved yeast-induced pyrexia and also suppressed paw swelling in an adjuvant-induced arthritis model. The effect on gastrointestinal (GI ulcer formation was also evaluated and compound A showed a lower GI adverse effect than indomethacin. However, compound A failed to relieve yeast-induced thermal hyperalgesia. These results suggest that selective inhibition of PGE2 synthesis may have anti-pyretic and anti-inflammatory properties without GI side effect, but lack the analgesic efficacy.

CAR-mediated repression of Foxo1 transcriptional activity regulates the cell cycle inhibitor p21 in mouse livers

International Nuclear Information System (INIS)

Kazantseva, Yuliya A.; Yarushkin, Andrei A.; Pustylnyak, Vladimir O.

2014-01-01

Highlights: • CAR activation decreased the level of Foxo1 in mouse livers. • CAR activation decreased the level of p21 in mouse livers. • CAR activation inhibited Foxo1 transcriptional activity in mouse livers. - Abstract: 1,4-Bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), an agonist of constitutive androstane receptor (CAR), is a well-known strong primary chemical mitogen for the mouse liver. Despite extensive investigation of the role of CAR in the regulation of cell proliferation, our knowledge of the intricate mediating mechanism is incomplete. In this study, we demonstrated that long-term CAR activation by TCPOBOP increased liver-to-body weight ratio and decreased tumour suppressor Foxo1 expression and transcriptional activity, which were correlated with reduced expression of genes regulated by Foxo1, including the cell-cycle inhibitor Cdkn1a(p21), and upregulation of the cell-cycle regulator Cyclin D1. Moreover, we demonstrated the negative regulatory effect of TCPOBOP-activated CAR on the association of Foxo1 with the target Foxo1 itself and Cdkn1a(p21) promoters. Thus, we identified CAR-mediated repression of cell cycle inhibitor p21, as mediated by repression of FOXO1 expression and transcriptional activity. CAR-FOXO1 cross-talk may provide new opportunities for understanding liver diseases and developing more effective therapeutic approaches to better drug treatments

Somite-Derived Retinoic Acid Regulates Zebrafish Hematopoietic Stem Cell Formation.

Directory of Open Access Journals (Sweden)

Laura M Pillay

Full Text Available Hematopoietic stem cells (HSCs are multipotent progenitors that generate all vertebrate adult blood lineages. Recent analyses have highlighted the importance of somite-derived signaling factors in regulating HSC specification and emergence from dorsal aorta hemogenic endothelium. However, these factors remain largely uncharacterized. We provide evidence that the vitamin A derivative retinoic acid (RA functions as an essential regulator of zebrafish HSC formation. Temporal analyses indicate that RA is required for HSC gene expression prior to dorsal aorta formation, at a time when the predominant RA synthesis enzyme, aldh1a2, is strongly expressed within the paraxial mesoderm and somites. Previous research implicated the Cxcl12 chemokine and Notch signaling pathways in HSC formation. Consequently, to understand how RA regulates HSC gene expression, we surveyed the expression of components of these pathways in RA-depleted zebrafish embryos. During somitogenesis, RA-depleted embryos exhibit altered expression of jam1a and jam2a, which potentiate Notch signaling within nascent endothelial cells. RA-depleted embryos also exhibit a severe reduction in the expression of cxcr4a, the predominant Cxcl12b receptor. Furthermore, pharmacological inhibitors of RA synthesis and Cxcr4 signaling act in concert to reduce HSC formation. Our analyses demonstrate that somite-derived RA functions to regulate components of the Notch and Cxcl12 chemokine signaling pathways during HSC formation.

Cox-2 inhibitors and the risk of cardiovascular thrombotic events.

LENUS (Irish Health Repository)

Khan, M

2012-04-01

In 1971, Vane showed that the analgesic action of traditional NSAIDs relies on inhibition of the cyclo-oxygenase (COX) enzyme, which in turn results in reduced synthesis of proalgesic prostaglandins. Two decades later COX was shown to exist as two distinct isoforms. The constitutive isoform COX-1, supports the beneficial homeostatic functions whereas the inducible isoform, COX-2 becomes up regulated by inflammatory mediators and its products cause many of the symptoms of inflammatory diseases such as rheumatoid and osteoarthritis. Despite the benefits of NSAIDs for acute and chronic pain one of the most clinically significant and well characterized adverse effect is on GI mucosa. The search for NSAIDs with less gastrointestinal toxicity led to the introduction of the selective cyclo-oxygenase-2 (COX-2) inhibitors. The COX-2 selective (COX-1 sparing) inhibitors are associated with reduced GI mucosal damage as demonstrated in several trials. In light of the overwhelming and sometimes contradictory information for patients and physicians regarding the safety of COX-2 agents this article will summarize the available evidence regarding cardiovascular (CV) safety data and contemporary recommendations for prescribing of COX-2-selective NSAIDs.

Abscisic acid regulates pinoresinol-lariciresinol reductase gene expression and secoisolariciresinol accumulation in developing flax (Linum usitatissimum L.) seeds.

Science.gov (United States)

Renouard, Sullivan; Corbin, Cyrielle; Lopez, Tatiana; Montguillon, Josiane; Gutierrez, Laurent; Lamblin, Frédéric; Lainé, Eric; Hano, Christophe

2012-01-01

Secoisolariciresinol diglucoside (SDG), the main phytoestrogenic lignan of Linum usitatissimum, is accumulated in the seed coat of flax during its development and pinoresinol-lariciresinol reductase (PLR) is a key enzyme in flax for its synthesis. The promoter of LuPLR1, a flax gene encoding a pinoresinol lariciresinol reductase, contains putative regulatory boxes related to transcription activation by abscisic acid (ABA). Gel mobility shift experiments evidenced an interaction of nuclear proteins extracted from immature flax seed coat with a putative cis-acting element involved in ABA response. As ABA regulates a number of physiological events during seed development and maturation we have investigated its involvement in the regulation of this lignan synthesis by different means. ABA and SDG accumulation time courses in the seed as well as LuPLR1 expression were first determined in natural conditions. These results showed that ABA timing and localization of accumulation in the flax seed coat could be correlated with the LuPLR1 gene expression and SDG biosynthesis. Experimental modulations of ABA levels were performed by exogenous application of ABA or fluridone, an inhibitor of ABA synthesis. When submitted to exogenous ABA, immature seeds synthesized 3-times more SDG, whereas synthesis of SDG was reduced in immature seeds treated with fluridone. Similarly, the expression of LuPLR1 gene in the seed coat was up-regulated by exogenous ABA and down-regulated when fluridone was applied. These results demonstrate that SDG biosynthesis in the flax seed coat is positively controlled by ABA through the transcriptional regulation of LuPLR1 gene.

Synthesis, biological assessment and molecular modeling of new dihydroquinoline-3-carboxamides and dihydroquinoline-3-carbohydrazide derivatives as cholinesterase inhibitors, and Ca channel antagonists.

Science.gov (United States)

Tomassoli, Isabelle; Ismaili, Lhassane; Pudlo, Marc; de Los Ríos, Cristóbal; Soriano, Elena; Colmena, Inés; Gandía, Luis; Rivas, Luis; Samadi, Abdelouahid; Marco-Contelles, José; Refouvelet, Bernard

2011-01-01

The synthesis, biological evaluation, and molecular modeling of new 4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxamides(4), 4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carbohydrazide (6), and some hexahydropyrimido[5,4-c]quinoline-2,5-diones (9) produced earlier by our laboratory, as AChE/BuChE inhibitors, is described. From these analyses compound 4c resulted equipotent regarding the inhibition of cholinesterases'; inhibitors 6k, 9a, 9b were selective for AChE, whereas product 4d proved selective for BuChE. Docking analysis has been carry out in order to identify the binding mode in the active site, and to explain the observed selectivities. Only compound 9a has been shown to decrease K(+)-induced calcium signals in bovine chromaffin cells. Copyright © 2010 Elsevier Masson SAS. All rights reserved.

Effect of DNA polymerase inhibitors on DNA repair in intact and permeable human fibroblasts: Evidence that DNA polymerases δ and β are involved in DNA repair synthesis induced by N-methyl-N'-nitro-N-nitrosoguanidine

International Nuclear Information System (INIS)

Hammond, R.A.; Miller, M.R.; McClung, J.K.

1990-01-01

The involvement of DNA polymerases α, β, and δ in DNA repair synthesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was investigated in human fibroblasts (HF). The effects of anti-(DNA polymerase α) monoclonal antibody, (p-n-butylphenyl)deoxyguanosine triphosphate (BuPdGTP), dideoxythymidine triphosphate (ddTTP), and aphidicolin on MNNG-induced DNA repair synthesis were investigated to dissect the roles of the different DNA polymerases. A subcellular system (permeable cells), in which DNA repair synthesis and DNA replication were differentiated by CsCl gradient centrifugation of BrdUMP density-labeled DNA, was used to examine the effects of the polymerase inhibitors. Another approach investigated the effects of several of these inhibitors of MNNG-induced DNA repair synthesis in intact cells by measuring the amount of [ 3 H]thymidine incorporated into repair DNA as determined by autoradiography and quantitation with an automated video image analysis system. In permeable cells, MNNG-induced DNA repair synthesis was inhibited 56% by 50 μg of aphidicolin/mL, 6% by 10 μM BuPdGTP, 13% by anti-(DNA polymerse α) monoclonal antibodies, and 29% by ddTTP. In intact cells, MNNG-induced DNA repair synthesis was inhibited 57% by 50 μg of aphidicolin/mL and was not significantly inhibited by microinjecting anti-(DNA polymerase α) antibodies into HF nuclei. These results indicate that both DNA polymerase δ and β are involved in repairing DNA damage caused by MNNG

Keratinocyte-derived laminin-332 protein promotes melanin synthesis via regulation of tyrosine uptake.

Science.gov (United States)

Chung, Heesung; Jung, Hyejung; Lee, Jung-Hyun; Oh, Hye Yun; Kim, Ok Bin; Han, Inn-Oc; Oh, Eok-Soo

2014-08-01

Melanocytes, which produce the pigment melanin, are known to be closely regulated by neighboring keratinocytes. However, how keratinocytes regulate melanin production is unclear. Here we report that melanin production in melanoma cells (B16F10 and MNT-1) was increased markedly on a keratinocyte-derived extracellular matrix compared with a melanoma cell-derived extracellular matrix. siRNA-mediated reduction of keratinocyte-derived laminin-332 expression decreased melanin synthesis in melanoma cells, and laminin-332, but not fibronectin, enhanced melanin content and α-melanocyte-stimulating hormone-regulated melanin production in melanoma cells. Similar effects were observed in human melanocytes. Interestingly, however, laminin-332 did not affect the expression or activity of tyrosinase. Instead, laminin-332 promoted the uptake of extracellular tyrosine and, subsequently, increased intracellular levels of tyrosine in both melanocytes and melanoma cells. Taken together, these data strongly suggest that keratinocyte-derived laminin-332 contributes to melanin production by regulating tyrosine uptake. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Effects of the aromatase inhibitor Letrozole on serum immunoglobulin and lysozyme levels in immunized rainbow trout (Oncorhynchus mykiss Walbaum females

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Paria Akbary

2013-12-01

Full Text Available Letrozole is a synthetic aromatase inhibitor and interfere in the committed step in the synthesis of endogenous estrogens from androgens. Also estrogens regulate the immune system in teleost. Changes of 17- β- esrtradiol (E2, serum immunoglobulin and lysozyme levels were measured using a method based on the ability of lysozyme to lyse the bacterium Micrococcus lysodeikticus, enzyme-linked immunosorbent assay (ELISA and ELISA respectively. Twelve broodstocks were injected weekly with 2.5 mg kg-1 letrozole (an endocrine disrupter component two months before spawning season and vaccinated intraperitoneally (i.p with a bacterin (inactivated L. garviae one month before spawning. Twelve broodstocks for vaccination and twelve female rainbow trout as control group were also immiunised (i.p with the bacterin and injected (i.p with PBS, respectively. In the group received 2.5 mg AI kg-1 per week, serum E2 levels were significantly lower than that of other groups. Total immunoglobulin level and lysozyme activity were significantly higher in the parents received 2.5 mg kg-1 per week and were immunized with 10-9 cells ml-1 Lactococcus garvieae  compared to the group which immunized with L. garvieae and the control (non- immunized. The present study, suggests that aromatase inhibitors such as letrozole may be a potential tool to regulate the synthesis of E2, is involved in the hormone- immune system interaction in rainbow trout.

Synthesis and bioevaluation of new tacrine-cinnamic acid hybrids as cholinesterase inhibitors against Alzheimer's disease.

Science.gov (United States)

Chen, Yao; Zhu, Jie; Mo, Jun; Yang, Hongyu; Jiang, Xueyang; Lin, Hongzhi; Gu, Kai; Pei, Yuqiong; Wu, Liang; Tan, Renxiang; Hou, Jing; Chen, Jingyi; Lv, Yang; Bian, Yaoyao; Sun, Haopeng

2018-12-01

Small molecule cholinesterases inhibitor (ChEI) provides an effective therapeutic strategy to treat Alzheimer's disease (AD). Currently, the discovery of new ChEI with multi-target effect is still of great importance. Herein, we report the synthesis, structure-activity relationship study and biological evaluation of a series of tacrine-cinnamic acid hybrids as new ChEIs. All target compounds are evaluated for their in vitro cholinesterase inhibitory activities. The representatives which show potent activity on cholinesterase, are evaluated for the amyloid β-protein self-aggregation inhibition and in vivo assays. The optimal compound 19, 27, and 30 (human AChE IC 50  = 10.2 ± 1.2, 16.5 ± 1.7, and 15.3 ± 1.8 nM, respectively) show good performance in ameliorating the scopolamine-induced cognition impairment and preliminary safety in hepatotoxicity evaluation. These compounds deserve further evaluation for the development of new therapeutic agents against AD.

The resistance of insects to plant proteinase inhibitors

NARCIS (Netherlands)

Jongsma, M.A.

1995-01-01

The research reported in this thesis describes the induction of proteinase inhibitor synthesis in solanaceous plants (tobacco and tomato), when lepidopteran larvae (Manduca sexta and Spodoptera exigua) are feeding on leaves. It is shown that the

Synthesis and evaluation of iodide uptake inhibitors in thyroid gland

International Nuclear Information System (INIS)

Lacotte, Pierre

2012-01-01

This work was intended to discover small organic molecules acting as iodide uptake inhibitors in thyroid cells. These compounds can indeed be derivatized into biochemical probes for further characterization of proteins involved in iodide transport mechanisms. On the long term, these inhibitors also appear as attractive drug candidates for treatment of thyroid pathologies or radioprotection against iodine isotopes. A similar strategy was adopted for both of the two inhibitor families. First, we synthesized a chemical library of around 100 analogues; we measured their IC50 against iodide uptake in FRTL-5 cells to get structure-activity relationships. Absolute configuration of stereo-genic centers was also investigated, and a preferential stereochemistry was found to be responsible for activity. From this basis, around twenty 'second-generation' analogues were synthesized by combining fragments contributing to biological activity. Biological evaluation indicated that nine were very potent inhibitors, with IC50 ≤ 6 nM and satisfying physicochemical properties required for drug candidates. Finally, one photoactivatable biotinylated probe was developed in each family and used for photoaffinity labeling. Several specifically labeled proteins are still under identification and constitute new potential therapeutic targets. (author)

Naringenin Regulates Expression of Genes Involved in Cell Wall Synthesis in Herbaspirillum seropedicae▿

Science.gov (United States)

Tadra-Sfeir, M. Z.; Souza, E. M.; Faoro, H.; Müller-Santos, M.; Baura, V. A.; Tuleski, T. R.; Rigo, L. U.; Yates, M. G.; Wassem, R.; Pedrosa, F. O.; Monteiro, R. A.

2011-01-01

Five thousand mutants of Herbaspirillum seropedicae SmR1 carrying random insertions of transposon pTnMod-OGmKmlacZ were screened for differential expression of LacZ in the presence of naringenin. Among the 16 mutants whose expression was regulated by naringenin were genes predicted to be involved in the synthesis of exopolysaccharides, lipopolysaccharides, and auxin. These loci are probably involved in establishing interactions with host plants. PMID:21257805

Naringenin regulates expression of genes involved in cell wall synthesis in Herbaspirillum seropedicae.

Science.gov (United States)

Tadra-Sfeir, M Z; Souza, E M; Faoro, H; Müller-Santos, M; Baura, V A; Tuleski, T R; Rigo, L U; Yates, M G; Wassem, R; Pedrosa, F O; Monteiro, R A

2011-03-01

Five thousand mutants of Herbaspirillum seropedicae SmR1 carrying random insertions of transposon pTnMod-OGmKmlacZ were screened for differential expression of LacZ in the presence of naringenin. Among the 16 mutants whose expression was regulated by naringenin were genes predicted to be involved in the synthesis of exopolysaccharides, lipopolysaccharides, and auxin. These loci are probably involved in establishing interactions with host plants.

Isolation, identification, and synthesis of 2-carboxyarabinitol 1-phosphate, a diurnal regulator of ribulase-bisphosphate carboxylase activity

International Nuclear Information System (INIS)

Berry, J.A.; Lorimer, G.H.; Pierce, J.; Seemann, J.R.; Meek, J.; Freas, S.

1987-01-01

The diurnal change in activity of ribulose 1,5-bisphosphate (Rbu-1,5-P 2 ) carboxylase [3-phospho-D-glycerate carboxy-lyase (dimerizing); EC 4.1.1.39] of leaves of Phaseolus vulgaris is regulated (in part) by mechanisms that control the level of an endogenous inhibitor that binds tightly to the activated (carbamoylated) form of Rbu-1,5-P 2 carboxylase. This inhibitor was extracted from leaves and copurified with the Rbu-1,5-P 2 carboxylase of the leaves. Further purification by ion-exchange chromatography, adsorption to purified Rbu-1,5-P 2 carboxylase, barium precipitation, and HPLC separation yielded a phosphorylated compound that was a strong inhibitor of Rbu-1,5-P 2 carboxylase. The compound was analyzed by GC/MS, 13 C NMR, and 1 H NMR and shown to be 2-carboxyarabinitol 1-phosphate [(2-C-phosphohydroxymethyl)-D-ribonic acid]. The structure of the isolated compound differs from the Rbu-1,5-P 2 carboxylase transition-state analogue 2-carboxyarabinitol 1,5-bisphosphate only by the lack of the C-5 phosphate group. This difference results in a higher binding constant for the monophosphate compared with the bisphosphate. The less tightly bound compound acts in a light-dependent, reversible regulation of Rbu-1,5-P 2 carboxylase activity in vivo

Development of a UV-Cleavable Protecting Group for Hydroxylamines, Synthesis of a StructurallyWide Variety of Hydroxamic Acids, and Identification of Histone Deacetylase Inhibitors

DEFF Research Database (Denmark)

Mortensen, Kim Thollund

Photo-cleavable protecting groups are highly applicable for the synthesis of structural complex and sensitive compounds, including biological important molecules. Herein, we present the development of a novel O-hydroxylamine photo-cleavable protecting group, based on the methyl-6-nitroveratryl...... moiety. We demonstrate the application of the protected hydroxylamine derivative for the synthesis of N-alkylated hydroxamic acids. We have shown that the construct is stable toward a diverse set of reaction conditions, as well as orthogonal with conventional protection groups. The O......-protected hydroxylamine derivative was applied to synthesize a small collection of N-alkylated hydroxamic acids as inhibitors of the histone deacetylase enzymes, an important class of enzymes for the treatment of a range of diseases, most importantly cancer. During my external stay at Nanyang Technological University...

Variations in Glycogen Synthesis in Human Pluripotent Stem Cells with Altered Pluripotent States

Science.gov (United States)

Chen, Richard J.; Zhang, Guofeng; Garfield, Susan H.; Shi, Yi-Jun; Chen, Kevin G.; Robey, Pamela G.; Leapman, Richard D.

2015-01-01

Human pluripotent stem cells (hPSCs) represent very promising resources for cell-based regenerative medicine. It is essential to determine the biological implications of some fundamental physiological processes (such as glycogen metabolism) in these stem cells. In this report, we employ electron, immunofluorescence microscopy, and biochemical methods to study glycogen synthesis in hPSCs. Our results indicate that there is a high level of glycogen synthesis (0.28 to 0.62 μg/μg proteins) in undifferentiated human embryonic stem cells (hESCs) compared with the glycogen levels (0 to 0.25 μg/μg proteins) reported in human cancer cell lines. Moreover, we found that glycogen synthesis was regulated by bone morphogenetic protein 4 (BMP-4) and the glycogen synthase kinase 3 (GSK-3) pathway. Our observation of glycogen bodies and sustained expression of the pluripotent factor Oct-4 mediated by the potent GSK-3 inhibitor CHIR-99021 reveals an altered pluripotent state in hPSC culture. We further confirmed glycogen variations under different naïve pluripotent cell growth conditions based on the addition of the GSK-3 inhibitor BIO. Our data suggest that primed hPSCs treated with naïve growth conditions acquire altered pluripotent states, similar to those naïve-like hPSCs, with increased glycogen synthesis. Furthermore, we found that suppression of phosphorylated glycogen synthase was an underlying mechanism responsible for altered glycogen synthesis. Thus, our novel findings regarding the dynamic changes in glycogen metabolism provide new markers to assess the energetic and various pluripotent states in hPSCs. The components of glycogen metabolic pathways offer new assays to delineate previously unrecognized properties of hPSCs under different growth conditions. PMID:26565809

Targeting RNA transcription and translation in ovarian cancer cells with pharmacological inhibitor CDKI-73.

Science.gov (United States)

Lam, Frankie; Abbas, Abdullahi Y; Shao, Hao; Teo, Theodosia; Adams, Julian; Li, Peng; Bradshaw, Tracey D; Fischer, Peter M; Walsby, Elisabeth; Pepper, Chris; Chen, Yi; Ding, Jian; Wang, Shudong

2014-09-15

Dysregulation of cellular transcription and translation is a fundamental hallmark of cancer. As CDK9 and Mnks play pivotal roles in the regulation of RNA transcription and protein synthesis, respectively, they are important targets for drug development. We herein report the cellular mechanism of a novel CDK9 inhibitor CDKI-73 in an ovarian cancer cell line (A2780). We also used shRNA-mediated CDK9 knockdown to investigate the importance of CDK9 in the maintenance of A2780 cells. This study revealed that CDKI-73 rapidly inhibited cellular CDK9 kinase activity and down-regulated the RNAPII phosphorylation. This subsequently caused a decrease in the eIF4E phosphorylation by blocking Mnk1 kinase activity. Consistently, CDK9 shRNA was also found to down-regulate the Mnk1 expression. Both CDKI-73 and CDK9 shRNA decreased anti-apoptotic proteins Mcl-1 and Bcl-2 and induced apoptosis. The study confirmed that CDK9 is required for cell survival and that ovarian cancer may be susceptible to CDK9 inhibition strategy. The data also implied a role of CDK9 in eIF4E-mediated translational control, suggesting that CDK9 may have important implication in the Mnk-eIF4E axis, the key determinants of PI3K/Akt/mTOR- and Ras/Raf/MAPK-mediated tumorigenic activity. As such, CDK9 inhibitor drug candidate CDKI-73 should have a major impact on these pathways in human cancers.

Synthesis of Novel Chalcones as Acetylcholinesterase Inhibitors

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Thanh-Dao Tran

2016-07-01

Full Text Available A new series of benzylaminochalcone derivatives with different substituents on ring B were synthesized and evaluated as inhibitors of acetylcholinesterase. The study is aimed at identification of novel benzylaminochalcones capable of blocking acetylcholinesterase activity for further development of an approach to Alzheimer’s disease treatment. These compounds were produced in moderate to good yields via Claisen-Schmidt condensation and subjected to an in vitro acetylcholinesterase inhibition assay, using Ellman’s method. The in silico docking procedure was also employed to identify molecular interactions between the chalcone compounds and the enzyme. Compounds with ring B bearing pyridin-4-yl, 4-nitrophenyl, 4-chlorophenyl and 3,4-dimethoxyphenyl moieties were discovered to exhibit significant inhibitory activities against acetylcholinesterase, with IC50 values ranging from 23 to 39 µM. The molecular modeling studies are consistent with the hypothesis that benzylaminochalcones could exert their effects as dual-binding-site acetylcholinesterase inhibitors, which might simultaneously enhance cholinergic neurotransmission and inhibit β-amyloid aggregation through binding to both catalytic and peripheral sites of the enzyme. These derivatives could be further developed to provide novel leads for the discovery of new anti-Alzheimer drugs in the future.

Role of cytoskeleton in regulating fusion of nucleoli: a study using the activated mouse oocyte model.

Science.gov (United States)

Lian, Hua-Yu; Jiao, Guang-Zhong; Wang, Hui-Li; Tan, Xiu-Wen; Wang, Tian-Yang; Zheng, Liang-Liang; Kong, Qiao-Qiao; Tan, Jing-He

2014-09-01

Although fusion of nucleoli was observed during pronuclear development of zygotes and the behavior of nucleoli in pronuclei has been suggested as an indicator of embryonic developmental potential, the mechanism for nucleolar fusion is unclear. Although both cytoskeleton and the nucleolus are important cellular entities, there are no special reports on the relationship between the two. Role of cytoskeleton in regulating fusion of nucleoli was studied using the activated mouse oocyte model. Mouse oocytes were cultured for 6 h in activating medium (Ca²⁺-free CZB medium containing 10 mM SrCl₂) supplemented with or without inhibitors for cytoskeleton or protein synthesis before pronuclear formation, nucleolar fusion, and the activity of maturation-promoting factor (MPF) were examined. Whereas treatment with microfilament inhibitor cytochalasin D or B or intermediate filament inhibitor acrylamide suppressed nucleolar fusion efficiently, treatment with microtubule inhibitor demecolcine or nocodazole or protein synthesis inhibitor cycloheximide had no effect. The cytochalasin D- or acrylamide-sensitive temporal window coincided well with the reported temporal window for nucleolar fusion in activated oocytes. Whereas a continuous incubation with demecolcine prevented pronuclear formation, pronuclei formed normally when demecolcine was excluded during the first hour of activation treatment when the MPF activity dropped dramatically. The results suggest that 1) microfilaments and intermediate filaments but not microtubules support nucleolar fusion, 2) proteins required for nucleolar fusion including microfilaments and intermediate filaments are not de novo synthesized, and 3) microtubule disruption prevents pronuclear formation by activating MPF. © 2014 by the Society for the Study of Reproduction, Inc.

Synthesis and modifications of heterocyclic derivatives of D-arabinose: potential inhibitors of glucose-6-phosphate isomerase and glucosamine-6-phosphate synthase

International Nuclear Information System (INIS)

Viana, Renato Marcio Ribeiro; Prado, Maria Auxiliadora Fontes; Alves, Ricardo Jose

2008-01-01

The synthesis of -5-(D-arabino-1,2,3,4-tetrahydroxybutyl)tetrazole and -2-(d-arabino-1,2,3,4-tetra-acetoxybutyl)-5-methyl-1,3,4-oxadiazole from d-arabinose is described. Attempts at removing the protecting groups of the oxadiazole derivative were unsuccessful, leading to products resulting from the opening of the oxadiazole ring. The unprotected tetrazole derivative was selectively phosphorylated at the primary hydroxyl group with diethyl phosphoryl chloride. The resulting 5-[d-arabino-4-(diethylphosphoryloxy)-1,2,3-trihydroxybutyl]tetrazole is a protected form of a potential inhibitor of the enzymes glucose-6-phosphate isomerase and glucosamine synthase. (author)

Synthetic strategy for bicyclic tetrapeptides HDAC inhibitors using ...

Indian Academy of Sciences (India)

Cyclic peptides show diverse biological activities and are considered as good therapeutic agents due to structural rigidity, receptor selectivity and biochemical stability. We have developed bicyclic tetrapeptide HDAC inhibitors based on different cyclic tetrapeptide scaffolds. For the synthesis of these bicyclic tetrapeptides, ...

Ethylenediamine diacetate (EDDA) mediated synthesis of aurones under ultrasound: their evaluation as inhibitors of SIRT1.

Science.gov (United States)

Manjulatha, Khanapur; Srinivas, S; Mulakayala, Naveen; Rambabu, D; Prabhakar, M; Arunasree, Kalle M; Alvala, Mallika; Basaveswara Rao, M V; Pal, Manojit

2012-10-01

An improved synthesis of functionalized aurones has been accomplished via the reaction of benzofuran-3(2H)-one with a range of benzaldehydes in the presence of a mild base EDDA under ultrasound. A number of aurones were synthesized (within 5-30min) and the molecular structure of a representative compound determined by single crystal X-ray diffraction study confirmed Z-geometry of the C-C double bond present within the molecule. Some of the compounds synthesized have shown SIRT1 inhibiting as well as anti proliferative properties against two cancer cell lines in vitro. Compound 3a [(Z)-2-(5-bromo-2-hydroxybenzylidene) benzofuran-3(2H)-one] was identified as a potent inhibitor of SIRT1 (IC(50)=1μM) which showed a dose dependent increase in the acetylation of p53 resulting in induction of apoptosis. Copyright © 2012 Elsevier Ltd. All rights reserved.

Emerging Corrosion Inhibitors for Interfacial Coating

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Mona Taghavikish

2017-12-01

Full Text Available Corrosion is a deterioration of a metal due to reaction with environment. The use of corrosion inhibitors is one of the most effective ways of protecting metal surfaces against corrosion. Their effectiveness is related to the chemical composition, their molecular structures and affinities for adsorption on the metal surface. This review focuses on the potential of ionic liquid, polyionic liquid (PIL and graphene as promising corrosion inhibitors in emerging coatings due to their remarkable properties and various embedment or fabrication strategies. The review begins with a precise description of the synthesis, characterization and structure-property-performance relationship of such inhibitors for anti-corrosion coatings. It establishes a platform for the formation of new generation of PIL based coatings and shows that PIL corrosion inhibitors with various heteroatoms in different form can be employed for corrosion protection with higher barrier properties and protection of metal surface. However, such study is still in its infancy and there is significant scope to further develop new structures of PIL based corrosion inhibitors and coatings and study their behaviour in protection of metals. Besides, it is identified that the combination of ionic liquid, PIL and graphene could possibly contribute to the development of the ultimate corrosion inhibitor based coating.

Synthesis of natural urolithin M6, a galloflavin mimetic, as a potential inhibitor of lactate dehydrogenase A.

Science.gov (United States)

Rupiani, Sebastiano; Guidotti, Laura; Manerba, Marcella; Di Ianni, Lorenza; Giacomini, Elisa; Falchi, Federico; Di Stefano, Giuseppina; Roberti, Marinella; Recanatini, Maurizio

2016-11-22

Glycolysis is the main route for energy production in tumors. LDH-A is a key enzyme of this process and its inhibition represents an attractive strategy to hamper cancer cell metabolism. Galloflavin is a reliable LDH-A inhibitor as previously identified by us; however, its poor physicochemical properties and chemical tractability render it unsuitable for further development. Therefore, a rational design was undertaken with the aim to reproduce the pharmacophore of galloflavin on simpler, potentially more soluble and synthetic accessible scaffolds. Following a process of structural simplification, natural urolithin M6 (UM6), which is an ellagitannin metabolite produced by gut microbiota, was identified as a putative galloflavin mimetic. In the present study, the synthesis of UM6 is described for the first time. An efficient synthetic pathway has been developed, which involved five steps from readily accessible starting materials. The key reaction steps, a Suzuki coupling and an intramolecular C-H oxygenation, have been optimized to improve the synthetic feasibility and provide the best conditions in terms of reaction time and yield. Moreover, this route would be suitable to obtain other analogs for SAR studies. Preliminary biological tests revealed that UM6 was able to smoothly reproduce the behavior of galloflavin, confirming that our approach was successful in providing a new and accessible structure in the search for new LDH-A inhibitors.

Control of density-dependent, cell state-specific signal transduction by the cell adhesion molecule CEACAM1, and its influence on cell cycle regulation

International Nuclear Information System (INIS)

Scheffrahn, Inka; Singer, Bernhard B.; Sigmundsson, Kristmundur; Lucka, Lothar; Oebrink, Bjoern

2005-01-01

Growth factor receptors, extracellular matrix receptors, and cell-cell adhesion molecules co-operate in regulating the activities of intracellular signaling pathways. Here, we demonstrate that the cell adhesion molecule CEACAM1 co-regulates growth-factor-induced DNA synthesis in NBT-II epithelial cells in a cell-density-dependent manner. CEACAM1 exerted its effects by regulating the activity of the Erk 1/2 MAP kinase pathway and the expression levels of the cyclin-dependent kinase inhibitor p27 Kip1 . Interestingly, both inhibitory and stimulatory effects were observed. Confluent cells continuously exposed to fetal calf serum showed little Erk activity and DNA synthesis compared with sparse cells. Under these conditions, anti-CEACAM1 antibodies strongly stimulated Erk activation, decreased p27 expression, and induced DNA synthesis. In serum-starved confluent cells, re-addition of 10% fetal calf serum activated the Erk pathway, decreased p27 expression, and stimulated DNA synthesis to the same levels as in sparse cells. Under these conditions anti-CEACAM1 antibodies de-activated Erk, restored the level of p27, and inhibited DNA synthesis. These data indicate that CEACAM1 mediates contact inhibition of proliferation in cells that are constantly exposed to growth factors, but co-activates growth-factor-induced proliferation in cells that have been starved for growth factors; exposure to extracellular CEACAM1 ligands reverts these responses

Identification of microRNAs linked to regulators of muscle protein synthesis and regeneration in young and old skeletal muscle.

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Evelyn Zacharewicz

Full Text Available BACKGROUND: Over the course of ageing there is a natural and progressive loss of skeletal muscle mass. The onset and progression of age-related muscle wasting is associated with an attenuated activation of Akt-mTOR signalling and muscle protein synthesis in response to anabolic stimuli such as resistance exercise. MicroRNAs (miRNAs are novel and important post-transcriptional regulators of numerous cellular processes. The role of miRNAs in the regulation of muscle protein synthesis following resistance exercise is poorly understood. This study investigated the changes in skeletal muscle miRNA expression following an acute bout of resistance exercise in young and old subjects with a focus on the miRNA species predicted to target Akt-mTOR signalling. RESULTS: Ten young (24.2±0.9 years and 10 old (66.6±1.1 years males completed an acute resistance exercise bout known to maximise muscle protein synthesis, with muscle biopsies collected before and 2 hours after exercise. We screened the expression of 754 miRNAs in the muscle biopsies and found 26 miRNAs to be regulated with age, exercise or a combination of both factors. Nine of these miRNAs are highly predicted to regulate targets within the Akt-mTOR signalling pathway and 5 miRNAs have validated binding sites within the 3' UTRs of several members of the Akt-mTOR signalling pathway. The miR-99/100 family of miRNAs notably emerged as potentially important regulators of skeletal muscle mass in young and old subjects. CONCLUSION: This study has identified several miRNAs that were regulated with age or with a single bout of resistance exercise. Some of these miRNAs were predicted to influence Akt-mTOR signalling, and therefore potentially skeletal muscle mass. These miRNAs should be considered as candidate targets for in vivo modulation.

Synthesis, biological evaluation and molecular docking studies of ...

African Journals Online (AJOL)

Synthesis, biological evaluation and molecular docking studies of Mannich bases derived from 1, 3, 4-oxadiazole- 2-thiones as potential urease inhibitors. ... Mannich bases (5-17) were subjected to in silico screening as urease inhibitors, using crystal structure of urease (Protein Data Bank ID: 5FSE) as a model enzyme.

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

Science.gov (United States)

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

2013-01-01

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

Chronic effects of fluoxetine, a selective inhibitor of serotonin uptake, on neurotransmitter receptors

International Nuclear Information System (INIS)

Wong, D.T.; Reid, L.R.; Bymaster, F.P.; Threlkeld, P.G.

1985-01-01

Fluoxetine administration to rats dose of 10mg/kg i.p. daily up to 12 or 24 days failed to change the concentration-dependent binding of [ 3 H]WB4101, [ 3 H]clonidine and [ 3 H]dihydroalprenolol to α 1 -, α 2 - and β-adrenergic receptors, respectively; [ 3 H]quinuclidinyl benzilate to muscarinic receptors; [ 3 H]pyrilamine to histamine H 1 receptors and [ 3 H]naloxone to opiate receptors. Persistent and significant decreases in receptor number (Bsub(max) value) without changes in the dissociation constant (Ksub(D) value) of [ 3 H]5-HT binding in cortical membranes were observed upon chronic treatment with fluoxetine administered either by intraperitoneal injection or incorporation in the diet. A detectable reduction of 5-HT 1 receptor number occured after once-daily injections of fluoxetine at 10mg/kg i.p. within 49 hours. After pretreatment for 3 days with p-chlorophenylalanine, an inhibitor of 5-HT synthesis, followed by repeated administration of fluoxetine, 5-HT 1 receptor numbers were higher than those of normal rats, suggesting a dependence on synaptic concentration of 5-HT for fluoxetine to affect a receptor down-regulation. These studies provide further evidence for the selectivity of fluoxetine as an inhibitor of 5-HT reuptake, resulting in a selective down-regulation of 5-HT 1 receptors in the cerebal cortex of rat brain. (Author)

Regulation of DNA synthesis and the cell cycle in human prostate cancer cells and lymphocytes by ovine uterine serpin

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Hansen Peter J

2008-01-01

Full Text Available Abstract Background Uterine serpins are members of the serine proteinase inhibitor superfamily. Like some other serpins, these proteins do not appear to be functional proteinase inhibitors. The most studied member of the group, ovine uterine serpin (OvUS, inhibits proliferation of several cell types including activated lymphocytes, bovine preimplantation embryos, and cell lines for lymphoma, canine primary osteosarcoma and human prostate cancer (PC-3 cells. The goal for the present study was to evaluate the mechanism by which OvUS inhibits cell proliferation. In particular, it was tested whether inhibition of DNA synthesis in PC-3 cells involves cytotoxic actions of OvUS or the induction of apoptosis. The effect of OvUS in the production of the autocrine and angiogenic cytokine interleukin (IL-8 by PC-3 cells was also determined. Finally, it was tested whether OvUS blocks specific steps in the cell cycle using both PC-3 cells and lymphocytes. Results Recombinant OvUS blocked proliferation of PC-3 cells at concentrations as low as 8 μg/ml as determined by measurements of [3H]thymidine incorporation or ATP content per well. Treatment of PC-3 cells with OvUS did not cause cytotoxicity or apoptosis or alter interleukin-8 secretion into medium. Results from flow cytometry experiments showed that OvUS blocked the entry of PC-3 cells into S phase and the exit from G2/M phase. In addition, OvUS blocked entry of lymphocytes into S phase following activation of proliferation with phytohemagglutinin. Conclusion Results indicate that OvUS acts to block cell proliferation through disruption of the cell cycle dynamics rather than induction of cytotoxicity or apoptosis. The finding that OvUS can regulate cell proliferation makes this one of only a few serpins that function to inhibit cell growth.

AbetaPP/APLP2 family of Kunitz serine proteinase inhibitors regulate cerebral thrombosis.

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Xu, Feng; Previti, Mary Lou; Nieman, Marvin T; Davis, Judianne; Schmaier, Alvin H; Van Nostrand, William E

2009-04-29

The amyloid beta-protein precursor (AbetaPP) is best recognized as the precursor to the Abeta peptide that accumulates in the brains of patients with Alzheimer's disease, but less is known about its physiological functions. Isoforms of AbetaPP that contain a Kunitz-type serine proteinase inhibitor (KPI) domain are expressed in brain and, outside the CNS, in circulating blood platelets. Recently, we showed that KPI-containing forms of AbetaPP regulates cerebral thrombosis in vivo (Xu et al., 2005, 2007). Amyloid precursor like protein-2 (APLP2), a closely related homolog to AbetaPP, also possesses a highly conserved KPI domain. Virtually nothing is known of its function. Here, we show that APLP2 also regulates cerebral thrombosis risk. Recombinant purified KPI domains of AbetaPP and APLP2 both inhibit the plasma clotting in vitro. In a carotid artery thrombosis model, both AbetaPP(-/-) and APLP2(-/-) mice exhibit similar significantly shorter times to vessel occlusion compared with wild-type mice indicating a prothrombotic phenotype. Similarly, in an experimental model of intracerebral hemorrhage, both AbetaPP(-/-) and APLP2(-/-) mice produce significantly smaller hematomas with reduced brain hemoglobin content compared with wild-type mice. Together, these results indicate that AbetaPP and APLP2 share overlapping anticoagulant functions with regard to regulating thrombosis after cerebral vascular injury.

Activating Transcription Factor 3 regulates in part the enhanced tumour cell cytotoxicity of the histone deacetylase inhibitor M344 and cisplatin in combination

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St Germain Carly

2010-09-01

Full Text Available Abstract Background Activating Transcription Factor (ATF 3 is a key regulator of the cellular integrated stress response whose expression has also been correlated with pro-apoptotic activities in tumour cell models. Combination treatments with chemotherapeutic drugs, such as cisplatin, and histone deacetylase (HDAC inhibitors have been demonstrated to enhance tumour cell cytotoxicity. We recently demonstrated a role for ATF3 in regulating cisplatin-induced apoptosis and others have shown that HDAC inhibition can also induce cellular stress. In this study, we evaluated the role of ATF3 in regulating the co-operative cytotoxicity of cisplatin in combination with an HDAC inhibitor. Results The HDAC inhibitor M344 induced ATF3 expression at the protein and mRNA level in a panel of human derived cancer cell lines as determined by Western blot and quantitative RT-PCR analyses. Combination treatment with M344 and cisplatin lead to increased induction of ATF3 compared with cisplatin alone. Utilizing the MTT cell viability assay, M344 treatments also enhanced the cytotoxic effects of cisplatin in these cancer cell lines. The mechanism of ATF3 induction by M344 was found to be independent of MAPKinase pathways and dependent on ATF4, a known regulator of ATF3 expression. ATF4 heterozygote (+/- and knock out (-/- mouse embryonic fibroblast (MEF as well as chromatin immunoprecipitation (ChIP assays were utilized in determining the mechanistic induction of ATF3 by M344. We also demonstrated that ATF3 regulates the enhanced cytotoxicity of M344 in combination with cisplatin as evidenced by attenuation of cytotoxicity in shRNAs targeting ATF3 expressing cells. Conclusion This study identifies the pro-apoptotic factor, ATF3 as a novel target of M344, as well as a mediator of the co-operative effects of cisplatin and M344 induced tumour cell cytotoxicity.

Anti-protozoal and anti-bacterial antibiotics that inhibit protein synthesis kill cancer subtypes enriched for stem cell-like properties.

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Cuyàs, Elisabet; Martin-Castillo, Begoña; Corominas-Faja, Bruna; Massaguer, Anna; Bosch-Barrera, Joaquim; Menendez, Javier A

2015-01-01

Key players in translational regulation such as ribosomes might represent powerful, but hitherto largely unexplored, targets to eliminate drug-refractory cancer stem cells (CSCs). A recent study by the Lisanti group has documented how puromycin, an old antibiotic derived from Streptomyces alboniger that inhibits ribosomal protein translation, can efficiently suppress CSC states in tumorspheres and monolayer cultures. We have used a closely related approach based on Biolog Phenotype Microarrays (PM), which contain tens of lyophilized antimicrobial drugs, to assess the chemosensitivity profiles of breast cancer cell lines enriched for stem cell-like properties. Antibiotics directly targeting active sites of the ribosome including emetine, puromycin and cycloheximide, inhibitors of ribosome biogenesis such as dactinomycin, ribotoxic stress agents such as daunorubicin, and indirect inhibitors of protein synthesis such as acriflavine, had the largest cytotoxic impact against claudin-low and basal-like breast cancer cells. Thus, biologically aggressive, treatment-resistant breast cancer subtypes enriched for stem cell-like properties exhibit exacerbated chemosensitivities to anti-protozoal and anti-bacterial antibiotics targeting protein synthesis. These results suggest that old/existing microbicides might be repurposed not only as new cancer therapeutics, but also might provide the tools and molecular understanding needed to develop second-generation inhibitors of ribosomal translation to eradicate CSC traits in tumor tissues.

Synthesis of [11C]dapoxetine·HCl, a serotonin re-uptake inhibitor: biodistribution in rat and preliminary PET imaging in the monkey

International Nuclear Information System (INIS)

Livni, E.; Satterlee, W.; Robey, R.L.

1994-01-01

[ 11 C]Dapoxetine · HCl, S-(+)-N,N-dimethyl-a-[2-(naphthalenyloxy)ethyl] benzenemethanamine hydrochloride, a potent serotonin re-uptake inhibitor was prepared from its mono-methyl precursor, S-(+)-N-methyl-a-[2-(naphthalenyloxy)ethyl]benzene methanamine hydrochloride. Biodistribution was determined in rats at 5, 30 and 60 min after injection and preliminary PET studies were performed in a Rhesus monkey. 11 CH 3 I was bubbled into a solution of S-(+)-N-methyl-α-[2-(naphthalenyloxy)ethyl]benzene methanamine hydrochloride (3.0 mg in DMSO) and the mixture was heated at 110 o C for 8 min. [ 11 C]Dapoxetine · HCl was purified by HPLC on a C 18 cartridge eluted with MeOH: phosphate buffer, pH 7.2 (75:25) with a 10% yield (end of synthesis). The time required for the synthesis was 40 min, from the end of bombardment. Radiochemical purity of the final product was > 99% and specific activity was routinely > 400 mCi/μmol [EOS]. (author)

Coordinated Regulations of mRNA Synthesis and Decay during Cold Acclimation in Arabidopsis Cells.

KAUST Repository

Arae, Toshihiro

2017-04-18

Plants possess a cold acclimation system to acquire freezing tolerance through pre-exposure to non-freezing low temperatures. The transcriptional cascade of C-repeat binding factors (CBFs)/dehydration response element-binding factors (DREBs) is considered a major transcriptional regulatory pathway during cold acclimation. However, little is known regarding the functional significance of mRNA stability regulation in the response of gene expression to cold stress. The actual level of individual mRNAs is determined by a balance between mRNA synthesis and degradation. Therefore, it is important to assess the regulatory steps to increase our understanding of gene regulation. Here, we analyzed temporal changes in mRNA amounts and half-lives in response to cold stress in Arabidopsis cell cultures based on genome-wide analysis. In this mRNA decay array method, mRNA half-life measurements and microarray analyses were combined. In addition, temporal changes in the integrated value of transcription rates were estimated from the above two parameters using a mathematical approach. Our results showed that several cold-responsive genes, including Cold-regulated 15a, were relatively destabilized, whereas the mRNA amounts were increased during cold treatment by accelerating the transcription rate to overcome the destabilization. Considering the kinetics of mRNA synthesis and degradation, this apparently contradictory result supports that mRNA destabilization is advantageous for the swift increase in CBF-responsive genes in response to cold stress.

Regulation of leptin synthesis in white adipose tissue of the female fruit bat, Cynopterus sphinx: role of melatonin with or without insulin.

Science.gov (United States)

Banerjee, A; Udin, S; Krishna, A

2011-02-01

Factors regulating leptin synthesis during adipogenesis in wild species are not well known. Studies in the female Cynopterus sphinx bat have shown that it undergoes seasonal changes in its fat deposition and serum leptin and melatonin levels. The aim of the present study was to investigate the hormonal regulation of leptin synthesis by the white adipose tissue during the period of fat deposition in female C. sphinx. This study showed a significant correlation between the seasonal changes in serum melatonin level with the circulating leptin level (r = 0.78; P sphinx. A significant correlation between circulating insulin and leptin levels (r = 0.65; P sphinx. The study showed MT(2) receptors in adipose tissue and a stimulatory effect of melatonin on leptin synthesis, which was blocked by treatment with an MT(2) receptor antagonist, suggesting that the effect of melatonin on leptin synthesis by adipose tissue is mediated through the MT(2) receptor in C. sphinx. The in vitro study showed that the synthesis of leptin is directly proportional to the amount of glucose uptake by the adipose tissue. It further showed that melatonin together with insulin synergistically enhanced the leptin synthesis by adipose tissue through phosphorylation of mitogen-activated protein kinase in C. sphinx.

Expression of ODC Antizyme Inhibitor 2 (AZIN2 in Human Secretory Cells and Tissues.

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Tiina Rasila

Full Text Available Ornithine decarboxylase (ODC antizyme inhibitor 2 (AZIN2, originally called ODCp, is a regulator of polyamine synthesis that we originally identified and cloned. High expression of ODCp mRNA was found in brain and testis. We reported that AZIN2 is involved in regulation of cellular vesicle transport and / or secretion, but the ultimate physiological role(s of AZIN2 is still poorly understood. In this study we used a peptide antibody (K3 to human AZIN2 and by immunohistochemistry mapped its expression in various normal tissues. We found high expression in the nervous system, in type 2 pneumocytes in the lung, in megakaryocytes, in gastric parietal cells co-localized with H,K-ATPase beta subunit, in selected enteroendocrine cells, in acinar cells of sweat glands, in podocytes, in macula densa cells and epithelium of collecting ducts in the kidney. The high expression of AZIN2 in various cells with secretory or vesicle transport activity indicates that the polyamine metabolism regulated by AZIN2 is more significantly involved in these events than previously appreciated.

IMD-4690, a novel specific inhibitor for plasminogen activator inhibitor-1, reduces allergic airway remodeling in a mouse model of chronic asthma via regulating angiogenesis and remodeling-related mediators.

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Toshifumi Tezuka

Full Text Available Plasminogen activator inhibitor (PAI-1 is the principal inhibitor of plasminogen activators, and is responsible for the degradation of fibrin and extracellular matrix. IMD-4690 is a newly synthesized inhibitor for PAI-1, whereas the effect on allergic airway inflammation and remodeling is still unclear. We examined the in vivo effects by using a chronic allergen exposure model of bronchial asthma in mice. The model was generated by an immune challenge for 8 weeks with house dust mite antigen, Dermatophagoides pteronyssinus (Dp. IMD-4690 was intraperitoneally administered during the challenge. Lung histopathology, hyperresponsiveness and the concentrations of mediators in lung homogenates were analyzed. The amount of active PAI-1 in the lungs was increased in mice treated with Dp. Administration with IMD-4690 reduced an active/total PAI-1 ratio. IMD-4690 also reduced the number of bronchial eosinophils in accordance with the decreased expressions of Th2 cytokines in the lung homogenates. Airway remodeling was inhibited by reducing subepithelial collagen deposition, smooth muscle hypertrophy, and angiogenesis. The effects of IMD-4690 were partly mediated by the regulation of TGF-β, HGF and matrix metalloproteinase. These results suggest that PAI-1 plays crucial roles in airway inflammation and remodeling, and IMD-4690, a specific PAI-1 inhibitor, may have therapeutic potential for patients with refractory asthma due to airway remodeling.

IMD-4690, a novel specific inhibitor for plasminogen activator inhibitor-1, reduces allergic airway remodeling in a mouse model of chronic asthma via regulating angiogenesis and remodeling-related mediators.

Science.gov (United States)

Tezuka, Toshifumi; Ogawa, Hirohisa; Azuma, Masahiko; Goto, Hisatsugu; Uehara, Hisanori; Aono, Yoshinori; Hanibuchi, Masaki; Yamaguchi, Yoichi; Fujikawa, Tomoyuki; Itai, Akiko; Nishioka, Yasuhiko

2015-01-01

Plasminogen activator inhibitor (PAI)-1 is the principal inhibitor of plasminogen activators, and is responsible for the degradation of fibrin and extracellular matrix. IMD-4690 is a newly synthesized inhibitor for PAI-1, whereas the effect on allergic airway inflammation and remodeling is still unclear. We examined the in vivo effects by using a chronic allergen exposure model of bronchial asthma in mice. The model was generated by an immune challenge for 8 weeks with house dust mite antigen, Dermatophagoides pteronyssinus (Dp). IMD-4690 was intraperitoneally administered during the challenge. Lung histopathology, hyperresponsiveness and the concentrations of mediators in lung homogenates were analyzed. The amount of active PAI-1 in the lungs was increased in mice treated with Dp. Administration with IMD-4690 reduced an active/total PAI-1 ratio. IMD-4690 also reduced the number of bronchial eosinophils in accordance with the decreased expressions of Th2 cytokines in the lung homogenates. Airway remodeling was inhibited by reducing subepithelial collagen deposition, smooth muscle hypertrophy, and angiogenesis. The effects of IMD-4690 were partly mediated by the regulation of TGF-β, HGF and matrix metalloproteinase. These results suggest that PAI-1 plays crucial roles in airway inflammation and remodeling, and IMD-4690, a specific PAI-1 inhibitor, may have therapeutic potential for patients with refractory asthma due to airway remodeling.

Predicting the Performance of Organic Corrosion Inhibitors

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David A. Winkler

2017-12-01

Full Text Available The withdrawal of effective but toxic corrosion inhibitors has provided an impetus for the discovery of new, benign organic compounds to fill that role. Concurrently, developments in the high-throughput synthesis of organic compounds, the establishment of large libraries of available chemicals, accelerated corrosion inhibition testing technologies, and the increased capability of machine learning methods have made discovery of new corrosion inhibitors much faster and cheaper than it used to be. We summarize these technical developments in the corrosion inhibition field and describe how data-driven machine learning methods can generate models linking molecular properties to corrosion inhibition that can be used to predict the performance of materials not yet synthesized or tested. We briefly summarize the literature on quantitative structure–property relationships models of small organic molecule corrosion inhibitors. The success of these models provides a paradigm for rapid discovery of novel, effective corrosion inhibitors for a range of metals and alloys in diverse environments.

The effect of intracellular calcium level regulators on the synthesis of pollen tube callose in Oenothera biennis L.

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Elżbieta Bednarska

2014-01-01

Full Text Available It is shown that callose synthesis in the Oenothera biennis pollen tube is regulated by the endogenous Ca2+ level. Calcium antagonists reduced the amount of callose in the wall above the tip of the pollen tube (Verapamil - calcium channels blocker and at the tube tip after stopping tube growth (La3+ - a Ca2+ substitute. Ruthenium red and ionophore A 23187, which raise the Ca 21 level in the cytoplasm, induced callose synthesis at the tip of pollen tube.

Cytochrome P450 regulation: the interplay between its heme and apoprotein moieties in synthesis, assembly, repair, and disposal.

Science.gov (United States)

Correia, Maria Almira; Sinclair, Peter R; De Matteis, Francesco

2011-02-01

Heme is vital to our aerobic universe. Heme cellular content is finely tuned through an exquisite control of synthesis and degradation. Heme deficiency is deleterious to cells, whereas excess heme is toxic. Most of the cellular heme serves as the prosthetic moiety of functionally diverse hemoproteins, including cytochromes P450 (P450s). In the liver, P450s are its major consumers, with >50% of hepatic heme committed to their synthesis. Prosthetic heme is the sine qua non of P450 catalytic biotransformation of both endo- and xenobiotics. This well-recognized functional role notwithstanding, heme also regulates P450 protein synthesis, assembly, repair, and disposal. These less well-appreciated aspects are reviewed herein.

Pregnenolone biosynthesis in C6-2B glioma cell mitochondria: regulation by a mitochondrial diazepam binding inhibitor receptor.

OpenAIRE

Papadopoulos, V; Guarneri, P; Kreuger, K E; Guidotti, A; Costa, E

1992-01-01

The C6-2B glioma cell line, rich in mitochondrial receptors that bind with high affinity to benzodiazepines, imidazopyridines, and isoquinolinecarboxamides (previously called peripheral-type benzodiazepine receptors), was investigated as a model to study the significance of the polypeptide diazepam binding inhibitor (DBI) and the putative DBI processing products on mitochondrial receptor-regulated steroidogenesis. DBI and its naturally occurring fragments have been found to be present in high...

Glucocorticoids and Polyamine Inhibitors Synergize to Kill Human Leukemic CEM Cells

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Aaron L. Miller

2002-01-01

Full Text Available Glucocorticoids are well-known apoptotic agents in certain classes of lymphoid cell malignancies. Reduction of intracellular polyamine levels by use of inhibitors that block polyamine synthesis slows or inhibits growth of many cells in vitro. Several such inhibitors have shown efficacy in clinical trials, though the toxicity of some compounds has limited their usefulness. We have tested the effects of combinations of the glucocorticoid dexamethasone. (20Dex and two polyamine inhibitors, difluoromethylornithine. (20DFMO and methyl glyoxal bis guanylhydrazone. (20MGBG, on the clonal line of human acute lymphoblastic leukemia cells, CEM-C7-14. Dex alone kills these cells, though only after a delay of at least 24 hours. We also evaluated a partially glucocorticoid-resistant c-Myc-expressing CEM-C7-14 clone. We show that Dex downregulates ornithine decarboxylase. (20ODC, the rate-limiting enzyme in polyamine synthesis. Pretreatment with the ODC inhibitor DFMO, followed by addition of Dex, enhances steroid-evoked kill slightly. The combination of pretreatment with sublethal concentrations of both DFMO and the inhibitor of S-adenosylmethionine decarboxylase, MGBG, followed by addition of Dex, results in strong synergistic cell kill. Both the rapidity and extent of cell kill are enhanced compared to the effects of Dex alone. These results suggest that use of such combinations in vivo may result in apoptosis of malignant cells with lower overall toxicity.

Farnesoid X receptor up-regulates expression of Lipid transfer inhibitor protein in liver cells and mice

Energy Technology Data Exchange (ETDEWEB)

Li, Liangpeng [Department of Biochemistry and Molecular Biology, College of Basic Medical Science, Third Military Medical University, Chongqing 400038 (China); Liu, Hong [Department of Hematology, Xinqiao Hospital, Third Military Medical University, Chongqing 400037 (China); Peng, Jiahe; Wang, Yongchao; Zhang, Yan; Dong, Jinyu; Liu, Xiaohua; Guo, Dongmei [Department of Biochemistry and Molecular Biology, College of Basic Medical Science, Third Military Medical University, Chongqing 400038 (China); Jiang, Yu, E-mail: yujiang61@gmail.com [Department of Biochemistry and Molecular Biology, College of Basic Medical Science, Third Military Medical University, Chongqing 400038 (China)

2013-11-29

Highlights: •FXR up-regulates apoF. •It binds to ER1 element. •It activates apoF gene promoter. -- Abstract: Apolipoprotein F is a component protein mainly secreted by liver and resides on several lipoprotein classes. It can inhibit lipids transfer between different lipoproteins. FXR is a member of the nuclear receptor superfamily which is also highly expressed in the liver. It modulates bile acids synthesis and lipids metabolism by transcriptional regulation. We aimed to determine whether apoF can be regulated by FXR. The FXR agonist Chenodeoxycholic acid (CDCA) and GW4064 both can activate the expression of apoF in liver cell lines and in C57/BL6 mouse liver. This is dependent on the binding of FXR to the FXR element ER1 (−2904 to −2892 bp) in the apoF gene promoter. Taken together, we have identified apoF as likely another target gene of FXR.

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

International Nuclear Information System (INIS)

Utsumi, Hiroshi; Elkind, M.M.

1994-01-01

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

Synthesis of a wild-type and three mutant Cucurbita maxima trypsin inhibitor-encoding genes by a single-strand approach.

Science.gov (United States)

Botes, D P; Qobose, M D; Corfield, V A

1991-09-15

A single-strand approach to gene assembly, based on a modification of an in vitro complementary oligodeoxyribonucleotide template-directed ligation of the desired sequence to a linearized vector [Chen et al., Nucleic Acids Res. 18 (1990) 871-878], is described. The gene coding for the wild-type Cucurbita maxima trypsin inhibitor of 29 amino acid residues [Bode et al., FEBS Lett. 242 (1989) 285-292], as well as three mutant forms of the gene, in which two of the three disulfide bonds have been replaced singly or as a pair, have been synthesized in a single synthesis run with minimal manual intervention. Subsequent to ligation to pUC9 and in vivo gapped duplex repair by Escherichia coli, their sequences have been verified.

The role of mTOR signaling in the regulation of protein synthesis and muscle mass during immobilization in mice

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You, Jae-Sung; Anderson, Garrett B.; Dooley, Matthew S.; Hornberger, Troy A.

2015-01-01

ABSTRACT The maintenance of skeletal muscle mass contributes substantially to health and to issues associated with the quality of life. It has been well recognized that skeletal muscle mass is regulated by mechanically induced changes in protein synthesis, and that signaling by mTOR is necessary for an increase in protein synthesis and the hypertrophy that occurs in response to increased mechanical loading. However, the role of mTOR signaling in the regulation of protein synthesis and muscle mass during decreased mechanical loading remains largely undefined. In order to define the role of mTOR signaling, we employed a mouse model of hindlimb immobilization along with pharmacological, mechanical and genetic means to modulate mTOR signaling. The results first showed that immobilization induced a decrease in the global rates of protein synthesis and muscle mass. Interestingly, immobilization also induced an increase in mTOR signaling, eIF4F complex formation and cap-dependent translation. Blocking mTOR signaling during immobilization with rapamycin not only impaired the increase in eIF4F complex formation, but also augmented the decreases in global protein synthesis and muscle mass. On the other hand, stimulating immobilized muscles with isometric contractions enhanced mTOR signaling and rescued the immobilization-induced decrease in global protein synthesis through a rapamycin-sensitive mechanism that was independent of ribosome biogenesis. Unexpectedly, the effects of isometric contractions were also independent of eIF4F complex formation. Similar to isometric contractions, overexpression of Rheb in immobilized muscles enhanced mTOR signaling, cap-dependent translation and global protein synthesis, and prevented the reduction in fiber size. Therefore, we conclude that the activation of mTOR signaling is both necessary and sufficient to alleviate the decreases in protein synthesis and muscle mass that occur during immobilization. Furthermore, these results indicate

Dependency on de novo protein synthesis and proteomic changes during metamorphosis of the marine bryozoan Bugula neritina

KAUST Repository

Wong, Yue Him

2010-05-24

Background: Metamorphosis in the bryozoan Bugula neritina (Linne) includes an initial phase of rapid morphological rearrangement followed by a gradual phase of morphogenesis. We hypothesized that the first phase may be independent of de novo synthesis of proteins and, instead, involves post-translational modifications of existing proteins, providing a simple mechanism to quickly initiate metamorphosis. To test our hypothesis, we challenged B. neritina larvae with transcription and translation inhibitors. Furthermore, we employed 2D gel electrophoresis to characterize changes in the phosphoproteome and proteome during early metamorphosis. Differentially expressed proteins were identified by liquid chromatography tandem mass spectrometry and their gene expression patterns were profiled using semi-quantitative real time PCR.Results: When larvae were incubated with transcription and translation inhibitors, metamorphosis initiated through the first phase but did not complete. We found a significant down-regulation of 60 protein spots and the percentage of phosphoprotein spots decreased from 15% in the larval stage to12% during early metamorphosis. Two proteins--the mitochondrial processing peptidase beta subunit and severin--were abundantly expressed and phosphorylated in the larval stage, but down-regulated during metamorphosis. MPPbeta and severin were also down-regulated on the gene expression level.Conclusions: The initial morphogenetic changes that led to attachment of B. neritina did not depend on de novo protein synthesis, but the subsequent gradual morphogenesis did. This is the first time that the mitochondrial processing peptidase beta subunit or severin have been shown to be down-regulated on both gene and protein expression levels during the metamorphosis of B. neritina. Future studies employing immunohistochemistry to reveal the expression locality of these two proteins during metamorphosis should provide further evidence of the involvement of these two

Heme exporter FLVCR1a regulates heme synthesis and degradation and controls activity of cytochromes P450.

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Vinchi, Francesca; Ingoglia, Giada; Chiabrando, Deborah; Mercurio, Sonia; Turco, Emilia; Silengo, Lorenzo; Altruda, Fiorella; Tolosano, Emanuela

2014-05-01

The liver has one of the highest rates of heme synthesis of any organ. More than 50% of the heme synthesized in the liver is used for synthesis of P450 enzymes, which metabolize exogenous and endogenous compounds that include natural products, hormones, drugs, and carcinogens. Feline leukemia virus subgroup C cellular receptor 1a (FLVCR1a) is plasma membrane heme exporter that is ubiquitously expressed and controls intracellular heme content in hematopoietic lineages. We investigated the role of Flvcr1a in liver function in mice. We created mice with conditional disruption of Mfsd7b, which encodes Flvcr1a, in hepatocytes (Flvcr1a(fl/fl);alb-cre mice). Mice were analyzed under basal conditions, after phenylhydrazine-induced hemolysis, and after induction of cytochromes P450 synthesis. Livers were collected and analyzed by histologic, quantitative real-time polymerase chain reaction, and immunoblot analyses. Hepatic P450 enzymatic activities were measured. Flvcr1a(fl/fl);alb-cre mice accumulated heme and iron in liver despite up-regulation of heme oxygenase 1, ferroportin, and ferritins. Hepatic heme export activity of Flvcr1a was closely associated with heme biosynthesis, which is required to sustain cytochrome induction. Upon cytochromes P450 stimulation, Flvcr1a(fl/fl);alb-cre mice had reduced cytochrome activity, associated with accumulation of heme in hepatocytes. The expansion of the cytosolic heme pool in these mice was likely responsible for the early inhibition of heme synthesis and increased degradation of heme, which reduced expression and activity of cytochromes P450. In livers of mice, Flvcr1a maintains a free heme pool that regulates heme synthesis and degradation as well as cytochromes P450 expression and activity. These findings have important implications for drug metabolism. Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.

Root jasmonic acid synthesis and perception regulate folivore-induced shoot metabolites and increase Nicotiana attenuata resistance.

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Fragoso, Variluska; Rothe, Eva; Baldwin, Ian T; Kim, Sang-Gyu

2014-06-01

While jasmonic acid (JA) signaling is widely accepted as mediating plant resistance to herbivores, and the importance of the roots in plant defenses is recently being recognized, the role of root JA in the defense of above-ground parts remains unstudied. To restrict JA impairment to the roots, we micrografted wildtype Nicotiana attenuata shoots to the roots of transgenic plants impaired in JA signaling and evaluated ecologically relevant traits in the glasshouse and in nature. Root JA synthesis and perception are involved in regulating nicotine production in roots. Strikingly, systemic root JA regulated local leaf JA and abscisic acid (ABA) concentrations, which were associated with differences in nicotine transport from roots to leaves via the transpiration stream. Root JA signaling also regulated the accumulation of other shoot metabolites; together these account for differences in resistance against a generalist, Spodoptera littoralis, and a specialist herbivore, Manduca sexta. In N. attenuata's native habitat, silencing root JA synthesis increased the shoot damage inflicted by Empoasca leafhoppers, which are able to select natural jasmonate mutants. Silencing JA perception in roots also increased damage by Tupiocoris notatus. We conclude that attack from above-ground herbivores recruits root JA signaling to launch the full complement of plant defense responses. © 2014 Max Planck Society. New Phytologist © 2014 New Phytologist Trust.

Multipotent MAO and cholinesterase inhibitors for the treatment of Alzheimer's disease: synthesis, pharmacological analysis and molecular modeling of heterocyclic substituted alkyl and cycloalkyl propargyl amine.

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Samadi, Abdelouahid; de los Ríos, Cristóbal; Bolea, Irene; Chioua, Mourad; Iriepa, Isabel; Moraleda, Ignacio; Bartolini, Manuela; Andrisano, Vincenza; Gálvez, Enrique; Valderas, Carolina; Unzeta, Mercedes; Marco-Contelles, José

2012-06-01

The synthesis, pharmacological evaluation and molecular modeling of heterocyclic substituted alkyl and cycloalkyl propargyl amines 1-7 of type I, and 9-12 of type II, designed as multipotent inhibitors able to simultaneously inhibit monoamine oxidases (MAO-A/B) as well as cholinesterase (AChE/BuChE) enzymes, as potential drugs for the treatment of Alzheimer's disease, are described. Indole derivatives 1-7 of type I are well known MAO inhibitors whose capacity to inhibit AChE and BuChE was here investigated for the first time. As a result, compound 7 was identified as a MAO-B inhibitor (IC(50) = 31 ± 2 nM) and a moderately selective eqBuChE inhibitor (IC(50) = 4.7 ± 0.2 μM). Conversely, the new and readily available 5-amino-7-(prop-2-yn-1-yl)-6,7,8,9-tetrahydropyrido[2,3-b][1,6]naphthyridine derivatives 9-13 of type II are poor MAO inhibitors, but showed AChE selective inhibition, compound 12 being the most attractive as it acts as a non-competitive inhibitor on EeAChE (IC(50) = 25 ± 3 nM, K(i) = 65 nM). The ability of this compound to interact with the AChE peripheral binding site was confirmed by kinetic studies and by molecular modeling investigation. Studies on human ChEs confirmed that 12 is a selective AChE inhibitor with inhibitory potency in the submicromolar range. Moreover, in agreement with its mode of action, 12 was shown to be able to inhibit Aβ aggregation induced by hAChE by 30.6%. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

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

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Ying Wang

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

Synthesis of deleobuvir, a potent hepatitis C virus polymerase inhibitor, and its major metabolites labeled with carbon-13 and carbon-14.

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Latli, Bachir; Hrapchak, Matt; Chevliakov, Maxim; Li, Guisheng; Campbell, Scot; Busacca, Carl A; Senanayake, Chris H

2015-05-30

Deleobuvir, (2E)-3-(2-{1-[2-(5-bromopyrimidin-2-yl)-3-cyclopentyl-1-methyl-1H-indole-6-carboxamido]cyclobutyl}-1-methyl-1H-benzimidazol-6-yl)prop-2-enoic acid (1), is a non-nucleoside, potent, and selective inhibitor of hepatitis C virus NS5B polymerase. Herein, we describe the detailed synthesis of this compound labeled with carbon-13 and carbon-14. The synthesis of its three major metabolites, namely, the reduced double bond metabolite (2) and the acyl glucuronide derivatives of (1) and (2), is also reported. Aniline-(13) C6 was the starting material to prepare butyl (E)-3-(3-methylamino-4-nitrophenyl-(13) C6 )acrylate [(13) C6 ]-(11) in six steps. This intermediate was then used to obtain [(13) C6 ]-(1) and [(13) C6 ]-(2) in five and four more steps, respectively. For the radioactive synthesis, potassium cyanide-(14) C was used to prepare 1-cylobutylaminoacid [(14) C]-(23) via Buchrer-Bergs reaction. The carbonyl chloride of this acid was then used to access both [(14) C]-(1) and [(14) C]-(2) in four steps. The acyl glucuronide derivatives [(13) C6 ]-(3), [(13) C6 ]-(4) and [(14) C]-(3) were synthesized in three steps from the acids [(13) C6 ]-(1), [(13) C6 ]-(2) and [(14) C]-(1) using known procedures. Copyright © 2015 John Wiley & Sons, Ltd.

Discovery of inhibitors of Leishmania β-1,2-mannosyltransferases using a click-chemistry-derived guanosine monophosphate library.

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Phillip van der Peet

Full Text Available Leishmania spp. are a medically important group of protozoan parasites that synthesize a novel intracellular carbohydrate reserve polymer termed mannogen. Mannogen is a soluble homopolymer of β-1,2-linked mannose residues that accumulates in the major pathogenic stages in the sandfly vector and mammalian host. While several steps in mannogen biosynthesis have been defined, none of the enzymes have been isolated or characterized. We report the development of a simple assay for the GDP-mannose-dependent β-1,2-mannosyltransferases involved in mannogen synthesis. This assay utilizes octyl α-D-mannopyranoside to prime the formation of short mannogen oligomers up to 5 mannose residues. This assay was used to screen a focussed library of 44 GMP-triazole adducts for inhibitors. Several compounds provided effective inhibition of mannogen β-1,2-mannosyltransferases in a cell-free membrane preparation. This assay and inhibitor compounds will be useful for dissecting the role of different mannosyltransferases in regulating de novo biosynthesis and elongation reactions in mannogen metabolism.

Polyamines Function in Stress Tolerance: From Synthesis to Regulation

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Ji-Hong eLiu

2015-10-01

Full Text Available Plants are challenged by a variety of biotic or abiotic stresses, which can affect their growth and development, productivity and geographic distribution. In order to survive adverse environmental conditions, plants have evolved various adaptive strategies, among which is the accumulation of metabolites that play protective roles. A well-established example of the metabolites that are involved in stress responses, or stress tolerance, is the low-molecular-weight aliphatic polyamines, including putrescine,spermidine and spermine. The critical role of polyamines in stress tolerance is suggested by several lines of evidence: firstly, the transcript levels of polyamine biosynthetic genes, as well as the activities of the corresponding enzymes, are induced by stresses; secondly, elevation of endogenous polyamine levels by exogenous supply of polyamines, or overexpression of polyamine biosynthetic genes, results in enhanced stress tolerance; and thirdly, a reduction of endogenous polyamines is accompanied by compromised stress tolerance. A number of studies have demonstrated that polyamines function in stress tolerance largely by modulating the homeostasis of reactive oxygen species (ROS due to their direct, or indirect, roles in regulating antioxidant systems or suppressing ROS production. The transcriptional regulation of polyamine synthesis by transcription factors is also reviewed here. Meanwhile, future perspectives on polyamine research are also suggested.

Synthesis and Biological Assessment of Racemic Benzochromenopyrimidinimines as Antioxidant, Cholinesterase, and Aβ1-42 Aggregation Inhibitors for Alzheimer's Disease Therapy.

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Dgachi, Youssef; Ismaili, Lhassane; Knez, Damijan; Benchekroun, Mohamed; Martin, Hélène; Szałaj, Natalia; Wehle, Sarah; Bautista-Aguilera, Oscar M; Luzet, Vincent; Bonnet, Alexandre; Malawska, Barbara; Gobec, Stanislav; Chioua, Mourad; Decker, Michael; Chabchoub, Fakher; Marco-Contelles, José

2016-06-20

Given the complex nature of Alzheimer's disease (AD), compounds that are able to simultaneously address two or more AD-associated targets show greater promise for development into drugs for AD therapy. Herein we report an efficient two-step synthesis and biological evaluation of new racemic benzochromene derivatives as antioxidants, inhibitors of cholinesterase and β-amyloid (Aβ1-42 ) aggregation. Based on the results of the primary screening, we identified 15-(3-methoxyphenyl)-9,11,12,15-tetrahydro-10H,14H-benzo[5,6]chromeno[2,3-d]pyrido[1,2-a]pyrimidin-14-imine (3 e) and 16-(3-methoxyphenyl)-9,10,11,12,13,16-hexahydro-15H-benzo[5',6']chromeno[2',3':4,5]pyrimido[1,2-a]azepin-15-imine (3 f) as new potential multitarget-directed ligands for AD therapy. Further in-depth biological analysis showed that compound 3 f is a good human acetylcholinesterase inhibitor [IC50 =(0.36±0.02) μm], has strong antioxidant activity (3.61 μmol Trolox equivalents), and moderate Aβ1-42 antiaggregating power (40.3 %). © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Roles of secretory leukocyte protease inhibitor amniotic membrane in oral wound healing

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Elly Munadziroh

2006-12-01

Full Text Available Secretory Leukocyte Protease Inhibitor (SLPI is serine protease inhibitor. Secretory Leukocyte Protease Inhibitor is a protein found in secretions such as whole saliva, seminal fluid, cervical mucus, synovial fluid, breast milk, tears, and cerebral spinal fluid, as in secretions from the nose and bronchi, amniotic fluid and amniotic membrane etc. These findings demonstrate that SLPI function as a potent anti protease, anti inflammatory, bactericidal, antifungal, tissue repair, extra cellular synthesis. Impaired healing states are characterized by excessive proteolysis and often bacterial infection, leading to the hypothesis that SLPI may have a role in the process. The objectives of this article are to investigate the role of SLPI in oral inflammation and how it contributes to tissue repair in oral mucosa. The oral wound healing responses are impaired in the SLPI sufficient mice and matrix synthesis and collagen deposition are delayed. This study indicated that SLPI is a povital factor necessary for optimal wound healing.

Synthesis and biological evaluation of glycogen synthase kinase 3 (GSK-3) inhibitors: an fast and atom efficient access to 1-aryl-3-benzylureas.

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Monte, Fabio Lo; Kramer, Thomas; Boländer, Alexander; Plotkin, Batya; Eldar-Finkelman, Hagit; Fuertes, Ana; Dominguez, Juan; Schmidt, Boris

2011-09-15

The glycogen synthase kinase 3 (GSK-3) is implicated in multiple cellular processes and has been linked to the pathogenesis of Alzheimer's disease (AD). In the course of our research topic we synthesized a library of potent GSK-3 inhibitors. We utilized the urea scaffold present in the potent and highly selective GSK-3 inhibitor AR-A014418 (AstraZeneca). This moiety suits both (a) a convergent approach utilizing readily accessible building blocks and (b) a divergent approach based on a microwave heating assisted Suzuki coupling. We established a chromatography-free purification method to generate products with sufficient purity for the biological assays. The structure-activity relationship of the library provided the rationale for the synthesis of the benzothiazolylurea 66 (IC(50)=140 nM) and the pyridylurea 62 (IC(50)=98 nM), which displayed two to threefold enhanced activity versus the reference compound 18 (AR-A014418: IC(50)=330 nM) in our assays. Copyright © 2011. Published by Elsevier Ltd.

A role for PPARα in the regulation of arginine metabolism and nitric oxide synthesis.

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Guelzim, Najoua; Mariotti, François; Martin, Pascal G P; Lasserre, Frédéric; Pineau, Thierry; Hermier, Dominique

2011-10-01

The pleiotropic effects of PPARα may include the regulation of amino acid metabolism. Nitric oxide (NO) is a key player in vascular homeostasis. NO synthesis may be jeopardized by a differential channeling of arginine toward urea (via arginase) versus NO (via NO synthase, NOS). This was studied in wild-type (WT) and PPARα-null (KO) mice fed diets containing either saturated fatty acids (COCO diet) or 18:3 n-3 (LIN diet). Metabolic markers of arginine metabolism were assayed in urine and plasma. mRNA levels of arginases and NOS were determined in liver. Whole-body NO synthesis and the conversion of systemic arginine into urea were assessed by using (15)N(2)-guanido-arginine and measuring urinary (15)NO(3) and [(15)N]-urea. PPARα deficiency resulted in a markedly lower whole-body NO synthesis, whereas the conversion of systemic arginine into urea remained unaffected. PPARα deficiency also increased plasma arginine and decreased citrulline concentration in plasma. These changes could not be ascribed to a direct effect on hepatic target genes, since NOS mRNA levels were unaffected, and arginase mRNA levels decreased in KO mice. Despite the low level in the diet, the nature of the fatty acids modulated some effects of PPARα deficiency, including plasma arginine and urea, which increased more in KO mice fed the LIN diet than in those fed the COCO diet. In conclusion, PPARα is largely involved in normal whole-body NO synthesis. This warrants further study on the potential of PPARα activation to maintain NO synthesis in the initiation of the metabolic syndrome.

Repositioning of Verrucosidin, a Purported Inhibitor of Chaperone Protein GRP78, as an Inhibitor of Mitochondrial Electron Transport Chain Complex I

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Gonzalez, Reyna; Pao, Peng-Wen; Hofman, Florence M.; Chen, Thomas C.; Louie, Stan G.; Pirrung, Michael C.; Schönthal, Axel H.

2013-01-01

Verrucosidin (VCD) belongs to a group of fungal metabolites that were identified in screening programs to detect molecules that preferentially kill cancer cells under glucose-deprived conditions. Its mode of action was proposed to involve inhibition of increased GRP78 (glucose regulated protein 78) expression during hypoglycemia. Because GRP78 plays an important role in tumorigenesis, inhibitors such as VCD might harbor cancer therapeutic potential. We therefore sought to characterize VCD’s anticancer activity in vitro. Triple-negative breast cancer cell lines MDA-MB-231 and MDA-MB-468 were treated with VCD under different conditions known to trigger increased expression of GRP78, and a variety of cellular processes were analyzed. We show that VCD was highly cytotoxic only under hypoglycemic conditions, but not in the presence of normal glucose levels, and VCD blocked GRP78 expression only when glycolysis was impaired (due to hypoglycemia or the presence of the glycolysis inhibitor 2-deoxyglucose), but not when GRP78 was induced by other means (hypoxia, thapsigargin, tunicamycin). However, VCD’s strictly hypoglycemia-specific toxicity was not due to the inhibition of GRP78. Rather, VCD blocked mitochondrial energy production via inhibition of complex I of the electron transport chain. As a result, cellular ATP levels were quickly depleted under hypoglycemic conditions, and common cellular functions, including general protein synthesis, deteriorated and resulted in cell death. Altogether, our study identifies mitochondria as the primary target of VCD. The possibility that other purported GRP78 inhibitors (arctigenin, biguanides, deoxyverrucosidin, efrapeptin, JBIR, piericidin, prunustatin, pyrvinium, rottlerin, valinomycin, versipelostatin) might act in a similar GRP78-independent fashion will be discussed. PMID:23755268

Repositioning of Verrucosidin, a purported inhibitor of chaperone protein GRP78, as an inhibitor of mitochondrial electron transport chain complex I.

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Simmy Thomas

Full Text Available Verrucosidin (VCD belongs to a group of fungal metabolites that were identified in screening programs to detect molecules that preferentially kill cancer cells under glucose-deprived conditions. Its mode of action was proposed to involve inhibition of increased GRP78 (glucose regulated protein 78 expression during hypoglycemia. Because GRP78 plays an important role in tumorigenesis, inhibitors such as VCD might harbor cancer therapeutic potential. We therefore sought to characterize VCD's anticancer activity in vitro. Triple-negative breast cancer cell lines MDA-MB-231 and MDA-MB-468 were treated with VCD under different conditions known to trigger increased expression of GRP78, and a variety of cellular processes were analyzed. We show that VCD was highly cytotoxic only under hypoglycemic conditions, but not in the presence of normal glucose levels, and VCD blocked GRP78 expression only when glycolysis was impaired (due to hypoglycemia or the presence of the glycolysis inhibitor 2-deoxyglucose, but not when GRP78 was induced by other means (hypoxia, thapsigargin, tunicamycin. However, VCD's strictly hypoglycemia-specific toxicity was not due to the inhibition of GRP78. Rather, VCD blocked mitochondrial energy production via inhibition of complex I of the electron transport chain. As a result, cellular ATP levels were quickly depleted under hypoglycemic conditions, and common cellular functions, including general protein synthesis, deteriorated and resulted in cell death. Altogether, our study identifies mitochondria as the primary target of VCD. The possibility that other purported GRP78 inhibitors (arctigenin, biguanides, deoxyverrucosidin, efrapeptin, JBIR, piericidin, prunustatin, pyrvinium, rottlerin, valinomycin, versipelostatin might act in a similar GRP78-independent fashion will be discussed.

Gene profile analysis of osteoblast genes differentially regulated by histone deacetylase inhibitors

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Lamblin Anne-Francoise

2007-10-01

Full Text Available Abstract Background Osteoblast differentiation requires the coordinated stepwise expression of multiple genes. Histone deacetylase inhibitors (HDIs accelerate the osteoblast differentiation process by blocking the activity of histone deacetylases (HDACs, which alter gene expression by modifying chromatin structure. We previously demonstrated that HDIs and HDAC3 shRNAs accelerate matrix mineralization and the expression of osteoblast maturation genes (e.g. alkaline phosphatase, osteocalcin. Identifying other genes that are differentially regulated by HDIs might identify new pathways that contribute to osteoblast differentiation. Results To identify other osteoblast genes that are altered early by HDIs, we incubated MC3T3-E1 preosteoblasts with HDIs (trichostatin A, MS-275, or valproic acid for 18 hours in osteogenic conditions. The promotion of osteoblast differentiation by HDIs in this experiment was confirmed by osteogenic assays. Gene expression profiles relative to vehicle-treated cells were assessed by microarray analysis with Affymetrix GeneChip 430 2.0 arrays. The regulation of several genes by HDIs in MC3T3-E1 cells and primary osteoblasts was verified by quantitative real-time PCR. Nine genes were differentially regulated by at least two-fold after exposure to each of the three HDIs and six were verified by PCR in osteoblasts. Four of the verified genes (solute carrier family 9 isoform 3 regulator 1 (Slc9a3r1, sorbitol dehydrogenase 1, a kinase anchor protein, and glutathione S-transferase alpha 4 were induced. Two genes (proteasome subunit, beta type 10 and adaptor-related protein complex AP-4 sigma 1 were suppressed. We also identified eight growth factors and growth factor receptor genes that are significantly altered by each of the HDIs, including Frizzled related proteins 1 and 4, which modulate the Wnt signaling pathway. Conclusion This study identifies osteoblast genes that are regulated early by HDIs and indicates pathways that

A Short Synthesis of (+)-Cyclophellitol

DEFF Research Database (Denmark)

Hansen, Flemming Gundorph; Bundgaard, Eva; Madsen, Robert

2005-01-01

A new synthesis of (+)-cyclophellitol, a potent b-glucosidase inhibitor, has been completed in nine steps from D-xylose. The key transformations involve a zinc-mediated fragmentation of benzyl-protected methyl 5-deoxy-5-iodo-xylofuranoside followed by a highly diastereoselective indium-mediated c......-mediated coupling with ethyl 4-bromocrotonate. Subsequent ring-closing olefin metathesis, ester reduction, olefin epoxidation, and deprotection then afford the natural product. This constitutes the shortest synthesis of (+)-cyclophellitol reported to date....

Molecular regulation of MICA expression after HDAC inhibitor treatment of cancer cells

DEFF Research Database (Denmark)

Jensen, Helle

and NKG2D-ligands are upregulated on the surface of abnormal cells. We have previously shown that cancer cells can be stimulated to express the NKG2D-ligands MICA/B after exposure to HDAC-inhibitors (HDAC-i), an occurrence that is not observed in healthy cells. Here we characterize the molecular signal...... pathways that lead to MICA expression after HDAC-inhibitor treatment of cancer cells. Chelating Calcium with Bapta-AM or EGTA potently inhibited HDAC-inhibitor and CMV mediated MICA/B expression. It was further observed that ER Calcium stores were depleted after HDAC-inhibitor treatment. NF-kB activity can...

A role for PPARa in the regulation of arginine metabolismand nitric oxide synthesis

OpenAIRE

2011-01-01

The pleiotropic effects of PPARa may includethe regulation of amino acid metabolism. Nitric oxide (NO)is a key player in vascular homeostasis. NO synthesis maybe jeopardized by a differential channeling of argininetoward urea (via arginase) versus NO (via NO synthase,NOS). This was studied in wild-type (WT) and PPARa-null(KO) mice fed diets containing either saturated fatty acids(COCO diet) or 18:3 n-3 (LIN diet). Metabolic markers ofarginine metabolism were assayed in urine and plasma.mRNA l...

On the use of triazines as inhibitors of steel corrosion

International Nuclear Information System (INIS)

Sizaya, O.I.; Andrushko, A.P.

2004-01-01

A possibility of using substandard pesticides as a raw materials for synthesis of a set of triazines and also using them as a inhibitors of acidic corrosion of steel 20, as well as additions to epoxy powder coatings is considered. It is shown that triazines studied are inhibitors of acidic corrosion of steel 20. 2,4-di(ethylamino)-6-phenylhydrazono-1,3,5-triazine (In 4) has a maximum inhibiting effect among the studied compounds [ru

Evidence for a systemic regulation of neurotrophin synthesis in response to peripheral nerve injury.

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Shakhbazau, Antos; Martinez, Jose A; Xu, Qing-Gui; Kawasoe, Jean; van Minnen, Jan; Midha, Rajiv

2012-08-01

Up-regulation of neurotrophin synthesis is an important mechanism of peripheral nerve regeneration after injury. Neurotrophin expression is regulated by a complex series of events including cell interactions and multiple molecular stimuli. We have studied neurotrophin synthesis at 2 weeks time-point in a transvertebral model of unilateral or bilateral transection of sciatic nerve in rats. We have found that unilateral sciatic nerve transection results in the elevation of nerve growth factor (NGF) and NT-3, but not glial cell-line derived neurotrophic factor or brain-derived neural factor, in the uninjured nerve on the contralateral side, commonly considered as a control. Bilateral transection further increased NGF but not other neurotrophins in the nerve segment distal to the transection site, as compared to the unilateral injury. To further investigate the distinct role of NGF in regeneration and its potential for peripheral nerve repair, we transduced isogeneic Schwann cells with NGF-encoding lentivirus and transplanted the over-expressing cells into the distal segment of a transected nerve. Axonal regeneration was studied at 2 weeks time-point using pan-neuronal marker NF-200 and found to directly correlate with NGF levels in the regenerating nerve. © 2012 The Authors. Journal of Neurochemistry © 2012 International Society for Neurochemistry.

AβPP/APLP2 Family of Kunitz Serine Proteinase Inhibitors Regulate Cerebral Thrombosis

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Xu, Feng; Previti, Mary Lou; Nieman, Marvin T.; Davis, Judianne; Schmaier, Alvin H.; Van Nostrand, William E.

2009-01-01

The amyloid β-protein precursor (AβPP) is best recognized as the precursor to the Aβ peptide that accumulates in the brains of patients with Alzheimer’s disease, but less is known about its physiological functions. Isoforms of AβPP that contain a Kunitz-type serine proteinase inhibitor (KPI) domain are expressed in brain and, outside the CNS, in circulating blood platelets. Recently, we showed that KPI-containing forms of AβPP regulates cerebral thrombosis in vivo (Xu et al., 2005 Proc. Natl. Acad. Sci. USA 102:18135–18140; Xu et al. 2007 Stroke 38:2598–2601). Amyloid precursor like protein-2 (APLP2), a closely related homolog to AβPP, also possesses a highly conserved KPI domain. Virtually nothing is known of its function. Here we show that APLP2 also regulates cerebral thrombosis risk. Recombinant purified KPI domains of AβPP and APLP2 both inhibit the plasma clotting in vitro. In a carotid artery thrombosis model both AβPP−/− and APLP2−/− mice exhibit similar significantly shorter times to vessel occlusion compared with wild-type mice indicating a pro-thrombotic phenotype. Similarly, in an experimental model of intracerebral hemorrhage both AβPP−/− and APLP2−/− mice produce significantly smaller hematomas with reduced brain hemoglobin content compared with wild-type mice. Together, these results indicate that AβPP and APLP2 share overlapping anticoagulant functions with regard to regulating thrombosis after cerebral vascular injury. PMID:19403832

High frequencies of chromatid aberrations produced during G/sub 2/ in human lymphocytes by very low doses (0. 025-0. 4 Gy) of X-rays in combination with inhibitors of DNA synthesis

Energy Technology Data Exchange (ETDEWEB)

Andersson, H.C.; Kihlman, B.A. (Uppsala Univ. (Sweden). Dept. of Genetics)

1984-09-01

Whole-blood cultures of human lymphocytes were exposed in the G/sub 2/-phase (3.5 h before harvesting) to various doses of X-rays and post-treated for 3 h with inhibitors of DNA synthesis. The inhibitors used were 2'-deoxyadenosine (dAdo), hydroxyurea (HU) and 1-..beta..-D-arabinofuranosylcytosine (ara-C). To prevent deamination of dAdo by adenosine deaminase (ADA), the dAdo treatments were carried out in the presence of the ADA inhibitor coformycin. HU and ara-C were used either alone or in combination. After the 3-h inhibitor treatments, the cultures were harvested and slides prepared and analyzed for chromatid aberrations in metaphase. When the inhibitors were used at concentrations high enough to cause marked chromosome damage by themselves, very low doses of X-rays (0.025-0.2 Gy) were sufficient to produce a dramatic increase in the frequency of chromatid aberrations. High frequencies of chromatid aberrations were also obtained when cultures that had received moderate doses of X-rays (0.4-0.8 Gy) were post-treated with low inhibitor concentrations that produce no or only a few aberrations by themselves.

A survey of cyclic replacements for the central diamide moiety of inhibitors of inosine monophosphate dehydrogenase.

Science.gov (United States)

Dhar, T G Murali; Liu, Chunjian; Pitts, William J; Guo, Junquing; Watterson, Scott H; Gu, Henry; Fleener, Catherine A; Rouleau, Katherine; Sherbina, N Z; Barrish, Joel C; Hollenbaugh, Diane; Iwanowicz, Edwin J

2002-11-04

A series of heterocyclic replacements for the central diamide moiety of 1, a potent small molecule inhibitor of inosine monophosphate dehydrogenase (IMPDH) were explored The synthesis and the structure-activity relationships (SARs), derived from in vitro studies, for these new series of inhibitors is given.

Vitamin B12–dependent taurine synthesis regulates growth and bone mass

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Roman-Garcia, Pablo; Quiros-Gonzalez, Isabel; Mottram, Lynda; Lieben, Liesbet; Sharan, Kunal; Wangwiwatsin, Arporn; Tubio, Jose; Lewis, Kirsty; Wilkinson, Debbie; Santhanam, Balaji; Sarper, Nazan; Clare, Simon; Vassiliou, George S.; Velagapudi, Vidya R.; Dougan, Gordon; Yadav, Vijay K.

2014-01-01

Both maternal and offspring-derived factors contribute to lifelong growth and bone mass accrual, although the specific role of maternal deficiencies in the growth and bone mass of offspring is poorly understood. In the present study, we have shown that vitamin B12 (B12) deficiency in a murine genetic model results in severe postweaning growth retardation and osteoporosis, and the severity and time of onset of this phenotype in the offspring depends on the maternal genotype. Using integrated physiological and metabolomic analysis, we determined that B12 deficiency in the offspring decreases liver taurine production and associates with abrogation of a growth hormone/insulin-like growth factor 1 (GH/IGF1) axis. Taurine increased GH-dependent IGF1 synthesis in the liver, which subsequently enhanced osteoblast function, and in B12-deficient offspring, oral administration of taurine rescued their growth retardation and osteoporosis phenotypes. These results identify B12 as an essential vitamin that positively regulates postweaning growth and bone formation through taurine synthesis and suggests potential therapies to increase bone mass. PMID:24911144

Synthesis, delivery and regulation of eukaryotic heme and Fe-S cluster cofactors.

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Barupala, Dulmini P; Dzul, Stephen P; Riggs-Gelasco, Pamela Jo; Stemmler, Timothy L

2016-02-15

In humans, the bulk of iron in the body (over 75%) is directed towards heme- or Fe-S cluster cofactor synthesis, and the complex, highly regulated pathways in place to accomplish biosynthesis have evolved to safely assemble and load these cofactors into apoprotein partners. In eukaryotes, heme biosynthesis is both initiated and finalized within the mitochondria, while cellular Fe-S cluster assembly is controlled by correlated pathways both within the mitochondria and within the cytosol. Iron plays a vital role in a wide array of metabolic processes and defects in iron cofactor assembly leads to human diseases. This review describes progress towards our molecular-level understanding of cellular heme and Fe-S cluster biosynthesis, focusing on the regulation and mechanistic details that are essential for understanding human disorders related to the breakdown in these essential pathways. Copyright © 2016 Elsevier Inc. All rights reserved.

The Impact of Selection with Diflubenzuron, a Chitin Synthesis Inhibitor, on the Fitness of Two Brazilian Aedes aegypti Field Populations

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Belinato, Thiago Affonso; Valle, Denise

2015-01-01

Several Aedes aegypti field populations are resistant to neurotoxic insecticides, mainly organophoshates and pyrethroids, which are extensively used as larvicides and adulticides, respectively. Diflubenzuron (DFB), a chitin synthesis inhibitor (CSI), was recently approved for use in drinking water, and is presently employed in Brazil for Ae. aegypti control, against populations resistant to the organophosphate temephos. However, tests of DFB efficacy against field Ae. aegypti populations are lacking. In addition, information regarding the dynamics of CSI resistance, and characterization of any potential fitness effects that may arise in conjunction with resistance are essential for new Ae. aegypti control strategies. Here, the efficacy of DFB was evaluated for two Brazilian Ae. aegypti populations known to be resistant to both temephos and the pyrethroid deltamethrin. Laboratory selection for DFB resistance was then performed over six or seven generations, using a fixed dose of insecticide that inhibited 80% of adult emergence in the first generation. The selection process was stopped when adult emergence in the diflubenzuron-treated groups was equivalent to that of the control groups, kept without insecticide. Diflubenzuron was effective against the two Ae. aegypti field populations evaluated, regardless of their resistance level to neurotoxic insecticides. However, only a few generations of DFB selection were sufficient to change the susceptible status of both populations to this compound. Several aspects of mosquito biology were affected in both selected populations, indicating that diflubenzuron resistance acquisition is associated with a fitness cost. We believe that these results can significantly contribute to the design of control strategies involving the use of insect growth regulators. PMID:26107715

Recent Advances in the Development of Mammalian Geranylgeranyl Diphosphate Synthase Inhibitors

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Staci L. Haney

2017-05-01

Full Text Available The enzyme geranylgeranyl diphosphate synthase (GGDPS catalyzes the synthesis of the 20-carbon isoprenoid geranylgeranyl diphosphate (GGPP. GGPP is the isoprenoid donor for protein geranylgeranylation reactions catalyzed by the enzymes geranylgeranyl transferase (GGTase I and II. Inhibitors of GGDPS result in diminution of protein geranylgeranylation through depletion of cellular GGPP levels, and there has been interest in GGDPS inhibitors as potential anti-cancer agents. Here we discuss recent advances in the development of GGDPS inhibitors, including insights gained by structure-function relationships, and review the preclinical data that support the continued development of this novel class of drugs.

Synthesis of S-linked oligoxylans

DEFF Research Database (Denmark)

Bonora, Beatrice

by several families of enzymes,collected under the name of Glycosyl Hydrolases (GHs). Among other methods,the use of enzyme inhibitors like thio-linked oligosaccharides has for a long timebeen a common tool to analyze and characterize these enzymes.In the present work the chemical synthesis of thio......-analogs of xylo- andarabinoxyloglycans is presented. Furthermore, the selection of a reliable method forthe incorporation of thiolinkages in the synthesis of oligoxylans is alsoinvestigated.Therefore, different strategies for assembling S-linked-disaccharides have beenapproached both involving 1-thioglycoside...... donors and thioacceptors. Advantagesand disadvantages concerning the different methods are described and evaluated inrelation to the synthesis of linear and branched oligoxylans....

Gardenia jasminoides Encodes an Inhibitor-2 Protein for Protein Phosphatase Type 1

Science.gov (United States)

Gao, Lan; Li, Hao-Ming

2017-08-01

Protein phosphatase-1 (PP1) regulates diverse, essential cellular processes such as cell cycle progression, protein synthesis, muscle contraction, carbohydrate metabolism, transcription and neuronal signaling. Inhibitor-2 (I-2) can inhibit the activity of PP1 and has been found in diverse organisms. In this work, a Gardenia jasminoides fruit cDNA library was constructed, and the GjI-2 cDNA was isolated from the cDNA library by sequencing method. The GjI-2 cDNA contains a predicted 543 bp open reading frame that encodes 180 amino acids. The bioinformatics analysis suggested that the GjI-2 has conserved PP1c binding motif, and contains a conserved phosphorylation site, which is important in regulation of its activity. The three-dimensional model structure of GjI-2 was buite, its similar with the structure of I-2 from mouse. The results suggest that GjI-2 has relatively conserved RVxF, FxxR/KxR/K and HYNE motif, and these motifs are involved in interaction with PP1.

Glucocorticoids and Polyamine Inhibitors Synergize to Kill Human Leukemic CEM Cells1

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Miller, Aaron L; Johnson, Betty H; Medh, Rheem D; Townsend, Courtney M; Thompson, E Brad

2002-01-01

Abstract Glucocorticoids are well-known apoptotic agents in certain classes of lymphoid cell malignancies. Reduction of intracellular polyamine levels by use of inhibitors that block polyamine synthesis slows or inhibits growth of many cells in vitro. Several such inhibitors have shown efficacy in clinical trials, though the toxicity of some compounds has limited their usefulness. We have tested the effects of combinations of the glucocorticoid dexamethasone (Dex) and two polyamine inhibitors, difluoromethylornithine (DFMO) and methyl glyoxal bis guanylhydrazone (MGBG), on the clonal line of human acute lymphoblastic leukemia cells, CEM-C7-14. Dex alone kills these cells, though only after a delay of at least 24 hours. We also evaluated a partially glucocorticoid-resistant c-Myc-expressing CEM-C7-14 clone. We show that Dex downregulates ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine synthesis. Pretreatment with the ODC inhibitor DFMO, followed by addition of Dex, enhances steroid-evoked kill slightly. The combination of pretreatment with sublethal concentrations of both DFMO and the inhibitor of S-adenosylmethionine decarboxylase, MGBG, followed by addition of Dex, results in strong synergistic cell kill. Both the rapidity and extent of cell kill are enhanced compared to the effects of Dex alone. These results suggest that use of such combinations in vivo may result in apoptosis of malignant cells with lower overall toxicity. PMID:11922393

Indanones as high-potency reversible inhibitors of monoamine oxidase.

Science.gov (United States)

Mostert, Samantha; Petzer, Anél; Petzer, Jacobus P

2015-05-01

Recent reports document that α-tetralone (3,4-dihydro-2H-naphthalen-1-one) is an appropriate scaffold for the design of high-potency monoamine oxidase (MAO) inhibitors. Based on the structural similarity between α-tetralone and 1-indanone, the present study involved synthesis of 34 1-indanone and related indane derivatives as potential inhibitors of recombinant human MAO-A and MAO-B. The results show that C6-substituted indanones are particularly potent and selective MAO-B inhibitors, with IC50 values ranging from 0.001 to 0.030 μM. C5-Substituted indanone and indane derivatives are comparatively weaker MAO-B inhibitors. Although the 1-indanone and indane derivatives are selective inhibitors of the MAO-B isoform, a number of homologues are also potent MAO-A inhibitors, with three homologues possessing IC50 values 1-indanone as a reversible MAO inhibitor with a competitive mode of inhibition. It may be concluded that 1-indanones are promising leads for the design of therapies for neurodegenerative and neuropsychiatric disorders such as Parkinson's disease and depression. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Catalase anabolism in yeast: loss of regulation by oxygen of catalase apoprotein synthesis after mutation.

Science.gov (United States)

Berte, C; Sels, A

1979-04-17

A mutant of Saccharomyces cerevisiae which displays catalase activity when grown under strictly anaerobic conditions has been selected on solid media. Although some preformed holoenzyme has accumulated in anaerobic cells, a sharp increase of activity is still measured during adaptation to oxygen in glucose-buffer; however, a striking difference with the wild-type strain is that in the mutant, catalase formation is observed in the presence of cycloheximide that totally inhibits cytoplasmic translation. It is concluded that kat 80 mutant has lost the regulatory control by oxygen of apocatalase synthesis; the later precursor, characterized as apocatalase synthesis; the latter precursor, characterized as apocatalase T, is thought to be activated in vivo, under aerobic conditions, by inclusion of prosthetic group. Regulation of enzyme synthesis by catabolite repression (glucose erfect) persists, unmodified by reference to the wild-type parental strain. Mutation kat 80 specifically hits catalase anabolism, as no significant variations were observed for the edification of the respiratory system and (apo)cytochrome c peroxidase production. Genetic analysis shows that kat 80 phenotype, recessive in heterozygotes, results from a single nuclear mutation.

Basic studies on I-123-beta-methyl-p-iodophenylpentadecanoic acid (BMIPP) for myocardial functional diagnosis. Effect of beta-oxidation inhibitor

Energy Technology Data Exchange (ETDEWEB)

Fujibayashi, Yasuhisa; Yonekura, Yoshiharu; Yamamoto, Kazutaka; Tamaki, Nagara; Konishi, Junji; Kawai, Keiichi; Yokoyama, Akira; Torizuka, Kanji.

1988-10-01

To clarify the availability of I-123-beta-methyl-p-iodophenylpentadecanoic acid (BMIPP) as myocardial metabolism diagnostic agent, the effect of beta-oxidation inhibitor on BMIPP metabolic behavior was studied in relation to lipid pool. As for inhibitor, tetradecylglycidic acid (TDGA), mitochondrial carnitine acyltransferase I inhibitor, was used. Both in TDGA pre-treated and control rats, BMIPP was found in TG fraction of the heart, showing no inhibitory effect of TDGA on TG-synthesis. In TDGA pre-treated rats, BMIPP accumulation in the heart was greatly increased together with triglyceride (TG) content; free fatty acid and diglyceride content had no remarkable change. So, TG synthesis, which acts as substrate-storage, can be evaluated as an index reflecting the changes of fatty acid metabolism. BMIPP is a plausible radiopharmaceutical for myocardial fatty acid metabolism study, as a substrate of triglyceride synthesis.

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

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Robert W Tilghman

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

Novel inhibitors of IMPDH: a highly potent and selective quinolone-based series.

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Watterson, Scott H; Carlsen, Marianne; Dhar, T G Murali; Shen, Zhongqi; Pitts, William J; Guo, Junqing; Gu, Henry H; Norris, Derek; Chorba, John; Chen, Ping; Cheney, Daniel; Witmer, Mark; Fleener, Catherine A; Rouleau, Katherine; Townsend, Robert; Hollenbaugh, Diane L; Iwanowicz, Edwin J

2003-02-10

A series of novel quinolone-based small molecule inhibitors of inosine monophosphate dehydrogenase (IMPDH) was explored. The synthesis and the structure-activity relationships (SARs) derived from in vitro studies are described.

Inhibitory effects of KN-93, an inhibitor of Ca2+ calmodulin-dependent protein kinase II, on light-regulated root gravitropism in maize

Science.gov (United States)

Feldman, L. J.; Hidaka, H.

1993-01-01

Light is essential for root gravitropism in Zea mays L., cultivar Merit. It is hypothesized that calcium mediates this light-regulated response. KN-93, an inhibitor of calcium/calmodulin kinase II (CaMK II), inhibits light-regulated root gravitropism but does not affect light perception. We hypothesize that CaMK II, or a homologue, operates late in the light/gravity signal transduction chain. Here we provide evidence suggesting a possible physiological involvement of CaMK II in root gravitropism in plants.

The roles of the cyclo-oxygenases types one and two in prostaglandin synthesis in human fetal membranes at term.

Science.gov (United States)

Sawdy, R J; Slater, D M; Dennes, W J; Sullivan, M H; Bennett, P R

2000-01-01

The aim of this study was to determine the relative contributions of cyclo-oxygenase (COX) types 1 and 2 to prostaglandin synthesis at term. Fetal membranes were collected from 6 pregnancies after elective caesarean section at term, prior to labour. The presence of COX-1 and COX-2 protein was determined using Western analysis. The relative contributions of the two isoforms of COX to prostaglandin synthesis were determined by incubation of fetal membrane discs with either a COX-2 selective inhibitor, SC236, or a COX-1 selective inhibitor, SC560, and measurement of prostaglandin release during 24 h using enzyme-linked immuno-sorbent assay (ELISA). Both COX-1 and COX-2 protein were demonstrated in amnion and chorion-decidua. The COX-2 selective inhibitor, SC-236, significantly reduced prostaglandin synthesis, both in its COX-2 specific and higher, non-specific concentration ranges. The COX-1 selective inhibitor, SC-560, had no effect upon prostaglandin synthesis in its COX-1 specific concentration range, but did significantly reduce prostaglandin synthesis at higher, non-selective concentrations. Fetal membranes contain both COX-1 and COX-2 at term, but only COX-2 contributes towards prostaglandin synthesis. COX-2 selective NSAI drugs will be as effective as non-selective agents in inhibition of fetal membrane prostaglandin synthesis and may represent a new strategy for tocolysis. Copyright 2000 Harcourt Publishers Ltd.

Sumoylation of the Tumor Suppressor Promyelocytic Leukemia Protein Regulates Arsenic Trioxide-Induced Collagen Synthesis in Osteoblasts.

Science.gov (United States)

Xu, Wen-Xiao; Liu, Sheng-Zhi; Wu, Di; Qiao, Guo-Fen; Yan, Jinglong

2015-01-01

Promyelocytic leukemia (PML) protein is a tumor suppressor that fuses with retinoic acid receptor-α (PML-RARα) to contribute to the initiation of acute promyelocytic leukemia (APL). Arsenic trioxide (ATO) upregulates expression of TGF-β1, promoting collagen synthesis in osteoblasts, and ATO binds directly to PML to induce oligomerization, sumoylation, and ubiquitination. However, how ATO upregulates TGF-β1 expression is uncertain. Thus, we suggested that PML sumoylation is responsible for regulation of TGF-β1 protein expression. Kunming mice were treated with ATO, and osteoblasts were counted under scanning electron microscopy. Masson's staining was used to quantify collagen content. hFOB1.19 cells were transfected with siRNA against UBC9 or RNF4, and then treated with ATO or FBS. TGF-β1, PML expression, and sumoylation were quantified with Western blot, and collagen quantified via immunocytochemistry. ATO enhanced osteoblast accumulation, collagen synthesis, and PML-NB formation in vivo. Knocking down UBC9 in hFOB1.19 cells inhibited ATO- and FBS-induced PML sumoylation, TGF-β1 expression, and collagen synthesis. Conversely, knocking down RNF4 enhanced ATO- and FBS-induced PML sumoylation, TGF-β1 expression, and collagen synthesis. These data suggest that PML sumoylation is required for ATO-induced collagen synthesis in osteoblasts. © 2015 S. Karger AG, Basel.

Dapagliflozin, a selective SGLT2 Inhibitor, attenuated cardiac fibrosis by regulating the macrophage polarization via STAT3 signaling in infarcted rat hearts.

Science.gov (United States)

Lee, Tsung-Ming; Chang, Nen-Chung; Lin, Shinn-Zong

2017-03-01

During myocardial infarction, infiltrated macrophages have pivotal roles in cardiac remodeling and delayed M1 toward M2 macrophage phenotype transition is considered one of the major factors for adverse ventricular remodeling. We investigated whether dapagliflozin, a sodium-glucose cotransporter 2 (SGLT2) inhibitor, attenuates cardiac fibrosis via regulating macrophage phenotype by a reactive oxygen and nitrogen species (RONS)/STAT3-dependent pathway in postinfarcted rats. Normoglycemic male Wistar rats were subjected to coronary ligation and then randomized to either saline, dapagliflozin (a specific SGLT2 inhibitor), phlorizin (a nonspecific SGLT1/2 inhibitor), dapagliflozin + S3I-201 (a STAT3 inhibitor), or phlorizin + S3I-201 for 4 weeks. There were similar infarct sizes among the infarcted groups at the acute and chronic stages of infarction. At day 3 after infarction, post-infarction was associated with increased levels of superoxide and nitrotyrosine, which can be inhibited by administering either dapagliflozin or phlorizin. SGLT2 inhibitors significantly increased STAT3 activity, STAT3 nuclear translocation, myocardial IL-10 levels and the percentage of M2 macrophage infiltration. At day 28 after infarction, SGLT2 inhibitors were associated with attenuated myofibroblast infiltration and cardiac fibrosis. Although phlorizin decreased myofibroblast infiltration, the effect of dapagliflozin on attenuated myofibroblast infiltration was significantly higher than phlorizin. The effects of SGLT2 inhibitors on cardiac fibrosis were nullified by adding S3I-201. Furthermore, the effects of dapagliflozin on STAT3 activity and myocardial IL-10 levels can be reversed by 3-morpholinosydnonimine, a peroxynitrite generator. Taken together, these observations provide a novel mechanism of SGLT2 inhibitors-mediated M2 polarization through a RONS-dependent STAT3-mediated pathway and selective SGLT2 inhibitors are more effective in attenuating myofibroblast infiltration during

Involvement of the ribose operon repressor RbsR in regulation of purine nucleotide synthesis in Escherichia coli.

Science.gov (United States)

Shimada, Tomohiro; Kori, Ayako; Ishihama, Akira

2013-07-01

Escherichia coli is able to utilize d-ribose as its sole carbon source. The genes for the transport and initial-step metabolism of d-ribose form a single rbsDACBK operon. RbsABC forms the ABC-type high-affinity d-ribose transporter, while RbsD and RbsK are involved in the conversion of d-ribose into d-ribose 5-phosphate. In the absence of inducer d-ribose, the ribose operon is repressed by a LacI-type transcription factor RbsR, which is encoded by a gene located downstream of this ribose operon. At present, the rbs operon is believed to be the only target of regulation by RbsR. After Genomic SELEX screening, however, we have identified that RbsR binds not only to the rbs promoter but also to the promoters of a set of genes involved in purine nucleotide metabolism. Northern blotting analysis indicated that RbsR represses the purHD operon for de novo synthesis of purine nucleotide but activates the add and udk genes involved in the salvage pathway of purine nucleotide synthesis. Taken together, we propose that RbsR is a global regulator for switch control between the de novo synthesis of purine nucleotides and its salvage pathway. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Heme Exporter FLVCR1a Regulates Heme Synthesis and Degradation and Controls Activity of Cytochromes P450

Science.gov (United States)

Vinchi, Francesca; Ingoglia, Giada; Chiabrando, Deborah; Mercurio, Sonia; Turco, Emilia; Silengo, Lorenzo; Altruda, Fiorella; Tolosano, Emanuela

2014-01-01

Background & Aims The liver has one of the highest rates of heme synthesis of any organ. More than 50% of the heme synthesized in the liver is used for synthesis of P450 enzymes, which metabolize exogenous and endogenous compounds that include natural products, hormones, drugs, and carcinogens. Feline leukemia virus subgroup C cellular receptor 1a (FLVCR1a) is plasma membrane heme exporter that is ubiquitously expressed and controls intracellular heme content in hematopoietic lineages. We investigated the role of Flvcr1a in liver function in mice. Methods We created mice with conditional disruption of Mfsd7b, which encodes Flvcr1a, in hepatocytes (Flvcr1afl/fl;alb-cre mice). Mice were analyzed under basal conditions, after phenylhydrazine-induced hemolysis, and after induction of cytochromes P450 synthesis. Livers were collected and analyzed by histologic, quantitative real-time polymerase chain reaction, and immunoblot analyses. Hepatic P450 enzymatic activities were measured. Results Flvcr1afl/fl;alb-cre mice accumulated heme and iron in liver despite up-regulation of heme oxygenase 1, ferroportin, and ferritins. Hepatic heme export activity of Flvcr1a was closely associated with heme biosynthesis, which is required to sustain cytochrome induction. Upon cytochromes P450 stimulation, Flvcr1afl/fl;alb-cre mice had reduced cytochrome activity, associated with accumulation of heme in hepatocytes. The expansion of the cytosolic heme pool in these mice was likely responsible for the early inhibition of heme synthesis and increased degradation of heme, which reduced expression and activity of cytochromes P450. Conclusions In livers of mice, Flvcr1a maintains a free heme pool that regulates heme synthesis and degradation as well as cytochromes P450 expression and activity. These findings have important implications for drug metabolism. PMID:24486949

Screening of protein kinase inhibitors identifies PKC inhibitors as inhibitors of osteoclastic acid secretion and bone resorption

Directory of Open Access Journals (Sweden)

Boutin Jean A

2010-10-01

Full Text Available Abstract Background Bone resorption is initiated by osteoclastic acidification of the resorption lacunae. This process is mediated by secretion of protons through the V-ATPase and chloride through the chloride antiporter ClC-7. To shed light on the intracellular signalling controlling extracellular acidification, we screened a protein kinase inhibitor library in human osteoclasts. Methods Human osteoclasts were generated from CD14+ monocytes. The effect of different kinase inhibitors on lysosomal acidification in human osteoclasts was investigated using acridine orange for different incubation times (45 minutes, 4 and 24 hours. The inhibitors were tested in an acid influx assay using microsomes isolated from human osteoclasts. Bone resorption by human osteoclasts on bone slices was measured by calcium release. Cell viability was measured using AlamarBlue. Results Of the 51 compounds investigated only few inhibitors were positive in both acidification and resorption assays. Rottlerin, GF109203X, Hypericin and Ro31-8220 inhibited acid influx in microsomes and bone resorption, while Sphingosine and Palmitoyl-DL-carnitine-Cl showed low levels of inhibition. Rottlerin inhibited lysosomal acidification in human osteoclasts potently. Conclusions In conclusion, a group of inhibitors all indicated to inhibit PKC reduced acidification in human osteoclasts, and thereby bone resorption, indicating that acid secretion by osteoclasts may be specifically regulated by PKC in osteoclasts.

Identification and development of novel indazole derivatives as potent bacterial peptidoglycan synthesis inhibitors

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Prasanthi Malapati

2018-01-01

Full Text Available Background: Tuberculosis is well-known airborne disease caused by Mycobacterium tuberculosis. Available treatment regimen was unsuccessful in eradicating the deaths caused by the disease worldwide. Owing to the drawbacks such as prolonged treatment period, side effects, and drug tolerance, there resulted in patient noncompliance. In the current study, we attempted to develop inhibitors against unexplored key target glutamate racemase. Methods: Lead identification was done using thermal shift assay from in-house library; inhibitors were developed by lead derivatization technique and evaluated using various biological assays. Results: In indazole series, compounds 11 (6.32 ± 0.35 μM and 22 (6.11 ± 0.51 μM were found to be most promising potent inhibitors among all. These compounds also showed their inhibition on replicating and nonreplicating bacteria. Conclusion: We have developed the novel inhibitors against M. tuberculosis capable of inhibiting active and dormant bacteria, further optimization of inhibitor derivatives can results in better compounds for eradicating tuberculosis.

Expression of a TGF-{beta} regulated cyclin-dependent kinase inhibitor in normal and immortalized airway epithelial cells

Energy Technology Data Exchange (ETDEWEB)

Tierney, L.A.; Bloomfield, C.; Johnson, N.F. [and others

1995-12-01

Tumors arising from epithelial cells, including lung cancers are frequently resistant to factors that regulate growth and differentiation in normal in normal cells. Once such factor is transforming growth factor-{Beta} (TGF-{Beta}). Escape from the growth-inhibitory effects of TGF-{Beta} is thought to be a key step in the transformation of airway epithelial cells. most lung cancer cell lines require serum for growth. In contrast, normal human bronchial epithelial (NHBE) cells are exquisitely sensitive to growth-inhibitory and differentiating effects of TGF-{Beta}. The recent identification of a novel cyclin-dependent kinase inhibitor, p15{sup INK4B}, which is regulated by TGF-{Beta}, suggests a mechanism by which TGF-{Beta} mediates growth arrest in NHBE cells. The purpose of this study was two-fold: (1) to determine if p15{sup INK4B} is induced by TGF-{Beta} in NHBE cells or immortalized bronchial epithelial (R.1) cells and if that induction corresponds to a G1/S cell-cycle arrest; (2) to determine the temporal relationship between p15{sup INK4B} induction, cell-cycle arrest, and the phosphorylation state of the pRB because it is thought that p15{sup INK4B} acts indirectly by preventing phosphorylation of the RB gene product. In this study, expression of p15{sup INK4B} was examined in NHBE cells and R.1 cells at different time intervals following TGF-{Beta} treatment. The expression of this kinase inhibitor and its relationship to the cell and the pRb phosphorylation state were examined in cells that were both sensitive (NHBE) and resistant (R.1) to the effects of TGF-{Beta}. These results suggest that the cyclin-dependent kinase inhibitor, p15{sup INK4B}, is involved in airway epithelial cell differentiation and that loss or reduction of expression plays a role in the resistance of transformed or neoplastic cells to the growth-inhibitory effects of TGF-{Beta}.

Adenosine-derived inhibitors of 78 kDa glucose regulated protein (Grp78) ATPase: insights into isoform selectivity.

Science.gov (United States)

Macias, Alba T; Williamson, Douglas S; Allen, Nicola; Borgognoni, Jenifer; Clay, Alexandra; Daniels, Zoe; Dokurno, Pawel; Drysdale, Martin J; Francis, Geraint L; Graham, Christopher J; Howes, Rob; Matassova, Natalia; Murray, James B; Parsons, Rachel; Shaw, Terry; Surgenor, Allan E; Terry, Lindsey; Wang, Yikang; Wood, Mike; Massey, Andrew J

2011-06-23

78 kDa glucose-regulated protein (Grp78) is a heat shock protein (HSP) involved in protein folding that plays a role in cancer cell proliferation. Binding of adenosine-derived inhibitors to Grp78 was characterized by surface plasmon resonance and isothermal titration calorimetry. The most potent compounds were 13 (VER-155008) with K(D) = 80 nM and 14 with K(D) = 60 nM. X-ray crystal structures of Grp78 bound to ATP, ADPnP, and adenosine derivative 10 revealed differences in the binding site between Grp78 and homologous proteins.

Dynamic Compression Promotes the Matrix Synthesis of Nucleus Pulposus Cells Through Up-Regulating N-CDH Expression in a Perfusion Bioreactor Culture.

Science.gov (United States)

Xu, Yichun; Yao, Hui; Li, Pei; Xu, Wenbin; Zhang, Junbin; Lv, Lulu; Teng, Haijun; Guo, Zhiliang; Zhao, Huiqing; Hou, Gang

2018-01-01

An adequate matrix production of nucleus pulposus (NP) cells is an important tissue engineering-based strategy to regenerate degenerative discs. Here, we mainly aimed to investigate the effects and mechanism of mechanical compression (i.e., static compression vs. dynamic compression) on the matrix synthesis of three-dimensional (3D) cultured NP cells in vitro. Rat NP cells seeded on small intestinal submucosa (SIS) cryogel scaffolds were cultured in the chambers of a self-developed, mechanically active bioreactor for 10 days. Meanwhile, the NP cells were subjected to compression (static compression or dynamic compression at a 10% scaffold deformation) for 6 hours once per day. Unloaded NP cells were used as controls. The cellular phenotype and matrix biosynthesis of NP cells were investigated by real-time PCR and Western blotting assays. Lentivirus-mediated N-cadherin (N-CDH) knockdown and an inhibitor, LY294002, were used to further investigate the role of N-CDH and the PI3K/Akt pathway in this process. Dynamic compression better maintained the expression of cell-specific markers (keratin-19, FOXF1 and PAX1) and matrix macromolecules (aggrecan and collagen II), as well as N-CDH expression and the activity of the PI3K/Akt pathway, in the 3D-cultured NP cells compared with those expression levels and activity in the cells grown under static compression. Further analysis showed that the N-CDH knockdown significantly down-regulated the expression of NP cell-specific markers and matrix macromolecules and inhibited the activation of the PI3K/Akt pathway under dynamic compression. However, inhibition of the PI3K/Akt pathway had no effects on N-CDH expression but down-regulated the expression of NP cell-specific markers and matrix macromolecules under dynamic compression. Dynamic compression increases the matrix synthesis of 3D-cultured NP cells compared with that of the cells under static compression, and the N-CDH-PI3K/Akt pathway is involved in this regulatory process

Design and synthesis of 3,3'-biscoumarin-based c-Met inhibitors.

Science.gov (United States)

Xu, Jimin; Ai, Jing; Liu, Sheng; Peng, Xia; Yu, Linqian; Geng, Meiyu; Nan, Fajun

2014-06-14

A library of biscoumarin-based c-Met inhibitors was synthesized, based on optimization of 3,3'-biscoumarin hit 3, which was identified as a non-ATP competitive inhibitor of c-Met from a diverse library of coumarin derivatives. Among these compounds, 38 and 40 not only showed potent enzyme activities with IC50 values of 107 nM and 30 nM, respectively, but also inhibited c-Met phosphorylation in BaF3/TPR-Met and EBC-1 cells.

Regulation of NAD(P)H:quininone oxidoreductase by glucocorticoids

International Nuclear Information System (INIS)

Pinaire, J.A.; Xiao, G.-H.; Falkner, K.C.; Prough, R.A.

2004-01-01

Previous studies in neonatal and adolescent rats as well as adrenalectomized rats have demonstrated that glucocorticoids regulate the expression of the rat NAD(P)H:quinone oxidoreductase gene (QOR). We used primary cultures of rat adult hepatocytes to document that added glucorticoids repress both the basal and 1,2-benzanthracene-induced expression of QOR mRNA by 65-70%. QOR enzyme activity and protein were concomitantly suppressed as well. The monotonic concentration response for repression of QOR gene products up to 100 μM DEX concentration demonstrated that the glucocorticoid receptor (GR) was most likely involved in this process. The lack of effect at higher concentration rules out a role for the Pregnane X receptor in this regulation by DEX. In addition, the anti-glucorticoid RU38486 blocked this negative regulation and the protein synthesis inhibitor cycloheximide had no effect on this repression process. Similar results of GR dependence were observed using a luciferase reporter construct containing the 5'-flanking region of the human QOR gene using HepG2 cells. Collectively, these results demonstrate that GR must directly participate in the negative regulation of QOR gene expression by dexamethasone and other glucocorticoids in vivo

Ecdysteroid-stimulated synthesis and secretion of an N-acetyl-D-glucosamine-rich glycopeptide in a lepidopteran cell line derived from imaginal discs.

Science.gov (United States)

Porcheron, P; Morinière, M; Coudouel, N; Oberlander, H

1991-01-01

Hormone-regulated processing of N-acetyl-D-glucosamine was studied in an insect cell line derived from imaginal wing discs of the Indian meal moth, Plodia interpunctella (Hübner). The cell line, IAL-PID2, responded to treatment with 20-hydroxyecdysone with increased incorporation of GlcNAc into glycoproteins. Cycloheximide and tunicamycin counteracted the action of the hormone. In particular, treatment with 20-hydroxyecdysone resulted in the secretion of a 5,000 dalton N-acetyl-D-glucosamine-rich glycopeptide by the IAL-PID2 cells. Accumulation of this peptide was prevented by the use of teflubenzuron, a potent chitin synthesis inhibitor. A glycopeptide of similar molecular weight was observed in imaginal discs of P. interpunctella treated with 20-hydroxyecdysone in vitro, under conditions that induce chitin synthesis. Although the function of the 5,000 dalton glycopeptide is not known, we believe that the PID2 cell line is a promising model for molecular analysis of ecdysteroid-regulated processing of aminosugars by epidermal cells during insect development.

Synthesis of gallinamide A analogues as potent falcipain inhibitors and antimalarials.

Science.gov (United States)

Conroy, Trent; Guo, Jin T; Elias, Nabiha; Cergol, Katie M; Gut, Jiri; Legac, Jennifer; Khatoon, Lubna; Liu, Yang; McGowan, Sheena; Rosenthal, Philip J; Hunt, Nicholas H; Payne, Richard J

2014-12-26

Analogues of the natural product gallinamide A were prepared to elucidate novel inhibitors of the falcipain cysteine proteases. Analogues exhibited potent inhibition of falcipain-2 (FP-2) and falcipain-3 (FP-3) and of the development of Plasmodium falciparum in vitro. Several compounds were equipotent to chloroquine as inhibitors of the 3D7 strain of P. falciparum and maintained potent activity against the chloroquine-resistant Dd2 parasite. These compounds serve as promising leads for the development of novel antimalarial agents.

Early steps in protein synthesis and their regulation: a background study related to the biological effects of radiation. Progress report, July 1, 1975--June 30, 1976

Energy Technology Data Exchange (ETDEWEB)

Zamecnik, P.C.

1976-03-01

This is a continuing study of protein synthesis, involving a search for the role of Ap/sub 4/A and other unusual nucleotides in growth regulation; studies of the mechanism of action of aminoacyl-tRNA ligases and the effect thereof on protein synthesis; a search for new regulators of the translation step, in cell-free systems; and an effort to improve the sensitivity and quantitation of chemical sequencing at the 3'-end of messenger RNA.

Sterol regulatory element binding protein and dietary lipid regulation of fatty acid synthesis in the mammary epithelium.

Science.gov (United States)

Rudolph, Michael C; Monks, Jenifer; Burns, Valerie; Phistry, Meridee; Marians, Russell; Foote, Monica R; Bauman, Dale E; Anderson, Steven M; Neville, Margaret C

2010-12-01

The lactating mammary gland synthesizes large amounts of triglyceride from fatty acids derived from the blood and from de novo lipogenesis. The latter is significantly increased at parturition and decreased when additional dietary fatty acids become available. To begin to understand the molecular regulation of de novo lipogenesis, we tested the hypothesis that the transcription factor sterol regulatory element binding factor (SREBF)-1c is a primary regulator of this system. Expression of Srebf1c mRNA and six of its known target genes increased ≥2.5-fold at parturition. However, Srebf1c-null mice showed only minor deficiencies in lipid synthesis during lactation, possibly due to compensation by Srebf1a expression. To abrogate the function of both isoforms of Srebf1, we bred mice to obtain a mammary epithelial cell-specific deletion of SREBF cleavage-activating protein (SCAP), the SREBF escort protein. These dams showed a significant lactation deficiency, and expression of mRNA for fatty acid synthase (Fasn), insulin-induced gene 1 (Insig1), mitochondrial citrate transporter (Slc25a1), and stearoyl-CoA desaturase 2 (Scd2) was reduced threefold or more; however, the mRNA levels of acetyl-CoA carboxylase-1α (Acaca) and ATP citrate lyase (Acly) were unchanged. Furthermore, a 46% fat diet significantly decreased de novo fatty acid synthesis and reduced the protein levels of ACACA, ACLY, and FASN significantly, with no change in their mRNA levels. These data lead us to conclude that two modes of regulation exist to control fatty acid synthesis in the mammary gland of the lactating mouse: the well-known SREBF1 system and a novel mechanism that acts at the posttranscriptional level in the presence of SCAP deletion and high-fat feeding to alter enzyme protein.

Regulation of pro-adrenocorticotropin-endorphin synthesis and secretion in cultured neonatal rat anterior pituitary

Energy Technology Data Exchange (ETDEWEB)

Sato, S.M.; Mains, R.E. (Johns Hopkins Univ. School of Medicine, Baltimore, MD (USA))

1987-08-01

Previous work demonstrated that newborn rat anterior pituitary corticotropes display processing patterns for pro-ACTH/endorphin that are different from the adult. The synthesis and release of beta-endorphin-related peptides was examined in dispersed cell and explant cultures of newborn anterior pituitary to investigate corticotrope development further. The temporal pattern of pro-ACTH/endorphin processing differed significantly from adult rat melanotropes and AtT-20 cells. While pro-ACTH/endorphin processing begins within 30 min of synthesis in adult melanotropes and AtT-20 cells, pulse-labeling of newborn corticotropes in culture indicated that pro-ACTH/endorphin remained uncleaved for at least 90 min after synthesis. With further incubation, there was a decrease in radioactivity associated with the precursor and an equivalent rise in the radioactivity associated with beta-endorphin and beta-lipotropin. However, unprocessed precursor still remained in the cultured newborn anterior pituitary cells after a 25-h chase. Although intact pro-ACTH/endorphin from newborn corticotropes was very long-lived, the precursor did undergo oligosaccharide maturation and became endoglycosidase H resistant within 1 h after synthesis. Similar to the adult, pro-ACTH/endorphin synthesis was doubled in cultures of newborn anterior pituitary chronically treated with 10 nM CRF resulting in a 3- to 4-fold stimulation of secretion over the basal rate. However, unlike the AtT-20 cell or adult rat corticotrope, the proteolytic processing of pro-ACTH/endorphin in the newborn corticotrope was altered by chronic secretagogue treatment; less pro-ACTH/endorphin was converted to beta-endorphin in secretagogue-treated corticotropes than in controls. Thus processing of pro-ACTH/endorphin in the corticotrope is not mature by birth and can be regulated by chronic CRF treatment.

Regulation of pro-adrenocorticotropin-endorphin synthesis and secretion in cultured neonatal rat anterior pituitary

International Nuclear Information System (INIS)

Sato, S.M.; Mains, R.E.

1987-01-01

Previous work demonstrated that newborn rat anterior pituitary corticotropes display processing patterns for pro-ACTH/endorphin that are different from the adult. The synthesis and release of beta-endorphin-related peptides was examined in dispersed cell and explant cultures of newborn anterior pituitary to investigate corticotrope development further. The temporal pattern of pro-ACTH/endorphin processing differed significantly from adult rat melanotropes and AtT-20 cells. While pro-ACTH/endorphin processing begins within 30 min of synthesis in adult melanotropes and AtT-20 cells, pulse-labeling of newborn corticotropes in culture indicated that pro-ACTH/endorphin remained uncleaved for at least 90 min after synthesis. With further incubation, there was a decrease in radioactivity associated with the precursor and an equivalent rise in the radioactivity associated with beta-endorphin and beta-lipotropin. However, unprocessed precursor still remained in the cultured newborn anterior pituitary cells after a 25-h chase. Although intact pro-ACTH/endorphin from newborn corticotropes was very long-lived, the precursor did undergo oligosaccharide maturation and became endoglycosidase H resistant within 1 h after synthesis. Similar to the adult, pro-ACTH/endorphin synthesis was doubled in cultures of newborn anterior pituitary chronically treated with 10 nM CRF resulting in a 3- to 4-fold stimulation of secretion over the basal rate. However, unlike the AtT-20 cell or adult rat corticotrope, the proteolytic processing of pro-ACTH/endorphin in the newborn corticotrope was altered by chronic secretagogue treatment; less pro-ACTH/endorphin was converted to beta-endorphin in secretagogue-treated corticotropes than in controls. Thus processing of pro-ACTH/endorphin in the corticotrope is not mature by birth and can be regulated by chronic CRF treatment

Stereoselective synthesis of an active metabolite of the potent PI3 kinase inhibitor PKI-179.

Science.gov (United States)

Chen, Zecheng; Venkatesan, Aranapakam M; Dos Santos, Osvaldo; Delos Santos, Efren; Dehnhardt, Christoph M; Ayral-Kaloustian, Semiramis; Ashcroft, Joseph; McDonald, Leonard A; Mansour, Tarek S

2010-03-05

The synthesis and stereochemical determination of 1-(4-(4-((1R,5R,6R)-6-hydroxy-3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-morpholino-1,3,5-triazin-2-yl)phenyl)-3-(pyridin-4-yl)urea (2), an active metabolite of the potent PI3 kinase inhibitor PKI-179 (1), is described. Stereospecific hydroboration of the double bond of 2,5-dihydro-1H-pyrrole 8 gave the 2,3-trans alcohol 9 exclusively. The configuration of the 3-hydroxyl group in 9 was inverted by an oxidation and stereoselective reduction sequence to give the corresponding 2,3-cis isomer 23. Both exo (21) and endo (27) isomers of the metabolite 2 were prepared via a practical synthetic route from 9 and 23, respectively, and the stereochemistry of 2 was determined to be endo. The endo isomer (27) was separated into two enantiomers 28 and 29 by chiral HPLC. Compound 2 was found to be enantiomerically pure and identical to the enantiomer 28. The absolute stereochemistry of the enantiomer 28 was determined by Mosher's method, thus establishing the stereochemistry of the active metabolite 2.

BET Bromodomain Inhibitors with One-Step Synthesis Discovered from Virtual Screen.

Science.gov (United States)

Ayoub, Alex M; Hawk, Laura M L; Herzig, Ryan J; Jiang, Jiewei; Wisniewski, Andrea J; Gee, Clifford T; Zhao, Peiliang; Zhu, Jin-Yi; Berndt, Norbert; Offei-Addo, Nana K; Scott, Thomas G; Qi, Jun; Bradner, James E; Ward, Timothy R; Schönbrunn, Ernst; Georg, Gunda I; Pomerantz, William C K

2017-06-22

Chemical inhibition of epigenetic regulatory proteins BrdT and Brd4 is emerging as a promising therapeutic strategy in contraception, cancer, and heart disease. We report an easily synthesized dihydropyridopyrimidine pan-BET inhibitor scaffold, which was uncovered via a virtual screen followed by testing in a fluorescence anisotropy assay. Dihydropyridopyimidine 3 was subjected to further characterization and is highly selective for the BET family of bromodomains. Structure-activity relationship data and ligand deconstruction highlight the importance of the substitution of the uracil moiety for potency and selectivity. Compound 3 was also cocrystallized with Brd4 for determining the ligand binding pose and rationalizing subsequent structure-activity data. An additional series of dihydropyridopyrimidines was synthesized to exploit the proximity of a channel near the ZA loop of Brd4, leading to compounds with submicromolar affinity and cellular target engagement. Given these findings, novel and easily synthesized inhibitors are being introduced to the growing field of bromodomain inhibitor development.

BET Bromodomain Inhibitors with One-Step Synthesis Discovered from Virtual Screen

Energy Technology Data Exchange (ETDEWEB)

Ayoub, Alex M.; Hawk, Laura M.L.; Herzig, Ryan J.; Jiang, Jiewei; Wisniewski, Andrea J.; Gee, Clifford T.; Zhao, Peiliang; Zhu, Jin-Yi; Berndt, Norbert; Offei-Addo, Nana K.; Scott, Thomas G.; Qi, Jun; Bradner, James E.; Ward, Timothy R.; Schönbrunn, Ernst; Georg, Gunda I.; Pomerantz, William C.K. (Moffitt); (UMM); (Harvard-Med)

2017-06-06

Chemical inhibition of epigenetic regulatory proteins BrdT and Brd4 is emerging as a promising therapeutic strategy in contraception, cancer, and heart disease. We report an easily synthesized dihydropyridopyrimidine pan-BET inhibitor scaffold, which was uncovered via a virtual screen followed by testing in a fluorescence anisotropy assay. Dihydropyridopyimidine 3 was subjected to further characterization and is highly selective for the BET family of bromodomains. Structure–activity relationship data and ligand deconstruction highlight the importance of the substitution of the uracil moiety for potency and selectivity. Compound 3 was also cocrystallized with Brd4 for determining the ligand binding pose and rationalizing subsequent structure–activity data. An additional series of dihydropyridopyrimidines was synthesized to exploit the proximity of a channel near the ZA loop of Brd4, leading to compounds with submicromolar affinity and cellular target engagement. Given these findings, novel and easily synthesized inhibitors are being introduced to the growing field of bromodomain inhibitor development.

Design, Synthesis and Biological Activity of Novel Reversible Peptidyl FVIIa Inhibitors Rh-Catalyzed Enantioselective Synthesis of Diaryl Amines

DEFF Research Database (Denmark)

Storgaard, Morten

functional group tolerance. Unfortunately, these -aryl tetramic acids were too unreactive and ring opening toward the synthesis of the building block did not succeed. However, -aryl tetramic acids are still interesting compounds due to their potential biological activity. The building block 3.15 (P1......-catalyzed enantioselective synthesis of diaryl amines, which is an important class of compounds (Chapter 4). For example it is found in the third generation anti-histaminic agent levocetirizine. Development of efficient synthetic routes is therefore of considerably interest. The rhodium-catalyzed enantioselective synthesis...

Co-evolution of insect proteases and plant protease inhibitors.

Science.gov (United States)

Jongsma, Maarten A; Beekwilder, Jules

2011-08-01

Plants are at the basis of the food chain, but there is no such thing as a "free lunch" for herbivores. To promote reproductive success, plants evolved multi-layered defensive tactics to avoid or discourage herbivory. To the detriment of plants, herbivores, in turn, evolved intricate strategies to find, eat, and successfully digest essential plant parts to raise their own offspring. In this battle the digestive tract is the arena determining final victory or defeat as measured by growth or starvation of the herbivore. Earlier, specific molecular opponents were identified as proteases and inhibitors: digestive proteases of herbivores evolved structural motifs to occlude plant protease inhibitors, or alternatively, the insects evolved proteases capable of specifically degrading the host plant inhibitors. In response plant inhibitors evolved hyper-variable and novel protein folds to remain active against potential herbivores. At the level of protease regulation in herbivorous insects, it was shown that inhibition-insensitive digestive proteases are up-regulated when sensitive proteases are inhibited. The way this regulation operates in mammals is known as negative feedback by gut-luminal factors, so-called 'monitor peptides' that are sensitive to the concentration of active enzymes. We propose that regulation of gut enzymes by endogenous luminal factors has been an open invitation to plants to "hijack" this regulation by evolving receptor antagonists, although yet these plant factors have not been identified. In future research the question of the co-evolution of insect proteases and plant inhibitors should, therefore, be better approached from a systems level keeping in mind that evolution is fundamentally opportunistic and that the plant's fitness is primarily improved by lowering the availability of essential amino acids to an herbivore by any available mechanism.

Synthesis of acrylic prepolymer

International Nuclear Information System (INIS)

Hussin bin Mohd Nor; Dahlan bin Haji Mohd; Mohamad Hilmi bin Mahmood.

1988-04-01

An acrylic prepolymer was synthesized from glycidyl methacrylate (GMA), butyl methacrylate (BMA), methyl methacrylate (MMA) and acrylic acid (AA). Butyl acetate (BAc), benzoyl peroxide (BzO), 4-methoxyphenol (MPh) and triethylamine (TEA) were used as solvent, initiator, inhibitor and catalyst respectively. Observations of the synthesis leading to the formation of acrylic prepolymer are described. (author)

Mutations in the Nonstructural Protein 3A Confer Resistance to the Novel Enterovirus Replication Inhibitor TTP-8307

NARCIS (Netherlands)

de Palma, Armando M.; Thibaut, Hendrik Jan; van der Linden, Lonneke; Lanke, Kjerstin; Heggermont, Ward; Ireland, Stephen; Andrews, Robert; Arimilli, Murty; Al-tel, Taleb H.; de Clercq, Erik; van Kuppeveld, Frank; Neyts, Johan

2009-01-01

A novel compound, TTP-8307, was identified as a potent inhibitor of the replication of several rhino- and enteroviruses. TTP-8307 inhibits viral RNA synthesis in a dose-dependent manner, without affecting polyprotein synthesis and/or processing. Drug-resistant variants of coxsackievirus B3 were all

Novel bioassay for the discovery of inhibitors of the 2-C-methyl-D-erythritol 4-phosphate (MEP and terpenoid pathways leading to carotenoid biosynthesis.

Directory of Open Access Journals (Sweden)

Natália Corniani

Full Text Available The 2-C-methyl-D-erythritol 4-phosphate (MEP pathway leads to the synthesis of isopentenyl diphosphate in plastids. It is a major branch point providing precursors for the synthesis of carotenoids, tocopherols, plastoquinone and the phytyl chain of chlorophylls, as well as the hormones abscisic acid and gibberellins. Consequently, disruption of this pathway is harmful to plants. We developed an in vivo bioassay that can measure the carbon flow through the carotenoid pathway. Leaf cuttings are incubated in the presence of a phytoene desaturase inhibitor to induce phytoene accumulation. Any compound reducing the level of phytoene accumulation is likely to interfere with either one of the steps in the MEP pathway or the synthesis of geranylgeranyl diphosphate. This concept was tested with known inhibitors of steps of the MEP pathway. The specificity of this in vivo bioassay was also verified by testing representative herbicides known to target processes outside of the MEP and carotenoid pathways. This assay enables the rapid screen of new inhibitors of enzymes preceding the synthesis of phytoene, though there are some limitations related to the non-specific effect of some inhibitors on this assay.

Evaluation of Enoyl-Acyl Carrier Protein Reductase Inhibitors as Pseudomonas aeruginosa Quorum-Quenching Reagents

DEFF Research Database (Denmark)

Yang, Liang; Liu, Yang; Sternberg, Claus

2010-01-01

Pseudomonas aeruginosa is an opportunistic pathogen which is responsible for a wide range of infections. Production of virulence factors and biofilm formation by P. aeruginosa are partly regulated by cell-to-cell communication quorum-sensing systems. Identification of quorum-quenching reagents...... which block the quorum-sensing process can facilitate development of novel treatment strategies for P. aeruginosa infections. We have used molecular dynamics simulation and experimental studies to elucidate the efficiencies of two potential quorum-quenching reagents, triclosan and green tea...... epigallocatechin gallate (EGCG), which both function as inhibitors of the enoyl-acyl carrier protein (ACP) reductase (ENR) from the bacterial type II fatty acid synthesis pathway. Our studies suggest that EGCG has a higher binding affinity towards ENR of P. aeruginosa and is an efficient quorum-quenching reagent...

The Azaindole Framework in the Design of Kinase Inhibitors

Directory of Open Access Journals (Sweden)

Jean-Yves Mérour

2014-11-01

Full Text Available This review article illustrates the growing use of azaindole derivatives as kinase inhibitors and their contribution to drug discovery and innovation. The different protein kinases which have served as targets and the known molecules which have emerged from medicinal chemistry and Fragment-Based Drug Discovery (FBDD programs are presented. The various synthetic routes used to access these compounds and the chemical pathways leading to their synthesis are also discussed. An analysis of their mode of binding based on X-ray crystallography data gives structural insights for the design of more potent and selective inhibitors.

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

Science.gov (United States)

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

2016-09-01

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

Tc-MYBPA an Arabidopsis TT2-like transcription factor and functions in the regulation of proanthocyanidin synthesis in Theobroma cacao.

Science.gov (United States)

Liu, Yi; Shi, Zi; Maximova, Siela N; Payne, Mark J; Guiltinan, Mark J

2015-06-25

The flavan-3-ols catechin and epicatechin, and their polymerized oligomers, the proanthocyanidins (PAs, also called condensed tannins), accumulate to levels of up to 15 % of the total weight of dry seeds of Theobroma cacao L. These compounds have been associated with several health benefits in humans. They also play important roles in pest and disease defense throughout the plant. In Arabidopsis, the R2R3 type MYB transcription factor TT2 regulates the major genes leading to the synthesis of PA. To explore the transcriptional regulation of the PA synthesis pathway in cacao, we isolated and characterized an R2R3 type MYB transcription factor MYBPA from cacao. We examined the spatial and temporal gene expression patterns of the Tc-MYBPA gene and found it to be developmentally expressed in a manner consistent with its involvement in PAs and anthocyanin synthesis. Functional complementation of an Arabidopsis tt2 mutant with Tc-MYBPA suggested that it can functionally substitute the Arabidopsis TT2 gene. Interestingly, in addition to PA accumulation in seeds of the Tc-MYBPA expressing plants, we also observed an obvious increase of anthocyanidin accumulation in hypocotyls. We observed that overexpression of the Tc-MYBPA gene resulted in increased expression of several key genes encoding the major structural enzymes of the PA and anthocyanidin pathway, including DFR (dihydroflavanol reductase), LDOX (leucoanthocyanidin dioxygenase) and BAN (ANR, anthocyanidin reductase). We conclude that the Tc-MYBPA gene that encodes an R2R3 type MYB transcription factor is an Arabidopsis TT2 like transcription factor, and may be involved in the regulation of both anthocyanin and PA synthesis in cacao. This research may provide molecular tools for breeding of cacao varieties with improved disease resistance and enhanced flavonoid profiles for nutritional and pharmaceutical applications.

Autocrine-paracrine regulation of the mammary gland.

Science.gov (United States)

Weaver, S R; Hernandez, L L

2016-01-01

The mammary gland has a remarkable capacity for regulation at a local level, particularly with respect to its main function: milk secretion. Regulation of milk synthesis has significant effects on animal and human health, at the level of both the mother and the neonate. Control by the mammary gland of its essential function, milk synthesis, is an evolutionary necessity and is therefore tightly regulated at a local level. For at least the last 60 yr, researchers have been interested in elucidating the mechanisms underpinning the mammary gland's ability to self-regulate, largely without the influence from systemic hormones or signals. By the 1960s, scientists realized the importance of milk removal in the capacity of the gland to produce milk and that the dynamics of this removal, including emptying of the alveolar spaces and frequency of milking, were controlled locally as opposed to traditional systemic hormonal regulation. Using both in vitro systems and various mammalian species, including goats, marsupials, humans, and dairy cows, it has been demonstrated that the mammary gland is largely self-regulating in its capacity to support the young, which is the evolutionary basis for milk production. Local control occurs at the level of the mammary epithelial cell through pressure and stretching negative-feedback mechanisms, and also in an autocrine fashion through bioactive factors within the milk which act as inhibitors, regulating milk secretion within the alveoli themselves. It is only within the last 20 to 30 yr that potential candidates for these bioactive factors have been examined at a molecular level. Several, including parathyroid hormone-related protein, growth factors (transforming growth factor, insulin-like growth factor, epidermal growth factor), and serotonin, are synthesized within and act upon the gland and possess dynamic receptor activity resulting in diverse effects on growth, calcium homeostasis, and milk composition. This review will focus on the

The Fatty Acid Synthase Inhibitor Platensimycin Improves Insulin Resistance without Inducing Liver Steatosis in Mice and Monkeys.

Directory of Open Access Journals (Sweden)

Sheo B Singh

Full Text Available Platensimycin (PTM is a natural antibiotic produced by Streptomyces platensis that selectively inhibits bacterial and mammalian fatty acid synthase (FAS without affecting synthesis of other lipids. Recently, we reported that oral administration of PTM in mouse models (db/db and db/+ with high de novo lipogenesis (DNL tone inhibited DNL and enhanced glucose oxidation, which in turn led to net reduction of liver triglycerides (TG, reduced ambient glucose, and improved insulin sensitivity. The present study was conducted to explore translatability and the therapeutic potential of FAS inhibition for the treatment of diabetes in humans.We tested PTM in animal models with different DNL tones, i.e. intrinsic synthesis rates, which vary among species and are regulated by nutritional and disease states, and confirmed glucose-lowering efficacy of PTM in lean NHPs with quantitation of liver lipid by MRS imaging. To understand the direct effect of PTM on liver metabolism, we performed ex vivo liver perfusion study to compare FAS inhibitor and carnitine palmitoyltransferase 1 (CPT1 inhibitor.The efficacy of PTM is generally reproduced in preclinical models with DNL tones comparable to humans, including lean and established diet-induced obese (eDIO mice as well as non-human primates (NHPs. Similar effects of PTM on DNL reduction were observed in lean and type 2 diabetic rhesus and lean cynomolgus monkeys after acute and chronic treatment of PTM. Mechanistically, PTM lowers plasma glucose in part by enhancing hepatic glucose uptake and glycolysis. Teglicar, a CPT1 inhibitor, has similar effects on glucose uptake and glycolysis. In sharp contrast, Teglicar but not PTM significantly increased hepatic TG production, thus caused liver steatosis in eDIO mice.These findings demonstrate unique properties of PTM and provide proof-of-concept of FAS inhibition having potential utility for the treatment of diabetes and related metabolic disorders.

Histone deacetylase inhibitors up-regulate LL-37 expression independent of toll-like receptor mediated signalling in airway epithelial cells

OpenAIRE

Liu, Q.; Liu, J.; Roschmann, K.I.L.; Egmond, D. van; Golebski, K.; Fokkens, W.J.; Wang, D.; Drunen, C.M. van

2013-01-01

HDAC inhibitors have been proposed as anticancer agents. However, their roles in innate genes expression remain not well known. Cathelicidin LL-37 is one of the few human bactericidal peptides, but the regulation of histone acetylation on LL-37 expression in airway epithelium remains largely unknown. Therefore, we investigated the effects of two non-selective HDACi, trichostatin A (TSA) and sodium butyrate (SB), on the expression of the cathelicidin LL-37 in human airway epithelial cells. LL3...

Lack of effect of inhibitors of DNA synthesis/repair on the ionizing radiation-induced chromosomal damage in G[sub 2] stage of ataxia telangiectasia cells

Energy Technology Data Exchange (ETDEWEB)

Antoccia, A. (Univ. ' La Sapienza' , Rome (Italy). Dipt. di Genetica e Biologia Molecolare); Palitti, F.; Raggi, T. (Univ. del Tuscia, Viterbo (Italy). Dipt. di Agrobiologia ed Agrochimica); Catena, C. (ENEA, Casaccia (Italy). Centro Ricerche Energia); Tanzarella, C. (Rome Univ. 3 (Italy). Dipt. di Biologia)

1994-09-01

The relationship between the repair processes occurring at the G[sub 2] phase of the cell cycle and cytogenetic damage in ataxia telangiectasia (AT) cells was studied. Lymphoblastoid cells derived from normal, heterozygote AT (HzAT) and three AT patients were exposed to X-rays or fission neutrons and post-treated with inhibitors of DNA synthesis/repair, such as inhibitors of DNA polymerases [alpha], [sigma] and [epsilon] (cytosine arabinoside, ara-C; aphidicolin, APC; buthylphenyl-guanine, BuPdG) or ribonucleotide reductase (hydroxyurea HU). A strong increase of radiation-induced chromosomal aberrations was observed in normal and HzAT cells post-treated with ara-C, APC and HU, but not in the presence of BuPdG. No enhancing effect was observed in cells derived from AT patients, except for HU post-irradiation treatment. These results suggest that the enzymes that can be inhibited by these agents are not directly involved in the repair of radiation damage induced in G[sub 2] cells from AT patients, indicating that probably the AT cells that we used lack the capability to transform the primary DNA lesions into reparable products, or that AT cells might contain a mutated form of DNA polymerase resistant to the inhibitors. (author).

Regulation of very-long acyl chain ceramide synthesis by acyl-CoA-binding protein

DEFF Research Database (Denmark)

Ferreira, Natalia Santos; Engelsby, Hanne; Neess, Ditte

2017-01-01

and cardiovascular diseases, as well as neurological disorders. Here we show that acyl-coenzyme A-binding protein (ACBP) potently facilitates very-long acyl chain ceramide synthesis. ACBP increases the activity of ceramide synthase 2 (CerS2) by more than 2-fold and CerS3 activity by 7-fold. ACBP binds very......-long-chain acyl-CoA esters, which is required for its ability to stimulate CerS activity. We also show that high-speed liver cytosol from wild-type mice activates CerS3 activity, whereas cytosol from ACBP knock-out mice does not. Consistently, CerS2 and CerS3 activities are significantly reduced in the testes...... of ACBP(-/-) mice, concomitant with a significant reduction in long- and very-long-chain ceramide levels. Importantly, we show that ACBP interacts with CerS2 and CerS3. Our data uncover a novel mode of regulation of very-long acyl chain ceramide synthesis by ACBP, which we anticipate is of crucial...

The small-molecule kinase inhibitor D11 counteracts 17-AAG-mediated up-regulation of HSP70 in brain cancer cells.

Science.gov (United States)

Schaefer, Susanne; Svenstrup, Tina H; Guerra, Barbara

2017-01-01

Many types of cancer express high levels of heat shock proteins (HSPs) that are molecular chaperones regulating protein folding and stability ensuring protection of cells from potentially lethal stress. HSPs in cancer cells promote survival, growth and spreading even in situations of growth factors deprivation by associating with oncogenic proteins responsible for cell transformation. Hence, it is not surprising that the identification of potent inhibitors of HSPs, notably HSP90, has been the primary research focus, in recent years. Exposure of cancer cells to HSP90 inhibitors, including 17-AAG, has been shown to cause resistance to chemotherapeutic treatment mostly attributable to induction of the heat shock response and increased cellular levels of pro-survival chaperones. In this study, we show that treatment of glioblastoma cells with 17-AAG leads to HSP90 inhibition indicated by loss of stability of the EGFR client protein, and significant increase in HSP70 expression. Conversely, co-treatment with the small-molecule kinase inhibitor D11 leads to suppression of the heat shock response and inhibition of HSF1 transcriptional activity. Beside HSP70, Western blot and differential mRNA expression analysis reveal that combination treatment causes strong down-regulation of the small chaperone protein HSP27. Finally, we demonstrate that incubation of cells with both agents leads to enhanced cytotoxicity and significantly high levels of LC3-II suggesting autophagy induction. Taken together, results reported here support the notion that including D11 in future treatment regimens based on HSP90 inhibition can potentially overcome acquired resistance induced by the heat shock response in brain cancer cells.

Up-regulation of melanin synthesis by the antidepressant fluoxetine.

Science.gov (United States)

Liao, Sha; Shang, Jing; Tian, Xiaoli; Fan, Xueqi; Shi, Xiupu; Pei, Siran; Wang, Qian; Yu, Boyang

2012-08-01

Fluoxetine, a member of the class of selective serotonin reuptake inhibitors, is a potent antidepressant commonly used in clinical practice. Here, we report that fluoxetine increases cellular tyrosinase (TYR) activity, enhances the protein levels of microphthalmia-associated transcription factor (MITF), TYR and tyrosinase-related protein-1 (TRP-1) and eventually leads to a dramatic increase in melanin production in both murine B16F10 melanoma cells and normal human melanocytes (NHMCs). In well-characterized C57BL/6 mouse models, systemic application of fluoxetine increased hair pigmentation by up-regulating hair follicular MITF, TYR, TRP-1 and tyrosinase-related protein-2 (TRP-2) protein levels. Using a serotonin 1A receptor (SR1A) antagonist and RNA interference (RNAi) technique, we revealed that SR1A appears to be one of the involved pathways in the fluoxetine-induced melanogenesis in B16F10 cells. These results suggest that fluoxetine may hold a significant therapeutic potential for treating skin hypopigmentation disorders, and SR1A may serve as a novel target in modulating melanogenesis. © 2012 John Wiley & Sons A/S.

Statins increase hepatic cholesterol synthesis and stimulate fecal cholesterol elimination in mice

NARCIS (Netherlands)

Schonewille, Marleen; de Boer, Jan Freark; Mele, Laura; Wolters, Henk; Bloks, Vincent W.; Wolters, Justina C.; Kuivenhoven, Jan A.; Tietge, Uwe J. F.; Brufau, Gemma; Groen, Albert K.

Statins are competitive inhibitors of HMG-CoA reductase, the rate-limiting enzyme of cholesterol synthesis. Statins reduce plasma cholesterol levels, but whether this is actually caused by inhibition of de novo cholesterol synthesis has not been clearly established. Using three different statins, we

Statins increase hepatic cholesterol synthesis and stimulate fecal cholesterol elimination in mice

NARCIS (Netherlands)

Schonewille, Marleen; de Boer, Jan Freark; Mele, Laura; Wolters, Henk; Bloks, Vincent W.; Wolters, Justina C.; Kuivenhoven, Jan A.; Tietge, Uwe J. F.; Brufau, Gemma; Groen, Albert K.

2016-01-01

Statins are competitive inhibitors of HMG-CoA reductase, the rate-limiting enzyme of cholesterol synthesis. Statins reduce plasma cholesterol levels, but whether this is actually caused by inhibition of de novo cholesterol synthesis has not been clearly established. Using three different statins, we

Down-regulation of inflammatory mediator synthesis and infiltration of inflammatory cells by MMP-3 in experimentally induced rat pulpitis.

Science.gov (United States)

Takimoto, Koyo; Kawashima, Nobuyuki; Suzuki, Noriyuki; Koizumi, Yu; Yamamoto, Mioko; Nakashima, Misako; Suda, Hideaki

2014-09-01

Matrix metalloproteinase (MMP)-3 is a member of the MMP family that degrades the extracellular matrix. Application of MMP-3 to injured pulp tissue induces angiogenesis and wound healing, but its anti-inflammatory effects are still unclear. Here, we evaluated the anti-inflammatory functions of MMP-3 in vitro and in vivo. Nitric oxide and inflammatory mediator synthesis in macrophages activated by lipopolysaccharide (LPS) was measured in the presence or absence of MMP-3. The mouse Mmp3 (mMmp3) expression vector containing full length cDNA sequence of mMmp3 or cDNA sequence of mMmp3 missing the signal peptide and pro-peptide regions was transfected to RAW264, a mouse macrophage cell line, and NO synthesis and inflammatory mediator expression were evaluated. Pulpal inflammation was histologically and immunohistochemically evaluated in a rat model of incisor pulpitis induced by the application of LPS for 9 hours in the presence or absence of MMP-3. NO and pro-inflammatory mediator synthesis promoted by LPS was significantly down-regulated by MMP-3 in vitro. The full length of mMmp3 down-regulated the LPS-induced NO synthesis and chemical mediator mRNA expression, however the mMmp3 missing the signal peptide failed to block the NO synthesis induced by LPS. The numbers of major histocompatibility complex class II+ and CD68+ cells, which infiltrated into the rat incisor pulp tissues in response to the topical application of LPS, were significantly decreased by the application of MMP-3 in vivo. These results indicate that MMP-3 possesses anti-inflammatory functions, suggesting its potential utility as an anti-inflammatory agent for pulpal inflammation. Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

ROLE OF NEUROTRANSMITTERS AND PROTEIN SYNTHESIS IN SHORT- AND LONG-TERM MEMORY

Energy Technology Data Exchange (ETDEWEB)

Bennett, E.L.; Rosenzweig, M.R.; Flood, J.F.

1978-10-01

Anisomycin is an effective inhibitor of cerebral protein synthesis in mice and is also an effective amnestic agent for both passive and active behavioral tasks. From use of anisomycin in combination with a variety of stimulant and depressant drugs, we conclude that the level of arousal following acquisition plays an important role in determining the duration and the rate of the biosynthetic phase of memory formation. While we have interpreted the experiments with anisomycin as evidence for an essential role of protein in memory storage, others have suggested that side effects of inhibitors of protein synthesis on catecholamine metabolism are the main cause of amnesia. Several experiments were therefore done to compare the effects of anisemycin and catecholamine inhibitors on memory. We conclude that anisomycin's principal amnestic mechanism does not involve inhibition of the catecholamine system. The results strengthen our conclusion that protein synthesis is an essential component for longterm memory trace formation. Also, it is suggested that proteins synthesized in the neuronal cell body are used, in conjunction with other molecules, to produce permanent and semi-permanent anatomical changes.

Feedback regulation of bile acid synthesis in human liver: Importance of HNF-4α for regulation of CYP7A1

International Nuclear Information System (INIS)

Abrahamsson, Anna; Gustafsson, Ulf; Ellis, Ewa; Nilsson, Lisa-Mari; Sahlin, Staffan; Bjoerkhem, Ingemar; Einarsson, Curt

2005-01-01

A great number of nuclear factors are involved in the negative feedback mechanism regulating bile acid synthesis. There are two major ways for the negative feedback to effect the synthesis; the SHP-dependent, involving FXR, and the SHP-independent way, affecting HNF-4α. We studied 23 patients with gallstone disease. Eight patients were treated with chenodeoxycholic acid, 7 with cholestyramine prior to operation, and 8 served as controls. Liver biopsies were analyzed with Real-time-PCR. In the cholestyramine-treated group mRNA levels of CYP7A1 were increased about 10-fold. Treatment with CDCA decreased the mRNA levels of CYP7A1 by about 70%. The mRNA levels of CYP8B1, CYP27A1, and CYP7B1 were not significantly altered in the treated groups. The analysis of mRNA levels for HNF-4α showed 64% higher levels in the cholestyramine-treated group compared to the controls. These levels showed positive and highly significant correlation to the levels of mRNA of CYP7A1 when studied in all three groups together. FXR, SHP, and LRH-1/FTF were not significantly affected by the different treatments. Our results indicate that when bile acid synthesis is upregulated by cholestyramine treatment the SHP-independent pathway for controlling CYP7A1 transcription dominates over the SHP-dependent pathway

Macrocyclic Peptoid–Peptide Hybrids as Inhibitors of Class I Histone Deacetylases

DEFF Research Database (Denmark)

Olsen, Christian Adam; Montero, Ana; Leman, Luke J.

2012-01-01

We report the design, synthesis, and biological evaluation of the first macrocyclic peptoid-containing histone deacetylase (HDAC) inhibitors. The compounds selectively inhibit human class I HDAC isoforms in vitro, with no inhibition of the tubulin deacetylase activity associated with class IIb HDAC...

On the mechanism of regulation of the catalase synthesis rate in the rat liver in the course of acute radiation disease

International Nuclear Information System (INIS)

Komov, V.P.; Rakhmanina, T.F.

1976-01-01

A method has been proposed to determine the activity of factors that regulate the rate of catalase synthesis in the rat liver at the stage of translation. The analysis of certain normal and pathologic parameters of these factors suggests a possibility of interpreting more definitely the effect of radiation on the catalase synthesis. Marked changes have been found both in the structure and the activity of the given factors in the course of the development of radiation damage

Effect of Phosphatase and Tensin Homologue on Chromosome 10 on Angiotensin II-Mediated Proliferation, Collagen Synthesis, and Akt/P27 Signaling in Neonatal Rat Cardiac Fibroblasts

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Ling Nie

2016-01-01

Full Text Available Cardiac fibroblasts (CFs play a key role in cardiac fibrosis by regulating the balance between extracellular matrix synthesis and breakdown. Although phosphatase and tensin homologue on chromosome 10 (PTEN has been found to play an important role in cardiovascular disease, it is not clear whether PTEN is involved in functional regulation of CFs. In the present study, PTEN was overexpressed in neonatal rat CFs via recombinant adenovirus-mediated gene transfer. The effects of PTEN overexpression on cell-cycle progression and angiotensin II- (Ang II- mediated regulation of collagen metabolism, synthesis of matrix metalloproteinases, and Akt/P27 signaling were investigated. Compared with uninfected cells and cells infected with green fluorescent protein-expressing adenovirus (Ad-GFP, cells infected with PTEN-expressing adenovirus (Ad-PTEN significantly increased PTEN protein and mRNA levels in CFs (P<0.05. The proportion of CFs in the G1/S cell-cycle phase was significantly higher for PTEN-overexpressing cells. In addition, Ad-PTEN decreased mRNA expression and the protein synthesis rate of collagen types I and III and antagonized Ang II-induced collagen synthesis. Overexpression of PTEN also decreased Ang II-induced matrix metalloproteinase-2 (MMP-2 and tissue inhibitor of metalloproteinase-1 (TIMP-1 production as well as gelatinase activity. Moreover, Ad-PTEN decreased Akt expression and increased P27 expression independent of Ang II stimulation. These results suggest that PTEN could regulate its functional effects in neonatal rat CFs partially via the Akt/P27 signaling pathway.

Epithelial tissue hyperplasia induced by the RAF inhibitor PF-04880594 is attenuated by a clinically well-tolerated dose of the MEK inhibitor PD-0325901.

Science.gov (United States)

Torti, Vince R; Wojciechowicz, Donald; Hu, Wenyue; John-Baptiste, Annette; Evering, Winston; Troche, Gabriel; Marroquin, Lisa D; Smeal, Tod; Yamazaki, Shinji; Palmer, Cynthia L; Burns-Naas, Leigh Ann; Bagrodia, Shubha

2012-10-01

Clinical trials of selective RAF inhibitors in patients with melanoma tumors harboring activated BRAFV600E have produced very promising results, and a RAF inhibitor has been approved for treatment of advanced melanoma. However, about a third of patients developed resectable skin tumors during the course of trials. This is likely related to observations that RAF inhibitors activate extracellular signal-regulated kinase (ERK) signaling, stimulate proliferation, and induce epithelial hyperplasia in preclinical models. Because these findings raise safety concerns about RAF inhibitor development, we further investigated the underlying mechanisms. We showed that the RAF inhibitor PF-04880594 induces ERK phosphorylation and RAF dimerization in those epithelial tissues that undergo hyperplasia. Hyperplasia and ERK hyperphosphorylation are prevented by treatment with the mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitor PD-0325901 at exposures that extrapolate to clinically well-tolerated doses. To facilitate mechanistic and toxicologic studies, we developed a three-dimensional cell culture model of epithelial layering that recapitulated the RAF inhibitor-induced hyperplasia and reversal by MEK inhibitor in vitro. We also showed that PF-04880594 stimulates production of the inflammatory cytokine interleukin 8 in HL-60 cells, suggesting a possible mechanism for the skin flushing observed in dogs. The complete inhibition of hyperplasia by MEK inhibitor in epithelial tissues does not seem to reduce RAF inhibitor efficacy and, in fact, allows doubling of the PF-04880594 dose without toxicity usually associated with such doses. These findings indicated that combination treatment with MEK inhibitors might greatly increase the safety and therapeutic index of RAF inhibitors for the treatment of melanoma and other cancers. ©2012 AACR.

Concise Synthesis of Thapsigargin from Nortrilobolide

DEFF Research Database (Denmark)

Crestey, François; Toma, Maddalena; Christensen, Søren Brøgger

2015-01-01

Herein, we wish to describe for the first time an expedient synthesis of the hexaoxygenated guaianolide thapsigargin (1), a potent inhibitor of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), from the natural product nortrilobolide (2). This innovative protocol involves three key steps: a on...

Discovery of a novel dual fungal CYP51/human 5-lipoxygenase inhibitor: implications for anti-fungal therapy.

Directory of Open Access Journals (Sweden)

Eric K Hoobler

Full Text Available We report the discovery of a novel dual inhibitor targeting fungal sterol 14α-demethylase (CYP51 or Erg11 and human 5-lipoxygenase (5-LOX with improved potency against 5-LOX due to its reduction of the iron center by its phenylenediamine core. A series of potent 5-LOX inhibitors containing a phenylenediamine core, were synthesized that exhibit nanomolar potency and >30-fold selectivity against the LOX paralogs, platelet-type 12-human lipoxygenase, reticulocyte 15-human lipoxygenase type-1, and epithelial 15-human lipoxygenase type-2, and >100-fold selectivity against ovine cyclooxygenase-1 and human cyclooxygnease-2. The phenylenediamine core was then translated into the structure of ketoconazole, a highly effective anti-fungal medication for seborrheic dermatitis, to generate a novel compound, ketaminazole. Ketaminazole was found to be a potent dual inhibitor against human 5-LOX (IC50 = 700 nM and CYP51 (IC50 = 43 nM in vitro. It was tested in whole blood and found to down-regulate LTB4 synthesis, displaying 45% inhibition at 10 µM. In addition, ketaminazole selectively inhibited yeast CYP51 relative to human CYP51 by 17-fold, which is greater selectivity than that of ketoconazole and could confer a therapeutic advantage. This novel dual anti-fungal/anti-inflammatory inhibitor could potentially have therapeutic uses against fungal infections that have an anti-inflammatory component.

Regulation of aortic extracellular matrix synthesis via noradrenergic system and angiotensin II in juvenile rats.

Science.gov (United States)

Dab, Houcine; Hachani, Rafik; Dhaouadi, Nedra; Sakly, Mohsen; Hodroj, Wassim; Randon, Jacques; Bricca, Giampiero; Kacem, Kamel

2012-10-01

Extracellular matrix (ECM) synthesis regulation by sympathetic nervous system (SNS) or angiotensin II (ANG II) was widely reported, but interaction between the two systems on ECM synthesis needs further investigation. We tested implication of SNS and ANG II on ECM synthesis in juvenile rat aorta. Sympathectomy with guanethidine (50 mg/kg, subcutaneous) and blockade of the ANG II AT1 receptors (AT1R) blocker with losartan (20 mg/kg/day in drinking water) were performed alone or in combination in rats. mRNA and protein synthesis of collagen and elastin were examined by Q-RT-PCR and immunoblotting. Collagen type I and III mRNA were increased respectively by 62 and 43% after sympathectomy and decreased respectively by 31 and 60% after AT1R blockade. Combined treatment increased collagen type III by 36% but not collagen type I. The same tendency of collagen expression was observed at mRNA and protein levels after the three treatments. mRNA and protein level of elastin was decreased respectively by 63 and 39% and increased by 158 and 15% after losartan treatment. Combined treatment abrogates changes induced by single treatments. The two systems act as antagonists on ECM expression in the aorta and combined inhibition of the two systems prevents imbalance of mRNA and protein level of collagen I and elastin induced by single treatment. Combined inhibition of the two systems prevents deposit or excessive reduction of ECM and can more prevent cardiovascular disorders.

Piperine analogs as potent Staphylococcus aureus NorA efflux pump inhibitors

DEFF Research Database (Denmark)

Sangwan, Payare L; Koul, Jawahir L; Koul, Surrinder

2008-01-01

Based on our recent findings that piperine is a potent Staphylococcus aureus NorA efflux pump inhibitor (EPI), 38 piperine analogs were synthesized and bioevaluated for their EPI activity. Twenty-five of them were found active with potentiating activity equivalent or more than known EPIs like...... reserpine, carsonic acid and verapamil. The inhibitory mechanism of the compounds was confirmed by efflux inhibition assay using ethidium bromide as NorA substrate. The present communication describes the synthesis, bioevaluation and structure related activity of these efflux pump inhibitors....

Combined effects of EGFR tyrosine kinase inhibitors and vATPase inhibitors in NSCLC cells

Energy Technology Data Exchange (ETDEWEB)

Jin, Hyeon-Ok [KIRAMS Radiation Biobank, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of); Hong, Sung-Eun [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of); Kim, Chang Soon [Department of Microbiological Engineering, Kon-Kuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul, 143–701 (Korea, Republic of); Park, Jin-Ah; Kim, Jin-Hee; Kim, Ji-Young; Kim, Bora [KIRAMS Radiation Biobank, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of); Chang, Yoon Hwan; Hong, Seok-Il; Hong, Young Jun [Department of Laboratory Medicine, Korea Cancer Center Hospital, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of); Park, In-Chul, E-mail: parkic@kirams.re.kr [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of); Lee, Jin Kyung, E-mail: jklee@kirams.re.kr [KIRAMS Radiation Biobank, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of); Department of Laboratory Medicine, Korea Cancer Center Hospital, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of)

2015-08-15

Despite excellent initial clinical responses of non-small cell lung cancer (NSCLC) patients to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), many patients eventually develop resistance. According to a recent report, vacuolar H + ATPase (vATPase) is overexpressed and is associated with chemotherapy drug resistance in NSCLC. We investigated the combined effects of EGFR TKIs and vATPase inhibitors and their underlying mechanisms in the regulation of NSCLC cell death. We found that combined treatment with EGFR TKIs (erlotinib, gefitinib, or lapatinib) and vATPase inhibitors (bafilomycin A1 or concanamycin A) enhanced synergistic cell death compared to treatments with each drug alone. Treatment with bafilomycin A1 or concanamycin A led to the induction of Bnip3 expression in an Hif-1α dependent manner. Knock-down of Hif-1α or Bnip3 by siRNA further enhanced cell death induced by bafilomycin A1, suggesting that Hif-1α/Bnip3 induction promoted resistance to cell death induced by the vATPase inhibitors. EGFR TKIs suppressed Hif-1α and Bnip3 expression induced by the vATPase inhibitors, suggesting that they enhanced the sensitivity of the cells to these inhibitors by decreasing Hif-1α/Bnip3 expression. Taken together, we conclude that EGFR TKIs enhance the sensitivity of NSCLC cells to vATPase inhibitors by decreasing Hif-1α/Bnip3 expression. We suggest that combined treatment with EGFR TKIs and vATPase inhibitors is potentially effective for the treatment of NSCLC. - Highlights: • Co-treatment with EGFR TKIs and vATPase inhibitors induces synergistic cell death • EGFR TKIs enhance cell sensitivity to vATPase inhibitors via Hif-1α downregulation • Co-treatment of these inhibitors is potentially effective for the treatment of NSCLC.

Chemoproteomics-Aided Medicinal Chemistry for the Discovery of EPHA2 Inhibitors.

Science.gov (United States)

Heinzlmeir, Stephanie; Lohse, Jonas; Treiber, Tobias; Kudlinzki, Denis; Linhard, Verena; Gande, Santosh Lakshmi; Sreeramulu, Sridhar; Saxena, Krishna; Liu, Xiaofeng; Wilhelm, Mathias; Schwalbe, Harald; Kuster, Bernhard; Médard, Guillaume

2017-06-21

The receptor tyrosine kinase EPHA2 has gained attention as a therapeutic drug target for cancer and infectious diseases. However, EPHA2 research and EPHA2-based therapies have been hampered by the lack of selective small-molecule inhibitors. Herein we report the synthesis and evaluation of dedicated EPHA2 inhibitors based on the clinical BCR-ABL/SRC inhibitor dasatinib as a lead structure. We designed hybrid structures of dasatinib and the previously known EPHA2 binders CHEMBL249097, PD-173955, and a known EPHB4 inhibitor in order to exploit both the ATP pocket entrance as well as the ribose pocket as binding epitopes in the kinase EPHA2. Medicinal chemistry and inhibitor design were guided by a chemical proteomics approach, allowing early selectivity profiling of the newly synthesized inhibitor candidates. Concomitant protein crystallography of 17 inhibitor co-crystals delivered detailed insight into the atomic interactions that underlie the structure-affinity relationship. Finally, the anti-proliferative effect of the inhibitor candidates was confirmed in the glioblastoma cell line SF-268. In this work, we thus discovered a novel EPHA2 inhibitor candidate that features an improved selectivity profile while maintaining potency against EPHA2 and anticancer activity in SF-268 cells. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Action of lovastatin, simvastatin, and pravastatin on sterol synthesis and their antiproliferative effect in cultured myoblasts from human striated muscle

NARCIS (Netherlands)

Vliet, A.K. van; Nègre-Arrariou, P.; Thiel, G.C.F. van; Bolhuis, P.A.; Cohen, L.H.

1996-01-01

Lovastatin, simvastatin, and pravastatin are fairly strong inhibitors of sterol synthesis in human myoblasts in culture. Lovastatin and simvastatin have IC50 values of 19 ± 6 nM and 4.0 ± 2.3 nM, respectively. Pravastatin is a weaker inhibitor of sterol synthesis (IC50 value of 110 ± 38 nM). Through

Natural inhibitors of tumor-associated proteases

International Nuclear Information System (INIS)

Magdolen, U.; Krol, J.; Sato, S.; Schmitt, M.; Magdolen, V.; Krueger, A.; Mueller, M.M.; Sperl, S.

2002-01-01

The turnover and remodelling of extracellular matrix (ECM) is an essential part of many normal biological processes including development, morphogenesis, and wound healing. ECM turnover also occurs in severe pathological situations like artherosclerosis, fibrosis, tumor invasion and metastasis. The major proteases involved in this turnover are serine proteases (especially the urokinase-type plasminogen activator/plasmin system), matrix metalloproteases (a family of about 20 zinc-dependent endopeptidases including collagenases, gelatinases, stromelysins, and membrane-type metalloproteases), and cysteine proteases. In vivo, the activity of these proteases is tightly regulated in the extracellular space by zymogen activation and/or controlled inhibition. In the present review, we give an overview on the structure and biochemical properties of important tumor-associated protease inhibitors such as plasminogen activator inhibitor type 1 and type 2 (PAI-1, PAI-2), tissue inhibitors of metalloproteinases (TIMP-1, -2, -3, and -4), and the cysteine protease inhibitor cystatin C. Interestingly, some of these inhibitors of tumor-associated proteases display multiple functions which rather promote than inhibit tumor progression, when the presence of inhibitors in the tumor tissue is not balanced. (author)

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

Science.gov (United States)

Emanuelsson, H; Heby, O

1978-01-01

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

Nucleotide synthesis is regulated by cytoophidium formation during neurodevelopment and adaptive metabolism

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Gabriel N. Aughey

2014-10-01

Full Text Available The essential metabolic enzyme CTP synthase (CTPsyn can be compartmentalised to form an evolutionarily-conserved intracellular structure termed the cytoophidium. Recently, it has been demonstrated that the enzymatic activity of CTPsyn is attenuated by incorporation into cytoophidia in bacteria and yeast cells. Here we demonstrate that CTPsyn is regulated in a similar manner in Drosophila tissues in vivo. We show that cytoophidium formation occurs during nutrient deprivation in cultured cells, as well as in quiescent and starved neuroblasts of the Drosophila larval central nervous system. We also show that cytoophidia formation is reversible during neurogenesis, indicating that filament formation regulates pyrimidine synthesis in a normal developmental context. Furthermore, our global metabolic profiling demonstrates that CTPsyn overexpression does not significantly alter CTPsyn-related enzymatic activity, suggesting that cytoophidium formation facilitates metabolic stabilisation. In addition, we show that overexpression of CTPsyn only results in moderate increase of CTP pool in human stable cell lines. Together, our study provides experimental evidence, and a mathematical model, for the hypothesis that inactive CTPsyn is incorporated into cytoophidia.

Social Recognition Memory Requires Two Stages of Protein Synthesis in Mice

Science.gov (United States)

Wolf, Gerald; Engelmann, Mario; Richter, Karin

2005-01-01

Olfactory recognition memory was tested in adult male mice using a social discrimination task. The testing was conducted to begin to characterize the role of protein synthesis and the specific brain regions associated with activity in this task. Long-term olfactory recognition memory was blocked when the protein synthesis inhibitor anisomycin was…

Hydrocortisone and triiodothyronine regulate hyaluronate synthesis in a tissue-engineered human dermal equivalent through independent pathways.

Science.gov (United States)

Deshpande, Madhura; Papp, Suzanne; Schaffer, Lana; Pouyani, Tara

2015-02-01

Hydrocortisone (HC) and triiodothyronine (T3) have both been shown to be capable of independently inhibiting hyaluronate (HA, hyaluronic acid) synthesis in a self-assembled human dermal equivalent (human dermal matrix). We sought to investigate the action of these two hormones in concert on extracellular matrix formation and HA inhibition in the tissue engineered human dermal matrix. To this end, neonatal human dermal fibroblasts were cultured in defined serum-free medium for 21 days in the presence of each hormone alone, or in combination, in varying concentrations. Through a process of self-assembly, a substantial dermal extracellular matrix formed that was characterized. The results of these studies demonstrate that combinations of the hormones T3 and hydrocortisone showed significantly higher levels of hyaluronate inhibition as compared to each hormone alone in the human dermal matrix. In order to gain preliminary insight into the genes regulating HA synthesis in this system, a differential gene array analysis was conducted in which the construct prepared in the presence of 200 μg/mL HC and 0.2 nM T3 was compared to the normal construct (0.4 μg/mL HC and 20 pM T3). Using a GLYCOv4 gene chip containing approximately 1260 human genes, we observed differential expression of 131 genes. These data suggest that when these two hormones are used in concert a different mechanism of inhibition prevails and a combination of degradation and inhibition of HA synthesis may be responsible for HA regulation in the human dermal matrix. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Regulation of protein translation initiation in response to ionizing radiation

International Nuclear Information System (INIS)

Trivigno, Donatella; Bornes, Laura; Huber, Stephan M; Rudner, Justine

2013-01-01

Proliferating tumor cells require continuous protein synthesis. De novo synthesis of most proteins is regulated through cap-dependent translation. Cellular stress such as ionizing radiation (IR) blocks cap-dependent translation resulting in shut-down of global protein translation which saves resources and energy needed for the stress response. At the same time, levels of proteins required for stress response are maintained or even increased. The study aimed to analyze the regulation of signaling pathways controlling protein translation in response to IR and the impact on Mcl-1, an anti-apoptotic and radioprotective protein, which levels rapidly decline upon IR. Protein levels and processing were analyzed by Western blot. The assembly of the translational pre-initiation complex was examined by Immunoprecipitation and pull-down experiments with 7-methyl GTP agarose. To analyze IR-induced cell death, dissipation of the mitochondrial membrane potential and DNA fragmentation were determined by flow cytometry. Protein levels of the different initiation factors were down-regulated using RNA interference approach. IR induced caspase-dependent cleavage of the translational initiation factors eIF4G1, eIF3A, and eIF4B resulting in disassembly of the cap-dependent initiation complex. In addition, DAP5-dependent initiation complex that regulates IRES-dependent translation was disassembled in response to IR. Moreover, IR resulted in dephosphorylation of 4EBP1, an inhibitor of cap-dependent translation upstream of caspase activation. However, knock-down of eIF4G1, eIF4B, DAP5, or 4EBP1 did not affect IR-induced decline of the anti-apoptotic protein Mcl-1. Our data shows that cap-dependent translation is regulated at several levels in response to IR. However, the experiments indicate that IR-induced Mcl-1 decline is not a consequence of translational inhibition in Jurkat cells

Regulation of protein translation initiation in response to ionizing radiation

Directory of Open Access Journals (Sweden)

Trivigno Donatella

2013-02-01

Full Text Available Abstract Background Proliferating tumor cells require continuous protein synthesis. De novo synthesis of most proteins is regulated through cap-dependent translation. Cellular stress such as ionizing radiation (IR blocks cap-dependent translation resulting in shut-down of global protein translation which saves resources and energy needed for the stress response. At the same time, levels of proteins required for stress response are maintained or even increased. The study aimed to analyze the regulation of signaling pathways controlling protein translation in response to IR and the impact on Mcl-1, an anti-apoptotic and radioprotective protein, which levels rapidly decline upon IR. Methods Protein levels and processing were analyzed by Western blot. The assembly of the translational pre-initiation complex was examined by Immunoprecipitation and pull-down experiments with 7-methyl GTP agarose. To analyze IR-induced cell death, dissipation of the mitochondrial membrane potential and DNA fragmentation were determined by flow cytometry. Protein levels of the different initiation factors were down-regulated using RNA interference approach. Results IR induced caspase-dependent cleavage of the translational initiation factors eIF4G1, eIF3A, and eIF4B resulting in disassembly of the cap-dependent initiation complex. In addition, DAP5-dependent initiation complex that regulates IRES-dependent translation was disassembled in response to IR. Moreover, IR resulted in dephosphorylation of 4EBP1, an inhibitor of cap-dependent translation upstream of caspase activation. However, knock-down of eIF4G1, eIF4B, DAP5, or 4EBP1 did not affect IR-induced decline of the anti-apoptotic protein Mcl-1. Conclusion Our data shows that cap-dependent translation is regulated at several levels in response to IR. However, the experiments indicate that IR-induced Mcl-1 decline is not a consequence of translational inhibition in Jurkat cells.

Metabolic Circuit Involving Free Fatty Acids, microRNA 122, and Triglyceride Synthesis in Liver and Muscle Tissues.

Science.gov (United States)

Chai, Chofit; Rivkin, Mila; Berkovits, Liav; Simerzin, Alina; Zorde-Khvalevsky, Elina; Rosenberg, Nofar; Klein, Shiri; Yaish, Dayana; Durst, Ronen; Shpitzen, Shoshana; Udi, Shiran; Tam, Joseph; Heeren, Joerg; Worthmann, Anna; Schramm, Christoph; Kluwe, Johannes; Ravid, Revital; Hornstein, Eran; Giladi, Hilla; Galun, Eithan

2017-11-01

Effective treatments are needed for hepatic steatosis characterized by accumulation of triglycerides in hepatocytes, which leads to hepatocellular carcinoma. MicroRNA 122 (MIR122) is expressed only in the liver, where it regulates lipid metabolism. We investigated the mechanism by which free fatty acids (FFAs) regulate MIR122 expression and the effect of MIR122 on triglyceride synthesis. We analyzed MIR122 promoter activity and validated its target mRNAs by transfection of Luciferase reporter plasmids into Huh7, BNL-1ME, and HEK293 cultured cell lines. We measured levels of microRNAs and mRNAs by quantitative real-time PCR analysis of RNA extracted from plasma, liver, muscle, and adipose tissues of C57BL/6 mice given the FFA-inducer CL316243. MIR122 was inhibited using an inhibitor of MIR122. Metabolic profiles of mice were determined using metabolic chambers and by histologic analyses of liver tissues. We performed RNA sequence analyses to identify metabolic pathways involving MIR122. We validated human Agpat1 and Dgat1 mRNAs, involved in triglyceride synthesis, as targets of MIR122. FFAs increased MIR122 expression in livers of mice by activating the retinoic acid-related orphan receptor alpha, and induced secretion of MIR122 from liver to blood. Circulating MIR122 entered muscle and adipose tissues of mice, reducing mRNA levels of genes involved in triglyceride synthesis. Mice injected with an inhibitor of MIR122 and then given CL316243, accumulated triglycerides in liver and muscle tissues, and had reduced rates of β-oxidation. There was a positive correlation between level of FFAs and level of MIR122 in plasma samples from 6 healthy individuals, collected before and during fasting. In biochemical and histologic studies of plasma, liver, muscle, and adipose tissues from mice, we found that FFAs increase hepatic expression and secretion of MIR122, which regulates energy storage vs expenditure in liver and peripheral tissues. Strategies to reduce

Synthesis, characterization and metal coordination of a potential β-lactamase inhibitor: 5-Methyl-2-phenoxymethyl-3-H-imidazole-4-carboxylic acid (PIMA

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Chiara Romagnoli

2017-12-01

Full Text Available Among relevant metal ions in biological systems, zinc and iron play a key role as active partners of the catalytic machinery. In particular, the inhibition of metal enzymes that are involved in physiological and pathological processes has been deeply investigated for the rational design of selective and efficient drugs based on chelators. Since imidazole histidine residue is one of the most versatile sites in proteins, especially in enzymes acting in the presence of metal ions as cofactors, in this work the synthesis and characterization of a new imidazole derivative, namely 5-methyl-2-phenoxymethyl-3-H-imidazole-4-carboxylic acid (PIMA is reported. PIMA was designed as metallo-β-lactamase inhibitor thanks to its similarity with penicillin V, a β-lactam antibiotic inactivated by metallo-β-lactamase, for which there are no commercially available inhibitors. The evaluation of PIMA coordinating ability toward iron, zinc, and gallium, these latter selected as a non-paramagnetic probe for iron, is performed by theoretical DFT calculations and in solution by experimental techniques, i.e. potentiometry, UV–vis and NMR spectroscopy. PIMA exhibits an efficient metal chelating ability; the prevailing species in physiological condition are ML3 for Fe3+ and Ga3+ and ML2 for Zn2+, in which chelation is due to deprotonated carboxylic oxygen and imidazole nitrogen in the N,O donor set. The demonstrated ability of PIMA to chelate zinc ion, combined with its structure similarity with penicillin V, supports further exploration of this imidazole-4-carboxylate as metallo-β-lactamase inhibitor.

Regulation of auxin transport during gravitropism

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Rashotte, A.; Brady, S.; Kirpalani, N.; Buer, C.; Muday, G.

Plants respond to changes in the gravity vector by differential growth across the gravity-stimulated organ. The plant hormone auxin, which is normally basipetally transported, changes in direction and auxin redistribution has been suggested to drive this differential growth or gravitropism. The mechanisms by which auxin transport directionality changes in response to a change in gravity vector are largely unknown. Using the model plant, Arabidopsis thaliana, we have been exploring several regulatory mechanisms that may control auxin transport. Mutations that alter protein phosphorylation suggest that auxin transport in arabidopsis roots may be controlled via phosphorylation and this signal may facilitate gravitropic bending. The protein kinase mutant pinoid (pid9) has reduced auxin transport; whereas the protein phosphatase mutant, rcn1, has elevated transport, suggesting reciprocal regulation of auxin transport by reversible protein phosphorylation. In both of these mutants, the auxin transport defects are accompanied by gravitropic defects, linking phosphorylation signaling to gravity-induced changes in auxin transport. Additionally, auxin transport may be regulated during gravity response by changes in an endogenous auxin efflux inhibitor. Flavonoids, such as quercetin and kaempferol, have been implicated in regulation of auxin transport in vivo and in vitro. Mutants that make no flavonoids have reduced root gravitropic bending. Furthermore, changes in auxin-induced gene expression and flavonoid accumulation patterns have been observed during gravity stimulation. Current studies are examining whether there are spatial and temporal changes in flavonoid accumulation that precede gravitropic bending and whether the absence of these changes are the cause of the altered gravity response in plants with mutations that block flavonoid synthesis. These results support the idea that auxin transport may be regulated during gravity response by several mechanisms including

An efficient method for synthesis of succinate-based MMP inhibitors.

Science.gov (United States)

Sibi, Mukund P; Hasegawa, Hikaru

2002-10-03

A differentially protected fumarate undergoes radical addition followed by allylstannane trapping to provide disubstituted succinates in good yields and high anti diastereoselectivity. The conversion of the succinate to a known MMP inhibitor has been accomplished. [reaction: see text

TOR-inhibitor insensitive-1 (TRIN1) regulates cotyledons greening in Arabidopsis.

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Li, Linxuan; Song, Yun; Wang, Kai; Dong, Pan; Zhang, Xueyan; Li, Fuguang; Li, Zhengguo; Ren, Maozhi

2015-01-01

Target of Rapamycin (TOR) is an eukaryotic protein kinase and evolutionally conserved from the last eukaryotic common ancestor (LECA) to humans. The growing evidences have shown that TOR signaling acts as a central controller of cell growth and development. The downstream effectors of TOR have been well-identified in yeast and animals by using the immunosuppression agent rapamycin. However, less is known about TOR in plants. This is largely due to the fact that plants are insensitive to rapamycin. In this study, AZD8055 (AZD), the novel ATP-competitive inhibitor of TOR, was employed to decipher the downstream effectors of TOR in Arabidopsis. One AZD insensitive mutant, T O R - i nhibitor i n sensitive- 1 (trin1), was screened from 10,000 EMS-induced mutation seeds. The cotyledons of trin1 can turn green when its seeds were germinated on ½ MS medium supplemented with 2 μM AZD, whereas the cotyledons greening of wild-type (WT) can be completely blocked at this concentration. Through genetic mapping, TRIN1 was mapped onto the long arm of chromosome 2, between markers SGCSNP26 and MI277. Positional cloning revealed that TRIN1 was an allele of ABI4, which encoded an ABA-regulated AP2 domain transcription factor. Plants containing P35S::TRIN1 or P35S::TRIN1-GUS were hypersensitive to AZD treatment and displayed the opposite phenotype observed in trin1. Importantly, GUS signaling was significantly enhanced in P35S::TRIN1-GUS transgenic plants in response to AZD treatment, indicating that suppression of TOR resulted in the accumulation of TRIN1. These observations revealed that TOR controlled seed-to-seedling transition by negatively regulating the stability of TRIN1 in Arabidopsis. For the first time, TRIN1, the downstream effector of TOR signaling, was identified through a chemical genetics approach.

A Ca2+-calmodulin-eEF2K-eEF2 signalling cascade, but not AMPK, contributes to the suppression of skeletal muscle protein synthesis during contractions

DEFF Research Database (Denmark)

Rose, Adam John; Alsted, Thomas Junker; Jensen, Thomas Elbenhardt

2009-01-01

Skeletal muscle protein synthesis rate decreases during contractions but the underlying regulatory mechanisms are poorly understood. It was hypothesised that there would be a coordinated regulation of eukaryotic elongation factor 2 (eEF2) and eukaryotic initiation factor 4E-binding protein 1 (4EBP1......) phosphorylation by signalling cascades downstream of rises in intracellular [Ca(2+)] and decreased energy charge via AMP activated protein kinase (AMPK) in contracting skeletal muscle. When fast-twitch skeletal muscles were contracted ex vivo using different protocols, the suppression of protein synthesis...... correlated more closely with changes in eEF2 rather than 4EBP1 phosphorylation. Using a combination of Ca(2+) release agents and ATPase inhibitors it was shown that the 60-70% suppression of fast-twitch skeletal muscle protein synthesis during contraction was equally distributed between Ca(2+) and energy...

Regulation of Hyaluronan Synthesis in Vascular Diseases and Diabetes

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Paola Moretto

2015-01-01

Full Text Available Cell microenvironment has a critical role determining cell fate and modulating cell responses to injuries. Hyaluronan (HA is a ubiquitous extracellular matrix glycosaminoglycan that can be considered a signaling molecule. In fact, interacting with several cell surface receptors can deeply shape cell behavior. In vascular biology, HA triggers smooth muscle cells (SMCs dedifferentiation which contributes to vessel wall thickening. Furthermore, HA is able to modulate inflammation by altering the adhesive properties of endothelial cells. In hyperglycemic conditions, HA accumulates in vessels and can contribute to the diabetic complications at micro- and macrovasculature. Due to the pivotal role in favoring atherogenesis and neointima formation after injuries, HA could be a new target for cardiovascular pathologies. This review will focus on the recent findings regarding the regulation of HA synthesis in human vascular SMCs. In particular, the effects of the intracellular HA substrates availability, adenosine monophosphate-activated protein kinase (AMPK, and protein O-GlcNAcylation on the main HA synthetic enzyme (i.e., HAS2 will be discussed.

Improved synthesis of (3E,6Z,9Z)-1,3,6,9-nonadecatetraene, attraction inhibitor of bruce spanworm, Operophtera bruceata, to pheromone traps for monitoring winter moth, Operophtera brumata.

Science.gov (United States)

Khrimian, Ashot; Lance, David R; Mastro, Victor C; Elkinton, Joseph S

2010-02-10

The winter moth, Operophtera brumata (Lepidoptera: Geometridae), is an early-season defoliator that attacks a wide variety of hardwoods and, in some cases, conifers. The insect is native to Europe but has become established in at least three areas of North America including southeastern New England. The female-produced sex attractant pheromone of the winter moth was identified as (3Z,6Z,9Z)-1,3,6,9-nonadecatetraene (1), which also attracts a native congener, the Bruce spanworm, Operophtera bruceata . Dissection, or (for certainty) DNA molecular testing, is required to differentiate between males of the two species. Thus, a trapping method that is selective for winter moth would be desirable. A geometric isomer of the pheromone, (3E,6Z,9Z)-1,3,6,9-nonadecatetraene (2), can reportedly inhibit attraction of Bruce spanworm to traps without affecting winter moth catch, but use of the pheromone and inhibitor together has not been optimized, nor has the synthesis of the inhibitor. This paper presents two new syntheses of the inhibitor (3E,6Z,9Z)-1,3,6,9-nonadecatetraene based on the intermediate (3Z,6Z)-3,6-hexadecadien-1-ol (4), which has also been utilized in the synthesis of the pheromone. The syntheses combine traditional acetylenic chemistry and Wittig olefination reactions. In one approach, 2 was synthesized in 80% purity (20% being pheromone 1), and in the second, tetraene 2 of 96% purity (and free of 1) was produced in 25% overall yield from dienol 4. The last method benefitted from a refined TEMPO-mediated PhI(OAc)(2) oxidation of 4 and a two-carbon homologation of the corresponding aldehyde 7.

N-acetylcysteine stimulates protein synthesis in enterocytes independently of glutathione synthesis.

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Yi, Dan; Hou, Yongqing; Wang, Lei; Long, Minhui; Hu, Shengdi; Mei, Huimin; Yan, Liqiong; Hu, Chien-An Andy; Wu, Guoyao

2016-02-01

Dietary supplementation with N-acetylcysteine (NAC) has been reported to improve intestinal health and treat gastrointestinal diseases. However, the underlying mechanisms are not fully understood. According to previous reports, NAC was thought to exert its effect through glutathione synthesis. This study tested the hypothesis that NAC enhances enterocyte growth and protein synthesis independently of cellular glutathione synthesis. Intestinal porcine epithelial cells were cultured for 3 days in Dulbecco's modified Eagle medium containing 0 or 100 μM NAC. To determine a possible role for GSH (the reduced form of glutathione) in mediating the effect of NAC on cell growth and protein synthesis, additional experiments were conducted using culture medium containing 100 μM GSH, 100 μM GSH ethyl ester (GSHee), diethylmaleate (a GSH-depletion agent; 10 μM), or a GSH-synthesis inhibitor (buthionine sulfoximine, BSO; 20 μM). NAC increased cell proliferation, GSH concentration, and protein synthesis, while inhibiting proteolysis. GSHee enhanced cell proliferation and GSH concentration without affecting protein synthesis but inhibited proteolysis. Conversely, BSO or diethylmaleate reduced cell proliferation and GSH concentration without affecting protein synthesis, while promoting protein degradation. At the signaling level, NAC augmented the protein abundance of total mTOR, phosphorylated mTOR, and phosphorylated 70S6 kinase as well as mRNA levels for mTOR and p70S6 kinase in IPEC-1 cells. Collectively, these results indicate that NAC upregulates expression of mTOR signaling proteins to stimulate protein synthesis in enterocytes independently of GSH generation. Our findings provide a hitherto unrecognized biochemical mechanism for beneficial effects of NAC in intestinal cells.

Synthesis and Docking Studies of 2,4,6-Trihydroxy-3-Geranylacetophenone Analogs as Potential Lipoxygenase Inhibitor

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Chean Hui Ng

2014-08-01

Full Text Available The natural product molecule 2,4,6-trihydroxy-3-geranyl-acetophenone (tHGA isolated from the medicinal plant Melicope ptelefolia was shown to exhibit potent lipoxygenase (LOX inhibitory activity. It is known that LOX plays an important role in inflammatory response as it catalyzes the oxidation of unsaturated fatty acids, such as linoleic acid to form hydroperoxides. The search for selective LOX inhibitors may provide new therapeutic approach for inflammatory diseases. Herein, we report the synthesis of tHGA analogs using simple Friedel-Craft acylation and alkylation reactions with the aim of obtaining a better insight into the structure-activity relationships of the compounds. All the synthesized analogs showed potent soybean 15-LOX inhibitory activity in a dose-dependent manner (IC50 = 10.31–27.61 μM where compound 3e was two-fold more active than tHGA. Molecular docking was then applied to reveal the important binding interactions of compound 3e in soybean 15-LOX binding site. The findings suggest that the presence of longer acyl bearing aliphatic chain (5Cs and aromatic groups could significantly affect the enzymatic activity.

An endogenous ribonuclease inhibitor regulates the antimicrobial activity of ribonuclease 7 in the human urinary tract

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Spencer, John David; Schwaderer, Andrew L.; Eichler, Tad; Wang, Huanyu; Kline, Jennifer; Justice, Sheryl S.; Cohen, Daniel M.; Hains, David S.

2013-01-01

Recent studies stress the importance of antimicrobial peptides in protecting the urinary tract from infection. Previously, we have shown that ribonuclease 7 (RNase 7) is a potent antimicrobial peptide that has broad-spectrum antimicrobial activity against uropathogenic bacteria. The urothelium of the lower urinary tract and intercalated cells of the kidney produce RNase 7 but regulation of its antimicrobial activity has not been well defined. Here we characterize the expression of an endogenous inhibitor, ribonuclease inhibitor (RI), in the urinary tract and evaluate its effect on RNase 7’s antimicrobial activity. Using RNA isolated from non-infected human bladder and kidney tissue, quantitative real-time PCR showed that RNH1, the gene encoding RI, is constitutively expressed throughout the urinary tract. With pyelonephritis, RNH1 expression and RI peptide production significantly decrease. Immunostaining localized RI production to the umbrella cells of the bladder and intercalated cells of the renal collecting tubule. In vitro assays showed that RI bound to RNase 7 and suppressed its antimicrobial activity by blocking its ability to bind the cell wall of uropathogenic bacteria. Thus, these results demonstrate a new immunomodulatory role for RI and identified a unique regulatory pathway that may affect how RNase 7 maintains urinary tract sterility. PMID:24107847

Synthesis and application of polyaminoamide as new paraffin inhibitor from vegetable oil

OpenAIRE

Chen, Gang; Tang, Ying; Zhang, Jie

2011-01-01

Abstract In this work, a series of novel paraffin inhibitor, polyaminoamide (PAA), was designed and prepared by aminolysis and poly-condensation using soybean oil and canola oil as the raw material. The property of the PAAs as paraffin inhibitor was investigated, the results show several PAA samples are potent in paraffin inhibition, and PPC-2 is the most effective one. Besides, the paraffin crystal morphology analysis was carried out to provide the mechanism of paraffin inhibition.

Molecular biological mechanism II. Molecular mechanisms of cell cycle regulation

International Nuclear Information System (INIS)

Jung, T.

2000-01-01

The cell cycle in eukaryotes is regulated by central cell cycle controlling protein kinase complexes. These protein kinase complexes consist of a catalytic subunit from the cyclin-dependent protein kinase family (CDK), and a regulatory subunit from the cyclin family. Cyclins are characterised by their periodic cell cycle related synthesis and destruction. Each cell cycle phase is characterised by a specific set of CDKs and cyclins. The activity of CDK/cyclin complexes is mainly regulated on four levels. It is controlled by specific phosphorylation steps, the synthesis and destruction of cyclins, the binding of specific inhibitor proteins, and by active control of their intracellular localisation. At several critical points within the cell cycle, named checkpoints, the integrity of the cellular genome is monitored. If damage to the genome or an unfinished prior cell cycle phase is detected, the cell cycle progression is stopped. These cell cycle blocks are of great importance to secure survival of cells. Their primary importance is to prevent the manifestation and heritable passage of a mutated genome to daughter cells. Damage sensing, DNA repair, cell cycle control and apoptosis are closely linked cellular defence mechanisms to secure genome integrity. Disregulation in one of these defence mechanisms are potentially correlated with an increased cancer risk and therefore in at least some cases with an increased radiation sensitivity. (orig.) [de

Beyond triglyceride synthesis: the dynamic functional roles of MGAT and DGAT enzymes in energy metabolism.

Science.gov (United States)

Shi, Yuguang; Cheng, Dong

2009-07-01

Monoacyglycerol acyltransferases (MGATs) and diacylglycerol acyltransferases (DGATs) catalyze two consecutive steps of enzyme reactions in the synthesis of triacylglycerols (TAGs). The metabolic complexity of TAG synthesis is reflected by the presence of multiple isoforms of MGAT and DGAT enzymes that differ in catalytic properties, subcellular localization, tissue distribution, and physiological functions. MGAT and DGAT enzymes play fundamental roles in the metabolism of monoacylglycerol (MAG), diacylglycerol (DAG), and triacylglycerol (TAG) that are involved in many aspects of physiological functions, such as intestinal fat absorption, lipoprotein assembly, adipose tissue formation, signal transduction, satiety, and lactation. The recent progress in the phenotypic characterization of mice deficient in MGAT and DGAT enzymes and the development of chemical inhibitors have revealed important roles of these enzymes in the regulation of energy homeostasis and insulin sensitivity. Consequently, selective inhibition of MGAT or DGAT enzymes by synthetic compounds may provide novel treatment for obesity and its related metabolic complications.

Clinical and pathogenetic interrelation between molecular regulation of apoptosis and cell differentiation in osteoarthritis

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M A Kabalyk

2018-02-01

Full Text Available Aim. To determine clinical and pathogenetic relationship between the levels of apoptosis and growth and differentiation regulation (growth inhibitor 1 induced by oxidative stress, growth/differentiation factor 5 in osteoarthritis. Methods. In a rheumatology office of Vladivostok polyclinic №3 65 patients with knee osteoarthritis Kellgren grade 1-4 aged 66.5±8.0 years were examined. 25 healthy volunteers matched by sex and age without clinical and radiologic manifestations of osteoarthritis were included into control group. To measure concentration of the studied molecules in study patients’ blood, ELISA method was used. Results. Patients with osteoarthritis compared to control group had statistically significantly increased levels of Fas, growth/differentiation factor 5 and ratio of growth/differentiation factor 5/growth inhibitor 1 induced by oxidative stress. Fas levels were significantly lower in late stages 2-4 of osteoarthritis compared to stages 1 and 2. Growth/differentiation factor 5 level was lower in patients with stage 3-4 of osteoarthritis compared to stages 1 and 2. As radiologic signs of osteoarthritis progressed, decrease of the ratio of growth/differentiation factor 5/growth inhibitor 1 induced by oxidative stress, was registered which was significantly lower in stages 2 and 3 compared to stage 1. Conclusion. Extrinsic pathway of apoptosis plays a big role in forming pain syndrome in osteoarthritis, and its maintenance is provided by other mechanisms which include influence of oxidative stress via inhibition of cell cycle mediated by growth inhibitor 1 induced by oxidative stress, reduced involvement of growth/differentiation factor 5 in differentiation processes and regulation of protein synthesis of extracellular cartilaginous tissue matrix.

Transport mechanisms and their pathology-induced regulation govern tyrosine kinase inhibitor delivery in rheumatoid arthritis.

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Christian Schmidt-Lauber

Full Text Available BACKGROUND: Tyrosine kinase inhibitors (TKIs are effective in treating malignant disorders and were lately suggested to have an impact on non-malignant diseases. However, in some inflammatory conditions like rheumatoid arthritis (RA the in vivo effect seemed to be moderate. As most TKIs are taken up actively into cells by cell membrane transporters, this study aimed to evaluate the role of such transporters for the accumulation of the TKI Imatinib mesylates in RA synovial fibroblasts as well as their regulation under inflammatory conditions. METHODOLOGY/PRINCIPAL FINDINGS: The transport and accumulation of Imatinib was investigated in transporter-transfected HEK293 cells and human RA synovial fibroblasts (hRASF. Transporter expression was quantified by qRT-PCR. In transfection experiments, hMATE1 showed the highest apparent affinity for Imatinib among all known Imatinib transporters. Experiments quantifying the Imatinib uptake in the presence of specific transporter inhibitors and after siRNA knockdown of hMATE1 indeed identified hMATE1 to mediate Imatinib transport in hRASF. The anti-proliferative effect of Imatinib on PDGF stimulated hRASF was quantified by cell counting and directly correlated with the uptake activity of hMATE1. Expression of hMATE1 was investigated by Western blot and immuno-fluorescence. Imatinib transport under disease-relevant conditions, such as an altered pH and following stimulation with different cytokines, was also investigated by HPLC. The uptake was significantly reduced by an acidic extracellular pH as well as by the cytokines TNFα, IL-1β and IL-6, which all decreased the expression of hMATE1-mRNA and protein. CONCLUSION/SIGNIFICANCE: The regulation of Imatinib uptake via hMATE1 in hRASF and resulting effects on their proliferation may explain moderate in vivo effects on RA. Moreover, our results suggest that investigating transporter mediated drug processing under normal and pathological conditions is important

Bombyx mori cyclin-dependent kinase inhibitor is involved in regulation of the silkworm cell cycle.

Science.gov (United States)

Tang, X-F; Zhou, X-L; Zhang, Q; Chen, P; Lu, C; Pan, M-H

2018-06-01

Cyclin-dependent kinase inhibitors (CKIs) are negative regulators of the cell cycle. They can bind to cyclin-dependent kinase (CDK)-cyclin complexes and inhibit CDK activities. We identified a single homologous gene of the CDK interacting protein/kinase inhibitory protein (Cip/Kip) family, BmCKI, in the silkworm, Bombyx mori. The gene transcribes two splice variants: a 654-bp-long BmCKI-L (the longer splice variant) encoding a protein with 217 amino acids and a 579-bp-long BmCKI-S (the shorter splice variant) encoding a protein with 192 amino acids. BmCKI-L and BmCKI-S contain the Cip/Kip family conserved cyclin-binding domain and the CDK-binding domain. They are localized in the nucleus and have an unconventional bipartite nuclear localization signal at amino acid residues 181-210. Overexpression of BmCKI-L or BmCKI-S affected cell cycle progression; the cell cycle was arrested in the first gap phase of cell cycle (G1). RNA interference of BmCKI-L or BmCKI-S led to cells accumulating in the second gap phase and the mitotic phase of cell cycle (G2/M). Both BmCKI-L and BmCKI-S are involved in cell cycle regulation and probably have similar effects. The transgenic silkworm with BmCKI-L overexpression (BmCKI-L-OE), exhibited embryonic lethal, larva developmental retardation and lethal phenotypes. These results suggest that BmCKI-L might regulate the growth and development of silkworm. These findings clarify the function of CKIs and increase our understanding of cell cycle regulation in the silkworm. © 2018 The Royal Entomological Society.

In vitro ischemia suppresses hypoxic induction of hypoxia-inducible factor-1α by inhibition of synthesis and not enhanced degradation.

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Karuppagounder, Saravanan S; Basso, Manuela; Sleiman, Sama F; Ma, Thong C; Speer, Rachel E; Smirnova, Natalya A; Gazaryan, Irina G; Ratan, Rajiv R

2013-08-01

Hypoxia-inducible factor (HIF) mediates a broad, conserved adaptive response to hypoxia, and the HIF pathway is a potential therapeutic target in cerebral ischemia. This study investigated the mechanism by which in vitro ischemia (oxygen-glucose deprivation; OGD) affects canonical hypoxic HIF-1α stabilization. We validated the use of a reporter containing the oxygen-dependent degradation domain of HIF-1α fused to firefly luciferase (ODD-luc) to monitor quantitatively distinct biochemical events leading to hypoxic HIF-1α expression or stabilization in a human neuroblastoma cell line (SH-SY5Y). When OGD was imposed following a 2-hr hypoxic stabilization of ODD-luc, the levels of the reporter were reduced, consistent with prior models proposing that OGD enhances HIF prolylhydroxylase (PHD) activity. Surprisingly, PHD inhibitors and proteasome inhibitors do not stabilize ODD-luc in OGD. Furthermore, OGD does not affect the half-life of ODD-luc protein following hypoxia, suggesting that OGD abrogates hypoxic HIF-1α induction by reducing HIF-1α synthesis rather than by enhancing its degradation. We observed ATP depletion under OGD vs. hypoxia and propose that ATP depletion enhances translational suppression, overcoming the selective synthesis of HIF concurrent with global decreases in protein synthesis in hypoxia. Taken together, these findings biochemically characterize a practical reporter for monitoring HIF-1α levels and support a novel model for HIF regulation in an in vitro model of human ischemia. Copyright © 2013 Wiley Periodicals, Inc.

Histone Deacetylase Inhibitors as Anticancer Drugs.

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Eckschlager, Tomas; Plch, Johana; Stiborova, Marie; Hrabeta, Jan

2017-07-01

Carcinogenesis cannot be explained only by genetic alterations, but also involves epigenetic processes. Modification of histones by acetylation plays a key role in epigenetic regulation of gene expression and is controlled by the balance between histone deacetylases (HDAC) and histone acetyltransferases (HAT). HDAC inhibitors induce cancer cell cycle arrest, differentiation and cell death, reduce angiogenesis and modulate immune response. Mechanisms of anticancer effects of HDAC inhibitors are not uniform; they may be different and depend on the cancer type, HDAC inhibitors, doses, etc. HDAC inhibitors seem to be promising anti-cancer drugs particularly in the combination with other anti-cancer drugs and/or radiotherapy. HDAC inhibitors vorinostat, romidepsin and belinostat have been approved for some T-cell lymphoma and panobinostat for multiple myeloma. Other HDAC inhibitors are in clinical trials for the treatment of hematological and solid malignancies. The results of such studies are promising but further larger studies are needed. Because of the reversibility of epigenetic changes during cancer development, the potency of epigenetic therapies seems to be of great importance. Here, we summarize the data on different classes of HDAC inhibitors, mechanisms of their actions and discuss novel results of preclinical and clinical studies, including the combination with other therapeutic modalities.

Histone Deacetylase Inhibitors as Anticancer Drugs

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Tomas Eckschlager

2017-07-01

Full Text Available Carcinogenesis cannot be explained only by genetic alterations, but also involves epigenetic processes. Modification of histones by acetylation plays a key role in epigenetic regulation of gene expression and is controlled by the balance between histone deacetylases (HDAC and histone acetyltransferases (HAT. HDAC inhibitors induce cancer cell cycle arrest, differentiation and cell death, reduce angiogenesis and modulate immune response. Mechanisms of anticancer effects of HDAC inhibitors are not uniform; they may be different and depend on the cancer type, HDAC inhibitors, doses, etc. HDAC inhibitors seem to be promising anti-cancer drugs particularly in the combination with other anti-cancer drugs and/or radiotherapy. HDAC inhibitors vorinostat, romidepsin and belinostat have been approved for some T-cell lymphoma and panobinostat for multiple myeloma. Other HDAC inhibitors are in clinical trials for the treatment of hematological and solid malignancies. The results of such studies are promising but further larger studies are needed. Because of the reversibility of epigenetic changes during cancer development, the potency of epigenetic therapies seems to be of great importance. Here, we summarize the data on different classes of HDAC inhibitors, mechanisms of their actions and discuss novel results of preclinical and clinical studies, including the combination with other therapeutic modalities.

Molecular Design, Synthesis and Trypanocidal Activity of Dipeptidyl Nitriles as Cruzain Inhibitors

Science.gov (United States)

Avelar, Leandro A. A.; Camilo, Cristian D.; de Albuquerque, Sérgio; Fernandes, William B.; Gonçalez, Cristiana; Kenny, Peter W.; Leitão, Andrei; McKerrow, James H.; Montanari, Carlos A.; Orozco, Erika V. Meñaca; Ribeiro, Jean F. R.; Rocha, Josmar R.; Rosini, Fabiana; Saidel, Marta E.

2015-01-01

A series of compounds based on the dipeptidyl nitrile scaffold were synthesized and assayed for their inhibitory activity against the T. cruzi cysteine protease cruzain. Structure activity relationships (SARs) were established using three, eleven and twelve variations respectively at the P1, P2 and P3 positions. A K i value of 16 nM was observed for the most potent of these inhibitors which reflects a degree of non-additivity in the SAR. An X-ray crystal structure was determined for the ligand-protein complex for the structural prototype for the series. Twenty three inhibitors were also evaluated for their anti-trypanosomal effects and an EC50 value of 28 μM was observed for the most potent of these. Although there remains scope for further optimization, the knowledge gained from this study is also transferable to the design of cruzain inhibitors based on warheads other than nitrile as well as alternative scaffolds. PMID:26173110

Increasing Malonyl-CoA Derived Product through Controlling the Transcription Regulators of Phospholipid Synthesis in Saccharomyces cerevisiae.

Science.gov (United States)

Chen, Xiaoxu; Yang, Xiaoyu; Shen, Yu; Hou, Jin; Bao, Xiaoming

2017-05-19

Malonyl-CoA is a precursor of a variety of compounds such as polyketides and flavonoids. In Saccharomyces cerevisiae, malonyl-CoA concentration is tightly regulated and therefore maintained at a very low level, limiting the production of malonyl-CoA-derived chemicals. Here we manipulated the phospholipid synthesis transcriptional regulators to control the malonyl-CoA levels and increase the downstream product. Through manipulating different regulators including Ino2p, Ino4p, Opi1p, and a series of synthetic Ino2p variants, combining with studying the inositol and choline effect, the engineered strain achieved a 9-fold increase of the titer of malonyl-CoA-derived product 3-hydroxypropionic acid, which is among the highest improvement relative to previously reported strategies. Our study provides a new strategy to regulate malonyl-CoA availability and will contribute to the production of other highly valued malonyl-CoA-derived chemicals.

Role for tryptophan in regulation of protein synthesis in porcine muscle

International Nuclear Information System (INIS)

Lin, F.D.; Smith, T.K.; Bayley, H.S.

1988-01-01

Experiments were conducted to determine the effect of varying concentrations of dietary tryptophan on growth rate and protein synthesis in edible muscle tissues of growing swine. A total of 45 immature swine (initial weight approximately 24 kg) were fed corn-gelatin diets containing 0.5 (n = 8), 0.8 (n = 10), 1.3 (n = 10), 1.5 (n = 7) or 2.0 (n = 10) g tryptophan/kg diet for 35 d. Animals fed 0.5 and 0.8 g tryptophan/kg grew more slowly, consumed less feed and had a lower efficiency of feed utilization than animals fed higher concentrations of tryptophan. Thirty similar animals were used in a second experiment. Diets containing 0.5, 0.8, 1.0, 1.5 or 2.0 g tryptophan/kg diet (n = 6) were fed for 14 d, after which all animals were killed and samples were taken of longissimus dorsi, triceps brachii and biceps femoris. Protein synthetic activity was determined by monitoring the incorporation of [ 14 C]phenylalanine into protein in vitro. There was no significant difference in synthetic activity between different muscle types. There was no effect of diet on the activity of the muscle soluble protein fraction. The activity of the muscle ribosomal fraction, however, was positively correlated with increasing concentrations of dietary tryptophan. It was concluded that tryptophan has the potential to regulate muscle protein synthesis in a manner beyond serving simply as a component of protein

Chromatin-associated regulation of sorbitol synthesis in flower buds of peach.

Science.gov (United States)

Lloret, Alba; Martínez-Fuentes, Amparo; Agustí, Manuel; Badenes, María Luisa; Ríos, Gabino

2017-11-01

PpeS6PDH gene is postulated to mediate sorbitol synthesis in flower buds of peach concomitantly with specific chromatin modifications. Perennial plants have evolved an adaptive mechanism involving protection of meristems within specialized structures named buds in order to survive low temperatures and water deprivation during winter. A seasonal period of dormancy further improves tolerance of buds to environmental stresses through specific mechanisms poorly known at the molecular level. We have shown that peach PpeS6PDH gene is down-regulated in flower buds after dormancy release, concomitantly with changes in the methylation level at specific lysine residues of histone H3 (H3K27 and H3K4) in the chromatin around the translation start site of the gene. PpeS6PDH encodes a NADPH-dependent sorbitol-6-phosphate dehydrogenase, the key enzyme for biosynthesis of sorbitol. Consistently, sorbitol accumulates in dormant buds showing higher PpeS6PDH expression. Moreover, PpeS6PDH gene expression is affected by cold and water deficit stress. Particularly, its expression is up-regulated by low temperature in buds and leaves, whereas desiccation treatment induces PpeS6PDH in buds and represses the gene in leaves. These data reveal the concurrent participation of chromatin modification mechanisms, transcriptional regulation of PpeS6PDH and sorbitol accumulation in flower buds of peach. In addition to its role as a major translocatable photosynthate in Rosaceae species, sorbitol is a widespread compatible solute and cryoprotectant, which suggests its participation in tolerance to environmental stresses in flower buds of peach.

SEARCH OF NEW SYNTHETIC INHIBITORS OF TYROSINASE

Directory of Open Access Journals (Sweden)

Yu. Shesterenko

2017-11-01

Full Text Available Melanin pigmentation of skin plays the most important role in the protection of organism against UV-irradiation, but the excessive accumulation of melanin brings to toxic melanodermia, melasma, lentigo and other skin lesions. Tyrosinase is the key enzyme of skin melanin pigment biosynthesis. In spite of certain progress in investigation of natural and synthetic tyrosinase inhibitors, actuality of such studies is of a high level, because the existing inhibitors are in some cases unstable, expensive, toxic, requires complex methods of synthesis or isolation from natural sources. The aim of the work is screening of new tyrosinase inhibitors, using the enzyme, isolated from Agaricus bisporus. Tyrosinase was isolated from Agaricus bisporus mushrooms by a modified method. It was found, that the introduction of polyethylene glycol 4000 in the extraction process promotes 3-fold reduction of polyphenol content, which leads to increase purity of enzyme with an increase in its activity by 25%. A search for new tyrosinase inhibitors among a wide range of compounds, including derivatives of 3-chloro-1,4-naphthoquinone, isatin, 3-hydroxy-2-naphthoic acid, etc was conducted. The studied substances did not displayed inhibitory effect at concentration of 0,1-0,5 mmol/dm3.

Structure-Based Design of Novel HIV-1 Protease Inhibitors to Combat Drug Resistance

Energy Technology Data Exchange (ETDEWEB)

Ghosh,A.; Sridhar, P.; Leshchenko, S.; Hussain, A.; Li, J.; Kovalevsky, A.; Walters, D.; Wedelind, J.; Grum-Tokars, V.; et al.

2006-01-01

Structure-based design and synthesis of novel HIV protease inhibitors are described. The inhibitors are designed specifically to interact with the backbone of HIV protease active site to combat drug resistance. Inhibitor 3 has exhibited exceedingly potent enzyme inhibitory and antiviral potency. Furthermore, this inhibitor maintains impressive potency against a wide spectrum of HIV including a variety of multi-PI-resistant clinical strains. The inhibitors incorporated a stereochemically defined 5-hexahydrocyclopenta[b]furanyl urethane as the P2-ligand into the (R)-(hydroxyethylamino)sulfonamide isostere. Optically active (3aS,5R,6aR)-5-hydroxy-hexahydrocyclopenta[b]furan was prepared by an enzymatic asymmetrization of meso-diacetate with acetyl cholinesterase, radical cyclization, and Lewis acid-catalyzed anomeric reduction as the key steps. A protein-ligand X-ray crystal structure of inhibitor 3-bound HIV-1 protease (1.35 Angstroms resolution) revealed extensive interactions in the HIV protease active site including strong hydrogen bonding interactions with the backbone. This design strategy may lead to novel inhibitors that can combat drug resistance.

How polyamine synthesis inhibitors and cinnamic acid affect tropane alkaloid production.

Science.gov (United States)

Marconi, Patricia L; Alvarez, María A; Pitta-Alvarez, Sandra I

2007-01-01

Hairy roots of Brugmansia candida produce the tropane alkaloids scopolamine and hyoscyamine. In an attempt to divert the carbon flux from competing pathways and thus enhance productivity, the polyamine biosynthesis inhibitors cyclohexylamine (CHA) and methylglyoxal-bis-guanylhydrazone (MGBG) and the phenylalanine-ammonia-lyase inhibitor cinnamic acid were used. CHA decreased the specific productivity of both alkaloids but increased significantly the release of scopolamine (approx 500%) when it was added in the mid-exponential phase. However, when CHA was added for only 48 h during the exponential phase, the specific productivity of both alkaloids increased (approx 200%), favoring scopolamine. Treatment with MGBG was detrimental to growth but promoted release into the medium of both alkaloids. However, when it was added for 48 h during the exponential phase, MGBG increased the specific productivity (approx 200%) and release (250- 1800%) of both alkaloids. Cinnamic acid alone also favored release but not specific productivity. When a combination of CHA or MGBG with cinnamic acid was used, the results obtained were approximately the same as with each polyamine biosynthesis inhibitor alone, although to a lesser extent. Regarding root morphology, CHA inhibited growth of primary roots and ramification. However, it had a positive effect on elongation of lateral roots.

Synthesis and PET studies of [(11)C-cyano]letrozole (Femara), an aromatase inhibitor drug.

Science.gov (United States)

Kil, Kun-Eek; Biegon, Anat; Ding, Yu-Shin; Fischer, Andre; Ferrieri, Richard A; Kim, Sung Won; Pareto, Deborah; Schueller, Michael J; Fowler, Joanna S

2009-02-01

Aromatase, a member of the cytochrome P450 family, converts androgens such as androstenedione and testosterone into estrone and estradiol, respectively. Letrozole (1-[bis-(4-cyanophenyl)methyl]-1H-1,2,4-triazole; Femara) is a high-affinity aromatase inhibitor (K(i)=11.5 nM) that has Food and Drug Administration approval for breast cancer treatment. Here we report the synthesis of carbon-11-labeled letrozole and its assessment as a radiotracer for brain aromatase in the baboon. Letrozole and its precursor (4-[(4-bromophenyl)-1H-1,2,4-triazol-1-ylmethyl]benzonitrile) were prepared in a two-step synthesis from 4-cyanobenzyl bromide and 4-bromobenzyl bromide, respectively. The [(11)C]cyano group was introduced via tetrakis(triphenylphosphine)palladium(0)-catalyzed coupling of [(11)C]cyanide with the bromo precursor. Positron emission tomography (PET) studies in the baboon brain were carried out to assess regional distribution and kinetics, reproducibility of repeated measures and saturability. Log D, the free fraction of letrozole in plasma and the [(11)C-cyano]letrozole fraction in arterial plasma were also measured. [(11)C-cyano]Letrozole was synthesized in 60 min with a radiochemical yield of 79-80%, with a radiochemical purity greater than 98% and a specific activity of 4.16+/-2.21 Ci/mumol at the end of bombardment (n=4). PET studies in the baboon revealed initial rapid and high uptake and initial rapid clearance, followed by slow clearance of carbon-11 from the brain, with no difference between brain regions. Brain kinetics was not affected by coinjection of unlabeled letrozole (0.1 mg/kg). The free fraction of letrozole in plasma was 48.9%, and log D was 1.84. [(11)C-cyano]Letrozole is readily synthesized via a palladium-catalyzed coupling reaction with [(11)C]cyanide. Although it is unsuitable as a PET radiotracer for brain aromatase, as revealed by the absence of regional specificity and saturability in brain regions such as amygdala, which are known to

Design, synthesis, and biological evaluation of 3-vinyl-quinoxalin-2(1H-one derivatives as novel antitumor inhibitors of FGFR1

Directory of Open Access Journals (Sweden)

Liu Z

2016-05-01

Full Text Available Zhiguo Liu,1,* Shufang Yu,1,* Di Chen,1 Guoliang Shen,1 Yu Wang,1 Leping Hou,2 Dan Lin,1 Jinsan Zhang,1 Faqing Ye1 1School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, 2Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, People’s Republic of China *These authors contributed equally to this work Abstract: FGFR1 is well known as a molecular target in anticancer drug design. TKI258 plays an important role in RTK inhibitors. Utilizing TKI258 as a lead compound that contains a quinazolinone nucleus, we synthesized four series of 3-vinyl-quinoxalin-2(1H-one derivatives, a total of 27 compounds. We further evaluated these compounds for FGFR1 inhibition ability as well as cytotoxicity against four cancer cell lines (H460, B16-F10, Hela229, and Hct116 in vitro. Some compounds displayed good-to-excellent potency against the four tested cancer cell lines compared with TKI258. Structure–activity relationship analyses indicated that small substituents at the side chain of the 3-vinyl-quinoxalin-2(1H-one were more effective than large substituents. Lastly, we used molecular docking to obtain further insight into the interactions between the compounds and FGFR1. Keywords: FGFR1, synthesis, quinoxaline, antitumor activity, kinase inhibitor

Cell-Free, De Nova Synthesis of Poliovirus

Science.gov (United States)

Molla, Akhteruzzaman; Paul, Aniko V.; Wimmer, Eckard

1991-12-01

Cell-free translation of poliovirus RNA in an extract of uninfected human (HeLa) cells yielded viral proteins through proteolysis of the polyprotein. In the extract, newly synthesized proteins catalyzed poliovirus-specific RNA synthesis, and formed infectious poliovirus de novo. Newly formed virions were neutralized by type-specific antiserum, and infection of human cells with them was prevented by poliovirus receptor-specific antibodies. Poliovirus synthesis was increased nearly 70-fold when nucleoside triphosphates were added, but it was abolished in the presence of inhibitors of translation or viral genome replication. The ability to conduct cell-free synthesis of poliovirus will aid in the study of picornavirus proliferation and in the search for the control of picornaviral disease.

Relative contributions of norspermidine synthesis and signaling pathways to the regulation of Vibrio cholerae biofilm formation.