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Sample records for activation protein inhibits

  1. Zinc ions bind to and inhibit activated protein C

    Zhu, Tianqing; Ubhayasekera, Wimal; Nickolaus, Noëlle;

    2010-01-01

    Zn2+ ions were found to efficiently inhibit activated protein C (APC), suggesting a potential regulatory function for such inhibition. APC activity assays employing a chromogenic peptide substrate demonstrated that the inhibition was reversible and the apparent K I was 13 +/- 2 microM. k cat was...... seven fold decreased whereas K M was unaffected in the presence of 10 microM Zn2+. The inhibitory effect of Zn2+ on APC activity was also observed when factor Va was used as a substrate in an assay coupled to a prothrombinase assay. The interaction of Zn2+ with APC was accompanied by a reversible...... fold enhanced, presumably due to the Ca2+-induced conformational change affecting the conformation of the Zn2+-binding site. The inhibition mechanism was non-competitive both in the absence and presence of Ca2+. Comparisons of sequences and structures suggested several possible sites for zinc binding...

  2. Haptoglobin inhibits phospholipid transfer protein activity in hyperlipidemic human plasma

    Leon Carlos G

    2009-07-01

    Full Text Available Abstract Background Haptoglobin is a plasma protein that scavenges haemoglobin during haemolysis. Phospholipid Transfer Protein (PLTP transfers lipids from Low Density Lipoproteins (LDL to High Density Lipoproteins (HDL. PLTP is involved in the pathogenesis of atherosclerosis which causes coronary artery disease, the leading cause of death in North America. It has been shown that Apolipoprotein-A1 (Apo-A1 binds and regulates PLTP activity. Haptoglobin can also bind to Apo-A1, affecting the ability of Apo-A1 to induce enzymatic activities. Thus we hypothesize that haptoglobin inhibits PLTP activity. This work tested the effect of Haptoglobin and Apo-A1 addition on PLTP activity in human plasma samples. The results will contribute to our understanding of the role of haptoglobin on modulating reverse cholesterol transport. Results We analyzed the PLTP activity and Apo-A1 and Haptoglobin content in six hyperlipidemic and six normolipidemic plasmas. We found that Apo-A1 levels are proportional to PLTP activity in hyperlipidemic (R2 = 0.66, p 2 = 0.57, p > 0.05. When the PLTP activity was graphed versus the Hp/Apo-A1 ratio in hyperlipidemic plasma there was a significant correlation (R2 = 0.69, p Conclusion These findings suggest an inhibitory effect of Haptoglobin over PLTP activity in hyperlipidemic plasma that may contribute to the regulation of reverse cholesterol transport.

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

    Richa Tyagi

    2015-02-01

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

  4. Effect of mitochondrial complex I inhibition on Fe-S cluster protein activity

    Highlights: → Mitochondrial complex I inhibition resulted in decreased activity of Fe-S containing enzymes mitochondrial aconitase and cytoplasmic aconitase and xanthine oxidase. → Complex I inhibition resulted in the loss of Fe-S clusters in cytoplasmic aconitase and of glutamine phosphoribosyl pyrophosphate amidotransferase. → Consistent with loss of cytoplasmic aconitase activity, an increase in iron regulatory protein 1 activity was found. → Complex I inhibition resulted in an increase in the labile cytoplasmic iron pool. -- Abstract: Iron-sulfur (Fe-S) clusters are small inorganic cofactors formed by tetrahedral coordination of iron atoms with sulfur groups. Present in numerous proteins, these clusters are involved in key biological processes such as electron transfer, metabolic and regulatory processes, DNA synthesis and repair and protein structure stabilization. Fe-S clusters are synthesized mainly in the mitochondrion, where they are directly incorporated into mitochondrial Fe-S cluster-containing proteins or exported for cytoplasmic and nuclear cluster-protein assembly. In this study, we tested the hypothesis that inhibition of mitochondrial complex I by rotenone decreases Fe-S cluster synthesis and cluster content and activity of Fe-S cluster-containing enzymes. Inhibition of complex I resulted in decreased activity of three Fe-S cluster-containing enzymes: mitochondrial and cytosolic aconitases and xanthine oxidase. In addition, the Fe-S cluster content of glutamine phosphoribosyl pyrophosphate amidotransferase and mitochondrial aconitase was dramatically decreased. The reduction in cytosolic aconitase activity was associated with an increase in iron regulatory protein (IRP) mRNA binding activity and with an increase in the cytoplasmic labile iron pool. Since IRP activity post-transcriptionally regulates the expression of iron import proteins, Fe-S cluster inhibition may result in a false iron deficiency signal. Given that inhibition of complex

  5. Protective effects of inhibition of adenosine monophosphate activated protein kinase activity against cerebral ischemia-reperfusion injury in mice

    补娟

    2013-01-01

    Objective To observe the effect of inhibition of adenosine monophosphate activated protein kinase (AMPK) on shape,function and inflammatory factor of microglia for mice after cerebral ischemia-reperfusion

  6. Thioredoxin interacting protein inhibits hypoxia-inducible factor transcriptional activity

    Farrell, Michael R; Rogers, Lynette K.; Liu, Yusen; Welty, Stephen E.; Tipple, Trent E.

    2010-01-01

    Vascular endothelial growth factor (VEGF) is required for proper lung development and is transcriptionally regulated in alveolar epithelial cells by hypoxia inducible factor (HIF). Previous findings in a newborn mouse model of bronchopulmonary dysplasia (BPD) suggest that thioredoxin interacting protein (Txnip) is a novel regulator of VEGF expression. The present studies were designed to test the hypothesis that Txnip negatively regulates VEGF through effects on HIF-mediated gene expression. ...

  7. Antipeptide antibody that specifically inhibits insulin receptor autophosphorylation and protein kinase activity

    Two site-specific antibodies that immunoprecipitate the human insulin receptor have been prepared by immunizing rabbits with chemically synthesized peptides derived from the cDNA-predicted amino acid sequence of the β subunit of the proreceptor. Antibodies to the carboxyl terminus (AbP5) and to a domain around tyrosine-960 (AbP4) specifically recognize the β subunit of the receptor on immunoblots. Both antibodies immunoprecipitated 125I-labeled insulin-receptor complexes and the autophosphorylated receptor. Although neither antibody inhibited insulin binding to the receptor, both insulin-dependent autophosphorylation and exogenous substrate phosphorylation were inhibited by AbP4. Inhibition by AbP4 was dependent upon the phosphorylation state of the receptor; it was not detected when the receptor was autophosphorylated prior to addition of AbP4. AbP4 did not inhibit activity of the related epidermal growth factor (EGF)-receptor tyrosine protein kinase nor did it inhibit the activity of cAMP-dependent kinase or protein kinase C. The observation that an antibody directed to residues 952-967 of the proreceptor neutralizes the protein kinase activity of the β subunit suggest that this region may play a critical role in the function of the hormone-dependent, protein tyrosine-specific kinase activity of the insulin receptor

  8. Antroquinonol blocks Ras and Rho signaling via the inhibition of protein isoprenyltransferase activity in cancer cells.

    Ho, Ching-Liang; Wang, Jui-Ling; Lee, Cheng-Chung; Cheng, Hsiu-Yi; Wen, Wu-Che; Cheng, Howard Hao-Yu; Chen, Miles Chih-Ming

    2014-10-01

    Antroquinonol is the smallest anticancer molecule isolated from Antrodia camphorata thus far. The ubiquinone-like structure of Antroquinonol exhibits a broad spectrum of activity against malignancies in vivo and in vitro. However, the mechanism of action of Antroquinonol remains unclear. Here, we provide evidence that Antroquinonol plays a role in the inhibition of Ras and Ras-related small GTP-binding protein functions through the inhibition of protein isoprenyl transferase activity in cancer cells. Using cell line-based assays, we found that the inactive forms of Ras and Rho proteins were significantly elevated after treatment with Antroquinonol. We also demonstrated that Antroquinonol binds directly to farnesyltransferase and geranylgeranyltransferase-I, which are key enzymes involved in activation of Ras-related proteins, and inhibits enzymes activities in vitro. Furthermore, a molecular docking analysis illustrated that the isoprenoid moiety of Antroquinonol binds along the hydrophobic cavity of farnesyltransferase similar to its natural substrate, farnesyl pyrophosphate. In contrast, the ring structure of Antroquinonol lies adjacent to the Ras-CAAX motif-binding site on farnesyltransferase. The molecular docking study also showed a reasonable correlation with the IC50 values of Antroquinonol analogues. We also found that the levels of LC3B-II and the autophagosome-associated LC3 form were also significantly increased in H838 after Antroquinonol administration. In conclusion, Antroquinonol inhibited Ras and Ras-related GTP-binding protein activation through inhibition of protein isoprenyl transferase activity, leading to activation of autophagy and associated mode of cell death in cancer cells. PMID:25312820

  9. INHIBITION OF IL-6-INDUCED STAT3 ACTIVATION IN MYELOMA CELLS BY PROTEIN KINASE A

    宋伦; 黎燕; 沈倍奋

    2001-01-01

    To investigate the regulation effect of protein kinase A on IL-6-induced STAT3 activation in myeloma cells. Methods: Two human myeloma cell lines-Sko-007 and U266 were pretreated with Forskolin, a protein kinase A antagonist, and then stimulated by IL-6. The activation state of STAT3 in these two cells were examined by electrophoretic mobility shift assay (EMSA). Results: Although PKA pathway itself doesn't participate in IL-6 signal transduction in Sko-007 and U266 cells, activation of protein kinase A can inhibit IL-6-induced STAT3 activation in these two cell lines. Conclusion: There exists an inhibitory effect of protein kinase A on STAT3 activation in human myeloma cells treated by IL-6.

  10. Mitochondrial thiol modification by a targeted electrophile inhibits metabolism in breast adenocarcinoma cells by inhibiting enzyme activity and protein levels.

    Smith, M Ryan; Vayalil, Praveen K; Zhou, Fen; Benavides, Gloria A; Beggs, Reena R; Golzarian, Hafez; Nijampatnam, Bhavitavya; Oliver, Patsy G; Smith, Robin A J; Murphy, Michael P; Velu, Sadanandan E; Landar, Aimee

    2016-08-01

    Many cancer cells follow an aberrant metabolic program to maintain energy for rapid cell proliferation. Metabolic reprogramming often involves the upregulation of glutaminolysis to generate reducing equivalents for the electron transport chain and amino acids for protein synthesis. Critical enzymes involved in metabolism possess a reactive thiolate group, which can be modified by certain oxidants. In the current study, we show that modification of mitochondrial protein thiols by a model compound, iodobutyl triphenylphosphonium (IBTP), decreased mitochondrial metabolism and ATP in MDA-MB 231 (MB231) breast adenocarcinoma cells up to 6 days after an initial 24h treatment. Mitochondrial thiol modification also depressed oxygen consumption rates (OCR) in a dose-dependent manner to a greater extent than a non-thiol modifying analog, suggesting that thiol reactivity is an important factor in the inhibition of cancer cell metabolism. In non-tumorigenic MCF-10A cells, IBTP also decreased OCR; however the extracellular acidification rate was significantly increased at all but the highest concentration (10µM) of IBTP indicating that thiol modification can have significantly different effects on bioenergetics in tumorigenic versus non-tumorigenic cells. ATP and other adenonucleotide levels were also decreased by thiol modification up to 6 days post-treatment, indicating a decreased overall energetic state in MB231 cells. Cellular proliferation of MB231 cells was also inhibited up to 6 days post-treatment with little change to cell viability. Targeted metabolomic analyses revealed that thiol modification caused depletion of both Krebs cycle and glutaminolysis intermediates. Further experiments revealed that the activity of the Krebs cycle enzyme, aconitase, was attenuated in response to thiol modification. Additionally, the inhibition of glutaminolysis corresponded to decreased glutaminase C (GAC) protein levels, although other protein levels were unaffected. This study

  11. Mitochondrial thiol modification by a targeted electrophile inhibits metabolism in breast adenocarcinoma cells by inhibiting enzyme activity and protein levels

    M. Ryan Smith

    2016-08-01

    Full Text Available Many cancer cells follow an aberrant metabolic program to maintain energy for rapid cell proliferation. Metabolic reprogramming often involves the upregulation of glutaminolysis to generate reducing equivalents for the electron transport chain and amino acids for protein synthesis. Critical enzymes involved in metabolism possess a reactive thiolate group, which can be modified by certain oxidants. In the current study, we show that modification of mitochondrial protein thiols by a model compound, iodobutyl triphenylphosphonium (IBTP, decreased mitochondrial metabolism and ATP in MDA-MB 231 (MB231 breast adenocarcinoma cells up to 6 days after an initial 24 h treatment. Mitochondrial thiol modification also depressed oxygen consumption rates (OCR in a dose-dependent manner to a greater extent than a non-thiol modifying analog, suggesting that thiol reactivity is an important factor in the inhibition of cancer cell metabolism. In non-tumorigenic MCF-10A cells, IBTP also decreased OCR; however the extracellular acidification rate was significantly increased at all but the highest concentration (10 µM of IBTP indicating that thiol modification can have significantly different effects on bioenergetics in tumorigenic versus non-tumorigenic cells. ATP and other adenonucleotide levels were also decreased by thiol modification up to 6 days post-treatment, indicating a decreased overall energetic state in MB231 cells. Cellular proliferation of MB231 cells was also inhibited up to 6 days post-treatment with little change to cell viability. Targeted metabolomic analyses revealed that thiol modification caused depletion of both Krebs cycle and glutaminolysis intermediates. Further experiments revealed that the activity of the Krebs cycle enzyme, aconitase, was attenuated in response to thiol modification. Additionally, the inhibition of glutaminolysis corresponded to decreased glutaminase C (GAC protein levels, although other protein levels were

  12. Normal protein content but abnormally inhibited enzyme activity in muscle carnitine palmitoyltransferase II deficiency.

    Lehmann, Diana; Zierz, Stephan

    2014-04-15

    The biochemical consequences of the disease causing mutations of muscle carnitine palmitoyltransferase II (CPT II) deficiency are still enigmatic. Therefore, CPT II was characterized in muscle biopsies of nine patients with genetically proven muscle CPT II deficiency. Total CPT activity (CPT I+CPT II) of patients was not significantly different from that of controls. Remaining activities upon inhibition by malonyl-CoA and Triton X-100 were significantly reduced in patients. Immunohistochemically CPT II protein was predominantly expressed in type-I-fibers with the same intensity in patients as in controls. Western blot showed the same CPT II staining intensity ratio in patients and controls. CPT I and CPT II protein concentrations estimated by ELISA were not significantly different in patients and in controls. Citrate synthase activity in patients was significantly increased. Total CPT activity significantly correlated with both CPT I and CPT II protein concentrations in patients and controls. This implies (i) that normal total CPT activity in patients with muscle CPT II deficiency is not due to compensatory increase of CPT I activity and that (ii) the mutant CPT II is enzymatically active. The data further support the notion that in muscle CPT II deficiency enzyme activity and protein content are not reduced, but rather abnormally inhibited when fatty acid metabolism is stressed. PMID:24602495

  13. Free Fatty Acids Inhibit Protein Tyrosine Phosphatase 1B and Activate Akt

    Eisuke Shibata

    2013-09-01

    Full Text Available Background/Aims: Accumulating evidence has suggested that free fatty acids (FFAs interact with protein kinases and protein phosphatases. The present study examined the effect of FFAs on protein phosphatases and Akt. Methods: Activities of protein phosphatase 1 (PP1, protein phosphatase 2A (PP2A, and protein tyrosine phosphatase 1B (PTP1B were assayed under the cell-free conditions. Phosphorylation of Akt was monitored in MSTO-211H human malignant pleural mesothelioma cells without and with knocking-down phosphatidylinositol 3 kinase (PI3K or 3-phosphoinositide-dependent protein kinase-1 (PDK1. Results: In the cell-free assay, unsaturated FFAs (uFFAs such as oleic, linoleic and linolenic acid and saturated FFAs (sFFAs such as stearic, palmitic, myristic, and behenic acid markedly reduced PTP1B activity, with the potential for uFFAs greater than that for sFFAs. All the investigated sFFAs inhibited PP2A activity, but otherwise no inhibition was obtained with uFFAs. Both uFFAs and sFFAs had no effect on PP1 activity. Oleic acid phosphorylated Akt both on Thr308 and Ser473, while stearic acid phosphorylated Akt on Thr308 alone. The effects of oleic and stearic acid on Akt phosphorylation were abrogated by the PI3K inhibitor wortmannin or the PDK1 inhibitor BX912 and also by knocking-down PI3K or PDK1. Conclusion: The results of the present study indicate that uFFAs and sFFAs could activate Akt through a pathway along a PI3K/PDK1/Akt axis in association with PTP1B inhibition.

  14. p-HPEA-EDA, a phenolic compound of virgin olive oil, activates AMP-activated protein kinase to inhibit carcinogenesis.

    Khanal, Prem; Oh, Won-Keun; Yun, Hyo Jeong; Namgoong, Gwang Mo; Ahn, Sang-Gun; Kwon, Seong-Min; Choi, Hoo-Kyun; Choi, Hong Seok

    2011-04-01

    Phenolic constituents of virgin olive oil are reported to have antitumor activity. However, the underlying molecular mechanisms and specific target proteins of virgin olive oil remain to be elucidated. Here, we report that dialdehydic form of decarboxymethyl ligstroside aglycone (p-HPEA-EDA), a phenolic compound of virgin olive oil, inhibits tumor promoter-induced cell transformation in JB6 Cl41 cells and suppress cyclooxygenase-2 (COX-2) and tumorigenicity by adenosine monophosphate-activated protein kinase (AMPK) activation in HT-29 cells. p-HPEA-EDA inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced phosphorylation of extracellular signal-regulated kinases 1/2 and p90RSK in JB6 Cl41 cells, resulting in the inhibition of cell proliferation, activator protein-1 transactivation and cell transformation promoted by TPA. Moreover, p-HPEA-EDA strongly inhibited the cell viability and COX-2 expression by activation of AMPK activity in HT-29 cells, resulted from depletion of intracellular adenosine triphosphate. p-HPEA-EDA-induced activation of caspase-3 and poly-adenosine diphosphate-ribose polymerase, phosphorylation of p53 (Ser15) and DNA fragmentation in HT-29 cells, leading to apoptosis. Importantly, p-HPEA-EDA suppressed the colony formation of HT-29 cells in soft agar. In contrast, Compound C, an AMPK inhibitor, and Z-DEVD-FMK, a caspase-3 inhibitor, blocked the p-HPEA-EDA-inhibited colony formation in HT-29 cells. In vivo chorioallantoic membrane assay also showed that p-HPEA-EDA-inhibited tumorigenicity of HT-29 cells. These findings revealed that targeted activation of AMPK and inhibition of COX-2 expression by p-HPEA-EDA contribute to the chemopreventive and chemotherapeutic potential of virgin olive oil against colon cancer cells. PMID:21216846

  15. Protein Synthesis Inhibition Activity by Strawberry Tissue Protein Extracts during Plant Life Cycle and under Biotic and Abiotic Stresses

    Walther Faedi

    2013-07-01

    Full Text Available Ribosome-inactivating proteins (RIPs, enzymes that are widely distributed in the plant kingdom, inhibit protein synthesis by depurinating rRNA and many other polynucleotidic substrates. Although RIPs show antiviral, antifungal, and insecticidal activities, their biological and physiological roles are not completely understood. Additionally, it has been described that RIP expression is augmented under stressful conditions. In this study, we evaluated protein synthesis inhibition activity in partially purified basic proteins (hereafter referred to as RIP activity from tissue extracts of Fragaria × ananassa (strawberry cultivars with low (Dora and high (Record tolerance to root pathogens and fructification stress. Association between the presence of RIP activity and the crop management (organic or integrated soil, growth stage (quiescence, flowering, and fructification, and exogenous stress (drought were investigated. RIP activity was found in every tissue tested (roots, rhizomes, leaves, buds, flowers, and fruits and under each tested condition. However, significant differences in RIP distribution were observed depending on the soil and growth stage, and an increase in RIP activity was found in the leaves of drought-stressed plants. These results suggest that RIP expression and activity could represent a response mechanism against biotic and abiotic stresses and could be a useful tool in selecting stress-resistant strawberry genotypes.

  16. Knockdown of Pokemon protein expression inhibits hepatocellular carcinoma cell proliferation by suppression of AKT activity.

    Zhu, Xiaosan; Dai, Yichen; Chen, Zhangxin; Xie, Junpei; Zeng, Wei; Lin, Yuanyuan

    2013-01-01

    Overexpression of Pokemon, which is an erythroid myeloid ontogenic factor protein, occurs in different cancers, including hepatocellular carcinoma (HCC). Pokemon is also reported to have an oncogenic activity in various human cancers. This study investigated the effect of Pokemon knockdown on the regulation of HCC growth. POK shRNA suppressed the expression of Pokemon protein in HepG2 cells compared to the negative control vector-transfected HCC cells. Pokemon knockdown also reduced HCC cell viability and enhanced cisplatin-induced apoptosis in HCC cells. AKT activation and the expression of various cell cycle-related genes were inhibited following Pokemon knockdown. These data demonstrate that Pokemon may play a role in HCC progression, suggesting that inhibition of Pokemon expression using Pokemon shRNA should be further evaluated as a novel target for the control of HCC. PMID:23924858

  17. Inhibition of g protein-activated inwardly rectifying k channels by phencyclidine.

    Kobayashi, Toru; Nishizawa, Daisuke; Ikeda, Kazutaka

    2011-03-01

    Addictive drugs, such as opioids, ethanol, cocaine, amphetamine, and phencyclidine (PCP), affect many functions of the nervous system and peripheral organs, resulting in severe health problems. G protein-activated inwardly rectifying K(+) (GIRK, Kir3) channels play an important role in regulating neuronal excitability through activation of various Gi/o protein-coupled receptors including opioid and CB(1) cannabinoid receptors. Furthermore, the channels are directly activated by ethanol and inhibited by cocaine at toxic levels, but not affected by methylphenidate, methamphetamine, and 3,4-methylenedioxymethamphetamine (MDMA) at toxic levels. The primary pharmacological action of PCP is blockade of N-methyl-D-aspartate (NMDA) receptor channels that are associated with its psychotomimetic effects. PCP also interacts with several receptors and channels at relatively high concentrations. However, the molecular mechanisms underlying the various effects of PCP remain to be clarified. Here, we investigated the effects of PCP on GIRK channels using the Xenopus oocyte expression system. PCP weakly but significantly inhibited GIRK channels at micromolar concentrations, but not Kir1.1 and Kir2.1 channels. The PCP concentrations effective in inhibiting GIRK channels overlap clinically relevant brain concentrations in severe intoxication. The results suggest that partial inhibition of GIRK channels by PCP may contribute to some of the toxic effects after overdose. PMID:21886598

  18. Transgenic Inhibition of Neuronal Protein Kinase A Activity Facilitates Fear Extinction

    Isiegas, Carolina; Park, Alice; Kandel, Eric R.; Abel, Ted; Lattal, K. Matthew

    2006-01-01

    Much is known about the neurobiology of memory storage for learned fear. In contrast, the molecular mechanisms underlying extinction of fear memory are just beginning to be delineated. Here, we investigate the role of protein kinase A (PKA) in extinction of memory for contextual fear by using conventional and temporally regulated transgenic approaches that allow us to inhibit PKA activity in neurons within brain regions thought to be involved in extinction. Strikingly, reduction of PKA activi...

  19. Silver nanoclusters-based fluorescence assay of protein kinase activity and inhibition.

    Shen, Congcong; Xia, Xiaodong; Hu, Shengqiang; Yang, Minghui; Wang, Jianxiu

    2015-01-01

    A simple and sensitive fluorescence method for monitoring the activity and inhibition of protein kinase (PKA) has been developed using polycytosine oligonucleotide (dC12)-templated silver nanoclusters (Ag NCs). Adenosine-5'-triphosphate (ATP) was found to enhance the fluorescence of Ag NCs, while the hydrolysis of ATP to adenosine diphosphate (ADP) by PKA decreased the fluorescence of Ag NCs. Compared to the existing methods for kinase activity assay, the developed method does not involve phosphorylation of the substrate peptides, which significantly simplifies the detection procedures. The method exhibits high sensitivity, good selectivity, and wide linear range toward PKA detection. The inhibition effect of kinase inhibitor H-89 on the activity of PKA was also studied. The sensing protocol was also applied to the assay of drug-stimulated activation of PKA in HeLa cell lysates. PMID:25517425

  20. Coumarins from Angelica decursiva inhibit α-glucosidase activity and protein tyrosine phosphatase 1B.

    Ali, Md Yousof; Jannat, Susoma; Jung, Hyun Ah; Jeong, Hyong Oh; Chung, Hae Young; Choi, Jae Sue

    2016-05-25

    In the present study, we investigated the anti-diabetic potential of six natural coumarins, 4-hydroxy Pd-C-III (1), 4'-methoxy Pd-C-I (2), decursinol (3), decursidin (4), umbelliferone 6-carboxylic acid (5), and 2'-isopropyl psoralene (6) isolated from Angelica decursiva and evaluated their inhibitory activities against protein tyrosine phosphatase 1B (PTP1B), α-glucosidase, and ONOO(-)-mediated protein tyrosine nitration. Coumarins 1-6 showed potent PTP1B and α-glucosidase inhibitory activities with ranges of IC50 values of 5.39-58.90 μM and 65.29-172.10 μM, respectively. In the kinetic study for PTP1B enzyme inhibition, compounds 1, 5, and 6 were competitive, whereas 2 and 4 showed mixed type, and 3 displayed noncompetitive type inhibition. For α-glucosidase enzyme inhibition, compounds 1 and 3 exhibited good mixed-type, while 2, 5, and 6 showed noncompetitive and 4 displayed competitive type inhibition. Furthermore, these coumarins also effectively suppressed ONOO(-)-mediated tyrosine nitration in a dose-dependent manner. To further investigate PTP1B inhibition, we generated a 3D structure of PTP1B using Autodock 4.2 and simulated the binding of compounds 1-6. Docking simulations showed that different residues of PTP1B interacted with different functional groups of compounds 1-6 through hydrogen and hydrophobic interactions. In addition, the binding energies of compounds 1-6 were negative, suggesting that hydrogen bonding may stabilize the open form of the enzyme and potentiate tight binding of the active site of PTP1B, thereby resulting in more effective PTP1B inhibition. These results demonstrate that the whole plant of A. decursiva and its coumarins are useful as potential functional food ingredients for the prevention and treatment of type 2 diabetes. PMID:27085377

  1. Major peptides from amaranth (Amaranthus cruentus) protein inhibit HMG-CoA reductase activity.

    Soares, Rosana Aparecida Manólio; Mendonça, Simone; de Castro, Luíla Ívini Andrade; Menezes, Amanda Caroline Cardoso Corrêa Carlos; Arêas, José Alfredo Gomes

    2015-01-01

    The objective of this study was to identify the major peptides generated by the in vitro hydrolysis of Amaranthus cruentus protein and to verify the effect of these peptides on the activity of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase), a key enzyme in cholesterol biosynthesis. A protein isolate was prepared, and an enzymatic hydrolysis that simulated the in vivo digestion of the protein was performed. After hydrolysis, the peptide mixture was filtered through a 3 kDa membrane. The peptide profile of this mixture was determined by reversed phase high performance chromatography (RP-HPLC), and the peptide identification was performed by LC-ESI MS/MS. Three major peptides under 3 kDa were detected, corresponding to more than 90% of the peptides of similar size produced by enzymatic hydrolysis. The sequences identified were GGV, IVG or LVG and VGVI or VGVL. These peptides had not yet been described for amaranth protein nor are they present in known sequences of amaranth grain protein, except LVG, which can be found in amaranth α‑amylase. Their ability to inhibit the activity of HMG-CoA reductase was determined, and we found that the sequences GGV, IVG, and VGVL, significantly inhibited this enzyme, suggesting a possible hypocholesterolemic effect. PMID:25690031

  2. Major Peptides from Amaranth (Amaranthus cruentus Protein Inhibit HMG-CoA Reductase Activity

    Rosana Aparecida Manólio Soares

    2015-02-01

    Full Text Available The objective of this study was to identify the major peptides generated by the in vitro hydrolysis of Amaranthus cruentus protein and to verify the effect of these peptides on the activity of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase, a key enzyme in cholesterol biosynthesis. A protein isolate was prepared, and an enzymatic hydrolysis that simulated the in vivo digestion of the protein was performed. After hydrolysis, the peptide mixture was filtered through a 3 kDa membrane. The peptide profile of this mixture was determined by reversed phase high performance chromatography (RP-HPLC, and the peptide identification was performed by LC-ESI MS/MS. Three major peptides under 3 kDa were detected, corresponding to more than 90% of the peptides of similar size produced by enzymatic hydrolysis. The sequences identified were GGV, IVG or LVG and VGVI or VGVL. These peptides had not yet been described for amaranth protein nor are they present in known sequences of amaranth grain protein, except LVG, which can be found in amaranth α‑amylase. Their ability to inhibit the activity of HMG-CoA reductase was determined, and we found that the sequences GGV, IVG, and VGVL, significantly inhibited this enzyme, suggesting a possible hypocholesterolemic effect.

  3. The Cytotoxicity of Elderberry Ribosome-Inactivating Proteins Is Not Solely Determined by Their Protein Translation Inhibition Activity.

    Chenjing Shang

    Full Text Available Although the protein translation inhibition activity of ribosome inactivating proteins (RIPs is well documented, little is known about the contribution of the lectin chain to the biological activity of these proteins. In this study, we compared the in vitro and intracellular activity of several S. nigra (elderberry RIPs and non-RIP lectins. Our data demonstrate that RIPs from elderberry are much more toxic to HeLa cells than to primary fibroblasts. Differences in the cytotoxicity between the elderberry proteins correlated with differences in glycan specificity of their lectin domain, cellular uptake efficiency and intracellular destination. Despite the fact that the bulk of the RIPs accumulated in the lysosomes and partly in the Golgi apparatus, we could demonstrate effective inhibition of protein synthesis in cellula. As we also observed cytotoxicity for non-RIP lectins, it is clear that the lectin chain triggers additional pathways heralding cell death. Our data suggest that one of these pathways involves the induction of autophagy.

  4. Inhibition of G protein-activated inwardly rectifying K+ channels by fluoxetine (Prozac)

    Kobayashi, Toru; Washiyama, Kazuo; Ikeda, Kazutaka

    2003-01-01

    The effects of fluoxetine, a commonly used antidepressant drug, on G protein-activated inwardly rectifying K+ channels (GIRK, Kir3) were investigated using Xenopus oocyte expression assays.In oocytes injected with mRNAs for GIRK1/GIRK2, GIRK2 or GIRK1/GIRK4 subunits, fluoxetine reversibly reduced inward currents through the basal GIRK activity. The inhibition by fluoxetine showed a concentration-dependence, a weak voltage-dependence and a slight time-dependence with a predominant effect on th...

  5. INHIBITION OF PROTEIN TYROSINE PHOSPHATASE ACTIVITY MEDIATES EPIDERMAL GROWTH FACTOR RECEPTOR SIGNALING IN HUMAN AIRWAY EPITHELIAL CELLS

    Epidemiological studies have implicated zinc in the toxicity of ambient particulate matter (PM) inhalation. We previously showed that exposure to metal-laden PM inhibits protein tyrosine phosphatase (PTP) activity in human primary bronchial epithelial cells (HAEC) and leads t...

  6. Protein-tyrosine phosphatase activity of Coxiella burnetii that inhibits human neutrophils

    Supernatants prepared from disrupted Coxiella burnetii posses acid phosphatase (ACP) activity that apparently accounts for the inhibition of the metabolic burst of formyl-Met-Leu-Phe(fMLP)-stimulated human neutrophils. Results are presented regarding purification and biochemical-biological characterization of the ACP. The highly purified enzyme, which exhibited an apparent M of 91 K and optimal activity at pH 5.0, also inhibited neutrophils. The enzyme retained full activity at pH 4.5, 5.5, and 7.4, when incubated overnight at 0 grad C and room temperature; at pH 5.5, it retained full activity after overnight incubation at 37 grad C. Apparently, the enzyme contains asparagine-linked but not serine- or threonine-liked glycan residues since its treatment with N-glycosidase F decreased its Mr to 87 K and no changes were detected with O-glycosidase. The enzyme's capacity to hydrolyze phosphate from a number of phosphate-containing compounds was examined; five phospho-compounds were significantly hydrolyzed: 5'-CMP > fructose 1,6-diphosphate > tyrosine phosphate > 3'-AMP >5'-AMP. The ACP also dephosphorylated 32P-Raytide, a phosphotyrosine-containing peptide. Dephosphorylation of Raytide was inhibited by the following phosphatase inhibitors: sodium molybdate, potassium fluoride, sodium ortho-vanadate and D2, a heteropolymolybdate compound. These results indicate that C.burnetii ACP may play a role in disrupting tyrosine phosphorylation/dephosphorylation reactions associated with the signal transduction pathway culminating in the metabolic burst. Interestingly, Western blot analysis of ACP-inhibited neutrophils showed a marked increase in tyrosine phosphorylation of a 44 K protein as compared to uninhibited cells. (author)

  7. Recrystallization inhibition in ice due to ice binding protein activity detected by nuclear magnetic resonance

    Jennifer R. Brown

    2014-09-01

    Full Text Available Liquid water present in polycrystalline ice at the interstices between ice crystals results in a network of liquid-filled veins and nodes within a solid ice matrix, making ice a low porosity porous media. Here we used nuclear magnetic resonance (NMR relaxation and time dependent self-diffusion measurements developed for porous media applications to monitor three dimensional changes to the vein network in ices with and without a bacterial ice binding protein (IBP. Shorter effective diffusion distances were detected as a function of increased irreversible ice binding activity, indicating inhibition of ice recrystallization and persistent small crystal structure. The modification of ice structure by the IBP demonstrates a potential mechanism for the microorganism to enhance survivability in ice. These results highlight the potential of NMR techniques in evaluation of the impact of IBPs on vein network structure and recrystallization processes; information useful for continued development of ice-interacting proteins for biotechnology applications.

  8. Qushi Huayu Decoction Inhibits Hepatic Lipid Accumulation by Activating AMP-Activated Protein Kinase In Vivo and In Vitro

    Qin Feng

    2013-01-01

    Full Text Available Qushi Huayu Decoction (QHD, a Chinese herbal formula, has been proven effective on alleviating nonalcoholic fatty liver disease (NAFLD in human and rats. The present study was conducted to investigate whether QHD could inhibit hepatic lipid accumulation by activating AMP-activated protein kinase (AMPK in vivo and in vitro. Nonalcoholic fatty liver (NAFL model was duplicated with high-fat diet in rats and with free fatty acid (FFA in L02 cells. In in vivo experimental condition, QHD significantly decreased the accumulation of fatty droplets in livers, lowered low-density lipoprotein cholesterol (LDL-c, alanine aminotransferase (ALT, and aspartate aminotransferase (AST levels in serum. Moreover, QHD supplementation reversed the HFD-induced decrease in the phosphorylation levels of AMPK and acetyl-CoA carboxylase (ACC and decreased hepatic nuclear protein expression of sterol regulatory element-binding protein-1 (SREBP-1 and carbohydrate-responsive element-binding protein (ChREBP in the liver. In in vitro, QHD-containing serum decreased the cellular TG content and alleviated the accumulation of fatty droplets in L02 cells. QHD supplementation reversed the FFA-induced decrease in the phosphorylation levels of AMPK and ACC and decreased the hepatic nuclear protein expression of SREBP-1 and ChREBP. Overall results suggest that QHD has significant effect on inhibiting hepatic lipid accumulation via AMPK pathway in vivo and in vitro.

  9. A cationic tetrapyrrole inhibits toxic activities of the cellular prion protein.

    Massignan, Tania; Cimini, Sara; Stincardini, Claudia; Cerovic, Milica; Vanni, Ilaria; Elezgarai, Saioa R; Moreno, Jorge; Stravalaci, Matteo; Negro, Alessandro; Sangiovanni, Valeria; Restelli, Elena; Riccardi, Geraldina; Gobbi, Marco; Castilla, Joaquín; Borsello, Tiziana; Nonno, Romolo; Biasini, Emiliano

    2016-01-01

    Prion diseases are rare neurodegenerative conditions associated with the conformational conversion of the cellular prion protein (PrP(C)) into PrP(Sc), a self-replicating isoform (prion) that accumulates in the central nervous system of affected individuals. The structure of PrP(Sc) is poorly defined, and likely to be heterogeneous, as suggested by the existence of different prion strains. The latter represents a relevant problem for therapy in prion diseases, as some potent anti-prion compounds have shown strain-specificity. Designing therapeutics that target PrP(C) may provide an opportunity to overcome these problems. PrP(C) ligands may theoretically inhibit the replication of multiple prion strains, by acting on the common substrate of any prion replication reaction. Here, we characterized the properties of a cationic tetrapyrrole [Fe(III)-TMPyP], which was previously shown to bind PrP(C), and inhibit the replication of a mouse prion strain. We report that the compound is active against multiple prion strains in vitro and in cells. Interestingly, we also find that Fe(III)-TMPyP inhibits several PrP(C)-related toxic activities, including the channel-forming ability of a PrP mutant, and the PrP(C)-dependent synaptotoxicity of amyloid-β (Aβ) oligomers, which are associated with Alzheimer's Disease. These results demonstrate that molecules binding to PrP(C) may produce a dual effect of blocking prion replication and inhibiting PrP(C)-mediated toxicity. PMID:26976106

  10. Inhibition of G protein-activated inwardly rectifying K+ channels by different classes of antidepressants.

    Toru Kobayashi

    Full Text Available Various antidepressants are commonly used for the treatment of depression and several other neuropsychiatric disorders. In addition to their primary effects on serotonergic or noradrenergic neurotransmitter systems, antidepressants have been shown to interact with several receptors and ion channels. However, the molecular mechanisms that underlie the effects of antidepressants have not yet been sufficiently clarified. G protein-activated inwardly rectifying K(+ (GIRK, Kir3 channels play an important role in regulating neuronal excitability and heart rate, and GIRK channel modulation has been suggested to have therapeutic potential for several neuropsychiatric disorders and cardiac arrhythmias. In the present study, we investigated the effects of various classes of antidepressants on GIRK channels using the Xenopus oocyte expression assay. In oocytes injected with mRNA for GIRK1/GIRK2 or GIRK1/GIRK4 subunits, extracellular application of sertraline, duloxetine, and amoxapine effectively reduced GIRK currents, whereas nefazodone, venlafaxine, mianserin, and mirtazapine weakly inhibited GIRK currents even at toxic levels. The inhibitory effects were concentration-dependent, with various degrees of potency and effectiveness. Furthermore, the effects of sertraline were voltage-independent and time-independent during each voltage pulse, whereas the effects of duloxetine were voltage-dependent with weaker inhibition with negative membrane potentials and time-dependent with a gradual decrease in each voltage pulse. However, Kir2.1 channels were insensitive to all of the drugs. Moreover, the GIRK currents induced by ethanol were inhibited by sertraline but not by intracellularly applied sertraline. The present results suggest that GIRK channel inhibition may reveal a novel characteristic of the commonly used antidepressants, particularly sertraline, and contributes to some of the therapeutic effects and adverse effects.

  11. Activated protein C inhibits neutrophil migration in allergic asthma: a randomised trial.

    de Boer, J Daan; Berger, Marieke; Majoor, Christof J; Kager, Liesbeth M; Meijers, Joost C M; Terpstra, Sanne; Nieuwland, Rienk; Boing, Anita N; Lutter, René; Wouters, Diana; van Mierlo, Gerard J; Zeerleder, Sacha S; Bel, Elisabeth H; van't Veer, Cornelis; de Vos, Alex F; van der Zee, Jaring S; van der Poll, Tom

    2015-12-01

    Asthma patients show evidence of a procoagulant state in their airways, accompanied by an impaired function of the anticoagulant protein C system. We aimed to study the effect of recombinant human activated protein C (rhAPC) in allergic asthma patients.We conducted a randomised, double-blind, placebo-controlled, proof-of-concept study in house dust mite (HDM) allergic asthma patients. Patients were randomised to receive intravenous rhAPC (24 µg·kg(-1)·h(-1); n=12) or placebo (n=12) for 11 h. 4 h after the start of infusion, a first bronchoscopy was performed to challenge one lung segment with saline (control) and a contralateral segment with a combination of HDM extract and lipopolysaccharide (HDM+LPS), thereby mimicking environmental house dust exposure. A second bronchoscopy was conducted 8 h after intrabronchial challenge to obtain bronchoalveolar lavage fluid (BALF).rhAPC did not influence HDM+LPS induced procoagulant changes in the lung. In contrast, rhAPC reduced BALF leukocyte counts by 43% relative to placebo, caused by an inhibitory effect on neutrophil influx (64% reduction), while leaving eosinophil influx unaltered. rhAPC also reduced neutrophil degranulation products in the airways.Intravenous rhAPC attenuates HDM+LPS-induced neutrophil migration and protein release in allergic asthma patients by an effect that does not rely on coagulation inhibition. PMID:26381519

  12. GABA/sub B/ receptor activation inhibits Ca2+-activated potassium channels in synaptosomes: involvement of G-proteins

    86Rb-efflux assay from preloaded synaptosomes of rat cerebral cortex was developed to study the effect of GABA/sub B/ receptor agonist baclofen on Ca2+-activated K+-channels. Depolarization of 86Rb-loaded synaptosomes in physiological buffer increased Ca2+-activated 86Rb-efflux by 400%. The 86Rb-efflux was blocked by quinine sulfate, tetraethylammonium, and La3+ indicating the involvement of Ca2+-activated K+-channels. (-)Baclofen inhibited Ca2+-activated 86Rb-efflux in a stereospecific manner. The inhibitory effect of (-)baclofen was mediated by GABA/sub B/ receptor activation, since it was blocked by GABA/sub B/ antagonist phaclofen, but not by bicuculline. Further, pertussis toxin also blocked the ability of baclofen or depolarizing action to affect Ca2+-activated K+-channels. These results suggest that baclofen inhibits Ca2+-activated K+-channels in synaptosomes and these channels are regulated by G-proteins. This assay may provide an ideal in vitro model to study GABA/sub B/ receptor pharmacology

  13. Betulinic acid selectively increases protein degradation and enhances prostate cancer-specific apoptosis: possible role for inhibition of deubiquitinase activity.

    Teresita Reiner

    Full Text Available Inhibition of the ubiquitin-proteasome system (UPS of protein degradation is a valid anti-cancer strategy and has led to the approval of bortezomib for the treatment of multiple myeloma. However, the alternative approach of enhancing the degradation of oncoproteins that are frequently overexpressed in cancers is less developed. Betulinic acid (BA is a plant-derived small molecule that can increase apoptosis specifically in cancer but not in normal cells, making it an attractive anti-cancer agent. Our results in prostate cancer suggested that BA inhibited multiple deubiquitinases (DUBs, which resulted in the accumulation of poly-ubiquitinated proteins, decreased levels of oncoproteins, and increased apoptotic cell death. In normal fibroblasts, however, BA did not inhibit DUB activity nor increased total poly-ubiquitinated proteins, which was associated with a lack of effect on cell death. In the TRAMP transgenic mouse model of prostate cancer, treatment with BA (10 mg/kg inhibited primary tumors, increased apoptosis, decreased angiogenesis and proliferation, and lowered androgen receptor and cyclin D1 protein. BA treatment also inhibited DUB activity and increased ubiquitinated proteins in TRAMP prostate cancer but had no effect on apoptosis or ubiquitination in normal mouse tissues. Overall, our data suggests that BA-mediated inhibition of DUBs and induction of apoptotic cell death specifically in prostate cancer but not in normal cells and tissues may provide an effective non-toxic and clinically selective agent for chemotherapy.

  14. Atheroprotective effects of antioxidants through inhibition of mitogen-activated protein kinases

    Moe KYAW; Masanori YOSHIZUMI; Koichiro TSUCHIYA; Yuki IZAWA; Yasuhisa KANEMATSU; Toshiaki TAMAKI

    2004-01-01

    Reactive oxygen species (ROS) have been known to play an important role in the pathogenesis of atherosclerosis and several other cardiovascular diseases. It is now apparent that ROS induce endothelial cell damage and vascular smooth muscle cell (VSMC) growth and cardiac remodeling, which are associated with hypertension,atherosclerosis, heart failure, and restenosis. Several lines of evidence have indicated that ROS and mitogenactivated protein (MAP) kinases were involved in vascular remodeling under various pathological conditions. Recenfiy,it was also reported that MAP kinases were sensitive to oxidative stress. MAP kinases play an important role in cell differentiation, growth, apoptosis, and the regulation of a variety of transcription factors and gene expressions.Bioflavonoids and polyphenolic compounds are believed to be beneficial for the prevention and treatment of atherosclerosis and cardiovascular diseases. One of the most widely distributed bioflavonoids, 3,3',4',5,7-pentahydroxyflavone (quercetin) and its metabolite quercetin 3-O-β-D-glucuronide (Q3GA) inhibited Angiotensin Ⅱstimulated JNK activation and resultant hypertrophy of VSMC. Several studies have suggested that various antioxidants including probucol, N-acetyl-L-cysteine, diphenylene iodonium, Trolox C (vitamin E analogue), and vitamin C inhibit VSMC growth, which is associated with pathogenesis of cardiovascular diseases. Therefore, inhibition of MAP kinases by antioxidant treatment may prove to be a therapeutic strategy for cardiovascular diseases. In contrast, some clinical studies have reported that antioxidant vitamins did not show beneficial effects in coronary artery disease or in a number of high-risk people. Thus, further studies are needed to clarify why antioxidants showed beneficial effects in vitro, whereas less satisfactory results were obtained in some clinical conditions.

  15. How anacetrapib inhibits the activity of the cholesteryl ester transfer protein? Perspective through atomistic simulations.

    Tarja Äijänen

    2014-11-01

    Full Text Available Cholesteryl ester transfer protein (CETP mediates the reciprocal transfer of neutral lipids (cholesteryl esters, triglycerides and phospholipids between different lipoprotein fractions in human blood plasma. A novel molecular agent known as anacetrapib has been shown to inhibit CETP activity and thereby raise high density lipoprotein (HDL-cholesterol and decrease low density lipoprotein (LDL-cholesterol, thus rendering CETP inhibition an attractive target to prevent and treat the development of various cardiovascular diseases. Our objective in this work is to use atomistic molecular dynamics simulations to shed light on the inhibitory mechanism of anacetrapib and unlock the interactions between the drug and CETP. The results show an evident affinity of anacetrapib towards the concave surface of CETP, and especially towards the region of the N-terminal tunnel opening. The primary binding site of anacetrapib turns out to reside in the tunnel inside CETP, near the residues surrounding the N-terminal opening. Free energy calculations show that when anacetrapib resides in this area, it hinders the ability of cholesteryl ester to diffuse out from CETP. The simulations further bring out the ability of anacetrapib to regulate the structure-function relationships of phospholipids and helix X, the latter representing the structural region of CETP important to the process of neutral lipid exchange with lipoproteins. Altogether, the simulations propose CETP inhibition to be realized when anacetrapib is transferred into the lipid binding pocket. The novel insight gained in this study has potential use in the development of new molecular agents capable of preventing the progression of cardiovascular diseases.

  16. Immunomodulatory effects of therapeutic gold compounds. Gold sodium thiomalate inhibits the activity of T cell protein kinase C.

    Hashimoto, K; Whitehurst, C. E.; Matsubara, T.; Hirohata, K; Lipsky, P E

    1992-01-01

    Previous studies have shown that the gold compounds, gold sodium thiomalate (GST) and auranofin (AUR), which are effective in the treatment of rheumatoid arthritis, inhibit functional activities of a variety of cells, but the biochemical basis of their effect is unknown. In the current studies, human T cell proliferation and interleukin 2 production by Jurkat cells were inhibited by GST or AUR at pharmacologically relevant concentrations. Because it has been documented that protein kinase C (...

  17. Phosphorylation of Human Choline Kinase Beta by Protein Kinase A: Its Impact on Activity and Inhibition

    Chang, Ching Ching; Few, Ling Ling; Konrad, Manfred; See Too, Wei Cun

    2016-01-01

    Choline kinase beta (CKβ) is one of the CK isozymes involved in the biosynthesis of phosphatidylcholine. CKβ is important for normal mitochondrial function and muscle development as the lack of the ckβ gene in human and mice results in the development of muscular dystrophy. In contrast, CKα is implicated in tumorigenesis and has been extensively studied as an anticancer target. Phosphorylation of human CKα was found to regulate the enzyme’s activity and its subcellular location. This study provides evidence for CKβ phosphorylation by protein kinase A (PKA). In vitro phosphorylation of CKβ by PKA was first detected by phosphoprotein staining, as well as by in-gel kinase assays. The phosphorylating kinase was identified as PKA by Western blotting. CKβ phosphorylation by MCF-7 cell lysate was inhibited by a PKA-specific inhibitor peptide, and the intracellular phosphorylation of CKβ was shown to be regulated by the level of cyclic adenosine monophosphate (cAMP), a PKA activator. Phosphorylation sites were located on CKβ residues serine-39 and serine-40 as determined by mass spectrometry and site-directed mutagenesis. Phosphorylation increased the catalytic efficiencies for the substrates choline and ATP about 2-fold, without affecting ethanolamine phosphorylation, and the S39D/S40D CKβ phosphorylation mimic behaved kinetically very similar. Remarkably, phosphorylation drastically increased the sensitivity of CKβ to hemicholinium-3 (HC-3) inhibition by about 30-fold. These findings suggest that CKβ, in concert with CKα, and depending on its phosphorylation status, might play a critical role as a druggable target in carcinogenesis. PMID:27149373

  18. Regulator of G protein signaling 8 inhibits protease-activated receptor 1/Gi/o signaling by forming a distinct G protein-dependent complex in live cells.

    Lee, Jinyong; Ghil, Sungho

    2016-05-01

    Activation of seven-transmembrane-domain-possessing G protein-coupled receptors (GPCRs) by extracellular stimuli elicits intracellular responses. One class of GPCRs-protease-activated receptors (PARs)-is activated by endogenous proteases, such as thrombin and trypsin. Members of the regulator of G protein signaling (RGS) family stimulate GTP hydrolysis of G protein alpha (Gα) subunits, thereby inhibiting GPCR/Gα-mediated signaling. We previously reported that RGS2 and RGS4 inhibit PAR1/Gα-mediated signaling by interacting with PAR1 in a Gα-dependent manner. Here, employing the bioluminescence resonance energy transfer (BRET) technique, we identified RGS8 as a novel PAR1-interacting protein. Very little BRET activity was observed between PAR1-Venus (PAR1-Ven) and RGS8-Luciferase (RGS8-Luc) in the absence of Gα. However, in the presence of Gαo, BRET activity was specifically and significantly increased. This interaction was confirmed by biochemical and immunofluorescence assays. Notably, RGS8 inhibited PAR1/Gαi/o-mediated adenylyl cyclase and ERK activation, and prevented Gαo-induced neurite outgrowth and activation of Necdin protein, a downstream target of Gαo. Our findings suggest a novel function of RGS8 and reveal cellular mechanisms by which RGS8 mediates PAR1 inhibition. PMID:26829215

  19. Inhibition of G Protein-Activated Inwardly Rectifying K+ Channels by Phencyclidine

    Kobayashi, Toru; Nishizawa, Daisuke; Ikeda, Kazutaka

    2011-01-01

    Addictive drugs, such as opioids, ethanol, cocaine, amphetamine, and phencyclidine (PCP), affect many functions of the nervous system and peripheral organs, resulting in severe health problems. G protein-activated inwardly rectifying K+ (GIRK, Kir3) channels play an important role in regulating neuronal excitability through activation of various Gi/o protein-coupled receptors including opioid and CB1 cannabinoid receptors. Furthermore, the channels are directly activated by ethanol and inhibi...

  20. L-Alanylglutamine inhibits signaling proteins that activate protein degradation, but does not affect proteins that activate protein synthesis after an acute resistance exercise.

    Wang, Wanyi; Choi, Ran Hee; Solares, Geoffrey J; Tseng, Hung-Min; Ding, Zhenping; Kim, Kyoungrae; Ivy, John L

    2015-07-01

    Sustamine™ (SUS) is a dipeptide composed of alanine and glutamine (AlaGln). Glutamine has been suggested to increase muscle protein accretion; however, the underlying molecular mechanisms of glutamine on muscle protein metabolism following resistance exercise have not been fully addressed. In the present study, 2-month-old rats climbed a ladder 10 times with a weight equal to 75 % of their body mass attached at the tail. Rats were then orally administered one of four solutions: placebo (PLA-glycine = 0.52 g/kg), whey protein (WP = 0.4 g/kg), low dose of SUS (LSUS = 0.1 g/kg), or high dose of SUS (HSUS = 0.5 g/kg). An additional group of sedentary (SED) rats was intubated with glycine (0.52 g/kg) at the same time as the ladder-climbing rats. Blood samples were collected immediately after exercise and at either 20 or 40 min after recovery. The flexor hallucis longus (FHL), a muscle used for climbing, was excised at 20 or 40 min post exercise and analyzed for proteins regulating protein synthesis and degradation. All supplements elevated the phosphorylation of FOXO3A above SED at 20 min post exercise, but only the SUS supplements significantly reduced the phosphorylation of AMPK and NF-kB p65. SUS supplements had no effect on mTOR signaling, but WP supplementation yielded a greater phosphorylation of mTOR, p70S6k, and rpS6 compared with PLA at 20 min post exercise. However, by 40 min post exercise, phosphorylation of mTOR and rpS6 in PLA had risen to levels not different than WP. These results suggest that SUS blocks the activation of intracellular signals for MPB, whereas WP accelerates mRNA translation. PMID:25837301

  1. Phosphorylation and inhibition of. gamma. -glutamyl transferase activity by cAMP-dependent protein kinase

    Kolesnichenko, L.S.; Chernov, N.N.

    1986-10-20

    It was shown that preparations of bovine kidney ..gamma..-glutamyl transferase of differing degrees of purity are phosphorylated by cAMP-dependent protein kinase. This is accompanied by a decrease in both the transferase and hydrolase activities of the enzyme. Consequently, ..gamma..-glutamyl transferase may serve as the substrate and target of the regulation of cAMP-dependent protein kinase.

  2. Platelet protein S directly inhibits procoagulant activity on platelets and microparticles

    Stavenuiter, Fabian; Davis, Nicole F.; Duan, Erning; Gale, Andrew J.; Heeb, Mary J.

    2012-01-01

    Anticoagulant plasma protein S (PS) is essential for maintaining hemostatic balance. About 2.5% of PS is stored in platelets and released upon platelet stimulation. So far, little is known about the functionality and importance of platelet (plt)PS. A platelet-associated protease cleaves plasma-derived (pd)PS and pltPS in the “thrombin-sensitive region”, abolishing activated protein C (APC) cofactor activity. However we showed that cleaved PS retains APC-independent anticoagulant activities (“...

  3. Protein-tyrosine phosphatase activity regulates osteoclast formation and function: inhibition by alendronate.

    Schmidt, A.; Rutledge, S J; Endo, N; Opas, E E; Tanaka, H; Wesolowski, G.; Leu, C T; Huang, Z; Ramachandaran, C; Rodan, S B; Rodan, G A

    1996-01-01

    Alendronate (ALN), an aminobisphosphonate used in the treatment of osteoporosis, is a potent inhibitor of bone resorption. Its molecular target is still unknown. This study examines the effects of ALN on the activity of osteoclast protein-tyrosine phosphatase (PTP; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48), called PTPepsilon. Using osteoclast-like cells generated by coculturing mouse bone marrow cells with mouse calvaria osteoblasts, we found by molecular cloning and RNA blot ...

  4. The HIV Protease Inhibitor Nelfinavir Downregulates Akt Phosphorylation by Inhibiting Proteasomal Activity and Inducing the Unfolded Protein Response

    Anjali K. Gupta

    2007-04-01

    Full Text Available HIV protease inhibitors (HPIs, which have been used to treat HIV patients since the mid 1990s, have been shown to downregulate the phosphatidylinositol 3kinase (PI3K-Akt pathway. Because this pathway is frequently activated in human malignancies and associated with resistance to ionizing radiation, we investigated and confirmed that HPIs could radiosensitize cells. However, the mechanism underlying this downregulation was unclear, prompting the investigations in this report. In this paper we show that nelfinavir inhibits proteasome activity. Inhibition of the proteasome leads to endoplasmic reticulum-based stress with accumulation of misfolded proteins, which triggers the unfolded protein response (UPR. As part of the UPR, the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α is phosphorylated, resulting in a decrease in global protein synthesis and induction of the feedback regulator growth arrest and DNA damageinducible protein (GADD34, which acts as a phosphatase in complex with protein phosphatase 1. This complex dephosphorylates eIF2α; however, our data also suggest that this phosphatase activity can dephosphorylate Akt. Furthermore, our data indicate that nelfinavir decreases Akt phosphorylation by triggering this response. These findings may have important implications in understanding how nelfinavir may increase radiation sensitivity and also result in downregulation of the PI3K/Akt pathway.

  5. Flavone inhibits nitric oxide synthase (NOS) activity, nitric oxide production and protein S-nitrosylation in breast cancer cells

    Zhu, Wenzhen; Yang, Bingwu; Fu, Huiling; Ma, Long; Liu, Tingting; Chai, Rongfei; Zheng, Zhaodi [Shandong Provincial Key Laboratory of Animal Resistant Biology, School of Life Sciences, Shandong Normal University, Jinan 250014 (China); Zhang, Qunye, E-mail: wz.zhangqy@sdu.edu.cn [Key Laboratory of Cardiovascular Remodeling and Function Research Chinese Ministry of Education and Ministry of Public Health, Qilu Hospital, Shandong University, Jinan, Shandong (China); Li, Guorong, E-mail: grli@sdnu.edu.cn [Shandong Provincial Key Laboratory of Animal Resistant Biology, School of Life Sciences, Shandong Normal University, Jinan 250014 (China)

    2015-03-13

    As the core structure of flavonoids, flavone has been proved to possess anticancer effects. Flavone's growth inhibitory functions are related to NO. NO is synthesized by nitric oxide synthase (NOS), and generally increased in a variety of cancer cells. NO regulates multiple cellular responses by S-nitrosylation. In this study, we explored flavone-induced regulations on nitric oxide (NO)-related cellular processes in breast cancer cells. Our results showed that, flavone suppresses breast cancer cell proliferation and induces apoptosis. Flavone restrains NO synthesis by does-dependent inhibiting NOS enzymatic activity. The decrease of NO generation was detected by fluorescence microscopy and flow cytometry. Flavone-induced inhibitory effect on NOS activity is dependent on intact cell structure. For the NO-induced protein modification, flavone treatment significantly down-regulated protein S-nitrosylation, which was detected by “Biotin-switch” method. The present study provides a novel, NO-related mechanism for the anticancer function of flavone. - Highlights: • Flavone inhibits proliferation and induces apoptosis in MCF-7 cells. • Flavone decreases nitric oxide production by inhibiting NOS enzymatic activity in breast cancer cells. • Flavone down-regulates protein S-nitrosylation.

  6. Flavone inhibits nitric oxide synthase (NOS) activity, nitric oxide production and protein S-nitrosylation in breast cancer cells

    As the core structure of flavonoids, flavone has been proved to possess anticancer effects. Flavone's growth inhibitory functions are related to NO. NO is synthesized by nitric oxide synthase (NOS), and generally increased in a variety of cancer cells. NO regulates multiple cellular responses by S-nitrosylation. In this study, we explored flavone-induced regulations on nitric oxide (NO)-related cellular processes in breast cancer cells. Our results showed that, flavone suppresses breast cancer cell proliferation and induces apoptosis. Flavone restrains NO synthesis by does-dependent inhibiting NOS enzymatic activity. The decrease of NO generation was detected by fluorescence microscopy and flow cytometry. Flavone-induced inhibitory effect on NOS activity is dependent on intact cell structure. For the NO-induced protein modification, flavone treatment significantly down-regulated protein S-nitrosylation, which was detected by “Biotin-switch” method. The present study provides a novel, NO-related mechanism for the anticancer function of flavone. - Highlights: • Flavone inhibits proliferation and induces apoptosis in MCF-7 cells. • Flavone decreases nitric oxide production by inhibiting NOS enzymatic activity in breast cancer cells. • Flavone down-regulates protein S-nitrosylation

  7. Mutant Forms of the Azotobacter vinelandii Transcriptional Activator NifA Resistant to Inhibition by the NifL Regulatory Protein

    Reyes-Ramirez, Francisca; Little, Richard; Dixon, Ray

    2002-01-01

    The Azotobacter vinelandii σ54-dependent transcriptional activator protein NifA is regulated by the NifL protein in response to redox, carbon, and nitrogen status. Under conditions inappropriate for nitrogen fixation, NifL inhibits transcription activation by NifA through the formation of the NifL-NifA protein complex. NifL inhibits the ATPase activity of the central AAA+ domain of NifA required to drive open complex formation by σ54-RNA polymerase and may also inhibit the activator-polymeras...

  8. Farnesoid X Receptor Inhibits the Transcriptional Activity of Carbohydrate Response Element Binding Protein in Human Hepatocytes

    Caron, Sandrine; Huaman Samanez, Carolina; Dehondt, Hélène; Ploton, Maheul; Briand, Olivier; Lien, Fleur; Dorchies, Emilie; Dumont, Julie; Postic, Catherine; Cariou, Bertrand; Lefebvre, Philippe; Staels, Bart

    2013-01-01

    The glucose-activated transcription factor carbohydrate response element binding protein (ChREBP) induces the expression of hepatic glycolytic and lipogenic genes. The farnesoid X receptor (FXR) is a nuclear bile acid receptor controlling bile acid, lipid, and glucose homeostasis. FXR negatively regulates hepatic glycolysis and lipogenesis in mouse liver. The aim of this study was to determine whether FXR regulates the transcriptional activity of ChREBP in human hepatocytes and to unravel the...

  9. Inhibition of protein kinase B activity induces cell cycle arrest and apoptosis during early G₁ phase in CHO cells.

    van Opstal, Angélique; Bijvelt, José; van Donselaar, Elly; Humbel, Bruno M; Boonstra, Johannes

    2012-04-01

    Inhibition of PKB (protein kinase B) activity using a highly selective PKB inhibitor resulted in inhibition of cell cycle progression only if cells were in early G1 phase at the time of addition of the inhibitor, as demonstrated by time-lapse cinematography. Addition of the inhibitor during mitosis up to 2 h after mitosis resulted in arrest of the cells in early G1 phase, as deduced from the expression of cyclins D and A and incorporation of thymidine. After 24 h of cell cycle arrest, cells expressed the cleaved caspase-3, a central mediator of apoptosis. These results demonstrate that PKB activity in early G1 phase is required to prevent the induction of apoptosis. Using antibodies, it was demonstrated that active PKB translocates to the nucleus during early G1 phase, while an even distribution of PKB was observed through cytoplasm and nucleus during the end of G1 phase. PMID:22251027

  10. Structural Basis of Response Regulator Inhibition by a Bacterial Anti-Activator Protein

    Melinda D Baker; Neiditch, Matthew B.

    2011-01-01

    The complex interplay between the response regulator ComA, the anti-activator RapF, and the signaling peptide PhrF controls competence development in Bacillus subtilis. More specifically, ComA drives the expression of genetic competence genes, while RapF inhibits the interaction of ComA with its target promoters. The signaling peptide PhrF accumulates at high cell density and upregulates genetic competence by antagonizing the interaction of RapF and ComA. How RapF functions mechanistically to...

  11. Tributyltin-binding protein type 1, a lipocalin, prevents inhibition of osteoblastic activity by tributyltin in fish scales.

    Satone, Hina; Lee, Jae Man; Oba, Yumi; Kusakabe, Takahiro; Akahoshi, Eriko; Miki, Shizuho; Suzuki, Nobuo; Sasayama, Yuichi; Nassef, Mohamed; Shimasaki, Yohei; Kawabata, Shun-Ichiro; Honjo, Tsuneo; Oshima, Yuji

    2011-05-01

    Tributyltin-binding protein type 1 (TBT-bp1) is a member of the lipocalin family of proteins which bind to small hydrophobic molecules. In this study, we expressed a recombinant TBT-bp1 (rTBT-bp1, ca. 35kDa) in a baculovirus expression system and purified the protein from the hemolymph of silkworm larvae injected with recombinant baculovirus. After incubation of a mixture of rTBT-bp1 and TBT and its fractionation by means of gel filtration chromatography, TBT was detected in the elution peak of rTBT-bp1, confirming the binding potential of rTBT-bp1 for TBT. An assay of the ability of rTBT-bp1 or native TBT-bp1 (nTBT-bp1) to restore osteoblastic activity inhibited by TBT showed that co-treatment of the scales with rTBT-bp1 or nTBT-bp1 in combination with TBT restored osteoblastic activity in goldfish scales, whereas treatment with TBT alone significantly inhibited osteoblastic activity. These results suggest that TBT-bp1 as a lipocalin member might function to decrease the toxicity of TBT by binding to TBT. PMID:21396342

  12. Activation of the Metabolic Sensor AMP-Activated Protein Kinase Inhibits Aquaporin-2 Function in Kidney Principal Cells

    Al-Bataineh, Mohammad M; Li, Hui; Ohmi, Kazuhiro;

    2016-01-01

    prevent AQP2 apical accumulation in response to the AVP analog desmopressin (dDAVP). Prolonged AMPK activation prevented AQP2 cell membrane accumulation in response to forskolin in mouse collecting duct mpkCCDc14 cells. Moreover, AMPK inhibition accelerated hypotonic lysis of Xenopus oocytes expressing...

  13. Mitochondrial thiol modification by a targeted electrophile inhibits metabolism in breast adenocarcinoma cells by inhibiting enzyme activity and protein levels

    M. Ryan Smith; Vayalil, Praveen K.; Fen Zhou; Benavides, Gloria A; Beggs, Reena R.; Hafez Golzarian; Bhavitavya Nijampatnam; Oliver, Patsy G.; Smith, Robin A.J.; Murphy, Michael P.; Velu, Sadanandan E.; Aimee Landar

    2016-01-01

    Many cancer cells follow an aberrant metabolic program to maintain energy for rapid cell proliferation. Metabolic reprogramming often involves the upregulation of glutaminolysis to generate reducing equivalents for the electron transport chain and amino acids for protein synthesis. Critical enzymes involved in metabolism possess a reactive thiolate group, which can be modified by certain oxidants. In the current study, we show that modification of mitochondrial protein thiols by a model compo...

  14. [The effect of physiologically active compounds on the production of ethylene and the activity of polygalacturonase inhibiting protein in fruits].

    Bulantseva, E A; Protsenko, M A; Toropkina, A S; Korableva, N P

    2011-01-01

    The treatment of apple and banana fruits with 2-CEFA and ethacyde induced the production of ethylene and accelerated the ripening and accumulation of ACC in apple fruits. Inhibitors AOA, AVG, and CoCl2 acted at the different steps of ethylene biosynthesis, inhibited the physiological aging process and increased storage longevity. Treatment with astaxantine and BOA delayed the pick of ethylene production by fruits. The content of PGIP was correlated with intensity of ethylene production. The infection of fruits with phytopathogenic microorganisms lowered as the result of the inhibition of pathogen PG. The dynamics of PGIP activity in fruits suggests its important role in the processes of ripening. PMID:22808745

  15. Inhibition of hepatic phosphatidylcholine synthesis by 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside is independent of AMP-activated protein kinase activation.

    Jacobs, René L; Lingrell, Susanne; Dyck, Jason R B; Vance, Dennis E

    2007-02-16

    5-Aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAr), a commonly used indirect activator of AMP-activated protein kinase (AMPK), inhibits phosphatidylcholine (PC) biosynthesis in freshly isolated hepatocytes. In all nucleated mammalian cells, PC is synthesized from choline via the Kennedy (CDP-choline) pathway. The purpose of our study was to provide direct evidence that AMPK regulates phospholipid biosynthesis and to elucidate the mechanism(s) by which AMPK inhibits hepatic PC synthesis. Incubations of hepatocytes with AICAr resulted in a dose-dependent activation of AMPK and inhibition of PC biosynthesis. Surprisingly, adenoviral delivery of constitutively active AMPK did not alter PC biosynthesis. In addition, expression of dominant negative mutants of AMPK was unable to block the AICAr-dependent inhibition of PC biosynthesis, indicating that AICAr was acting independently of AMPK activation. Determination of aqueous intermediates of the CDP-choline pathway indicated that choline kinase, the first enzyme in the pathway, was inhibited by AICAr administration. Flux through the CDP-choline pathway was directly correlated to the level of intracellular ATP concentrations. Therefore, it is possible that inhibition of PC biosynthesis is another process by which the cell can reduce ATP consumption in times of energetic stress. However, unlike cholesterol and triacylglycerol biosynthesis, PC production is not regulated by AMPK. PMID:17179149

  16. Proteolytic Inhibition of Salmonella enterica Serovar Typhimurium-Induced Activation of the Mitogen-Activated Protein Kinases ERK and JNK in Cultured Human Intestinal Cells

    Mynott, Tracey L.; Crossett, Ben; Prathalingam, S. Radhika

    2002-01-01

    Bromelain, a mixture of cysteine proteases from pineapple stems, blocks signaling by the mitogen-activated protein (MAP) kinases extracellular regulated kinase 1 (ERK-1) and ERK-2, inhibits inflammation, and protects against enterotoxigenic Escherichia coli infection. In this study, we examined the effect of bromelain on Salmonella enterica serovar Typhimurium infection, since an important feature of its pathogenesis is its ability to induce activation of ERK-1 and ERK-2, which leads to inter...

  17. Decreased activity of neutrophils in the presence of diferuloylmethane (curcumin) involves protein kinase C inhibition.

    Jancinová, Viera; Perecko, Tomás; Nosál, Radomír; Kostálová, Daniela; Bauerová, Katarína; Drábiková, Katarína

    2009-06-10

    Diferuloylmethane (curcumin) has been shown to act beneficially in arthritis, particularly through downregulated expression of proinflammatory cytokines and collagenase as well as through the modulated activities of T lymphocytes and macrophages. In this study its impact on activated neutrophils was investigated both in vitro and in experimental arthritis. Formation of reactive oxygen species in neutrophils was recorded on the basis of luminol- or isoluminol-enhanced chemiluminescence. Phosphorylation of neutrophil protein kinases C alpha and beta II was assessed by Western blotting, using phosphospecific antibodies. Adjuvant arthritis was induced in Lewis rats by heat-killed Mycobacterium butyricum. Diferuloylmethane or methotrexate was administered over a period of 28 days after arthritis induction. Under in vitro conditions, diferuloylmethane (1-100 microM) reduced dose-dependently oxidant formation both at extra- and intracellular level and it effectively reduced protein kinase C activation. Adjuvant arthritis was accompanied by an increased number of neutrophils in blood and by a more pronounced spontaneous as well as PMA (phorbol myristate acetate) stimulated chemiluminescence. Whereas the arthritis-related alterations in neutrophil count and in spontaneous chemiluminescence were not modified by diferuloylmethane, the increased reactivity of neutrophils to PMA was less evident in diferuloylmethane-treated animals. The effects of diferuloylmethane were comparable with those of methotrexate. Diferuloylmethane was found to be a potent inhibitor of neutrophil functions both in vitro and in experimental arthritis. As neutrophils are considered to be cells with the greatest capacity to inflict damage within diseased joints, the observed effects could represent a further mechanism involved in the antirheumatic activity of diferuloylmethane. PMID:19371737

  18. The G Protein-Coupled Estrogen Receptor Agonist G-1 Inhibits Nuclear Estrogen Receptor Activity and Stimulates Novel Phosphoproteomic Signatures.

    Smith, L Cody; Ralston-Hooper, Kimberly J; Ferguson, P Lee; Sabo-Attwood, Tara

    2016-06-01

    Estrogen exerts cellular effects through both nuclear (ESR1 and ESR2) and membrane-bound estrogen receptors (G-protein coupled estrogen receptor, GPER); however, it is unclear if they act independently or engage in crosstalk to influence hormonal responses. To investigate each receptor's role in proliferation, transcriptional activation, and protein phosphorylation in breast cancer cells (MCF-7), we employed selective agonists for ESR1 propyl-pyrazole-triol (PPT), ESR2 diarylpropionitrile (DPN), and GPER (G-1) and also determined the impact of xenoestrogens bisphenol-A (BPA) and genistein on these effects. As anticipated, 17β-estradiol (E2), PPT, DPN, BPA, and genistein each enhanced proliferation and activation of an ERE-driven reporter gene whereas G-1 had no significant impact. However, G-1 significantly reduced E2-, PPT-, DPN-, BPA-, and genistein-induced proliferation and ERE activation at doses greater than 500 nM indicating that G-1 mediated inhibition is not ESR isotype specific. As membrane receptors initiate cascades of phosphorylation events, we performed a global phosphoproteomic analysis on cells exposed to E2 or G-1 to identify potential targets of receptor crosstalk via downstream protein phosphorylation targets. Of the 211 phosphorylated proteins identified, 40 and 13 phosphoproteins were specifically modified by E2 and G-1, respectively. Subnetwork enrichment analysis revealed several processes related to cell cycle were specifically enriched by G-1 compared with E2. Further there existed a number of newly identified proteins that were specifically phosphorylated by G-1. These phosphorylation networks highlight specific proteins that may modulate the inhibitory effects of G-1 and suggest a novel role for interference with nuclear receptor activity driven by E2 and xenoestrogens. PMID:27026707

  19. Suramin inhibits helicase activity of NS3 protein of dengue virus in a fluorescence-based high throughput assay format.

    Basavannacharya, Chandrakala; Vasudevan, Subhash G

    2014-10-24

    Dengue fever is a major health concern worldwide. The virus encoded non-structural protein 3 (NS3) is a multifunctional protein endowed with protease, helicase, nucleoside triphosphatase (NTPase) and RNA 5' triphosphatase (RTPase) activities. Helicase activity of NS3 catalyzes the unwinding of double stranded polynucleotides by utilizing the energy released from ATP hydrolysis. As this activity is essential for replication, NS3 helicase represents an attractive drug target for developing a dengue antiviral drug. Here, we report fluorescence based molecular beacon helicase assay using a duplex RNA substrate that contains a fluorophore on the 5' end and a quencher on the 3' end of one of the strands. The assay was optimized with respect to several parameters and adapted to 384-well high-throughput screening format, with an average Z' factor of 0.65. Assay validation with a small diverse set library of 1600 compounds identified, suramin as a significant inhibitor of the helicase activity of NS3. Helicase activity deficient NS3 K199A was used in a counter-screen to identify compounds interfering with the assay. Suramin inhibited DENV (dengue virus) NS3 helicase activity with a Ki of 0.75±0.03μM as a non-competitive inhibitor. The molecular beacon helicase assay together with the counter screen and suramin as a tool compound can be used to identify novel inhibitors of DENV helicase. PMID:25281902

  20. Thanatos-associated protein 7 associates with template activating factor-Ibeta and inhibits histone acetylation to repress transcription.

    Macfarlan, Todd; Parker, J Brandon; Nagata, Kyosuke; Chakravarti, Debabrata

    2006-02-01

    The posttranslational modifications of histones on chromatin or a lack thereof is critical in transcriptional regulation. Emerging studies indicate a role for histone-binding proteins in transcriptional activation and repression. We have previously identified template-activating factor-Ibeta (TAF-Ibeta, also called PHAPII, SET, and I(2)(pp2A)) as a component of a cellular complex called inhibitor of acetyltransferases (INHAT) that masks histone acetylation in vitro and blocks histone acetyltransferase (HAT)-dependent transcription in living cells. TAF-Ibeta has also been shown to associate with transcription factors, including nuclear receptors, to regulate their activities. To identify novel interactors of TAF-Ibeta, we employed a yeast two-hybrid screen and identified a previously uncharacterized human protein called thanatos-associated protein-7 (THAP7), a member of a large family of THAP domain-containing putative DNA-binding proteins. In this study we demonstrate that THAP7 associates with TAF-Ibeta in vitro and map their association domains to a C-terminal predicted coiled-coil motif on THAP7 and the central region of TAF-Ibeta. Similarly, stably transfected THAP7 associates with endogenous TAF-Ibeta in intact cells. Like TAF-Ibeta, THAP7 associates with histone H3 and histone H4 and inhibits histone acetylation. The histone-interacting domain of THAP7 is sufficient for this activity in vitro. Promoter-targeted THAP7 can also recruit TAF-Ibeta and silencing mediator of retinoid and thyroid receptors/nuclear hormone receptor corepressor (NCoR) proteins to promoters, and knockdown of TAF-Ibeta by small interfering RNA relieves THAP7-mediated repression, indicating that, like nuclear hormone receptors, THAP7 may represent a novel class of transcription factor that uses TAF-Ibeta as a corepressor to maintain histones in a hypoacetylated, repressed state. PMID:16195249

  1. Mitogen-activated protein kinase kinase 1/2 inhibition and angiotensin II converting inhibition in mice with cardiomyopathy caused by lamin A/C gene mutation

    Highlights: • Both ACE and MEK1/2 inhibition are beneficial on cardiac function in Lmna cardiomyopathy. • MEK1/2 inhibitor has beneficial effects beyond ACE inhibition for Lmna cardiomyopathy. • These results provide further preclinical rationale for a clinical trial of a MEK1/2 inhibitor. - Abstract: Background: Mutations in the LMNA gene encoding A-type nuclear lamins can cause dilated cardiomyopathy with or without skeletal muscular dystrophy. Previous studies have shown abnormally increased extracellular signal-regulated kinase 1/2 activity in hearts of LmnaH222P/H222P mice, a small animal model. Inhibition of this abnormal signaling activity with a mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor has beneficial effects on heart function and survival in these mice. However, such treatment has not been examined relative to any standard of care intervention for dilated cardiomyopathy or heart failure. We therefore examined the effects of an angiotensin II converting enzyme (ACE) inhibitor on left ventricular function in LmnaH222P/H222P mice and assessed if adding a MEK1/2 inhibitor would provide added benefit. Methods: Male LmnaH222P/H222P mice were treated with the ACE inhibitor benazepril, the MEK1/2 inhibitor selumetinib or both. Transthoracic echocardiography was used to measure left ventricular diameters and fractional shortening was calculated. Results: Treatment of LmnaH222P/H222P mice with either benazepril or selumetinib started at 8 weeks of age, before the onset of detectable left ventricular dysfunction, lead to statistically significantly increased fractional shortening compared to placebo at 16 weeks of age. There was a trend towards a great value for fractional shortening in the selumetinib-treated mice. When treatment was started at 16 weeks of age, after the onset of left ventricular dysfunction, the addition of selumetinib treatment to benazepril lead to a statistically significant increase in left ventricular fractional

  2. Mitogen-activated protein kinase kinase 1/2 inhibition and angiotensin II converting inhibition in mice with cardiomyopathy caused by lamin A/C gene mutation

    Muchir, Antoine, E-mail: a.muchir@institut-myologie.org [Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY (United States); Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY (United States); Wu, Wei [Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY (United States); Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY (United States); Sera, Fusako; Homma, Shunichi [Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY (United States); Worman, Howard J., E-mail: hjw14@columbia.edu [Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY (United States); Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY (United States)

    2014-10-03

    Highlights: • Both ACE and MEK1/2 inhibition are beneficial on cardiac function in Lmna cardiomyopathy. • MEK1/2 inhibitor has beneficial effects beyond ACE inhibition for Lmna cardiomyopathy. • These results provide further preclinical rationale for a clinical trial of a MEK1/2 inhibitor. - Abstract: Background: Mutations in the LMNA gene encoding A-type nuclear lamins can cause dilated cardiomyopathy with or without skeletal muscular dystrophy. Previous studies have shown abnormally increased extracellular signal-regulated kinase 1/2 activity in hearts of Lmna{sup H222P/H222P} mice, a small animal model. Inhibition of this abnormal signaling activity with a mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor has beneficial effects on heart function and survival in these mice. However, such treatment has not been examined relative to any standard of care intervention for dilated cardiomyopathy or heart failure. We therefore examined the effects of an angiotensin II converting enzyme (ACE) inhibitor on left ventricular function in Lmna{sup H222P/H222P} mice and assessed if adding a MEK1/2 inhibitor would provide added benefit. Methods: Male Lmna{sup H222P/H222P} mice were treated with the ACE inhibitor benazepril, the MEK1/2 inhibitor selumetinib or both. Transthoracic echocardiography was used to measure left ventricular diameters and fractional shortening was calculated. Results: Treatment of Lmna{sup H222P/H222P} mice with either benazepril or selumetinib started at 8 weeks of age, before the onset of detectable left ventricular dysfunction, lead to statistically significantly increased fractional shortening compared to placebo at 16 weeks of age. There was a trend towards a great value for fractional shortening in the selumetinib-treated mice. When treatment was started at 16 weeks of age, after the onset of left ventricular dysfunction, the addition of selumetinib treatment to benazepril lead to a statistically significant increase in left

  3. Neuropeptide Y1 receptor inhibits cell growth through inactivating mitogen-activated protein kinase signal pathway in human hepatocellular carcinoma.

    Lv, Xiufang; Zhao, Fengbo; Huo, Xisong; Tang, Weidong; Hu, Baoying; Gong, Xiu; Yang, Juan; Shen, Qiujin; Qin, Wenxin

    2016-07-01

    Hepatocellular carcinoma (HCC) is one of the most common cancers, and its incidence is increasing worldwide. Neuropeptide Y (NPY) broadly expressed in the central and peripheral nervous system. It participates in multiple physiological and pathological processes through specific receptors. Evidences are accumulating that NPY is involved in development and progression in neuro- or endocrine-related cancers. However, little is known about the potential roles and underlying mechanisms of NPY receptors in HCC. In this study, we analyzed the expression of NPY receptors by real-time polymerase chain reaction, Western blot, and immunohistochemical staining. Correlation between NPY1R levels and clinicopathological characteristics, and survival of HCC patients were explored, respectively. Cell proliferation was researched by CCK-8 in vitro, and tumor growth was studied by nude mice xenografts in vivo. We found that mRNA and protein level of NPY receptor Y1 subtype (NPY1R) significantly decreased in HCC tissues. Low expression of NPY1R closely correlated with poor prognosis in HCC patients. Proliferation of HCC cells was significantly inhibited by recombinant NPY protein in vitro. This inhibitory effect could be blocked by selected NPY1R antagonist BIBP3226. Furthermore, overexpression of NPY1R could significantly inhibit HCC cell proliferation. Knockdown of NPY1R promoted cell multiplication in vitro and increased tumorigenicity and tumor growth in vivo. NPY1R was found to participate in the inhibition of cell proliferation via inactivating mitogen-activated protein kinase signal pathway in HCC cells. Collectively, NPY1R plays an inhibitory role in tumor growth and may be a promising therapeutic target for HCC. PMID:27262566

  4. Activation of double-stranded RNA-dependent protein kinase inhibits proliferation of pancreatic β-cells

    Highlights: •PKR can be activated by glucolipitoxicity and pro-inflammatory cytokines in β-cells. •Activated PKR inhibited β-cell proliferation by arresting cell cycle at G1 phase. •Activated PKR fully abrogated the pro-proliferative effects of IGF-I on β-cells. -- Abstract: Double-stranded RNA-dependent protein kinase (PKR) is revealed to participate in the development of insulin resistance in peripheral tissues in type 2 diabetes (T2DM). Meanwhile, PKR is also characterized as a critical regulator of cell proliferation. To date, no study has focused on the impact of PKR on the proliferation of pancreatic β-cells. Here, we adopted insulinoma cell lines and mice islet β-cells to investigate: (1) the effects of glucolipotoxicity and pro-inflammatory cytokines on PKR activation; (2) the effects of PKR on proliferation of pancreatic β-cells and its underlying mechanisms; (3) the actions of PKR on pro-proliferative effects of IGF-I and its underlying pathway. Our results provided the first evidence that PKR can be activated by glucolipitoxicity and pro-inflammatory cytokines in pancreatic β-cells, and activated PKR significantly inhibited cell proliferation by arresting cell cycle at G1 phase. Reductions in cyclin D1 and D2 as well as increases in p27 and p53 were associated with the anti-proliferative effects of PKR, and proteasome-dependent degradation took part in the reduction of cyclin D1 and D2. Besides, PKR activation abrogated the pro-proliferative effects of IGF-I by activating JNK and disrupting IRS1/PI3K/Akt signaling pathway. These findings indicate that the anti-proliferative actions of PKR on pancreatic β-cells may contribute to the pathogenesis of T2DM

  5. Activation of double-stranded RNA-dependent protein kinase inhibits proliferation of pancreatic β-cells

    Chen, Shan-Shan [Key Laboratory of Human Functional Genomics of Jiangsu Province, Nanjing Medical University, Nanjing (China); Department of Biochemistry and Molecular Biology, Nanjing Medical University, Nanjing (China); Jiang, Teng [Department of Neurology, Qingdao Municipal Hospital, Nanjing Medical University, Nanjing (China); Wang, Yi; Gu, Li-Ze [Key Laboratory of Human Functional Genomics of Jiangsu Province, Nanjing Medical University, Nanjing (China); Department of Biochemistry and Molecular Biology, Nanjing Medical University, Nanjing (China); Wu, Hui-Wen [Laboratory Center for Basic Medical Sciences, Nanjing Medical University, Nanjing (China); Tan, Lan [Department of Neurology, Qingdao Municipal Hospital, Nanjing Medical University, Nanjing (China); Guo, Jun, E-mail: Guoj@njmu.edu.cn [Key Laboratory of Human Functional Genomics of Jiangsu Province, Nanjing Medical University, Nanjing (China); Department of Biochemistry and Molecular Biology, Nanjing Medical University, Nanjing (China)

    2014-01-17

    Highlights: •PKR can be activated by glucolipitoxicity and pro-inflammatory cytokines in β-cells. •Activated PKR inhibited β-cell proliferation by arresting cell cycle at G1 phase. •Activated PKR fully abrogated the pro-proliferative effects of IGF-I on β-cells. -- Abstract: Double-stranded RNA-dependent protein kinase (PKR) is revealed to participate in the development of insulin resistance in peripheral tissues in type 2 diabetes (T2DM). Meanwhile, PKR is also characterized as a critical regulator of cell proliferation. To date, no study has focused on the impact of PKR on the proliferation of pancreatic β-cells. Here, we adopted insulinoma cell lines and mice islet β-cells to investigate: (1) the effects of glucolipotoxicity and pro-inflammatory cytokines on PKR activation; (2) the effects of PKR on proliferation of pancreatic β-cells and its underlying mechanisms; (3) the actions of PKR on pro-proliferative effects of IGF-I and its underlying pathway. Our results provided the first evidence that PKR can be activated by glucolipitoxicity and pro-inflammatory cytokines in pancreatic β-cells, and activated PKR significantly inhibited cell proliferation by arresting cell cycle at G1 phase. Reductions in cyclin D1 and D2 as well as increases in p27 and p53 were associated with the anti-proliferative effects of PKR, and proteasome-dependent degradation took part in the reduction of cyclin D1 and D2. Besides, PKR activation abrogated the pro-proliferative effects of IGF-I by activating JNK and disrupting IRS1/PI3K/Akt signaling pathway. These findings indicate that the anti-proliferative actions of PKR on pancreatic β-cells may contribute to the pathogenesis of T2DM.

  6. Stanniocalcin-1 Potently Inhibits the Proteolytic Activity of the Metalloproteinase Pregnancy-associated Plasma Protein-A

    Kløverpris, Søren; Mikkelsen, Jakob Hauge; Pedersen, Josefine Hvidkjær;

    2015-01-01

    regulation in these species. Several physiological functions of STC1 have been reported, although many molecular details are still lacking. We here demonstrate that STC1 is an inhibitor of the metzincin metalloproteinase, pregnancy-associated plasma protein-A (PAPP-A), which modulates insulin-like growth...... that the homologous STC2 inhibits PAPP-A proteolytic activity, and that this depends on the formation of a covalent complex between the inhibitor and the proteinase, mediated by Cys-120 of STC2. We find that STC1 is unable to bind covalently to PAPP-A, in agreement with the absence of a corresponding...... cysteine residue. It rather binds to PAPP-A with high affinity (KD = 75 pm). We further demonstrate that both STC1 and STC2 show inhibitory activity toward PAPP-A2, but not selected serine proteinases and metalloproteinases. We therefore conclude that the STCs are proteinase inhibitors, probably restricted...

  7. Inhibition of the liver enriched protein FOXA2 recovers HNF6 activity in human colon carcinoma and liver hepatoma cells.

    Frank Lehner

    Full Text Available Recently, we demonstrated that the transcription factors HNF6 and FOXA2 function as key regulators in human colorectal liver metastases. To better understand their proposed inhibitory crosstalk, the consequences of functional knockdown of FOXA2 on HNF6 and C/EBPα activity were investigated in the human colon Caco-2 and HepG2 carcinoma cell lines. Specifically, siRNA-mediated gene silencing of FOXA2 repressed transcript expression by >80%. This resulted in a statistically significant 6-, 3-, 4-, and 8-fold increase in mRNA expression of HNF6 and of genes targeted by this transcription factor, e.g., HSP105B, CYP51, and C/EBPα, as determined by qRT-PCR. Thus, functional knockdown of FOXA2 recovered HNF6 activity. Furthermore, with nuclear extracts of Caco-2 cells no HNF6 DNA binding was observed, but expression of HNF1α, FOXA2, FOXA3, and HNF4α protein was abundant. We therefore transfected a plasmid encoding HNF6 into Caco-2 cells but also employed a retroviral vector to transfect HNF6 into HepG2 cells. This resulted in HNF6 protein expression with DNA binding activity being recovered as determined by EMSA band shift assays. Furthermore, by flow cytometry the consequences of HNF6 expression on cell cycle regulation in transfected cells was studied. Essentially, HNF6 inhibited cell cycle progression in the G2/M and G1 phase in Caco-2 and HepG2 cell lines, respectively. Here, proliferation was reduced by 80% and 50% in Caco-2 and HepG2 cells, respectively, as determined by the BrdU labeling assay. Therefore functional knockdown of FOXA2 recovered HNF6 activity and inhibited growth of tumor-cells and may possibly represent a novel therapeutic target in primary and secondary liver malignancies.

  8. Synthesis and protein tyrosine phosphatase 1B inhibition activities of two new synthetic bromophenols and their methoxy derivatives

    Cui, Yongchao; Shi, Dayong; Hu, Zhiqiang

    2011-11-01

    3-bromo-4,5-bis(2,3-dibromo-4,5-dihydroxybenzyl)-1,2-benzenediol ( 1) is a natural bromophenol isolated from the red algae Rhodomela confervoides that exhibits significant inhibition against protein tyrosine phosphatase 1B (PTP1B). Based on its activity, we synthesized two new synthetic bromophenols and their methoxy derivatives from vanillin using the structure of natural bromophenol 1 as a scaffold. The structures of these bromophenols were elucidated from 1H NMR, 13C NMR, and high resolution electron ionization mass spectrometry as 2,3-dibromo-1-(2'-bromo-6'-(3″,4″-dimethoxybenzyl)-3',4'-dimethoxybenzyl)-4,5-dimethoxybenzene ( 2), 2,3-dibromo-1-(2'-bromo-6'-(2″-bromo-4″,5″-dimethoxybenzyl)-3',4'-dimethoxybenzyl)-4,5-dimethoxybenzene ( 3), 3,4-dibromo-5-(2'-bromo-6'-(2″-bromo-4″,5″-dihydroxybenzyl)-3',4'-dihydroxybenzyl)pyrocatechol ( 4) and 3,4-dibromo-5-(2'-bromo-6'-(3″,4″-dihydroxybenzyl)-3',4'-dihydroxybenzyl)pyrocatechol ( 5). PTP1B inhibition activities of these compounds were evaluated using a colorimetric assay, and compounds 3 and 4 demonstrated interesting activity against PTP1B.

  9. In Silico Elucidation and Inhibition Studies of Selected Phytoligands Against Mitogen-Activated Protein Kinases of Protozoan Parasites.

    Gupta, Chhedi Lal; Akhtar, Salman; Kumar, Nilesh; Ali, Jasarat; Pathak, Neelam; Bajpai, Preeti

    2016-03-01

    Parasitic MAPKs exhibiting significant divergence with humans and playing an imperative role in parasitic metabolic activities have been exploited from several years as important targets for development of novel therapeutics. In addition, the emergence of the drug-resistant variants of parasitic diseases in the recent years has aroused a great need for the development of potent inhibitors against them. In the present study, we selected the metabolically active MAPKs LmxMPK4, PfMAP2 and TbMAPK5 of the three parasitic protozoans Leishmania mexicana, Plasmodium falciparum and Trypanosoma brucei, respectively. The homology modeling technique was used to develop the 3D structures of these proteins, and the same was validated by PROCHECK, ERRAT, ProQ and ProSA web servers to check the reliability. Ten phytoligands were employed for molecular docking studies with these proteins to search for potent phytoligand as a broad spectrum inhibitor. In this regard, two phytoligands (aspidocarpine for LmxMPK4 and TbMAPK5 and cubebin for PfMAP2) were found to be more effective inhibitors, in terms of robust binding energy, strong inhibition constant and better interactions between protein-ligand complexes. Furthermore, predicted ADME and toxicity properties suggested that these identified phytoligands exhibited comparable results to control drugs potentiating them as persuasive therapeutic agents for Leishmania, Trypanosoma and Plasmodium sp. PMID:26264054

  10. Upstream mitogen-activated protein kinase (MAPK) pathway inhibition: MEK inhibitor followed by a BRAF inhibitor in advanced melanoma patients.

    Goldinger, Simone M; Zimmer, Lisa; Schulz, Carsten; Ugurel, Selma; Hoeller, Christoph; Kaehler, Katharina C; Schadendorf, Dirk; Hassel, Jessica C; Becker, Juergen; Hauschild, Axel; Dummer, Reinhard

    2014-01-01

    BRAF-mutant melanoma can be successfully treated by BRAF kinase inhibitors (BRAFi) and MEK kinase inhibitors (MEKi). However, the administration of BRAFi followed by MEKi did not generate promising response rate (RR). The purpose of this investigation was to evaluate the time to progression (TTP) with a mitogen-activated protein kinase (MAPK) pathway upstream inhibition strategy in BRAF mutated melanoma patients. BRAF mutation positive metastatic melanoma patients were identified within the Dermatology Cooperative Oncology Group (DeCOG) network and were treated first with a MEKi and upon progression with a selective BRAFi. A total of 23 melanoma patients (six females, 17 males, aged 47-80 years) were retrospectively analysed for TTP. The total median TTP was 8.9 months. The median TTP for MEKi was 4.8 (1.2-23.2) and subsequent for BRAFi 4.5 (1.2-15.7) months, respectively. A higher RR for MEKi (39%, nine partial responses and 0 complete responses) than previously reported was observed. Our analysis suggests that the reversed inhibition of the MAPK pathway is feasible in BRAF mutated melanoma. The median TTP (8.9 months) is close to the promising BRAF- and MEKi combination therapy (median progression-free survival (PFS) 9.4 months). The total treatment duration of the MAPK inhibition when a MEKi is administered first is similar compared to the reversed sequence, but TTP shifts in favour to the MEKi. This approach is feasible with reasonable tolerability. This clinical investigation encourages further studies in prospective clinical trials to define the optimal treatment schedule for the MAPK pathway inhibition and should be accompanied by molecular monitoring using repeated biopsies. PMID:24183461

  11. INHIBITION OF MELATONIN BIOSYNTHESIS ACTIVATES PROTEIN KINASE A AND INDUCES ALZHEIMER-LIKE TAU HYPERPHOSPHORYLATION IN RATS

    Ling-qiang Zhu; Shao-hui Wang; Zhi-qun Ling; Qun Wang; Mao-qiong Hu; Jian-zhi Wang

    2005-01-01

    Objective To investigate effect of inhibiting melatonin biosynthesis on activities of protein kinase A (PKA), glycogen synthase kinase-3 (GSK-3) and tau phosphorylation at PS214 and M4 epitopes using haloperidol, a specific inhibitor of 5-hydroxyindole-O-methyltransferase. Methods Brain ventricular and intraperitoneal injections were used for haloperidol administration, Western blots for tau phosphorylation, 32p-labeling for PKA and GSK-3 activity, and high performance liquid chromatograph for detection of serum melatonin levels.Results Haloperidol injection through the lateral ventricle and intraperitoneal reinforcement significantly stimulated PKA activity with a concurrent hyperphosphorylation of tau at M4 (Thr231/Ser235) and PS214 (Ser214) sites. Prior treatment of the rats using melatonin supplement for one week and reinforcement during the haloperidol administration arrested PKA activity and attenuated tau hyperphosphorylation. GSK-3 activity showed no obvious change after haloperidol injection, however, melatonin supplements and reinforcements during haloperidol infusion inactivated basal activity of GSK-3. Conclusion Decreased melatonin may be involved in Alzheimer-like tau hyperphosphorylation, and overactivation of PKA may play a crucial role in this process.

  12. GNL3L Inhibits Estrogen Receptor-Related Protein Activities by Competing for Coactivator Binding

    Yasumoto, Hiroaki; Meng, Lingjun; Lin, Tao; Zhu, Qubo; Tsai, Robert Y.L.

    2007-01-01

    Guanine-nucleotide binding protein 3-like (GNL3L) is the closest homologue of a stem cell-enriched factor nucleostemin in vertebrates. They share the same yeast orthologue, Grn1p, but only GNL3L can rescue the growth-deficient phenotype in Grn1p-null yeasts. To determine the unique function of GNL3L, we identified estrogen receptor-related protein-γ (ERRγ) as a GNL3L-specific binding protein. GNL3L and ERRγ are coexpressed in the eye, kidney and muscle, and co-reside in the nucleoplasm. The i...

  13. Chlamydiaphage φCPG1 Capsid Protein Vp1 Inhibits Chlamydia trachomatis Growth via the Mitogen-Activated Protein Kinase Pathway.

    Guo, Yuanli; Guo, Rui; Zhou, Quan; Sun, Changgui; Zhang, Xinmei; Liu, Yuanjun; Liu, Quanzhong

    2016-01-01

    Chlamydia trachomatis is the most common cause of curable bacterial sexually transmitted infections worldwide. Although the pathogen is well established, the pathogenic mechanisms remain unclear. Given the current challenges of antibiotic resistance and blocked processes of vaccine development, the use of a specific chlamydiaphage may be a new treatment solution. φCPG1 is a lytic phage specific for Chlamydia caviae, and shows over 90% nucleotide sequence identity with other chlamydiaphages. Vp1 is the major capsid protein of φCPG1. Purified Vp1 was previously confirmed to inhibit Chlamydia trachomatis growth. We here report the first attempt at exploring the relationship between Vp1-treated C. trachomatis and the protein and gene levels of the mitogen-activated/extracellular regulated protein kinase (MAPK/ERK) pathway by Western blotting and real-time PCR, respectively. Moreover, we evaluated the levels of pro-inflammatory cytokines interleukin (IL)-8 and IL-1 by enzyme-linked immunosorbent assay after Vp1 treatment. After 48 h of incubation, the p-ERK level of the Vp1-treated group decreased compared with that of the Chlamydia infection group. Accordingly, ERK1 and ERK2 mRNA expression levels of the Vp1-treated group also decreased compared with the Chlamydia infection group. IL-8 and IL-1 levels were also decreased after Vp1 treatment compared with the untreated group. Our results demonstrate that the inhibition effect of the chlamydiaphage φCPG1 capsid protein Vp1 on C. trachomatis is associated with the MAPK pathway, and inhibits production of the pro-inflammatory cytokines IL-8 and IL-1. The bacteriophages may provide insight into a new signaling transduction mechanism to influence their hosts, in addition to bacteriolysis. PMID:27089359

  14. Chlamydiaphage φCPG1 Capsid Protein Vp1 Inhibits Chlamydia trachomatis Growth via the Mitogen-Activated Protein Kinase Pathway

    Yuanli Guo

    2016-04-01

    Full Text Available Chlamydia trachomatis is the most common cause of curable bacterial sexually transmitted infections worldwide. Although the pathogen is well established, the pathogenic mechanisms remain unclear. Given the current challenges of antibiotic resistance and blocked processes of vaccine development, the use of a specific chlamydiaphage may be a new treatment solution. φCPG1 is a lytic phage specific for Chlamydia caviae, and shows over 90% nucleotide sequence identity with other chlamydiaphages. Vp1 is the major capsid protein of φCPG1. Purified Vp1 was previously confirmed to inhibit Chlamydia trachomatis growth. We here report the first attempt at exploring the relationship between Vp1-treated C. trachomatis and the protein and gene levels of the mitogen-activated/extracellular regulated protein kinase (MAPK/ERK pathway by Western blotting and real-time PCR, respectively. Moreover, we evaluated the levels of pro-inflammatory cytokines interleukin (IL-8 and IL-1 by enzyme-linked immunosorbent assay after Vp1 treatment. After 48 h of incubation, the p-ERK level of the Vp1-treated group decreased compared with that of the Chlamydia infection group. Accordingly, ERK1 and ERK2 mRNA expression levels of the Vp1-treated group also decreased compared with the Chlamydia infection group. IL-8 and IL-1 levels were also decreased after Vp1 treatment compared with the untreated group. Our results demonstrate that the inhibition effect of the chlamydiaphage φCPG1 capsid protein Vp1 on C. trachomatis is associated with the MAPK pathway, and inhibits production of the pro-inflammatory cytokines IL-8 and IL-1. The bacteriophages may provide insight into a new signaling transduction mechanism to influence their hosts, in addition to bacteriolysis.

  15. Novel Antiplatelet Activity of Minocycline Involves Inhibition of MLK3-p38 Mitogen Activated Protein Kinase Axis.

    Joseph W Jackson

    Full Text Available Platelets play an essential role in hemostasis and wound healing by facilitating thrombus formation at sites of injury. Platelets also mediate inflammation and contain several pro-inflammatory molecules including cytokines and chemokines that mediate leukocyte recruitment and activation. Not surprisingly, platelet dysfunction is known to contribute to several inflammatory disorders. Antiplatelet therapies, such as aspirin, adenosine diphosphate (ADP antagonists, glycoprotein IIb/IIIa (GPIIb/IIIa inhibitors, and anticoagulants such as warfarin, dampen platelet activity at the risk of unwarranted bleeding. Thus, the development of drugs that reduce platelet-mediated inflammation without interfering with thrombus formation is of importance to combat platelet-associated disorders. We have shown here for the first time that the tetracycline antibiotic, minocycline, administered to HIV-infected individuals reduces plasma levels of soluble CD40L and platelet factor 4 levels, host molecules predominately released by platelets. Minocycline reduced the activation of isolated platelets in the presence of the potent platelet activator, thrombin, as measured by ELISA and flow cytometry. Platelet degranulation was reduced upon exposure to minocycline as shown by mepacrine retention and flow cytometry. However, minocycline had no effect on spreading, aggregation, GPIIb/IIIa activation, or in vivo thrombus formation. Lastly, immunoblot analysis suggests that the antiplatelet activity of minocycline is likely mediated by inhibition of mixed lineage kinase 3 (MLK3-p38 MAPK signaling axis and loss of p38 activity. Our findings provide a better understanding of platelet biology and a novel repurposing of an established antibiotic, minocycline, to specifically reduce platelet granule release without affecting thrombosis, which may yield insights in generating novel, specific antiplatelet therapies.

  16. Novel Antiplatelet Activity of Minocycline Involves Inhibition of MLK3-p38 Mitogen Activated Protein Kinase Axis

    Jackson, Joseph W.; Singh, Meera V.; Singh, Vir B.; Jones, Letitia D.; Davidson, Gregory A.; Ture, Sara; Morrell, Craig N.; Schifitto, Giovanni; Maggirwar, Sanjay B.

    2016-01-01

    Platelets play an essential role in hemostasis and wound healing by facilitating thrombus formation at sites of injury. Platelets also mediate inflammation and contain several pro-inflammatory molecules including cytokines and chemokines that mediate leukocyte recruitment and activation. Not surprisingly, platelet dysfunction is known to contribute to several inflammatory disorders. Antiplatelet therapies, such as aspirin, adenosine diphosphate (ADP) antagonists, glycoprotein IIb/IIIa (GPIIb/IIIa) inhibitors, and anticoagulants such as warfarin, dampen platelet activity at the risk of unwarranted bleeding. Thus, the development of drugs that reduce platelet-mediated inflammation without interfering with thrombus formation is of importance to combat platelet-associated disorders. We have shown here for the first time that the tetracycline antibiotic, minocycline, administered to HIV-infected individuals reduces plasma levels of soluble CD40L and platelet factor 4 levels, host molecules predominately released by platelets. Minocycline reduced the activation of isolated platelets in the presence of the potent platelet activator, thrombin, as measured by ELISA and flow cytometry. Platelet degranulation was reduced upon exposure to minocycline as shown by mepacrine retention and flow cytometry. However, minocycline had no effect on spreading, aggregation, GPIIb/IIIa activation, or in vivo thrombus formation. Lastly, immunoblot analysis suggests that the antiplatelet activity of minocycline is likely mediated by inhibition of mixed lineage kinase 3 (MLK3)-p38 MAPK signaling axis and loss of p38 activity. Our findings provide a better understanding of platelet biology and a novel repurposing of an established antibiotic, minocycline, to specifically reduce platelet granule release without affecting thrombosis, which may yield insights in generating novel, specific antiplatelet therapies. PMID:27270236

  17. Structural basis of the zinc- and terbium-mediated inhibition of ferroxidase activity in Dps ferritin-like proteins

    Havukainen, Heli; Haataja, Sauli; Kauko, Anni; Pulliainen, Arto T.; Salminen, Annika; Haikarainen, Teemu; Finne, Jukka; Papageorgiou, Anastassios C.

    2008-01-01

    Streptococcus suis Dpr is an iron-binding protein involved in oxidative stress resistance. It belongs to the bacterial Dps protein family whose members form dodecameric assemblies. Previous studies have shown that zinc and terbium inhibit iron incorporation in Listeria innocua Dps protein. In order to gain structural insights into the inhibitory effect of zinc and terbium, the crystal structures of Streptococcus suis Dpr complexes with these ions were determined at 1.8 Å and 2.1 Å, respective...

  18. Phage Lambda P Protein: Trans-Activation, Inhibition Phenotypes and their Suppression

    Connie Hayes

    2013-02-01

    Full Text Available The initiation of bacteriophage λ replication depends upon interactions between the oriλ DNA site, phage proteins O and P, and E. coli host replication proteins. P exhibits a high affinity for DnaB, the major replicative helicase for unwinding double stranded DNA. The concept of P-lethality relates to the hypothesis that P can sequester DnaB and in turn prevent cellular replication initiation from oriC. Alternatively, it was suggested that P-lethality does not involve an interaction between P and DnaB, but is targeted to DnaA. P-lethality is assessed by examining host cells for transformation by ColE1-type plasmids that can express P, and the absence of transformants is attributed to a lethal effect of P expression. The plasmid we employed enabled conditional expression of P, where under permissive conditions, cells were efficiently transformed. We observed that ColE1 replication and plasmid establishment upon transformation is extremely sensitive to P, and distinguish this effect from P-lethality directed to cells. We show that alleles of dnaB protect the variant cells from P expression. P-dependent cellular filamentation arose in ΔrecA or lexA[Ind-] cells, defective for SOS induction. Replication propagation and restart could represent additional targets for P interference of E. coli replication, beyond the oriC-dependent initiation step.

  19. Anti-melanogenic activity of the novel herbal medicine, MA128, through inhibition of tyrosinase activity mediated by the p38 mitogen-activated protein kinases and protein kinase signaling pathway in B16F10 cells

    Aeyung Kim

    2014-01-01

    Full Text Available Background: Recently, our research group developed MA128, a novel herbal medicine, and demonstrated that MA128 is effective for the treatment of asthma and atopic dermatitis (AD. In particular, postinflammatory hyper-pigmentation in AD mice was improved with MA128 treatment. Thus, in this study, we determined the effect of MA128 on melanogenesis and its underlying mechanism in murine B16F10 melanoma cells. Materials and Methods: After treatment with MA128 at 100 and 250 μg/mL and/or alpha-melanocyte stimulating hormone (α-MSH (1 μM, cellular melanin content and tyrosinase activity in B16F10 cells were measured. Using western blotting, expression levels of tyrosinase, tyrosinase-related protein-1 (TRP-1, TRP-2, microphthalmia-associated transcription factor (MITF, and activation of c-AMP-dependent protein kinase (PKA, c-AMP-related element binding protein (CREB and mitogen-activated protein kinases (MAPKs were examined. Results: MA128 significantly inhibited melanin synthesis and tyrosinase activity in a resting state as well as α-MSH-stimulating condition, and significantly decreased the expression of tyrosinase, TRP-1, TRP-2 and MITF. In addition, phosphorylation of PKA and CREB by α-MSH stimulation was efficiently blocked by MA128 pretreatment. Moreover, MA128 as an herbal mixture showed synergistic anti-melanogenic effects compared with each single constituent herb. Conclusion: MA128 showed anti-melanogenic activity through inhibition of tyrosinase activity mediated by p38 MAPK and PKA signaling pathways in B16F10 cells. These results suggest that MA128 may be useful as an herbal medicine for controlling hyper-pigmentation and as a skin-whitening agent.

  20. GABA/sub B/ receptor activation inhibits Ca/sup 2 +/-activated potassium channels in synaptosomes: involvement of G-proteins

    Ticku, M.K.; Delgado, A.

    1989-01-01

    /sup 86/Rb-efflux assay from preloaded synaptosomes of rat cerebral cortex was developed to study the effect of GABA/sub B/ receptor agonist baclofen on Ca/sup 2 +/-activated K/sup +/-channels. Depolarization of /sup 86/Rb-loaded synaptosomes in physiological buffer increased Ca/sup 2 +/-activated /sup 86/Rb-efflux by 400%. The /sup 86/Rb-efflux was blocked by quinine sulfate, tetraethylammonium, and La/sup 3 +/ indicating the involvement of Ca/sup 2 +/-activated K/sup +/-channels. (-)Baclofen inhibited Ca/sup 2 +/-activated /sup 86/Rb-efflux in a stereospecific manner. The inhibitory effect of (-)baclofen was mediated by GABA/sub B/ receptor activation, since it was blocked by GABA/sub B/ antagonist phaclofen, but not by bicuculline. Further, pertussis toxin also blocked the ability of baclofen or depolarizing action to affect Ca/sup 2 +/-activated K/sup +/-channels. These results suggest that baclofen inhibits Ca/sup 2 +/-activated K/sup +/-channels in synaptosomes and these channels are regulated by G-proteins. This assay may provide an ideal in vitro model to study GABA/sub B/ receptor pharmacology.

  1. Identification and Evaluation of Cryoprotective Peptides from Chicken Collagen: Ice-Growth Inhibition Activity Compared to That of Type I Antifreeze Proteins in Sucrose Model Systems.

    Du, Lihui; Betti, Mirko

    2016-06-29

    The ability of chicken collagen peptides to inhibit the growth of ice crystals was evaluated and compared to that of fish antifreeze proteins (AFPs). This ice inhibition activity was assessed using a polarized microscope by measuring ice crystal dimensions in a sucrose model system with and without collagen peptides after seven thermal cycles. The system was stabilized at -25 °C and cycled between -16 and -12 °C. Five candidate peptides with ice inhibition activity were identified using liquid chromatography and tandem mass spectrometry and were then synthesized. Their ice inhibition capacity was compared to that of type I AFPs in a 23% sucrose model system. Specific collagen peptides with certain amino acid sequences reduced the extent of ice growth by approximately 70% at a relatively low concentration (1 mg/mL). These results suggest that specific collagen peptides may act in a noncolligative manner, inhibiting ice crystal growth like type I AFPs, but less efficiently. PMID:27293017

  2. G Protein-Coupled Bile Acid Receptor TGR5 Activation Inhibits Kidney Disease in Obesity and Diabetes.

    Wang, Xiaoxin X; Edelstein, Michal Herman; Gafter, Uzi; Qiu, Liru; Luo, Yuhuan; Dobrinskikh, Evgenia; Lucia, Scott; Adorini, Luciano; D'Agati, Vivette D; Levi, Jonathan; Rosenberg, Avi; Kopp, Jeffrey B; Gius, David R; Saleem, Moin A; Levi, Moshe

    2016-05-01

    Obesity and diabetes mellitus are the leading causes of renal disease. In this study, we determined the regulation and role of the G protein-coupled bile acid receptor TGR5, previously shown to be regulated by high glucose and/or fatty acids, in obesity-related glomerulopathy (ORG) and diabetic nephropathy (DN). Treatment of diabetic db/db mice with the selective TGR5 agonist INT-777 decreased proteinuria, podocyte injury, mesangial expansion, fibrosis, and CD68 macrophage infiltration in the kidney. INT-777 also induced renal expression of master regulators of mitochondrial biogenesis, inhibitors of oxidative stress, and inducers of fatty acid β-oxidation, including sirtuin 1 (SIRT1), sirtuin 3 (SIRT3), and Nrf-1. Increased activity of SIRT3 was evidenced by normalization of the increased acetylation of mitochondrial superoxide dismutase 2 (SOD2) and isocitrate dehydrogenase 2 (IDH2) observed in untreated db/db mice. Accordingly, INT-777 decreased mitochondrial H2O2 generation and increased the activity of SOD2, which associated with decreased urinary levels of H2O2 and thiobarbituric acid reactive substances. Furthermore, INT-777 decreased renal lipid accumulation. INT-777 also prevented kidney disease in mice with diet-induced obesity. In human podocytes cultured with high glucose, INT-777 induced mitochondrial biogenesis, decreased oxidative stress, and increased fatty acid β-oxidation. Compared with normal kidney biopsy specimens, kidney specimens from patients with established ORG or DN expressed significantly less TGR5 mRNA, and levels inversely correlated with disease progression. Our results indicate that TGR5 activation induces mitochondrial biogenesis and prevents renal oxidative stress and lipid accumulation, establishing a role for TGR5 in inhibiting kidney disease in obesity and diabetes. PMID:26424786

  3. Inhibition of the neutrophil oxidative burst by sphingoid long-chain bases: role of protein kinase C in the activation of the burst

    The neutrophil oxidative burst is triggered by a variety of both particulate (opsonized zymosan) and soluble agonists [formylmethionylleucylphenylalanine (FMLP), arachidonate, short-chained diacylglycerols (DAG) and phorbol myristate acetate (PMA)]. The authors show that the long-chain lipid bases sphinganine and sphingosine block activation of the burst in human neutrophils. Inhibition is reversible, does not alter cell viability, and does not affect phagocytosis. The inhibition affects the activation mechanism rather than the NADPH-oxidase enzyme. The structural requirements for inhibition include a hydrophobic carbon chain and an amino-containing headgroup, and the naturally occurring erythro sphinganine was more potent than the threo isomer. Activation of the oxidative burst by a variety of agonists was blocked by the same concentration of sphinganine indicating a common inhibited step. The authors suggest that the common step is protein kinase C, as evidenced by the following: 1) long-chain bases inhibit PKC in a micelle reconstituted system, 2) PMA-induced phophorylation is inhibited by sphinganine, and 3) sphinganine competes with (3H)-phorbol dibutyrate for its cytosolic receptor (i.e. protein kinase C). The authors suggest that sphingoid long-chain bases play a role in the cellular regulations

  4. Down syndrome critical region 2 protein inhibits the transcriptional activity of peroxisome proliferator-activated receptor β in HEK293 cells

    Down syndrome is mainly caused by a trisomy of chromosome 21. The Down syndrome critical region 2 (DSCR2) gene is located within a part of chromosome 21, the Down syndrome critical region (DSCR). To investigate the function of DSCR2, we sought to identify DSCR2-interacting proteins using yeast two-hybrid assays. A human fetal brain cDNA library was screened, and DSCR2 was found to interact with a member of the nuclear receptor superfamily, peroxisome proliferator-activated receptor β, (PPARβ). A co-immunoprecipitation assay demonstrated that DSCR2 physically interacts with PPARβ in mammalian HEK293 cells. DSCR2 also inhibited the ligand-induced transcriptional activity of PPARβ. Furthermore, PPARβ also decreased the solubility of DSCR2, which increased levels of insoluble DSCR2

  5. Hepatitis C virus non-structural protein 3 interacts with cytosolic 5'(3'-deoxyribonucleotidase and partially inhibits its activity.

    Chiu-Ping Fang

    Full Text Available Infection with hepatitis C virus (HCV is etiologically involved in liver cirrhosis, hepatocellular carcinoma and B-cell lymphomas. It has been demonstrated previously that HCV non-structural protein 3 (NS3 is involved in cell transformation. In this study, a yeast two-hybrid screening experiment was conducted to identify cellular proteins interacting with HCV NS3 protein. Cytosolic 5'(3'-deoxyribonucleotidase (cdN, dNT-1 was found to interact with HCV NS3 protein. Binding domains of HCV NS3 and cellular cdN proteins were also determined using the yeast two-hybrid system. Interactions between HCV NS3 and cdN proteins were further demonstrated by co-immunoprecipitation and confocal analysis in cultured cells. The cellular cdN activity was partially repressed by NS3 protein in both the transiently-transfected and the stably-transfected systems. Furthermore, HCV partially repressed the cdN activity while had no effect on its protein expression in the systems of HCV sub-genomic replicons and infectious HCV virions. Deoxyribonucleotidases are present in most mammalian cells and involve in the regulation of intracellular deoxyribonucleotides pools by substrate cycles. Control of DNA precursor concentration is essential for the maintenance of genetic stability. Reduction of cdN activity would result in the imbalance of DNA precursor concentrations. Thus, our results suggested that HCV partially reduced the cdN activity via its NS3 protein and this may in turn cause diseases.

  6. Gecko proteins induce the apoptosis of bladder cancer 5637 cells by inhibiting Akt and activating the intrinsic caspase cascade.

    Kim, Geun-Young; Park, Soon Yong; Jo, Ara; Kim, Mira; Leem, Sun-Hee; Jun, Woo-Jin; Shim, Sang In; Lee, Sang Chul; Chung, Jin Woong

    2015-09-01

    Gecko proteins have long been used as anti-tumor agents in oriental medicine, without any scientific background. Although anti-tumor effects of Gecko proteins on several cancers were recently reported, their effect on bladder cancer has not been investigated. Thus, we explored the anti-tumor effect of Gecko proteins and its cellular mechanisms in human bladder cancer 5637 cells. Gecko proteins significantly reduced the viability of 5637 cells without any cytotoxic effect on normal cells. These proteins increased the Annexin-V staining and the amount of condensed chromatin, demonstrating that the Gecko proteinsinduced cell death was caused by apoptosis. Gecko proteins suppressed Akt activation, and the overexpression of constitutively active form of myristoylated Akt prevented Gecko proteins-induced death of 5637 cells. Furthermore, Gecko proteins activated caspase 9 and caspase 3/7. Taken together, our data demonstrated that Gecko proteins suppressed the Akt pathway and activated the intrinsic caspase pathway, leading to the apoptosis of bladder cancer cells. [BMB Reports 2015; 48(9): 531-536]. PMID:26246284

  7. Ribosome Inactivating Proteins from Plants Inhibiting Viruses

    Inderdeep Kaur; R C Gupta; Munish Puri

    2011-01-01

    Many plants contain ribosome inactivating proteins (RIPs) with N-glycosidase activity,which depurinate large ribosomal RNA and arrest protein synthesis.RIPs so far tested inhibit replication of mRNA as well as DNA viruses and these proteins,isolated from plants,are found to be effective against a broad range of viruses such as human immunodeficiency virus (HIV),hepatitis B virus (HBV) and herpes simplex virus (HSV).Most of the research work related to RIPs has been focused on antiviral activity against HIV; however,the exact mechanism of antiviral activity is still not clear.The mechanism of antiviral activity was thought to follow inactivation of the host cell ribosome,leading to inhibition of viral protein translation and host cell death.Enzymatic activity of RIPs is not hmited to depurination of the large rRNA,in addition they can depurinate viral DNA as well as RNA.Recently,Phase Ⅰ/Ⅱ clinical trials have demonstrated the potential use of RIPs for treating patients with HIV disease.The aim of this review is to focus on various RIPs from plants associated with anti-HIV activity.

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

    Mikael S Lindström

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

  9. Dexamethasone Causes Sustained Expression of Mitogen-Activated Protein Kinase (MAPK) Phosphatase 1 and Phosphatase-Mediated Inhibition of MAPK p38

    Lasa, Marina; Abraham, Sonya M.; Boucheron, Christine; Saklatvala, Jeremy; Clark, Andrew R.

    2002-01-01

    The stress-activated protein kinase p38 stabilizes a number of mRNAs encoding inflammatory mediators, such as cyclooxygenase 2 (Cox-2). In HeLa cells the anti-inflammatory glucocorticoid dexamethasone destabilizes Cox-2 mRNA by inhibiting p38 function. Here we demonstrate that this effect is phosphatase dependent. Furthermore, in HeLa cells dexamethasone induced the sustained expression of mitogen-activated protein kinase phosphatase 1 (MKP-1), a potent inhibitor of p38 function. The inhibiti...

  10. Inhibition of fibroblast growth factor 2-induced apoptosis involves survivin expression, protein kinase Cα activation and subcellular translocation of Smac in human small cell lung cancer cells

    Desheng Xiao; Kuansong Wang; Jianhua Zhou; Huiqiu Cao; Zhenghao Deng; Yongbin Hu; Xiahui Qu; Jifang Wen

    2008-01-01

    To investigate the mechanism by which fibroblast growth factor 2 (FGF-2) inhibits apoptosis in the human small cell lung cancer cell line H446 subjected to serum starvation,apoptosis was evaluated by flow cytometry, Hoechst 33258 staining, caspase-3 activity, and DNA fragmentation.Survivin expression induced by FGF-2 and protein kinase Cα (PKCα) translocation was detected by subcellular fractionation and Western blot analysis. In addition, FGF-2-induced release of Smac from mitochondria to the cytoplasm was analyzed by Western blotting and immunofluorescence.FGF-2 reduced apoptosis induced by serum starvation and up-regulated survivin expression in H446 cells in a dosedependent and time-dependent manner, and inhibited caspase-3 activity. FGF-2 also inhibited the release of Smac from mitochondria to the cytoplasm induced by serum starvation and increased PKCα translocation from the cytoplasm to the cell membrane. In addition, PKC inhibitor inhibited the expression of survivin. FGF-2 up-regulates the expression of survivin protein in H446 cells and blocks the release of Smac from mitochondria to the cytoplasm. PKCα regulated FGF-2-induced survivin expression. Thus, survivin, Smac,and PKCα might play important roles in the inhibition of apoptosis by FGF-2 in human small cell lung cancer cells.

  11. Apoptotic cell death through inhibition of protein kinase CKII activity by 3,4-dihydroxybenzaldehyde purified from Xanthium strumarium.

    Lee, Bang Hyo; Yoon, Soo-Hyun; Kim, Yun-Sook; Kim, Sang Kook; Moon, Byong Jo; Bae, Young-Seuk

    2008-01-01

    The CKII inhibitory compound was purified from the fruit of Xanthium strumarium by organic solvent extraction and silica gel chromatography. The inhibitory compound was identified as 3,4-dihydroxybenzaldehyde by analysis with FT-IR, FAB-Mass, EI-Mass, (1)H-NMR and (13)C-NMR. 3,4-dihydroxybenzaldehyde inhibited the phosphotransferase activity of CKII with IC(50) of about 783 microM. Steady-state studies revealed that the inhibitor acts as a competitive inhibitor with respect to the substrate ATP. A value of 138.6 microM was obtained for the apparent K(i). Concentration of 300 microM 3,4-dihydroxybenzaldehyde caused 50% growth inhibition of human cancer cell U937. 3,4-dihydroxybenzaldehyde-induced cell death was characterised with the cleavage of poly(ADP-ribose) polymerase and procaspase-3. Furthermore, the inhibitor induced the fragmentation of DNA into multiples of 180 bp, indicating that it triggered apoptosis. This induction of apoptosis by 3,4-dihydroxybenzaldehyde was also confirmed by using flow cytometry analysis. Since CKII is involved in cell proliferation and oncogenesis, these results suggest that 3,4-dihydroxybenzaldehyde may function by inhibiting oncogenic disease, at least in part, through the inhibition of CKII activity. PMID:19023807

  12. Human T-cell leukemia virus type-1-encoded protein HBZ represses p53 function by inhibiting the acetyltransferase activity of p300/CBP and HBO1

    Hoang, Kimson; Ankney, John A.; Nguyen, Stephanie T.; Rushing, Amanda W.; Polakowski, Nicholas; Miotto, Benoit; Lemasson, Isabelle

    2016-01-01

    Adult T-cell leukemia (ATL) is an often fatal malignancy caused by infection with the complex retrovirus, human T-cell Leukemia Virus, type 1 (HTLV-1). In ATL patient samples, the tumor suppressor, p53, is infrequently mutated; however, it has been shown to be inactivated by the viral protein, Tax. Here, we show that another HTLV-1 protein, HBZ, represses p53 activity. In HCT116 p53+/+ cells treated with the DNA-damaging agent, etoposide, HBZ reduced p53-mediated activation of p21/CDKN1A and GADD45A expression, which was associated with a delay in G2 phase-arrest. These effects were attributed to direct inhibition of the histone acetyltransferase (HAT) activity of p300/CBP by HBZ, causing a reduction in p53 acetylation, which has be linked to decreased p53 activity. In addition, HBZ bound to, and inhibited the HAT activity of HBO1. Although HBO1 did not acetylate p53, it acted as a coactivator for p53 at the p21/CDKN1A promoter. Therefore, through interactions with two separate HAT proteins, HBZ impairs the ability of p53 to activate transcription. This mechanism may explain how p53 activity is restricted in ATL cells that do not express Tax due to modifications of the HTLV-1 provirus, which accounts for a majority of patient samples. PMID:26625199

  13. Astaxanthin can alter CYP1A-dependent activities via two different mechanisms: induction of protein expression and inhibition of NADPH P450 reductase dependent electron transfer.

    Ohno, Marumi; Darwish, Wageh S; Ikenaka, Yoshinori; Miki, Wataru; Ishizuka, Mayumi

    2011-06-01

    Astaxanthin (Ax), a xanthophyll carotenoid, is reported to induce cytochrome P450 (CYP) 1A-dependent activity. CYP1A is one of the most important enzymes participating in phase I metabolism for chemicals, and it can activate various mutagens. To investigate the effect of Ax on the metabolic activation of a typical promutagen, benzo[a]pyrene by CYP1A, we orally administrated Ax-containing oil (100 mg Ax/kg body weight/day for 3 days) to male Wistar rats. In the treated rat liver, expression of CYP1A1 mRNA, protein, and its activity were significantly increased (5.5-, 8.5-, and 2.5-fold, respectively). In contrast, the activities of phase II enzymes (glutathione S-transferase and glucuronosyl-transferase) were not modulated by Ax-containing oil. As a consequence, the mutagenicity of benzo[a]pyrene was more enhanced in Ax-treated rats, compared with controls in the Ames assay. On the other hand, NADPH P450 reductase activity was decreased in liver microsomes from the treated group. This result suggests the possibility that Ax inhibits the electron supply necessary for CYP catalytic activities and decreases CYP1A activity indirectly. In conclusion, Ax-containing oil intake can alter CYP1A-dependent activities through two different mechanisms: (1) induction of CYP1A1 mRNA, protein expression, and activity; and (2) inhibition of the electron supply for the enzyme. PMID:21414371

  14. Islet-activating protein inhibits leukotriene D4- and leukotriene C4- but not bradykinin- or calcium ionophore-induced prostacyclin synthesis in bovine endothelial cells.

    Clark, M. A.; Conway, T.M.; Bennett, C F; Crooke, S T; Stadel, J M

    1986-01-01

    Incubation of the bovine endothelial cell line, CPAE, with leukotriene D4, leukotriene C4, bradykinin, or the calcium ionophore A23187 results in the release of arachidonic acid metabolites including 6-keto-prostaglandin F1 alpha, the stable metabolite of prostacyclin. Pretreatment of these cells with the pertussis toxin islet-activating protein (IAP) results in a dose-dependent inhibition of the release of arachidonic acid metabolites and prostacyclin in response to leukotriene D4 and leukot...

  15. TAFII170 Interacts with the Concave Surface of TATA-Binding Protein To Inhibit Its DNA Binding Activity

    Pereira, Lloyd A.; van der Knaap, Jan A.; van den Boom, Vincent; van den Heuvel, Fiona A. J.; Timmers, H. T. Marc

    2001-01-01

    The human RNA polymerase II transcription factor B-TFIID consists of TATA-binding protein (TBP) and the TBP-associated factor (TAF) TAFII170 and can rapidly redistribute over promoter DNA. Here we report the identification of human TBP-binding regions in human TAFII170. We have defined the TBP interaction domain of TAFII170 within three amino-terminal regions: residues 2 to 137, 290 to 381, and 380 to 460. Each region contains a pair of Huntington-elongation-A subunit-Tor repeats and exhibits species-specific interactions with TBP family members. Remarkably, the altered-specificity TBP mutant (TBPAS) containing a triple mutation in the concave surface is defective for binding the TAFII170 amino-terminal region of residues 1 to 504. Furthermore, within this region the TAFII170 residues 290 to 381 can inhibit the interaction between Drosophila TAFII230 (residues 2 to 81) and TBP through competition for the concave surface of TBP. Biochemical analyses of TBP binding to the TATA box indicated that TAFII170 region 290-381 inhibits TBP-DNA complex formation. Importantly, the TBPAS mutant is less sensitive to TAFII170 inhibition. Collectively, our results support a mechanism in which TAFII170 induces high-mobility DNA binding by TBP through reversible interactions with its concave DNA binding surface. PMID:11585931

  16. An activation domain within the walleye dermal sarcoma virus retroviral cyclin protein is essential for inhibition of the viral promoter

    Walleye dermal sarcoma virus (WDSV) is a complex retrovirus associated with seasonal dermal sarcomas. Developing tumors have low levels of accessory gene transcripts, A1 and B, and regressing tumors have high levels of full-length and spliced transcripts. Transcript A1 encodes a retroviral cyclin (rv-cyclin) with limited homology to host cyclins. The rv-cyclin is physically linked to components of the transcriptional co-activator complex, Mediator, and regulates transcription. In walleye fibroblasts, it inhibits the WDSV promoter independently of cis-acting DNA sequences. The rv-cyclin activates transcription from GAL4 promoters when fused to the GAL4 DNA binding domain. A 30 a.a. activation domain in the carboxy region can be inactivated by single point mutations, and these mutations diminish the ability of the rv-cyclin to inhibit the WDSV promoter. When fused to glutathione S-transferase, the rv-cyclin, its carboxy region, and the activation domain pull down components of transcription complexes from nuclear extracts, and pulldown is lost by mutation of the activation domain

  17. Aldehyde dehydrogenase type 2 activation by adenosine and histamine inhibits ischemic norepinephrine release in cardiac sympathetic neurons: mediation by protein kinase Cε.

    Robador, Pablo A; Seyedi, Nahid; Chan, Noel Yan-Ki; Koda, Kenichiro; Levi, Roberto

    2012-10-01

    During myocardial ischemia/reperfusion, lipid peroxidation leads to the formation of toxic aldehydes that contribute to ischemic dysfunction. Mitochondrial aldehyde dehydrogenase type 2 (ALDH2) alleviates ischemic heart damage and reperfusion arrhythmias via aldehyde detoxification. Because excessive norepinephrine release in the heart is a pivotal arrhythmogenic mechanism, we hypothesized that neuronal ALDH2 activation might diminish ischemic norepinephrine release. Incubation of cardiac sympathetic nerve endings with acetaldehyde, at concentrations achieved in myocardial ischemia, caused a concentration-dependent increase in norepinephrine release. A major increase in norepinephrine release also occurred when sympathetic nerve endings were incubated in hypoxic conditions. ALDH2 activation substantially reduced acetaldehyde- and hypoxia-induced norepinephrine release, an action prevented by inhibition of ALDH2 or protein kinase Cε (PKCε). Selective activation of G(i/o)-coupled adenosine A(1), A(3), or histamine H(3) receptors markedly inhibited both acetaldehyde- and hypoxia-induced norepinephrine release. These effects were also abolished by PKCε and/or ALDH2 inhibition. Moreover, A(1)-, A(3)-, or H(3)-receptor activation increased ALDH2 activity in a sympathetic neuron model (differentiated PC12 cells stably transfected with H(3) receptors). This action was prevented by the inhibition of PKCε and ALDH2. Our findings suggest the existence in sympathetic neurons of a protective pathway initiated by A(1)-, A(3)-, and H(3)-receptor activation by adenosine and histamine released in close proximity of these terminals. This pathway comprises the sequential activation of PKCε and ALDH2, culminating in aldehyde detoxification and inhibition of hypoxic norepinephrine release. Thus, pharmacological activation of PKCε and ALDH2 in cardiac sympathetic nerves may have significant protective effects by alleviating norepinephrine-induced life-threatening arrhythmias that

  18. Human Bocavirus NS1 and NS1-70 Proteins Inhibit TNF-α-Mediated Activation of NF-κB by targeting p65.

    Liu, Qingshi; Zhang, Zhenfeng; Zheng, Zhenhua; Zheng, Caishang; Liu, Yan; Hu, Qinxue; Ke, Xianliang; Wang, Hanzhong

    2016-01-01

    Human bocavirus (HBoV), a parvovirus, is a single-stranded DNA etiologic agent causing lower respiratory tract infections in young children worldwide. Nuclear factor kappa B (NF-κB) transcription factors play crucial roles in clearance of invading viruses through activation of many physiological processes. Previous investigation showed that HBoV infection could significantly upregulate the level of TNF-α which is a strong NF-κB stimulator. Here we investigated whether HBoV proteins modulate TNF-α-mediated activation of the NF-κB signaling pathway. We showed that HBoV NS1 and NS1-70 proteins blocked NF-κB activation in response to TNF-α. Overexpression of TNF receptor-associated factor 2 (TRAF2)-, IκB kinase alpha (IKKα)-, IκB kinase beta (IKKβ)-, constitutively active mutant of IKKβ (IKKβ SS/EE)-, or p65-induced NF-κB activation was inhibited by NS1 and NS1-70. Furthermore, NS1 and NS1-70 didn't interfere with TNF-α-mediated IκBα phosphorylation and degradation, nor p65 nuclear translocation. Coimmunoprecipitation assays confirmed the interaction of both NS1 and NS1-70 with p65. Of note, NS1 but not NS1-70 inhibited TNF-α-mediated p65 phosphorylation at ser536. Our findings together indicate that HBoV NS1 and NS1-70 inhibit NF-κB activation. This is the first time that HBoV has been shown to inhibit NF-κB activation, revealing a potential immune-evasion mechanism that is likely important for HBoV pathogenesis. PMID:27329558

  19. A Cell-Based Fluorescent Assay to Detect the Activity of Shiga Toxin and Other Toxins That Inhibit Protein Synthesis

    Escherichia coli O157:H7, a major cause of food-borne illness, produces Shiga toxins that block protein synthesis by inactivating the ribosome. In this chapter we describe a simple cell-based fluorescent assay to detect Shiga toxins and inhibitors of toxin activity. The assay can also be used to d...

  20. Radiation inhibits proteasomes and increases ubiquitinated proteins

    Full text: Exposure of cells to ionizing radiation results in accumulation of a number of short lived proteins that mediate cell survival/death, proliferation, repair, and differentiation. Expression of most of these proteins, including p53, mdm2, p21, c-jun, IkB-a, bcl-2, bax, cyclins A, B, E, Cdc25A, DNA-PKcs, and caspase-3 is regulated at the post-transcriptional level through ubiquitin/26S proteasome pathway. Several previous studies have shown that inhibition of proteasome activity by drugs leads to accumulation of ubiquitinated proteins. In this study we show that irradiation can do the same due to its inhibitory effect on 26S, but not 20S, proteasome activity. Two prostate cancer cell lines, murine TRAMP-C1 and human PC3, were used to examine the effect of ionizing radiation on the catalytic activity of the 26S proteasome. Cells were irradiated with different doses ranging from 0.25 to 20 Gy and lysed at different time points after irradiation. Crude extracts of both cell lines showed a rapid 30-50% decrease in chymotryptic activity of the 26S proteasome, as measured by a fluorogenic assay. The same level of inhibition was observed if purified 26S proteasomes were themselves irradiated, indicating that radiation has direct effects on this multicatalytic enzyme complex. Neither direct irradiation of proteasomes or cells had effect on 20S catalytic activity, suggesting that radiation selectively acts on 26S structure. Next, we examined whether this partial inhibition had any effect on ability of 26S proteasome to efficiently remove ubiquitinated proteins. Cells were irradiated with 10Gy and lysed at different time points. Ubiquitinated proteins were precipitated and examined by Western blot. Levels of ubiquitinated conjugates slowly increased over time and peaked at 7h post-irradiation. Accumulation of ubiquitinated conjugates has been shown to lead to formation of protein aggregates which can induce cell death. It has also been shown that monoubiquitination

  1. Vitamin D Binding Protein-Macrophage Activating Factor (DBP-maf Inhibits Angiogenesis and Tumor Growth in Mice

    Oliver Kisker

    2003-01-01

    Full Text Available We have isolated a selectively deglycosylated form of vitamin D binding protein (DBP-maf generated from systemically available DBP by a human pancreatic cancer cell line. DBP-maf is anti proliferative for endothelial cells and antiangiogenic in the chorioallantoic membrane assay. DBP-maf administered daily was able to potently inhibit the growth of human pancreatic cancer in immune compromised mice (T/C=0.09. At higher doses, DBP-maf caused tumor regression. Histological examination revealed that treated tumors had a higher number of infiltrating macrophages as well as reduced microvessel density, and increased levels of apoptosis relative to untreated tumors. Taken together, these data suggest that DBP-maf is an antiangiogenic molecule that can act directly on endothelium as well as stimulate macrophages to attack both the endothelial and tumor cell compartment of a growing malignancy.

  2. Receptor-mediated inhibition of adenylate cyclase and stimulation of arachidonic acid release in 3T3 fibroblasts. Selective susceptibility to islet-activating protein, pertussis toxin

    Thrombin exhibited diverse effects on mouse 3T3 fibroblasts. It (a) decreased cAMP in the cell suspension, (b) inhibited adenylate cyclase in the Lubrol-permeabilized cell suspension in a GTP-dependent manner, increased releases of (c) arachidonic acid and (d) inositol from the cell monolayer prelabeled with these labeled compounds, (e) increased 45Ca2+ uptake into the cell monolayer, and (f) increased 86Rb+ uptake into the cell monolayer in a ouabain-sensitive manner. Most of the effects were reproduced by bradykinin, platelet-activating factor, and angiotensin II. The receptors for these agonists are thus likely to be linked to three separate effector systems: the adenylate cyclase inhibition, the phosphoinositide breakdown leading to Ca2+ mobilization and phospholipase A2 activation, and the Na,K-ATPase activation. Among the effects of these agonists, (a), (b), (c), and (e) were abolished, but (d) and (f) were not, by prior treatment of the cells with islet-activating protein (IAP), pertussis toxin, which ADP-ribosylates the Mr = 41,000 protein, the alpha-subunit of the inhibitory guanine nucleotide regulatory protein (Ni), thereby abolishing receptor-mediated inhibition of adenylate cyclase. The effects (a), (c), (d), and (e) of thrombin, but not (b), were mimicked by A23187, a calcium ionophore. The effects of A23187, in contrast to those of receptor agonists, were not affected by the treatment of cells with IAP. Thus, the IAP substrate, the alpha-subunit of Ni, or the protein alike, may play an additional role in signal transduction arising from the Ca2+-mobilizing receptors, probably mediating process(es) distal to phosphoinositide breakdown and proximal to Ca2+ gating

  3. AMPK-Activated Protein Kinase Suppresses Ccr2 Expression by Inhibiting the NF-κB Pathway in RAW264.7 Macrophages

    Kumase, Fumiaki; Takeuchi, Kimio; Morizane, Yuki; Suzuki, Jun; Matsumoto, Hidetaka; Kataoka, Keiko; Al-Moujahed, Ahmad; Maidana, Daniel E.; Miller, Joan W.; Vavvas, Demetrios G.

    2016-01-01

    C-C chemokine receptor 2 (Ccr2) is a key pro-inflammatory marker of classic (M1) macrophage activation. Although Ccr2 is known to be expressed both constitutively and inductively, the full regulatory mechanism of its expression remains unclear. AMP-activated protein kinase (AMPK) is not only a master regulator of energy homeostasis but also a central regulator of inflammation. In this study, we sought to assess AMPK’s role in regulating RAW264.7 macrophage Ccr2 protein levels in resting (M0) or LPS-induced M1 states. In both M0 and M1 RAW264.7 macrophages, knockdown of the AMPKα1 subunit by siRNA led to increased Ccr2 levels whereas pharmacologic (A769662) activation of AMPK, attenuated LPS-induced increases in Ccr2 expression in an AMPK dependent fashion. The increases in Ccr2 levels by AMPK downregulation were partially reversed by NF-κB inhibition whereas TNF-a inhibition had minimal effects. Our results indicate that AMPK is a negative regulator of Ccr2 expression in RAW264.7 macrophages, and that the mechanism of action of AMPK inhibition of Ccr2 is mediated, in part, through the NF-κB pathway. PMID:26799633

  4. A novel human BTB-kelch protein KLHL31, strongly expressed in muscle and heart, inhibits transcriptional activities of TRE and SRE.

    Yu, Weishi; Li, Yongqing; Zhou, Xijin; Deng, Yun; Wang, Zequn; Yuan, Wuzhou; Li, Dali; Zhu, Chuanbing; Zhao, Xueying; Mo, Xiaoyang; Huang, Wen; Luo, Na; Yan, Yan; Ocorr, Karen; Bodmer, Rolf; Wang, Yuequn; Wu, Xiushan

    2008-11-30

    The Bric-a-brac, Tramtrack, Broad-complex (BTB) domain is a protein-protein interaction domain that is found in many zinc finger transcription factors. BTB containing proteins play important roles in a variety of cellular functions including regulation of transcription, regulation of the cytoskeleton, protein ubiquitination, angiogenesis, and apoptosis. Here, we report the cloning and characterization of a novel human gene, KLHL31, from a human embryonic heart cDNA library. The cDNA of KLHL31 is 5743 bp long, encoding a protein product of 634 amino acids containing a BTB domain. The protein is highly conserved across different species. Western blot analysis indicates that the KLHL31 protein is abundantly expressed in both embryonic skeletal and heart tissue. In COS-7 cells, KLHL31 proteins are localized to both the nucleus and the cytoplasm. In primary cultures of nascent mouse cardiomyocytes, the majority of endogenous KLHL31 proteins are localized to the cytoplasm. KLHL31 acts as a transcription repressor when fused to GAL4 DNA-binding domain and deletion analysis indicates that the BTB domain is the main region responsible for this repression. Overexpression of KLHL31 in COS-7 cells inhibits the transcriptional activities of both the TPA-response element (TRE) and serum response element (SRE). KLHL31 also significantly reduces JNK activation leading to decreased phosphorylation and protein levels of the JNK target c-Jun in both COS-7 and Hela cells. These results suggest that KLHL31 protein may act as a new transcriptional repressor in MAPK/JNK signaling pathway to regulate cellular functions. PMID:18719355

  5. Zinc-L-carnosine binds to molecular chaperone HSP70 and inhibits the chaperone activity of the protein.

    Haga, Asami; Okamoto, Tomoya; Yamada, Shintaroh; Kubota, Toshihiko; Sanpei, Ann; Takahashi, Shota; Nakayama, Masahiro; Nagai, Miki; Otaka, Michiro; Miyazaki, Toshio; Nunomura, Wataru; Grave, Ewa; Itoh, Hideaki

    2013-09-01

    In this study, we have investigated the specific binding proteins of Zinc-L-carnosine (Polaprezinc) using Polaprezinc-affinity column chromatography in vitro. A protein having a 70-kDa molecular mass was eluted by the linear gradient of 0-1.0 mM Polaprezinc from the affinity column and the protein was identified as the molecular chaperone HSP70 by immunoblotting. The chaperone activity of HSP70 was completely suppressed by Polaprezinc. The ATPase activity of HSP70 was affected to some extent by the reagent. In the circular dichroism (CD) spectrum, the secondary structure of HSP70 was changed in the presence of Polaprezinc, i.e. it decreased in the α-helix. We have determined the Polaprezinc-binding domain of HSP70 by using recombinant HSP70N- and C-domains. Although Polaprezinc could bind to both the N-terminal and the C-terminal of HSP70, the HSP70N-domain has a high affinity to the drug. Regarding the peptide cleavage of the HSP70N- and C-domains with proteinase K, the intact HSP70N still remained in the presence of Polaprezinc. On the other hand, the quantity of the intact C-domain slightly decreased under the same conditions along with the newly digested small peptides appeared. It has been suggested that Polaprezinc binds to HSP70 especially in the N-domains, suppresses the chaperone activity and delays an ATPase activities of HSP70. PMID:23687308

  6. Tandutinib (MLN518) reverses multidrug resistance by inhibiting the efflux activity of the multidrug resistance protein 7 (ABCC10)

    Deng, Wen; Dai, Chun-ling; Chen, Jun-Jiang; KATHAWALA, RISHIL J.; SUN, YUE-LI; CHEN, HAI-FAN; Fu, Li-wu; Chen, Zhe-Sheng

    2013-01-01

    It is well established that ATP-binding cassette (ABC) transporter-mediated multidrug resistance (MDR) is one of the major mechanisms that causes resistance to antineoplastic drugs in cancer cells. ABC transporters can significantly decrease the intracellular concentration of antineoplastic drugs by increasing their efflux, thereby lowering their cytotoxic activity. One of these transporters, the multidrug resistance protein 7 (MRP7/ABCC10), has already been shown to produce resistance to ant...

  7. Gold Nanoparticle Aggregation as a Probe of Antifreeze (Glyco) Protein-Inspired Ice Recrystallization Inhibition and Identification of New IRI Active Macromolecules

    Mitchell, Daniel E.; Congdon, Thomas; Rodger, Alison; Gibson, Matthew I.

    2015-10-01

    Antifreeze (glyco)proteins are found in polar fish species and act to slow the rate of growth of ice crystals; a property known as ice recrystallization inhibition. The ability to slow ice growth is of huge technological importance especially in the cryopreservation of donor cells and tissue, but native antifreeze proteins are often not suitable, nor easily available. Therefore, the search for new materials that mimic this function is important, but currently limited by the low-throughout assays associated with the antifreeze properties. Here 30 nm gold nanoparticles are demonstrated to be useful colorimetric probes for ice recrystallization inhibition, giving a visible optical response and is compatible with 96 well plates for high-throughout studies. This method is faster, requires less infrastructure, and has easier interpretation than the currently used ‘splat’ methods. Using this method, a series of serum proteins were identified to have weak, but specific ice recrystallization inhibition activity, which was removed upon denaturation. It is hoped that high-throughput tools such as this will accelerate the discovery of new antifreeze mimics.

  8. Human Cytomegalovirus UL97 Kinase Activity Is Required for the Hyperphosphorylation of Retinoblastoma Protein and Inhibits the Formation of Nuclear Aggresomes

    Prichard, Mark N.; Sztul, Elizabeth; Daily, Shannon L.; Perry, Amie L.; Frederick, Samuel L.; Gill, Rachel B.; Hartline, Caroll B.; Streblow, Daniel N.; Varnum, Susan M.; Smith, Richard D.; Kern, Earl R.

    2008-05-01

    Cells infected with human cytomegalovirus in the absence of UL97 kinase activity produce large nuclear aggregates that sequester considerable quantities of viral proteins. A transient expression assay suggested that pp71 and IE1 were also involved in this process, and this suggestion was significant, since both proteins have been reported to interact with components of promyelocytic leukemia (PML) bodies (ND10) and also interact functionally with retinoblastoma pocket proteins (RB). PML bodies have been linked to the formation of nuclear aggresomes, and colocalization studies suggested that viral proteins were recruited to these structures and that UL97 kinase activity inhibited their formation. Proteins associated with PML bodies were examined by Western blot analysis, and pUL97 appeared to specifically affect the phosphorylation of RB in a kinasedependent manner. Three consensus RB binding motifs were identified in the UL97 kinase, and recombinant viruses were constructed in which each was mutated to assess a potential role in the phosphorylation of RB and the inhibition of nuclear aggresome formation. The mutation of either the conserved LxCxE RB binding moti for the lysine required for kinase activity impaired the ability of the virus to stabilize and phosphorylate RB. We concluded from these studies that both UL97 kinase activity and the LxCxE RB binding motif are required for the phosphorylation and stabilization of RB in infected cells and that this effect can be antagonized by the antiviral drug maribavir. These data also suggest a potential link between RB function and the formation of aggresomes.

  9. Sri Lankan black tea (Camellia sinensis L.) inhibits the methylglyoxal mediated protein glycation and potentiates its reversing activity in vitro

    Wanigasekara Daya Ratnasooriya

    2016-01-01

    Objective: To evaluate inhibitory activity of methylglyoxal (MGO) mediated protein glycation and ability to potentiate its reversing activity and range of antioxidant properties of Sri Lankan low grown orthodox orange pekoe grade black tea. Methods: Freeze dried black tea brew (BTB) was used as the sample in this study. Anti-glycation and glycation reversing activity was studied in bovine serum albumin (BSA)-MGO model. Antioxidant properties were studied using total polyphenolic content, total flavonoid content, 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid, 1,1-diphenyl-2-picrylhydrazine and ferric reducing antioxidant power in vitro antioxidant assays. Results: The results demonstrated significant (P Conclusions: The novel properties observed for Sri Lankan orange pekoe grade black tea indicate its usefulness as a supplementary beverage in managing MGO and advanced glycation end products related diseases and ailments.

  10. Transforming growth factor-β inhibits CCAAT/enhancer-binding protein expression and PPARγ activity in unloaded bone marrow stromal cells

    The molecular mechanisms regulating the adipogenic differentiation of bone marrow stromal cells in vivo remain largely unknown. In this study, we investigated the regulatory effects of transforming growth factor beta-2 (TGF-β2) on transcription factors involved in adipogenic differentiation induced by hind limb suspension in rat bone marrow stromal cells in vivo. Time course real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis of gene expression showed that skeletal unloading progressively increases the expression of CCAAT/enhancer-binding protein (C/EBP)α and C/EBPβ α at 5 days in bone marrow stromal cells resulting in increased peroxisome proliferator-activated receptor γ (PPARγ2) transcripts at 7 days. TGF-β2 administration in unloaded rats corrected the rise in C/EBPα and C/EBPβ transcripts induced by unloading in bone marrow stromal cells. This resulted in inhibition of PPARγ2 expression that was associated with increased Runx2 expression. Additionally, the inhibition of C/EBPα and C/EBPβ expression by TGF-β2 was associated with increased PPARγ serine phosphorylation in bone marrow stromal cells, a mechanism that inhibits PPARγ transactivating activity. The sequential inhibitory effect of TGF-β2 on C/EBPα, C/EBPβ, and PPARγ2 resulted in reduced LPL expression and abolition of bone marrow stromal cell adipogenic differentiation, which contributed to prevent bone loss induced by skeletal unloading. We conclude that TGF-β2 inhibits the excessive adipogenic differentiation of bone marrow stromal cells induced by skeletal unloading by inhibiting C/EBPα, C/EBPβ, and PPARγ expression and activity, which provides a sequential mechanism by which TGF-β2 regulates adipogenic differentiation of bone marrow stromal cells in vivo

  11. ArhGAP9, a novel MAP kinase docking protein, inhibits Erk and p38 activation through WW domain binding

    2007-01-01

    We have identified human ArhGAP9 as a novel MAP kinase docking protein that interacts with Erk2 and p38α through complementarily charged residues in the WW domain of ArhGAP9 and the CD domains of Erk2 and p38α. This interaction sequesters the MAP kinases in their inactive states through displacement of MAP kinase kinases targeting the same sites. While over-expression of wild type ArhGAP9 caused MAP kinase activation by the epidermal growth factor receptor (EGFR) to be suppressed and preserve...

  12. Activation of human monocytes by streptococcal rhamnose glucose polymers is mediated by CD14 antigen, and mannan binding protein inhibits TNF-alpha release.

    Soell, M; Lett, E; Holveck, F; Schöller, M; Wachsmann, D; Klein, J P

    1995-01-15

    The present work was initiated to define mechanisms that account for the binding on human monocytes of streptococcal cell wall polysaccharides formed by rhamnose glucose polymers (RGPs), and subsequent stimulatory activities. We show here that RGPs bind to and stimulate human monocytes to produce TNF-alpha in a dose-dependent manner. To detect cell surface RGPs binding proteins, intact monocytes were biotinylated before lysis with Nonidet P-40 and solubilized proteins were incubated with RGPs Affi-Prep beads. One major membrane protein of 55 kDa was specifically detected and identified as CD14 because it reacted with anti-CD14 mAbs. Furthermore, anti-CD14 mAbs were able to perform a dose-dependent inhibition of RGPs binding, and suppressed TNF-alpha release from RGPs-stimulated monocytes. Moreover, we demonstrated that RGPs also bind to CD11b; however, this binding is not implicated in synthesis of TNF-alpha. Interestingly, RGPs binding to monocytes was enhanced by human normal serum (HNS) whereas HNS inhibits the TNF-alpha-stimulating activity of RGPs. Western blotting analysis of HNS proteins purified on RGPs Affi-prep beads revealed three specific bands of 75, 55, and 32 kDa reactive with anti-C3 Abs, anti-CD14 mAbs (TUK4), and anti-human mannan binding protein (hMBP)-derived peptide IgG, respectively. These results suggest that C3, soluble CD14, and hMBP form complexes that are probably active in enhancing the binding of RGPs to monocytes. Additional studies have shown that hMBP that recognizes RGPs prevents, unlike the LPS binding protein, TNF-alpha release by inhibiting the binding of RGPs to CD14 Ag. By incubating cells with a constant amount of RGPs-hMBP complexes in the presence or absence of increasing concentrations of C1q, we also demonstrated that C1q receptor mediates the binding and probably the uptake of RGPs-hMBP complexes by human monocytes. PMID:7529289

  13. Avian renal proximal tubule urate secretion is inhibited by cellular stress-induced AMP-activated protein kinase.

    Bataille, Amy M; Maffeo, Carla L; Renfro, J Larry

    2011-06-01

    Urate is a potent antioxidant at high concentrations but it has also been associated with a wide variety of health risks. Plasma urate concentration is determined by ingestion, production, and urinary excretion; however, factors that regulate urate excretion remain uncertain. The objective of this study was to determine whether cellular stress, which has been shown to affect other renal transport properties, modulates urate secretion in the avian renal proximal tubule. Chick kidney proximal tubule epithelial cell primary culture monolayers were used to study the transepithelial transport of radiolabeled urate. This model allowed examination of the processes, such as multidrug resistance protein 4 (Mrp4, Abcc4), which subserve urate secretion in a functional, intact, homologous system. Our results show that the recently implicated urate efflux transporter, breast cancer resistance protein (ABCG2), does not significantly contribute to urate secretion in this system. Exposure to a high concentration of zinc for 6 h induced a cellular stress response and a striking decrease in transepithelial urate secretion. Acute exposure to zinc had no effect on transepithelial urate secretion or isolated membrane vesicle urate transport, suggesting involvement of a cellular stress adaptation. Activation of AMP-activated protein kinase (AMPK), a candidate modulator of ATP-dependent urate efflux, by 5'-aminoimidazole-4-carboxamide 1-β-d-ribo-furanoside caused a decrease in urate secretion similar to that seen with zinc-induced cellular stress. This effect was prevented with the AMPK inhibitor compound C. Notably, the decrease in urate secretion seen with zinc-induced cellular stress was also prevented by compound C, implicating AMPK in regulation of renal uric acid excretion. PMID:21429974

  14. Recombinant human activated protein C inhibits local and systemic activation of coagulation without influencing inflammation during Pseudomonas aeruginosa pneumonia in rats

    Choi, Goda; Hofstra, Jorrit-Jan H; Roelofs, Joris J T H; Florquin, Sandrine; Bresser, Paul; Levi, Marcel; van der Poll, Tom; Schultz, Marcus J

    2007-01-01

    OBJECTIVE: Alveolar fibrin deposition is a hallmark of pneumonia. It has been proposed that recombinant human activated protein C exerts lung-protective effects via anticoagulant and anti-inflammatory pathways. We investigated the role of the protein C system in pneumonia caused by Pseudomonas aerug

  15. Theophylline Represses IL-8 Secretion from Airway Smooth Muscle Cells Independently of Phosphodiesterase Inhibition. Novel Role as a Protein Phosphatase 2A Activator.

    Patel, Brijeshkumar S; Rahman, Md Mostafizur; Rumzhum, Nowshin N; Oliver, Brian G; Verrills, Nicole M; Ammit, Alaina J

    2016-06-01

    Theophylline is an old drug experiencing a renaissance owing to its beneficial antiinflammatory effects in chronic respiratory diseases, such as asthma and chronic obstructive pulmonary disease. Multiple modes of antiinflammatory action have been reported, including inhibition of the enzymes that degrade cAMP-phosphodiesterase (PDE). Using primary cultures of airway smooth muscle (ASM) cells, we recently revealed that PDE4 inhibitors can potentiate the antiinflammatory action of β2-agonists by augmenting cAMP-dependent expression of the phosphatase that deactivates mitogen-activated protein kinase (MAPK)-MAPK phosphatase (MKP)-1. Therefore, the aim of this study was to address whether theophylline repressed cytokine production in a similar, PDE-dependent, MKP-1-mediated manner. Notably, theophylline did not potentiate cAMP release from ASM cells treated with the long-acting β2-agonist formoterol. Moreover, theophylline (0.1-10 μM) did not increase formoterol-induced MKP-1 messenger RNA expression nor protein up-regulation, consistent with the lack of cAMP generation. However, theophylline (at 10 μM) was antiinflammatory and repressed secretion of the neutrophil chemoattractant cytokine IL-8, which is produced in response to TNF-α. Because theophylline's effects were independent of PDE4 inhibition or antiinflammatory MKP-1, we then wished to elucidate the novel mechanisms responsible. We investigated the impact of theophylline on protein phosphatase (PP) 2A, a master controller of multiple inflammatory signaling pathways, and show that theophylline increases TNF-α-induced PP2A activity in ASM cells. Confirmatory results were obtained in A549 lung epithelial cells. PP2A activators have beneficial effects in ex vivo and in vivo models of respiratory disease. Thus, our study is the first to link theophylline with PP2A activation as a novel mechanism to control respiratory inflammation. PMID:26574643

  16. High Glucose Inhibits the Aspirin-Induced Activation of the Nitric Oxide/cGMP/cGMP-Dependent Protein Kinase Pathway and Does Not Affect the Aspirin-Induced Inhibition of Thromboxane Synthesis in Human Platelets

    Russo, Isabella; Viretto, Michela; Barale, Cristina; Mattiello, Luigi; Doronzo, Gabriella; Pagliarino, Andrea; Cavalot, Franco; Trovati, Mariella; Anfossi, Giovanni

    2012-01-01

    Since hyperglycemia is involved in the “aspirin resistance” occurring in diabetes, we aimed at evaluating whether high glucose interferes with the aspirin-induced inhibition of thromboxane synthesis and/or activation of the nitric oxide (NO)/cGMP/cGMP-dependent protein kinase (PKG) pathway in platelets. For this purpose, in platelets from 60 healthy volunteers incubated for 60 min with 5–25 mmol/L d-glucose or iso-osmolar mannitol, we evaluated the influence of a 30-min incubation with lysine...

  17. Fluvastatin inhibits activation of JAK and STAT proteins in diabetic rat glomeruli and mesangial cells under high glucose conditions

    Yong-hong SHI; Song ZHAO; Chen WANG; Ying LI; Hui-jun DUAN

    2007-01-01

    Aim: The aim of the present study was to further elucidate the mechanism of the protective role of fluvastatin on diabetic nephropathy. Methods: Streptozotocin- induced diabetic rats were treated daily with fluvastatin (4 mg/kg body weight) by gavage. The animals were killed 4 weeks later and urine and blood samples were collected. The kidney tissues were removed and subjected to the following experiments. Rat glomerular mesangial cells (GMC) were cultured under normal glucose (5.5 mmol/L), high glucose (HG, 30 mmol/L), HG+AG490 (10 μmol/L), or HG with fluvastatin (1 pmol/L). Glomeruli or the GMC lysate was immunoprecipi- tated and/or immunoblotted with antibodies against Janus kinase 2 (JAK2), SH2- domain containing tyrosine phosphatase-1 (SHP-1), phosphospecific SHP-2, and signal transducer and activators of transcription (STAT), respectively. Trans- forming growth factor-β (TGF-β) mRNA was measured by RT-PCR. The protein synthesis of TGF-β1 and fibronectin in the culture medium of GMC was detected by ELISA. Results: The phosphorylation levels of JAK2, STAT1, STAT3, and SHP-2 increased significantly, and SHP-1 phosphorylation was reduced in glom- eruli of diabetic rats. Treatment with fluvastatin reduced phosphorylation levels of JAK2, STAT1, STAT3, and SHP-2 in glomeruli of diabetic rats, but it had no effect on the dephosphorylation of SHP-1. The exposure of GMC to 30 mmol/L glucose caused the activation of JAK2, STAT1, STAT3, and SHP-2. It upregulated TGF-β1 expression and increased protein synthesis of fibronectin. These high glucose-induced changes were suppressed by fluvastatin, as well as AG490, a JAK2 inhibitor. Conclusion: The regulation of the phosphorylation of JAK/STAT by fluvastatin may be responsible for its renal protective effects on diabetic nephropathy.

  18. Inhibition of protein tyrosine phosphatase activity mediates epidermal growth factor receptor signaling in human airway epithelial cells exposed to Zn2+

    Epidemiological studies have implicated zinc (Zn2+) in the toxicity of ambient particulate matter (PM) inhalation. We previously showed that exposure to metal-laden PM inhibits protein tyrosine phosphatase (PTP) activity in human primary bronchial epithelial cells (HAEC) and leads to Src-dependent activation of EGFR signaling in B82 and A431 cells. In order to elucidate the mechanism of Zn2+-induced EGFR activation in HAEC, we treated HAEC with 500 μM ZnSO4 for 5-20 min and measured the state of activation of EGFR, c-Src and PTPs. Western blots revealed that exposure to Zn2+ results in increased phosphorylation at both trans- and autophosphorylation sites in the EGFR. Zn2+-mediated EGFR phosphorylation did not require ligand binding and was ablated by the EGFR kinase inhibitor PD153035, but not by the Src kinase inhibitor PP2. Src activity was inhibited by Zn2+ treatment of HAEC, consistent with Src-independent EGFR transactivation in HAEC exposed to Zn2+. The rate of exogenous EGFR dephosphorylation in lysates of HAEC exposed to Zn2+ or V4+ was significantly diminished. Moreover, exposure of HAEC to Zn2+ also resulted in a significant impairment of dephosphorylation of endogenous EGFR. These data show that Zn2+-induced activation of EGFR in HAEC involves a loss of PTP activities whose function is to dephosphorylate EGFR in opposition to baseline EGFR kinase activity. These findings also suggest that there are marked cell-type-specific differences in the mechanism of EGFR activation induced by Zn2+ exposure

  19. Gecko proteins induce the apoptosis of bladder cancer 5637 cells by inhibiting Akt and activating the intrinsic caspase cascade

    Kim, Geun-Young; Park, Soon Yong; Jo, Ara; Kim, Mira; Leem, Sun-Hee; Jun, Woo-Jin; Shim, Sang In; Lee, Sang Chul; Chung, Jin Woong

    2015-01-01

    Gecko proteins have long been used as anti-tumor agents in oriental medicine, without any scientific background. Although anti-tumor effects of Gecko proteins on several cancers were recently reported, their effect on bladder cancer has not been investigated. Thus, we explored the anti-tumor effect of Gecko proteins and its cellular mechanisms in human bladder cancer 5637 cells. Gecko proteins significantly reduced the viability of 5637 cells without any cytotoxic effect on normal cells. Thes...

  20. Mungbean yellow mosaic Indian virus encoded AC2 protein suppresses RNA silencing by inhibiting Arabidopsis RDR6 and AGO1 activities.

    Kumar, Vikash; Mishra, Sumona Karjee; Rahman, Jamilur; Taneja, Jyoti; Sundaresan, Geethaa; Mishra, Neeti Sanan; Mukherjee, Sunil K

    2015-12-01

    RNA silencing refers to a conserved RNA-directed gene regulatory mechanism in a wide range of eukaryotes. It plays an important role in many processes including growth, development, genome stability, and antiviral defense in the plants. Geminivirus encoded AC2 is identified as an RNA silencing suppressor protein, however, the mechanism of action has not been characterized. In this paper, we elucidate another mechanism of AC2-mediated suppression activity of Mungbean Yellow Mosaic India Virus (MYMIV). The AC2 protein, unlike many other suppressors, does not bind to siRNA or dsRNA species and its suppression activity is mediated through interaction with key components of the RNA silencing pathway, viz., RDR6 and AGO1. AC2 interaction inhibits the RDR6 activity, an essential component of siRNA and tasi-RNA biogenesis and AGO1, the major slicing factor of RISC. Thus the study identifies dual sites of MYMIV-AC2 interference and probably accounts for its strong RNA silencing suppression activity. PMID:26433748

  1. Terpenic fraction of Pterodon pubescens inhibits nuclear factor kappa B and extracellular signal-regulated protein Kinase 1/2 activation and deregulates gene expression in leukemia cells

    Pereira Monica Farah

    2012-11-01

    Full Text Available Abstract Background Plant derived compounds have been shown to be important sources of several anti-cancer agents. As cell cycle deregulation and tumor growth are intimately linked, the discovery of new substances targeting events in this biochemical pathway would be of great value. The anti-leukemic effect of an ethanolic extract of Pterodon pubescens seeds (EEPp has been previously demonstrated and now we show that a terpenic subfraction (SF5 of EEPp containing farnesol, geranylgeraniol and vouacapan derivatives induces apoptosis in the human chronic myelogenous leukemia cell line K562. This work addresses SF5’s antiproliferative mechanisms in these cells since they are still unclear. Methods DNA synthesis in K562 cells was assessed by [3H]-methyl-thymidine incorporation and cell cycle status by flow cytometry. The expression of cyclins D1 and E2, of the cell cycle inhibitor p21 and of the proto-oncogene c-myc was evaluated by semi-quantitative RT-PCR. Extracellular-signal-regulated kinases (ERK 1/2 and nuclear factor kappa B (NF-κB activation was evaluated by western blotting. Results In K562 cells, SF5 treatment induced a higher inhibition of DNA synthesis and cell growth than the original EEPp hexanic fraction from which SF5 originated, and also arrested the cell cycle in G1. Exposure of these cells to SF5 led to a decrease in cyclin E2 and c-myc expression while p21 mRNA levels were increased. Furthermore, SF5 inhibited the activation of mitogen-activated protein kinase (MAPK ERK 1/2 and NF-κB. Conclusions This work suggests that the anti-leukemic action of SF5 is linked to the inhibition of ERKs, NF-κB and c-myc signaling pathways resulting in reduced cyclin E2 mRNA expression and cell cycle arrest in the G1 phase.

  2. Antiviral activities of whey proteins.

    Ng, Tzi Bun; Cheung, Randy Chi Fai; Wong, Jack Ho; Wang, Yan; Ip, Denis Tsz Ming; Wan, David Chi Cheong; Xia, Jiang

    2015-09-01

    Milk contains an array of proteins with useful bioactivities. Many milk proteins encompassing native or chemically modified casein, lactoferrin, alpha-lactalbumin, and beta-lactoglobulin demonstrated antiviral activities. Casein and alpha-lactalbumin gained anti-HIV activity after modification with 3-hydroxyphthalic anhydride. Many milk proteins inhibited HIV reverse transcriptase. Bovine glycolactin, angiogenin-1, lactogenin, casein, alpha-lactalbumin, beta-lactoglobulin, bovine lactoferrampin, and human lactoferrampin inhibited HIV-1 protease and integrase. Several mammalian lactoferrins prevented hepatitis C infection. Lactoferrin, methylated alpha-lactalbumin and methylated beta-lactoglobulin inhibited human cytomegalovirus. Chemically modified alpha-lactalbumin, beta-lactoglobulin and lysozyme, lactoferrin and lactoferricin, methylated alpha-lactalbumin, methylated and ethylated beta-lactoglobulins inhibited HSV. Chemically modified bovine beta-lactoglobulin had antihuman papillomavirus activity. Beta-lactoglobulin, lactoferrin, esterified beta-lactoglobulin, and esterified lactoferrindisplayed anti-avian influenza A (H5N1) activity. Lactoferrin inhibited respiratory syncytial virus, hepatitis B virus, adenovirus, poliovirus, hantavirus, sindbis virus, semliki forest virus, echovirus, and enterovirus. Milk mucin, apolactoferrin, Fe(3+)-lactoferrin, beta-lactoglobulin, human lactadherin, bovine IgG, and bovine kappa-casein demonstrated antihuman rotavirus activity. PMID:26198883

  3. Enzymatic Hydrolysis of Salmon By-products: Effect of Process Conditions on ACE Inhibiting Activities of Fish Protein Hydrolysates

    Five, Kathrine

    2013-01-01

    By-products from the salmon farming industry contain valuable components, such as proteins and lipids. By-products like frames, heads and viscera can be used as raw material for the production of fish protein hydrolysates with high nutritional value, but also bioactive properties. The hydrolysates are produced by enzymatic hydrolysis using endogenous and commercial enzymes, and the process conditions and raw material influence the properties of the hydrolysate. The first aim of this thesis wa...

  4. Polyene antibiotic that inhibits membrane transport proteins.

    te Welscher, Yvonne Maria; van Leeuwen, Martin Richard; de Kruijff, Ben; Dijksterhuis, Jan; Breukink, Eefjan

    2012-07-10

    The limited therapeutic arsenal and the increase in reports of fungal resistance to multiple antifungal agents have made fungal infections a major therapeutic challenge. The polyene antibiotics are the only group of antifungal antibiotics that directly target the plasma membrane via a specific interaction with the main fungal sterol, ergosterol, often resulting in membrane permeabilization. In contrast to other polyene antibiotics that form pores in the membrane, the mode of action of natamycin has remained obscure but is not related to membrane permeabilization. Here, we demonstrate that natamycin inhibits growth of yeasts and fungi via the immediate inhibition of amino acid and glucose transport across the plasma membrane. This is attributable to ergosterol-specific and reversible inhibition of membrane transport proteins. It is proposed that ergosterol-dependent inhibition of membrane proteins is a general mode of action of all the polyene antibiotics, of which some have been shown additionally to permeabilize the plasma membrane. Our results imply that sterol-protein interactions are fundamentally important for protein function even for those proteins that are not known to reside in sterol-rich domains. PMID:22733749

  5. Stanniocalcin-1 inhibits renal ischemia/reperfusion injury via an AMP-activated protein kinase-dependent pathway

    AKI is associated with increased morbidity, mortality, and cost of care, and therapeutic options remain limited. Reactive oxygen species are critical for the genesis of ischemic AKI. Stanniocalcin-1 (STC1) suppresses superoxide generation through induction of uncoupling proteins (UCPs), and transgen...

  6. p42/p44 mitogen-activated protein kinases inhibit atrial natriuretic peptide mRNA transcription in gp130-mediated hypertrophic ventricular myocytes

    Zhan-Ling Dong; Yang Wang; Tian-Fa Li; Shao-Jiang Zheng; Yue-Qiong Kong; You-Ling Lan; Jun-Li Guo; Shi-Gan Fu

    2014-01-01

    Objective:To understand the role ofANP mRNA transcription regulation in gp130-mediated cardiomyocyte hypertrophy, and the involved mitogen-activated protein kinase kinase(MEK)-extracellular signal-regulated kinase(ERK, also called p42/p44MAPK) signaling pathway. Methods:Isolated neonatal ventricular myocytes were treated with different concentrations of CT-1(10-9,10-8 and10-7 mol/L).MTT was used to analyze the viability andRT-PCR was used to detectANP mRNA levels in cardiomyocyte.To inhibit p42/p44MAPK activity in hypertrophic cardiomyocytes, the cells were pretreated with a specificMEK1 inhibitor.Results:CT-1 significantly inducedANP mRNA expression and the viability of cardiomyocytes in a dose- and time-dependent manner.Furthermore, blocking p42/p44MAPK activity by the special MEK1 inhibitor upregulated theANP mRNA.Conclusions: p42/p44MAPK have an important role in suppressingANP mRNA transcription and cell activity in gp130-mediated hypertrophic ventricular myocytes.

  7. Histamine H4-receptors inhibit mast cell renin release in ischemia/reperfusion via protein kinase C ε-dependent aldehyde dehydrogenase type-2 activation.

    Aldi, Silvia; Takano, Ken-ichi; Tomita, Kengo; Koda, Kenichiro; Chan, Noel Y-K; Marino, Alice; Salazar-Rodriguez, Mariselis; Thurmond, Robin L; Levi, Roberto

    2014-06-01

    Renin released by ischemia/reperfusion (I/R) from cardiac mast cells (MCs) activates a local renin-angiotensin system (RAS) causing arrhythmic dysfunction. Ischemic preconditioning (IPC) inhibits MC renin release and consequent activation of this local RAS. We postulated that MC histamine H4-receptors (H4Rs), being Gαi/o-coupled, might activate a protein kinase C isotype-ε (PKCε)-aldehyde dehydrogenase type-2 (ALDH2) cascade, ultimately eliminating MC-degranulating and renin-releasing effects of aldehydes formed in I/R and associated arrhythmias. We tested this hypothesis in ex vivo hearts, human mastocytoma cells, and bone marrow-derived MCs from wild-type and H4R knockout mice. We found that activation of MC H4Rs mimics the cardioprotective anti-RAS effects of IPC and that protection depends on the sequential activation of PKCε and ALDH2 in MCs, reducing aldehyde-induced MC degranulation and renin release and alleviating reperfusion arrhythmias. These cardioprotective effects are mimicked by selective H4R agonists and disappear when H4Rs are pharmacologically blocked or genetically deleted. Our results uncover a novel cardioprotective pathway in I/R, whereby activation of H4Rs on the MC membrane, possibly by MC-derived histamine, leads sequentially to PKCε and ALDH2 activation, reduction of toxic aldehyde-induced MC renin release, prevention of RAS activation, reduction of norepinephrine release, and ultimately to alleviation of reperfusion arrhythmias. This newly discovered protective pathway suggests that MC H4Rs may represent a new pharmacologic and therapeutic target for the direct alleviation of RAS-induced cardiac dysfunctions, including ischemic heart disease and congestive heart failure. PMID:24696042

  8. Activating transcription factor-3 (ATF3) functions as a tumor suppressor in colon cancer and is up-regulated upon heat-shock protein 90 (Hsp90) inhibition

    Activating transcription factor-3 (ATF3) is involved in the complex process of cellular stress response. However, its exact role in cancer is discussed controversially because both tumor suppressive and oncogenic effects have been described. Here we followed-up on our previous observation that inhibition of Hsp90 may increase ATF3 expression and sought to determine the role of ATF3 in colon cancer. Regulation of ATF3 was determined in cancer cells using signaling inhibitors and a heat-shock protein-90 (Hsp90) antagonist. Human HCT116 cancer cells were stably transfected with an ATF3-shRNA or a luciferase-shRNA expression plasmid and alterations in cell motility were assessed in migration assays. The impact of ATF3 down-regulation on cancer growth and metastasis were investigated in a subcutaneous tumor model, a model of hepatic tumor growth and in a model of peritoneal carcinomatosis. Human colon cancer tissues were analyzed for ATF3 expression. The results show that therapeutic Hsp90 inhibition substantially up-regulates the expression of ATF3 in various cancer cells, including colon, gastric and pancreatic cancer. This effect was evident both in vitro and in vivo. RNAi mediated knock-down of ATF3 in HCT116 colon cancer cells significantly increased cancer cell migration in vitro. Moreover, in xenogenic mouse models, ATF3 knock-down promoted subcutaneous tumor growth and hepatic metastasis, as well as peritoneal carcinomatosis. Importantly, ATF3 expression was lower in human colon cancer specimens, as compared to corresponding normal surrounding tissues, suggesting that ATF3 may represent a down-regulated tumor suppressor in colon cancer. In conclusion, ATF3 down-regulation in colon cancer promotes tumor growth and metastasis. Considering that blocking Hsp90 induces ATF3 expression, Hsp90 inhibition may represent a valid strategy to treat metastatic colon cancer by up-regulating this anti-metastatic transcription factor

  9. Pharmacological Inhibition of Protein Kinase G1 Enhances Bone Formation by Human Skeletal Stem Cells Through Activation of RhoA-Akt Signaling

    Kermani, Abbas Jafari; Siersbaek, Majken S; Chen, Li;

    2015-01-01

    for several malignant and nonmalignant conditions. We screened a library of kinase inhibitors to identify small molecules that enhance bone formation by human skeletal (stromal or mesenchymal) stem cells (hMSC). We identified H-8 (known to inhibit protein kinases A, C, and G) as a potent enhancer of...... differentiation and suggest that pharmacological inhibition of PRKG1 in hMSC implanted at the site of bone defect can enhance bone regeneration. Stem Cells 2015....

  10. N-ethylmaleimide inhibition of the DNA-binding activity of the herpes simplex virus type 1 major DNA-binding protein

    The major herpes simplex virus DNA-binding protein, designated ICP8, binds tightly to single-stranded DNA and is required for replication of viral DNA. The sensitivity of the DNA-binding activity of ICP8 to the action of the sulfhydryl reagent N-ethylmaleimide has been examined by using nitrocellulose filter-binding and agarose gel electrophoresis assays. Incubation of ICP8 with N-ethylmaleimide results in a rapid loss of DNA-binding activity. Preincubation of ICP8 with single-stranded DNA markedly inhibits this loss of binding activity. These results imply that a free sulfhydryl group is involved in the interaction of ICP8 with single-stranded DNA and that this sulfhydryl group becomes less accessible to the environment upon binding. Agarose gel electrophoretic analysis of the binding interaction in the presence and absence of N-ethylmaleimide indicates that the cooperative binding exhibited by ICP8 is lost upon treatment with this reagent but that some residual noncooperative binding may remain. This last result was confirmed by equilibrium dialysis experiments with the 32P-labeled oligonucleotide dT10 and native and N-ethylmaleimide-treated ICP8

  11. Inhibition of p38 Mitogen-Activated Protein Kinase Enhances the Apoptosis Induced by Oxidized Low-Density Lipoprotein in Endothelial Progenitor Cells.

    Tie, Guodong; Yan, Jinglian; Messina, Julia A; Raffai, Robert L; Messina, Louis M

    2015-01-01

    Oxidized low-density lipoprotein (oxLDL) is an important risk factor in the development of atherosclerosis. oxLDL has been shown to decrease endothelial progenitor cell (EPC) number by inducing apoptosis. p38 mitogen-activated protein kinase (MAPK) was shown to be activated by oxLDL and participated in the regulation of EPC number and function. However, the role of p38 remains unknown. Here, we show that oxLDL-induced p38 phosphorylation in EPCs is time and dose dependent. Treatment with antioxidant N-acetyl cysteine restored oxLDL-induced p38 phosphorylation to basal levels. LOX-1-blocking antibody also significantly decreased oxLDL-induced p38 phosphorylation. Interestingly, TUNEL staining showed that pretreatment with the p38 inhibitor SB203580 further increased oxLDL-induced apoptosis in EPCs. In accordance with these findings, pretreatment with SB203580 further attenuated Akt phosphorylation in EPCs challenged with oxLDL, indicating an interaction between Akt and p38 MAPK pathways. In agreement, inhibition of p38 MAPK further attenuated Akt phosphorylation and increased apoptosis in EPCs isolated from hypercholesterolemic ApoE-/- mice. In conclusion, p38 MAPK serves as an anti-apoptotic pathway by supporting Akt activity when EPCs are challenged with oxLDL. PMID:27031525

  12. AMP-activated protein kinase inhibits alkaline pH- and PKA-induced apical vacuolar H+-ATPase accumulation in epididymal clear cells.

    Hallows, Kenneth R; Alzamora, Rodrigo; Li, Hui; Gong, Fan; Smolak, Christy; Neumann, Dietbert; Pastor-Soler, Núria M

    2009-04-01

    Acidic luminal pH and low [HCO(3)(-)] maintain sperm quiescent during maturation in the epididymis. The vacuolar H(+)-ATPase (V-ATPase) in clear cells is a major contributor to epididymal luminal acidification. We have shown previously that protein kinase A (PKA), acting downstream of soluble adenylyl cyclase stimulation by alkaline luminal pH or HCO(3)(-), induces V-ATPase apical membrane accumulation in clear cells. Here we examined whether the metabolic sensor AMP-activated protein kinase (AMPK) regulates this PKA-induced V-ATPase apical membrane accumulation. Immunofluorescence labeling of rat and non-human primate epididymides revealed specific AMPK expression in epithelial cells. Immunofluorescence labeling of rat epididymis showed that perfusion in vivo with the AMPK activators 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) or A-769662 induced a redistribution of the V-ATPase into subapical vesicles, even in the presence of a luminal alkaline (pH 7.8) buffer compared with that of controls perfused without drug. Moreover, preperfusion with AICAR blocked the PKA-mediated V-ATPase translocation to clear cell apical membranes induced by N(6)-monobutyryl-cAMP (6-MB-cAMP). Purified PKA and AMPK both phosphorylated V-ATPase A subunit in vitro. In HEK-293 cells [(32)P]orthophosphate in vivo labeling of the A subunit increased following PKA stimulation and decreased following RNA interference-mediated knockdown of AMPK. Finally, the extent of PKA-dependent in vivo phosphorylation of the A subunit increased with AMPK knockdown. In summary, our findings suggest that AMPK inhibits PKA-mediated V-ATPase apical accumulation in epididymal clear cells, that both kinases directly phosphorylate the V-ATPase A subunit in vitro and in vivo, and that AMPK inhibits PKA-dependent phosphorylation of this subunit. V-ATPase activity may be coupled to the sensing of acid-base status via PKA and to metabolic status via AMPK. PMID:19211918

  13. Lupine protein hydrolysates inhibit enzymes involved in the inflammatory pathway

    Millán-Linares, María del Carmen; Yust, María del Mar; Alcaide-Hidalgo, J. M.; Millán, Francisco; Pedroche, Justo

    2014-01-01

    Lupine protein hydrolysates (LPHs) were obtained from a lupine protein isolate (LPI) by enzymatic hydrolysis using two proteases, Izyme AL and Alcalase 2.4 L, and their potential anti-inflammatory capacities were studied by determining their in vitro inhibition of the following enzymes that are involved in the inflammatory process: phospholipase A2 (PLA2), cyclooxygenase 2 (COX-2), thrombin, and transglutaminase (TG). The strongest inhibitory activities toward PLA2 and TG were found in the hy...

  14. Renin inhibition activity by chitooligosaccharides.

    Park, Pyo-Jam; Ahn, Chang-Bum; Jeon, You-Jin; Je, Jae-Young

    2008-04-01

    Six kinds of chitooligosaccharides (COSs) with different molecular weight (MW) and degree of deacetylation (DD) were prepared using ultrafiltration membrane reactor, and their renin inhibition modes were evaluated. All the COSs showed the renin-inhibitory activities with dose-dependent manner, and 90-COSs had the potent renin-inhibitory activity than that of 50-COSs. Among them, 90-MMWCOS (1000-5000Da) exhibits the highest activity with IC(50) value of 0.51mg/mL and acts as competitive inhibitor with K(i) value of 0.28mg/mL by Lineweaver-Burk and Dixon plots. These results indicated that DD value and MW of COSs are important factors affecting renin-inhibitory activity. PMID:18313296

  15. Activation of AML1-mediated transcription by MOZ and inhibition by the MOZ–CBP fusion protein

    Kitabayashi, Issay; Aikawa, Yukiko; Nguyen, Lan Anh; Yokoyama, Akihiko; Ohki, Misao

    2001-01-01

    The AML1–CBFβ transcription factor complex is the most frequent target of specific chromosome translocations in human leukemia. The MOZ gene, which encodes a histone acetyltransferase (HAT), is also involved in some leukemia-associated translocations. We report here that MOZ is part of the AML1 complex and strongly stimulates AML1-mediated transcription. The stimulation of AML1-mediated transcription is independent of the inherent HAT activity of MOZ. Rather, a potent transactivation domain w...

  16. Inhibition of p38 mitogen-activated protein kinase may decrease intestinal epithelial cell apoptosis and improve intestinal epithelial barrier function after ischemia- reperfusion injury

    Shu-Yun Zheng; Xiao-Bing Fu; Jian-Guo Xu; Jing-Yu Zhao; Tong-Zhu Sun; Wei Chen

    2005-01-01

    AIM: To investigate the role of p38 mitogen-activated protein kinase in rat small intestine after ischemia-reperfusion (I/R)insult and the relationship between activation of p38 MAPK and apoptotic cell death of intestine.METHODS: Ninety Wistar rats were divided randomly into three groups, namely sham-operated group (C), I/R vehicle group (R) and SB203580 pre-treated group(S).In groups R and S, the superior mesenteric artery(SMA)was separated and occluded for 45 min, then released for reperfusion for0.25, 0.5, 1, 2, 6, 12 and 24 h. In group C, SMA was separated without occlusion. Plasma D-lactate levels were examined and histological changes were observed under a light microscope. The activity of p38 MAPK was determined by Western immunoblotting and apoptotic cells were detected by the terminal deoxynucleotidyl transferase (TdT)-mediated dUDP-biotin nick end labeling (TUNEL).RESULTS: Intestinal ischemia followed by reperfusion activated p38 MAPK, and the maximal level of activation (7.3-fold vs sham-operated group) was reached 30 min after I/R. Treatment with SB 203580, a p38 MAPK inhibitor,reduced intestinal apoptosis (26.72±3.39% vs62.50±3.08%in I/R vehicle, P<0.01) and decreased plasma D-lactate level (0.78±0.15 mmol/L in I/R vehicle vs0.42±0.17 mmol/L in SB-treated group) and improved post-ischemic intestinal histological damage.CONCLUSION: p38 MAPK plays a crucial role in the signal transduction pathway mediating post-ischemic intestinal apoptosis, and inhibition of p38 MAPK may attenuate ischemia-reperfusion injury.

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

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

  18. Enhancement of fibroblast activation protein α-based vaccines and adenovirus boost immunity by cyclophosphamide through inhibiting IL-10 expression in 4T1 tumor bearing mice.

    Xia, Qiu; Geng, Fei; Zhang, Fang-Fang; Liu, Chen-Lu; Xu, Ping; Lu, Zhen-Zhen; Zhang, Hai-Hong; Kong, Wei; Yu, Xiang-Hui

    2016-08-31

    Fibroblast activation protein α (FAPα) is expressed in cancer-associated fibroblasts (CAFs) of more than 90% of malignant epithelia carcinomas. CAFs are the main type of cells in the tumor microenvironment which offer nutrition and protection to the tumor and regulate immunosuppression. To eliminate CAFs, a vaccine targeting FAPα may be used with a heterologous prime-boost strategy to enhance the FAPα-specific cellular immunity. Here, a FAP vaccine using a recombinant adenovirus (rAd) vector was constructed as well as a DNA vaccine reported in our previous work. Although the DNA prime-rAd boost strategy enhanced FAPα-specific immune responses, improvement of anti-tumor immunity effects was not observed. Examination of immunosuppressive factors revealed that high expression of the IL-10 cytokine was considered the main cause of the failure of the prime-boost strategy. However, heterologous vaccination in combination with a low-dose of cyclophosphamide (CY), which was reported to reduce IL-10 production and promote a shift from immunosuppression to immunopotentiation, resulted in enhanced effects in terms of numbers of effector T cells and tumor growth inhibition rates, compared to the CY alone or DNA alone group. Tumor growth was inhibited markedly when the prime-boost strategy was combined with CY in both the prophylactic and therapeutic settings and the survival time of 4T1 tumor bearing mice was also prolonged significantly. With the reduction of IL-10, enhancement of the anti-tumor effect by the prime-boost strategy was observed. These results suggest that FAPα-targeted rAd boosting in combination with CY is an attractive approach to overcoming immunosuppression in cancer vaccines. PMID:27498213

  19. Brain hyaluronan binding protein inhibits tumor growth

    高锋; 曹曼林; 王蕾

    2004-01-01

    Background Great efforts have been made to search for the angiogenic inhibitors in avascular tissues. Several proteins isolated from cartilage have been proved to have anti-angiogenic or anti-tumour effects. Because cartilage contains a great amount of hyaluronic acid (HA) oligosaccharides and abundant HA binding proteins (HABP), therefore, we speculated that HABP might be one of the factors regulating vascularization in cartilage or anti-angiogenesis in tumours. The purpose of this research was to evaluale the effects of hyaluronan binding protein on inhibiting tumour growth both in vivo and vitro. Methods A unique protein termed human brain hyaluronan (HA) binding protein (b-HABP) was cloned from human brain cDNA library. MDA-435 human breast cancer cell line was chosen as a transfectant. The in vitro underlying mechanisms were investigated by determining the possibilities of MDA-435/b-HABP colony formation on soft agar, the effects of the transfectant on the proliferation of endothelial cells and the expression levels of caspase 3 and FasL from MDA-435/b-HABP. The in vivo study included tumour growth on the chorioallantoic membrane (CAM) of chicken embryos and nude mice. Results Colony formation assay revealed that the colonies formed by MDA-435/b-HABP were greatly reduced compared to mock transfectants. The conditioned media from MDA-435/b-HABP inhibited the growth of endothelial cells in culture. Caspase 3 and FasL expressions were induced by MDA-435/b-HABP. The size of tumours of MDA-435/b-HABP in both CAM and nude mice was much smaller than that of MDA-435 alone. Conclusions Human brain hyaluronan binding protein (b-HABP) may represent a new kind of naturally existing anti-tumour substance. This brain-derived glycoprotein may block tumour growth by inducing apoptosis of cancer cells or by decreasing angiogenesis in tumour tissue via inhibiting proliferation of endothelial cells.

  20. Tetrathiomolybdate Inhibits Copper Trafficking Proteins Through Metal Cluster Formation

    Alvarez, Hamsell M.; Xue, Yi; Robinson, Chandler D.; Canalizo-Hernández, Mónica A.; Marvin, Rebecca G.; Kelly, Rebekah A.; Mondragón, Alfonso; Penner-Hahn, James E.; O’Halloran, Thomas V. (Michigan); (NWU)

    2010-05-06

    Tetrathiomolybdate (TM) is an orally active agent for treatment of disorders of copper metabolism. Here we describe how TM inhibits proteins that regulate copper physiology. Crystallographic results reveal that the surprising stability of the drug complex with the metallochaperone Atx1 arises from formation of a sulfur-bridged copper-molybdenum cluster reminiscent of those found in molybdenum and iron sulfur proteins. Spectroscopic studies indicate that this cluster is stable in solution and corresponds to physiological clusters isolated from TM-treated Wilson's disease animal models. Finally, mechanistic studies show that the drug-metallochaperone inhibits metal transfer functions between copper-trafficking proteins. The results are consistent with a model wherein TM can directly and reversibly down-regulate copper delivery to secreted metalloenzymes and suggest that proteins involved in metal regulation might be fruitful drug targets.

  1. Non-steroidal anti-inflammatory drugs activate NADPH oxidase in adipocytes and raise the H2O2 pool to prevent cAMP-stimulated protein kinase a activation and inhibit lipolysis

    Vázquez-Meza, Héctor; de Piña, Martha Zentella; Pardo, Juan Pablo; Riveros-Rosas, Héctor; Villalobos-Molina, Rafael; Piña, Enrique

    2013-01-01

    Background Non-steroidal anti-inflammatory drugs (NSAIDs) —aspirin, naproxen, nimesulide, and piroxicam— lowered activation of type II cAMP-dependent protein kinase A (PKA-II) in isolated rat adipocytes, decreasing adrenaline- and dibutyryl cAMP (Bt2cAMP)-stimulated lipolysis. The molecular bases of insulin-like actions of NSAID were studied. Results Based on the reported inhibition of lipolysis by H2O2, catalase was successfully used to block NSAID inhibitory action on Bt2cAMP-stimulated lip...

  2. p38 mitogen-activated protein kinase inhibition modulates nucleus pulposus cell apoptosis in spontaneous resorption of herniated intervertebral discs: An experimental study in rats.

    Zhu, Yu; Liu, Jin-Tao; Yang, Li-Yan; Du, Wen-Pei; Li, Xiao-Chun; Qian, Xiang; Yu, Peng-Fei; Liu, Jian-Wen; Jiang, Hong

    2016-05-01

    The present study was performed to investigate the role of p38 mitogen‑activated protein kinase (MAPK) in the resorption of herniated intervertebral discs in 30 rats. In the non‑contained and p38 MAPK inhibition (p38i) groups, two coccygeal intervertebral discs (IVDs) were removed and wounded prior to relocation into the subcutaneous space of the skin of the back. In the contained group, the cartilage endplates maintained their integrity. Furthermore, SB203580 was injected intraperitoneally into the p38i group, whereas saline was injected into the other two groups. In the non‑contained group, the weight of the relocated IVDs decreased to a greater extent over time when compared with the contained and p38i groups. Phosphorylated p38, tumor necrosis factor‑α, and interleukin‑1β were observed to exhibit higher expression levels in the non‑contained group compared with the contained and p38i groups, at weeks 1 and 4 post‑surgery. The expression level of caspase‑3 and the densities of apoptotic disc cells were significantly higher in the non‑contained group compared with the contained and p38i groups at 4 weeks post‑surgery. In conclusion, p38 MAPK induces apoptosis in IVDs, while also accelerating the resorption of the relocated IVDs. Thus, p38 MAPK may be important in spontaneous resorption of IVDs. PMID:27035219

  3. Exercise preconditioning reduces neonatal incision surgery-induced enhanced hyperalgesia via inhibition of P38 mitogen-activated protein kinase and IL-1β, TNF-α release.

    Gong, Xingrui; Jiang, Jing; Zhang, Mazhong

    2016-08-01

    Neonatal surgery leads to enhanced hyperalgesia to noxious stimulation in adulthood via a mechanism caused by enhanced phosphorylated (p)-p38 expression in microglia. We tested the effect of exercise on reducing enhanced hypersensitivity primed by neonatal incision surgery. Adult female Wistar rats, with or without neonatal incision surgery at postnatal day (P) 3, received right hind paw plantar incision surgery under anesthesia at P44. The rats performed wheel-running exercise from P22 to P41. Paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were measured and ipsilateral spinal cords were collected for protein quantification. For PWT and PWL, exercise reduced the pain index after incision surgery at P44 in rats with neonatal surgery (Pexercise suppressed P-p38 expression relative to adult rats without neonatal surgery (Pexercise reduced IL-1β and TNF-α (PExercise preconditioning is an effective approach to reducing enhanced adult hyperalgesia primed by neonatal surgery. The mechanism may be explained by exercise-induced inhibition of P-p38 activation and IL-1β, TNF-α release. PMID:27235543

  4. Expression of tetraspan protein CD63 activates protein-tyrosine kinase (PTK) and enhances the PTK-induced inhibition of ROMK channels.

    Lin, D.; Kamsteeg, E.J.; Zhang, Y.; Jin, Y.; Sterling, H.; Yue, P.; Roos, M.; Duffield, A.; Spencer, J.; Caplan, M.; Wang, W.H.

    2008-01-01

    In the present study, we tested the role of CD63 in regulating ROMK1 channels by protein-tyrosine kinase (PTK). Immunocytochemical staining shows that CD63 and receptor-linked tyrosine phosphatase alpha (RPTPalpha) are expressed in the cortical collecting duct and outer medulla collecting duct. Immu

  5. Inhibition of benzopyrene-diol-epoxide (BPDE)-induced bax and caspase-9 by cadmium: Role of mitogen activated protein kinase

    Cadmium, a major metal constituent of tobacco smoke, elicits synergistic enhancement of cell transformation when combined with benzo[a]pyrene (BP) or other polynuclear aromatic hydrocarbons (PAHs). The mechanism underlying this synergism is not clearly understood. Present study demonstrates that (+/-)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), an ultimate carcinogen of BP, induces apoptosis in human leukemic HL-60 cells and others, and cadmium at non-cytotoxic concentration inhibits BPDE-induced apoptosis. We observed that BPDE treatment also activates all three MAP kinases e.g. ERK1/2, p38 and JNK in HL-60 cells, and inhibition of BPDE-induced apoptosis by cadmium is associated with down-regulation of pro-apoptotic bax induction/caspase-9 activation and up-regulation of ERK phosphorylation, whereas p38 MAP kinase and c-Jun phosphorylation (indicative of JNK activation) remain unaffected. Inhibition of ERKs by prior treatment of cells with 10 μM U0126 relieves cadmium-mediated inhibition of apoptosis/bax induction/caspase-9 activation. Our results suggest that cadmium inhibits BPDE-induced apoptosis by modulating apoptotic signaling through up-regulation of ERK, which is known to promote cell survival

  6. Simvastatin inhibits protein isoprenylation in the brain.

    Ostrowski, Stephen M; Johnson, Kachael; Siefert, Matthew; Shank, Sam; Sironi, Luigi; Wolozin, Benjamin; Landreth, Gary E; Ziady, Assem G

    2016-08-01

    Evidence suggests that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, or statins, may reduce the risk of Alzheimer's disease (AD). Statin action in patients with AD, as in those with heart disease, is likely to be at least partly independent of the effects of statins on cholesterol. Statins can alter cellular signaling and protein trafficking through inhibition of isoprenylation of Rho, Cdc42, and Rab family GTPases. The effects of statins on protein isoprenylation in vivo, particularly in the central nervous system, are poorly studied. We utilized two-dimensional gel electrophoresis approaches to directly monitor the levels of isoprenylated and non-isoprenylated forms of Rho and Rab family GTPases. We report that simvastatin significantly inhibits RhoA and Rab4, and Rab6 isoprenylation at doses as low as 50nM in vitro. We also provide the first in vivo evidence that statins inhibit the isoprenylation of RhoA in the brains of rats and RhoA, Cdc42, and H-Ras in the brains of mice treated with clinically relevant doses of simvastatin. PMID:27180285

  7. p38γ Mitogen-Activated Protein Kinase Contributes to Oncogenic Properties Maintenance and Resistance to Poly (ADP-Ribose-Polymerase-1 Inhibition in Breast Cancer

    Fanyan Meng

    2011-05-01

    Full Text Available p38γ MAPK, one of the four members of p38 mitogen-activated protein kinases (MAPKs, has previously been shown to harbor oncogenic functions. However, the biologic function of p38γ MAPK in breast cancer has not been well defined. In this study, we have shown that p38γ MAPK is overexpressed in highly metastatic human and mouse breast cancer cell lines and p38γ MAPK expression is preferentially associated with basal-like and metastatic phenotypes of breast tumor samples. Ectopic expression of p38γ MAPK did not lead to an increase in oncogenic properties in vitro in most tested mammary epithelial cells. However, knockdown of p38γ MAPK expression resulted in a dramatic decrease in cell proliferation, colony formation, cell migration, invasion in vitro and significant retardation of tumorigenesis, and long-distance metastasis to the lungs in vivo. Moreover, knockdown of p38γ MAPK triggered the activation of AKT signaling. Inhibition of this feedback loop with various PI3K/AKT signaling inhibitors facilitated the effect of targeting p38γ MAPK. We further found that overexpression of p38γ MAPK did not promote cell resistance to chemotherapeutic agents doxorubicin and paclitaxel but significantly increased cell resistance to PJ-34, a DNA damage agent poly (ADP-ribose-polymerase-1 (PARP inhibitor in vitro and in vivo. Finally, we identified that p38γ MAPK overexpression led to marked cell cycle arrest in G2/M phase. Our study for the first time clearly demonstrates that p38γ MAPK is a promising target for the design of targeted therapies for basal-like breast cancer with metastatic characteristics and for overcoming potential resistance against the PARP inhibitor.

  8. A novel p38 mitogen activated protein kinase (MAPK) specific inhibitor suppresses respiratory syncytial virus and influenza A virus replication by inhibiting virus-induced p38 MAPK activation.

    Choi, Myung-Soo; Heo, Jinyuk; Yi, Chae-Min; Ban, Junsu; Lee, Noh-Jin; Lee, Na-Rae; Kim, Sang Won; Kim, Nam-Jung; Inn, Kyung-Soo

    2016-08-26

    Respiratory syncytial virus (RSV) and influenza A virus are leading causes of acute lower respiratory infectious disease. Respiratory diseases caused by RSV and influenza A virus result in serious economic burden and life-threatening disease for immunocompromised people. With the revelation that p38 mitogen-activated protein kinase (MAPK) activity in host cells is crucial for infection and replication of RSV and influenza A virus, inhibition of p38 MAPK activity has been suggested as a potential antiviral therapeutic strategy. However, the low selectivity and high toxicity of the p38 MAPK inhibitors necessitate the development of better inhibitors. Herein, we report the synthesis of a novel p38 MAPK inhibitor, NJK14047, with high kinase selectivity. In this work, it was demonstrated that NJK14047 inhibits RSV- and influenza A-mediated p38 MAPK activation in epithelial cells. Subsequently, NJK14047 treatment resulted in decreased viral replication and viral mRNA synthesis. In addition, secretion of interleukin-6 from infected cells was greatly diminished by NJK14047, suggesting that it can ameliorate immunopathological responses to RSV and influenza A. Collectively, the results suggest that NJK14047 has therapeutic potential to treat respiratory viral infection through the suppression of p38 MAPK activation, which is suggested to be an essential step for respiratory virus infection. PMID:27346133

  9. IL-1-induced ERK1/2 activation up-regulates p21Waf1/Cip1 protein by inhibition of degradation via ubiquitin-independent pathway in human melanoma cells A375

    IL-1 inhibits the proliferation of human melanoma cells A375 by arresting the cell cycle at G0/G1 phase, which accompanies the increase of p21Waf1/Cip1 (p21) protein. Here, we demonstrate that IL-1 induces the stabilization of p21 protein via ERK1/2 pathway. The degradation of p21 was inhibited by IL-1, however the ubiquitination level of p21 was not affected. In addition, the degradation of non-ubiquitinated form of lysine less mutant p21-K6R was also inhibited by IL-1, suggesting that IL-1 stabilized p21 protein via ubiquitin-independent pathway. Furthermore, the inhibition of p21 protein degradation was prevented by a selective inhibitor of ERK1/2 pathway, PD98059. These results suggest that IL-1-induced ERK1/2 activation leads to the up-regulation of p21 by inhibiting degradation via ubiquitin-independent pathway in human melanoma cells A375.

  10. High mobility group box-1 protein inhibits regulatory T cell immune activity in liver failure in patients with chronic hepatitis B

    Lu-WenWang; Hui Chen; Zuo-Jiong Gong

    2010-01-01

    BACKGROUND: Liver failure in chronic hepatitis B (CHB) patients is a severe, life-threatening condition. Intestinal endotoxemia plays a significant role in the progress to liver failure. High mobility group box-1 (HMGB1) protein is involved in the process of endotoxemia. Regulatory T (Treg) cells maintain immune tolerance and contribute to the immunological hyporesponsiveness against HBV infection. However, the roles of HMGB1 and Treg cells in the pathogenesis of liver failure in CHB patients, and whether HMGB1 affects the immune activity of Treg cells are poorly known at present, and so were explored in this study. METHODS: The levels of HMGB1 expression were detected by ELISA, real-time RT-PCR, and Western blotting, and the percentage of CD4+CD25+CD127low Treg cells among CD4+cells was detected by flow cytometry in liver failure patients with chronic HBV infection, CHB patients, and healthy controls. Then, CD4+CD25+CD127low Treg cells isolated from the peripheral blood mononuclear cells from CHB patients were stimulated with HMGB1 at different concentrations or at various intervals. The effect of HMGB1 on the immune activity of Treg cells was assessed by a suppression assay of the allogeneic mixed lymphocyte response. The levels of forkhead box P3 (Foxp3) expression in Treg cells treated with HMGB1 were detected by RT-PCR and Western blotting. RESULTS: A higher level of HMGB1 expression and a lower percentage of Treg cells within the population of CD4+ cells were found in liver failure patients than in CHB patients (82.6±20.1 μg/L vs. 34.2±13.7 μg/L; 4.55±1.34% vs. 9.52± 3.89%, respectively). The immune activity of Treg cells was significantly weakened and the levels of Foxp3 expression were reduced in a dose- or time-dependent manner when Treg cells were stimulated with HMGB1 in vitro. CONCLUSIONS: The high level of HMGB1 and the low percentage of Treg cells play an important role in the pathogenesis of liver failure in patients with chronic HBV infection

  11. Saponins from Platycodon grandiflorum inhibit hepatic lipogenesis through induction of SIRT1 and activation of AMP-activated protein kinase in high-glucose-induced HepG2 cells.

    Hwang, Yong Pil; Choi, Jae Ho; Kim, Hyung Gyun; Lee, Hyun-Sun; Chung, Young Chul; Jeong, Hye Gwang

    2013-09-01

    Saponins from the roots of Platycodon grandiflorum (Changkil saponins, CKS) have antioxidant and hepatoprotective properties. This study investigated the effects of CKS on AMP-activated protein kinase (AMPK) activation and hepatic lipogenesis in HepG2 cells. CKS suppressed high-glucose-induced lipid accumulation and inhibited high-glucose-induced fatty acid synthase (FAS) and sterol regulatory element binding protein-1c (SREBP-1c) expression in HepG2 cells. Moreover, the use of a pharmacological AMPK inhibitor revealed that AMPK is essential for the suppression of SREBP-1c expression in CKS-treated cells. Finally, the activation of calcium/calmodulin-dependent kinase kinase β (CaMKKβ) and SIRT1 was necessary for CKS-enhanced activation of AMPK. These results indicate that CKS prevents lipid accumulation in HepG2 cells by blocking the expression of SREBP-1c and FAS through SIRT1 and CaMKKβ/AMPK activation. Using CKS to target AMPK activation may provide a promising approach for the prevention lipogenesis. PMID:23578622

  12. Inhibition of Voltage-Gated Calcium Channels by RGK Proteins.

    Buraei, Zafir; Yang, Jian

    2015-01-01

    Due to their essential biological roles, voltage-gated calcium channels (VGCCs) are regulated by a myriad of molecules and mechanisms. Fifteen years ago, RGK proteins were discovered to bind the VGCC β subunit (Cavβ) and potently inhibit high-voltage activated Ca(2+) channels. RGKs (Rad, Rem, Rem2 and Gem/Kir) are a family of monomeric small GTPases belonging to the superfamily of Ras GTPases. They exert dual inhibitory effects on VGCCs, decreasing surface expression and suppressing surface channels through immobilization of the voltage sensor or reduction of channel open probability. While Cavβ is required for all forms of RGK inhibition, not all inhibition is mediated by the RGK-Cavβ interaction. Some RGK proteins also interact directly with the pore-forming α1 subunit of some types of VGCCs (Cavα1). Importantly, RGK proteins tonically inhibit VGCCs in native cells, regulating cardiac and neural functions. This minireview summarizes the mechanisms, molecular determinants, and physiological impact of RGK inhibition of VGCCs. PMID:25966691

  13. Soluble guanylyl cyclase-activated cyclic GMP-dependent protein kinase inhibits arterial smooth muscle cell migration independent of VASP-serine 239 phosphorylation.

    Holt, Andrew W; Martin, Danielle N; Shaver, Patti R; Adderley, Shaquria P; Stone, Joshua D; Joshi, Chintamani N; Francisco, Jake T; Lust, Robert M; Weidner, Douglas A; Shewchuk, Brian M; Tulis, David A

    2016-09-01

    Coronary artery disease (CAD) accounts for over half of all cardiovascular disease-related deaths. Uncontrolled arterial smooth muscle (ASM) cell migration is a major component of CAD pathogenesis and efforts aimed at attenuating its progression are clinically essential. Cyclic nucleotide signaling has long been studied for its growth-mitigating properties in the setting of CAD and other vascular disorders. Heme-containing soluble guanylyl cyclase (sGC) synthesizes cyclic guanosine monophosphate (cGMP) and maintains vascular homeostasis predominantly through cGMP-dependent protein kinase (PKG) signaling. Considering that reactive oxygen species (ROS) can interfere with appropriate sGC signaling by oxidizing the cyclase heme moiety and so are associated with several CVD pathologies, the current study was designed to test the hypothesis that heme-independent sGC activation by BAY 60-2770 (BAY60) maintains cGMP levels despite heme oxidation and inhibits ASM cell migration through phosphorylation of the PKG target and actin-binding vasodilator-stimulated phosphoprotein (VASP). First, using the heme oxidant ODQ, cGMP content was potentiated in the presence of BAY60. Using a rat model of arterial growth, BAY60 significantly reduced neointima formation and luminal narrowing compared to vehicle (VEH)-treated controls. In rat ASM cells BAY60 significantly attenuated cell migration, reduced G:F actin, and increased PKG activity and VASP Ser239 phosphorylation (pVASP·S239) compared to VEH controls. Site-directed mutagenesis was then used to generate overexpressing full-length wild type VASP (FL-VASP/WT), VASP Ser239 phosphorylation-mimetic (FL-VASP/239D) and VASP Ser239 phosphorylation-resistant (FL-VASP/239A) ASM cell mutants. Surprisingly, FL-VASP/239D negated the inhibitory effects of FL-VASP/WT and FL-VASP/239A cells on migration. Furthermore, when FL-VASP mutants were treated with BAY60, only the FL-VASP/239D group showed reduced migration compared to its VEH controls

  14. Enzymatic Treatment of Whey Proteins in Cow's Milk Results in Differential Inhibition of IgE-Mediated Mast Cell Activation Compared to T-Cell Activation

    Knipping, Karen; van Esch, Betty C. A. M.; van Ieperen-van Dijk, Adrie G.; van Hoffen, Els; van Baalen, Ton; Knippels, Leon M. J.; van der Heide, Sicco; Dubois, Anthony E. J.; Garssen, Johan; Knol, Edward F.

    2012-01-01

    Background: Cow's milk (CM) hydrolysates are frequently used as milk substitutes for children with CM allergy. In hydrolysates, allergenic epitopes within CM proteins are diminished by enzymatic treatment. The aim of this study was to examine the allergenic and immunogenic properties of whey protein

  15. Hsp90 inhibition decreases mitochondrial protein turnover.

    Daciana H Margineantu

    Full Text Available BACKGROUND: Cells treated with hsp90 inhibitors exhibit pleiotropic changes, including an expansion of the mitochondrial compartment, accompanied by mitochondrial fragmentation and condensed mitochondrial morphology, with ultimate compromise of mitochondrial integrity and apoptosis. FINDINGS: We identified several mitochondrial oxidative phosphorylation complex subunits, including several encoded by mtDNA, that are upregulated by hsp90 inhibitors, without corresponding changes in mRNA abundance. Post-transcriptional accumulation of mitochondrial proteins observed with hsp90 inhibitors is also seen in cells treated with proteasome inhibitors. Detailed studies of the OSCP subunit of mitochondrial F1F0-ATPase revealed the presence of mono- and polyubiquitinated OSCP in mitochondrial fractions. We demonstrate that processed OSCP undergoes retrotranslocation to a trypsin-sensitive form associated with the outer mitochondrial membrane. Inhibition of proteasome or hsp90 function results in accumulation of both correctly targeted and retrotranslocated mitochondrial OSCP. CONCLUSIONS: Cytosolic turnover of mitochondrial proteins demonstrates a novel connection between mitochondrial and cytosolic compartments through the ubiquitin-proteasome system. Analogous to defective protein folding in the endoplasmic reticulum, a mitochondrial unfolded protein response may play a role in the apoptotic effects of hsp90 and proteasome inhibitors.

  16. Tim-4 inhibition of T-cell activation and T helper type 17 differentiation requires both the immunoglobulin V and mucin domains and occurs via the mitogen-activated protein kinase pathway.

    Cao, Wei

    2011-06-01

    Emerging experimental data suggest an important role for the T-cell immunoglobulin mucin 1 (Tim-1):Tim-4 pathway in autoimmune and alloimmune responses in vivo. Using a Tim-4 ectodomain human IgG Fc fusion protein we studied the role of Tim-4 in T-cell activation, signalling and differentiation responses in vitro. We demonstrate that Tim-4Fc can inhibit naive and pre-activated T-cell activation, proliferation and cytokine secretion via a Tim-1-independent pathway. Tim-4 contains immunoglobulin variable (IgV) and mucin domains; to identify which domain accounts for the inhibitory effect novel Tim-4 fusion proteins containing either the IgV or mucin domain were generated. We demonstrate that both IgV and mucin domains are required for the inhibitory effects and that they are mediated at least in part by inhibition of extracellular signal-regulated kinase pathway activity. Given the emerging interest in the role of the Tim family in T helper type 17 (Th17) cells, which play an important role in autoimmune disease and transplantation tolerance, our data show that Tim-4Fc can prevent polarization of CD4(+) T cells to the Th17 phenotype. Collectively, our results highlight an inhibitory role for Tim-4Fc in vitro, which we propose is mediated by a receptor other than Tim-1. In addition, this study provides new insights into the role of Tim-4Fc in regulating Th17 immune responses and may open a new avenue for autoimmune therapy.

  17. Radiation sensitization by inhibition of activated ras

    Background and purpose: Ras has been identified as a significant contributor to radiation resistance. This article reviews preclinical and phase I clinical studies that reported on combining inhibition of activated Ras and downstream effectors of Ras with radiotherapy. Material and methods: transfection studies and RNA interference were used to check the role of the Ras isoforms for intrinsic radiation sensibility. Western blotting was used to control for prenylation inhibition of the respective Ras isoforms and for changes in activity of downstream proteins. Clonogenic assays with human and rodent tumor cell lines served for testing radiosensitivity. In vivo, farnesyltransferase inhibitors (FTIs) and irradiation were used to treat xenograft tumors. Ex vivo plating efficiency measurements, regrowth of tumors, and EF5 staining for detection of hypoxia were endpoints in these studies. Simultaneous treatment with L-778,123 and irradiation was performed in non-small cell lung cancer, head and neck cancer, and pancreatic cancer patients. Results: radiation sensitization was achieved in vitro and in vivo blocking the prenylation of Ras proteins in cell lines with Ras activated by mutations or receptor signaling. Among the many Ras downstream pathways the phosphoinositide 3 (PI3) kinase-Akt pathway was identified as a contributor to Ras-mediated radiation resistance. Furthermore, increased oxygenation was observed in xenograft tumors after FTI treatment. Combined treatment in a phase I study was safe and effective. Conclusion: the rational combination of FTIs with radiotherapy may improve the clinical results of patients with tumors who bear mutant or receptor-signaling activated Ras. (orig.)

  18. Curcumin inhibits HIV-1 by promoting Tat protein degradation

    Ali, Amjad; Banerjea, Akhil C.

    2016-01-01

    HIV-1 Tat is an intrinsically unfolded protein playing a pivotal role in viral replication by associating with TAR region of viral LTR. Unfolded proteins are degraded by 20S proteasome in an ubiquitin independent manner. Curcumin is known to activate 20S proteasome and promotes the degradation of intrinsically unfolded p53 tumor suppressor protein. Since HIV-1 Tat protein is largerly unfolded, we hypothesized that Tat may also be targeted through this pathway. Curcumin treated Tat transfected HEK-293T cells showed a dose and time dependent degradation of Tat protein. Contrary to this HIV-1 Gag which is a properly folded protein, remained unaffected with curcumin. Semi-quantitative RT-PCR analysis showed that curcumin treatment did not affect Tat gene transcription. Curcumin increased the rate of Tat protein degradation as shown by cycloheximide (CHX) chase assay. Degradation of the Tat protein is accomplished through proteasomal pathway as proteasomal inhibitor MG132 blocked Tat degradation. Curcumin also decreased Tat mediated LTR promoter transactivation and inhibited virus production from HIV-1 infected cells. Taken together our study reveals a novel observation that curcumin causes potent degradation of Tat which may be one of the major mechanisms behind its anti HIV activity. PMID:27283735

  19. Ribosome-inhibiting proteins from in vitro cultures of Phytolacca dodecandra

    Thomsen, S.; Hansen, Harald S.; Nyman, U.

    1991-01-01

    initiated from leaf and cotyledon exhibited protein synthesis-inhibiting activity. Ribosome-inhibiting proteins were purified at least 14 times from suspension cells of P. dodecandra. The purified protein fraction contained two proteins as seen by sodium dodecyl sulphate polyacrylamide gel electrophoresis....... The relative molecular masses were 30,000 and 31,000 and they showed a pI > 9.3. These new RIP's were shown to be different from dodecandrin with respect to molecular mass....

  20. Solid-phase classical complement activation by C-reactive protein (CRP) is inhibited by fluid-phase CRP-C1q interaction

    Sjöwall, Christoffer; Wetterö, Jonas; Bengtsson, Torbjörn; Askendal, Agneta; Skogh, Thomas; Tengvall, Pentti

    2007-01-01

    C-reactive protein (CRP) interacts with phosphorylcholine (PC), Fcγ receptors, complement factor C1q and cell nuclear constituents, yet its biological roles are insufficiently understood. The aim was to characterize CRP-induced complement activation by ellipsometry. PC conjugated with keyhole limpet hemocyanin (PC-KLH) was immobilized to cross-linked fibrinogen. A low-CRP serum with different amounts of added CRP was exposed to the PC-surfaces. The total serum protein deposition was quantifie...

  1. Identification of Peptides That Inhibit the DNA Binding, trans-Activator, and DNA Replication Functions of the Human Papillomavirus Type 11 E2 Protein

    Deng, Su-Jun; Pearce, Kenneth H.; Dixon, Eric P.; Hartley, Kelly A.; Stanley, Thomas B.; Lobe, David C.; Garvey, Edward P.; Kost, Thomas A.; Petty, Regina L.; Rocque, Warren J.; Alexander, Kenneth A.; Underwood, Mark R.

    2004-01-01

    Peptide antagonists of the human papillomavirus type 11 (HPV-11) E2-DNA association were identified using a filamentous bacteriophage random peptide library. Synthetic peptides antagonized the E2-DNA interaction, effectively blocked E2-mediated transcriptional activation of a reporter gene in cell culture, and inhibited E1-E2-mediated HPV-11 DNA replication in vitro. These peptides may prove to be useful tools for characterizing E2 function and for exploring the effectiveness of E2-inhibitor-...

  2. The Cellular TAR RNA Binding Protein, TRBP, Promotes HIV-1 Replication Primarily by Inhibiting the Activation of Double-Stranded RNA-Dependent Kinase PKR▿

    Sanghvi, Viraj R.; Steel, Laura F

    2011-01-01

    The TAR RNA binding protein, TRBP, is a cellular double-stranded RNA (dsRNA) binding protein that can promote the replication of HIV-1 through interactions with the viral TAR element as well as with cellular proteins that affect the efficiency of translation of viral transcripts. The structured TAR element, present on all viral transcripts, can impede efficient translation either by sterically blocking access of translation initiation factors to the 5′-cap or by activating the dsRNA-dependent...

  3. Ubiquitylation of terminal deoxynucleotidyltransferase inhibits its activity.

    So Maezawa

    Full Text Available Terminal deoxynucleotidyltransferase (TdT, which template-independently synthesizes DNA during V(DJ recombination in lymphoid cells, is ubiquitylated by a BPOZ-2/Cul3 complex, as the ubiquitin ligase, and then degraded by the 26 S proteasome. We show here that TdT is ubiquitylated by the Cul3-based ubiquitylation system in vitro. Because TdT could also be ubiquitylated in the absence of Cul/BPOZ-2, we determined that it could also be directly ubiquitylated by the E2 proteins UbcH5a/b/c and UbcH6, E3-independently. Furthermore, the ubiquitylated TdT inhibited its nucleotidyltransferase activity.

  4. Farnesoid X receptor inhibits the transcriptional activity of carbohydrate response element binding protein in human hepatocytes. : Transrepression of ChREBP by FXR

    Caron, Sandrine; Samanez, Carolina Huaman; Dehondt, Hélène; Ploton, Maheul; Briand, Olivier; Lien, Fleur; Dorchies, Emilie; Dumont, Julie; Postic, Catherine; Cariou, Bertrand; Lefebvre, Philippe; Staels, Bart

    2013-01-01

    The glucose-activated transcription factor carbohydrate response element binding protein (ChREBP) induces the expression of hepatic glycolytic and lipogenic genes. The farnesoid X receptor (FXR) is a nuclear bile acid receptor controlling bile acid, lipid, and glucose homeostasis. FXR negatively regulates hepatic glycolysis and lipogenesis in mouse liver. The aim of this study was to determine whether FXR regulates the transcriptional activity of ChREBP in human hepatocytes and to unravel the...

  5. Inhibition of nucleoside diphosphate kinase activity by in vitro phosphorylation by protein kinase CK2. Differential phosphorylation of NDP kinases in HeLa cells in culture

    Biondi, R M; Engel, M; Sauane, M;

    1996-01-01

    Although a number of nucleoside diphosphate kinases (NDPKs) have been reported to act as inhibitors of metastasis or as a transcription factor in mammals, it is not known whether these functions are linked to their enzymatic activity or how this protein is regulated. In this report, we show that ...... on histidine residues, however, only the B isoform appeared to be serine phosphorylated....

  6. Essential oil from the heartwood of Taiwan fir ameliorates LPS-induced inflammatory response by inhibiting the activation of mitogen-activated protein kinase.

    Liu, May-Lan; Hua, Kuo-Feng; Yang, Tzu-Jung; Chiu, Huan-Wen; Ho, Chen-Lung

    2014-10-01

    The essential oil from the heartwood of Taiwan fir (EOTC) was demonstrated to exhibit anti-inflammatory activity in lipopolysaccharide (LPS)-activated mouse macrophages. EOTC reduced nitrite oxide levels and inducible nitrite oxide synthase expression in, and tumor necrosis factor-α and interleukin-6 secretion by, LPS-activated macrophages without affecting cyclooxygenase-2 expression. EOTC reduced the levels of interleukin-lβ precursor induced by LPS and decreased the NLRP3 inflammasome-derived interleukin-lβ secretion induced by LPS and adenosine triphosphate. In addition, the phosphorylation levels of ERKI/2, JNK1/2, and p38 in LPS-activated macrophages were reduced by EOTC. Furthermore, EOTC was composed of oxygenated sesquiterpenes (68.4%), sesquiterpene hydrocarbons (28.9%) and diterpenes (0.9%). The major compounds of the oxygenated sesquiterpenes were τ-cadinol (23.9%), α-cadinol (21.1%) and cedrol (16.9%). These findings suggest that EOTC may be a candidate for the development of anti-inflammatory agents for preventing and ameliorating inflammation-related diseases. PMID:25522551

  7. Transcriptomic analysis of mouse EL4 T cells upon T cell activation and in response to protein synthesis inhibition via cycloheximide treatment.

    Lim, Pek Siew; Hardy, Kristine; Peng, Kaiman; Shannon, Frances M

    2016-03-01

    T cell activation involves the recognition of a foreign antigen complexed to the major histocompatibility complex on the antigen presenting T cell to the T cell receptor. This leads to activation of signaling pathways, which ultimately leads to induction of key cytokine genes responsible for eradication of foreign antigens. We used the mouse EL4 T cell as a model system to study genes that are induced as a result of T cell activation using phorbol myristate acetate (PMA) and calcium ionomycin (I) as stimuli. We were also interested to examine the importance of new protein synthesis in regulating the expression of genes involved in T cell activation. Thus we have pre-treated mouse EL4 T cells with cycloheximide, a protein synthesis inhibitor, and left the cells unstimulated or stimulated with PMA/I for 4 h. We performed microarray expression profiling of these cells to correlate the gene expression with chromatin state of T cells upon T cell activation [1]. Here, we detail further information and analysis of the microarray data, which shows that T cell activation leads to differential expression of genes and inducible genes can be further classified as primary and secondary response genes based on their protein synthesis dependency. The data is available in the Gene Expression Omnibus under accession number GSE13278. PMID:26981393

  8. Inability of NS1 protein from an H5N1 influenza virus to activate PI3K/Akt signaling pathway correlates to the enhanced virus replication upon PI3K inhibition

    Li Weizhong

    2012-04-01

    Full Text Available Abstract Background Phosphatidylinositol 3-kinase (PI3K/Akt signaling pathway, activated during influenza A virus infection, can promote viral replication via multiple mechanisms. Direct binding of NS1 protein to p85β subunit of PI3K is required for activation of PI3K/Akt signaling. Binding and subsequent activation of PI3K is believed to be a conserved character of influenza A virus NS1 protein. Sequence variation of NS1 proteins in different influenza A viruses led us to investigate possible deviation from the conservativeness. Results In the present study, NS1 proteins from four different influenza A virus subtypes/strains were tested for their ability to bind p85β subunit of PI3K and to activate PI3K/Akt. All NS1 proteins efficiently bound to p85β and activated PI3K/Akt, with the exception of NS1 protein from an H5N1 virus (A/Chicken/Guangdong/1/05, abbreviated as GD05, which bound to p85β but failed to activate PI3K/Akt, implying that as-yet-unidentified domain(s in NS1 may alternatively mediate the activation of PI3K. Moreover, PI3K inhibitor, LY294002, did not suppress but significantly increased the replication of GD05 virus. Conclusions Our study indicates that activation of PI3K/Akt by NS1 protein is not highly conserved among influenza A viruses and inhibition of the PI3K/Akt pathway as an anti-influenza strategy may not work for all influenza A viruses.

  9. AMP-activated protein kinase inhibits alkaline pH- and PKA-induced apical vacuolar H+-ATPase accumulation in epididymal clear cells

    Hallows, Kenneth R.; Alzamora, Rodrigo; Li, Hui; Gong, Fan; Smolak, Christy; Neumann, Dietbert; Pastor-Soler, Núria M.

    2009-01-01

    Acidic luminal pH and low [HCO3−] maintain sperm quiescent during maturation in the epididymis. The vacuolar H+-ATPase (V-ATPase) in clear cells is a major contributor to epididymal luminal acidification. We have shown previously that protein kinase A (PKA), acting downstream of soluble adenylyl cyclase stimulation by alkaline luminal pH or HCO3−, induces V-ATPase apical membrane accumulation in clear cells. Here we examined whether the metabolic sensor AMP-activated protein kinase (AMPK) reg...

  10. Inhibition of endogenous heat shock protein 70 attenuates inducible nitric oxide synthase induction via disruption of heat shock protein 70/Na(+) /H(+) exchanger 1-Ca(2+) -calcium-calmodulin-dependent protein kinase II/transforming growth factor β-activated kinase 1-nuclear factor-κB signals in BV-2 microglia.

    Huang, Chao; Lu, Xu; Wang, Jia; Tong, Lijuan; Jiang, Bo; Zhang, Wei

    2015-08-01

    Inducible nitric oxide synthase (iNOS) critically contributes to inflammation and host defense. The inhibition of heat shock protein 70 (Hsp70) prevents iNOS induction in lipopolysaccharide (LPS)-stimulated macrophages. However, the role and mechanism of endogenous Hsp70 in iNOS induction in microglia remains unclear. This study addresses this issue in BV-2 microglia, showing that Hsp70 inhibition or knockdown prevents LPS-induced iNOS protein expression and nitric oxide production. Real-time PCR experiments showed that LPS-induced iNOS mRNA transcription was blocked by Hsp70 inhibition. Further studies revealed that the inhibition of Hsp70 attenuated LPS-stimulated nuclear translocation and phosphorylation of nuclear factor (NF)-κB as well as the degradation of inhibitor of κB (IκB)-α and phosphorylation of IκB kinase β (IKKβ). This prevention effect of Hsp70 inhibition on IKKβ-NF-κB activation was found to be dependent on the Ca(2+) /calcium-calmodulin-dependent protein kinase II (CaMKII)/transforming growth factor β-activated kinase 1 (TAK1) signals based on the following observations: 1) chelation of intracellular Ca(2+) or inhibition of CaMKII reduced LPS-induced increases in TAK1 phosphorylation and 2) Hsp70 inhibition reduced LPS-induced increases in CaMKII/TAK1 phosphorylation, intracellular pH value, [Ca(2+) ]i , and CaMKII/TAK1 association. Mechanistic studies showed that Hsp70 inhibition disrupted the association between Hsp70 and Na(+) /H(+) exchanger 1 (NHE1), which is an important exchanger responsible for Ca(2+) influx in LPS-stimulated cells. These studies demonstrate that the inhibition of endogenous Hsp70 attenuates the induction of iNOS, which likely occurs through the disruption of NHE1/Hsp70-Ca(2+) -CaMKII/TAK1-NF-κB signals in BV-2 microglia, providing further insight into the functions of Hsp70 in the CNS. PMID:25691123

  11. Inhibition of Plasmepsin V activity demonstrates its essential role in protein export, PfEMP1 display, and survival of malaria parasites

    Sleebs, Brad E; Lopaticki, Sash; Marapana, Danushka S;

    2014-01-01

    The malaria parasite Plasmodium falciparum exports several hundred proteins into the infected erythrocyte that are involved in cellular remodeling and severe virulence. The export mechanism involves the Plasmodium export element (PEXEL), which is a cleavage site for the parasite protease, Plasmep......The malaria parasite Plasmodium falciparum exports several hundred proteins into the infected erythrocyte that are involved in cellular remodeling and severe virulence. The export mechanism involves the Plasmodium export element (PEXEL), which is a cleavage site for the parasite protease...... surface, and cytoadherence. The inhibitor killed parasites at the trophozoite stage and knockdown of PMV enhanced sensitivity to the inhibitor, while overexpression of PMV increased resistance. This provides the first direct evidence that PMV activity is essential for protein export in Plasmodium spp. and...... for parasite survival in human erythrocytes and validates PMV as an antimalarial drug target....

  12. Inhibition of protein kinase CK2 reduces CYP24A1 expression and enhances 1,25-dihydroxyvitamin D3 anti-tumor activity in human prostate cancer cells

    Luo, Wei; Yu, Wei-Dong; Ma, Yingyu; Chernov, Mikhail; Trump, Donald L.; Johnson, Candace S.

    2013-01-01

    Vitamin D has broad range of physiological functions and anti-tumor effects. 24-hydroxylase, encoded by the CYP24A1 gene, is the key enzyme for degrading many forms of vitamin D including the most active form, 1,25D3. Inhibition of CYP24A1 enhances 1,25D3 anti-tumor activity. In order to isolate regulators of CYP24A1 expression in prostate cancer cells, we established a stable prostate cancer cell line PC3 with CYP24A1 promoter driving luciferase expression to screen a small molecular library for compounds that inhibit CYP24A1 promoter activity. From this screening, we identified, 4,5,6,7-tetrabromobenzimidazole (TBBz), a protein kinase CK2 selective inhibitor as a disruptor of CYP24A1 promoter activity. We show that TBBz inhibits CYP24A1 promoter activity induced by 1,25D3 in prostate cancer cells. In addition, TBBz downregulates endogenous CYP24A1 mRNA level in TBBz treated PC3 cells. Furthermore, siRNA-mediated CK2 knockdown reduces 1,25D3 induced CYP24A1 mRNA expression in PC3 cells. These results suggest that CK2 contributes to 1,25D3 mediated target gene expression. Lastly, inhibition of CK2 by TBBz or CK2 siRNA significantly enhanced 1,25D3 mediated anti-proliferative effect in vitro and in vivo in a xenograft model. In summary, our findings reveal that protein kinase CK2 is involved in the regulation of CYP24A1 expression by 1,25D3 and CK2 inhibitor enhances 1,25D3 mediated anti-tumor effect. PMID:23358686

  13. Downregulation of Cellular c-Jun N-Terminal Protein Kinase and NF-κB Activation by Berberine May Result in Inhibition of Herpes Simplex Virus Replication

    Song, Siwei; Qiu, Min; Chu, Ying; Chen, Deyan; Wang, Xiaohui; Su, Airong; Wu, Zhiwei

    2014-01-01

    Berberine is a quaternary ammonium salt from the protoberberine group of isoquinoline alkaloids. Some reports show that berberine exhibits anti-inflammatory, antitumor, and antiviral properties by modulating multiple cellular signaling pathways, including p53, nuclear factor κB (NF-κB), and mitogen-activated protein kinase. In the present study, we investigated the antiviral effect of berberine against herpes simplex virus (HSV) infection. Current antiherpes medicines such as acyclovir can le...

  14. Inhibition of Naja kaouthia venom activities by plant polyphenols.

    Pithayanukul, Pimolpan; Ruenraroengsak, Pakatip; Bavovada, Rapepol; Pakmanee, Narumol; Suttisri, Rutt; Saen-oon, Suwipa

    2005-03-21

    Plant polyphenols from the aqueous extracts of Pentace burmanica, Pithecellobium dulce, Areca catechu and Quercus infectoria were tested for their inhibitory activities against Naja kaouthia (NK) venom by in vitro neutralization method. The first three extracts could completely inhibit the lethality of the venom at 4 LD50 concentration and the venom necrotizing activity at the minimum necrotizing dose while also inhibited up to 90% of the acetylcholinesterase activity of NK venom at much lower tannin concentrations than that of Quercus infectoria. The ED50 of plant tannins in inhibiting NK venom activities varied according to condensed tannins and their content in the extracts. Molecular docking of the complexes between alpha-cobratoxin and either hydrolysable or condensed tannins at their lowest energetic conformations were proposed. The anti-venom activities of these plant polyphenols by selectively blocking the nicotinic acetylcholine receptor and non-selectively by precipitation of the venom proteins were suggested. PMID:15740891

  15. The cellular TAR RNA binding protein, TRBP, promotes HIV-1 replication primarily by inhibiting the activation of double-stranded RNA-dependent kinase PKR.

    Sanghvi, Viraj R; Steel, Laura F

    2011-12-01

    The TAR RNA binding protein, TRBP, is a cellular double-stranded RNA (dsRNA) binding protein that can promote the replication of HIV-1 through interactions with the viral TAR element as well as with cellular proteins that affect the efficiency of translation of viral transcripts. The structured TAR element, present on all viral transcripts, can impede efficient translation either by sterically blocking access of translation initiation factors to the 5'-cap or by activating the dsRNA-dependent kinase, PKR. Several mechanisms by which TRBP can facilitate translation of viral transcripts have been proposed, including the binding and unwinding of TAR and the suppression of PKR activation. Further, TRBP has been identified as a cofactor of Dicer in the processing of microRNAs (miRNAs), and sequestration of TRBP by TAR in infected cells has been proposed as a viral countermeasure to potential host cell RNA interference-based antiviral activities. Here, we have addressed the relative importance of these various roles for TRBP in HIV-1 replication. Using Jurkat T cells, primary human CD4(+) T cells, and additional cultured cell lines, we show that depletion of TRBP has no effect on viral replication when PKR activation is otherwise blocked. Moreover, the presence of TAR-containing mRNAs does not affect the efficacy of cellular miRNA silencing pathways. These results establish that TRBP, when expressed at physiological levels, promotes HIV-1 replication mainly by suppressing the PKR-mediated antiviral response, while its contribution to HIV-1 replication through PKR-independent pathways is minimal. PMID:21937648

  16. Linking algal growth inhibition to chemical activity

    Schmidt, Stine N.; Mayer, Philipp

    chemical activity, as opposed to e.g. the total concentration. Baseline toxicity (narcosis) for neutral hydrophobic organic compounds has been shown to initiate in the narrow chemical activity range of 0.01 to 0.1. This presentation focuses on linking algal growth inhibition to chemical activity with the...

  17. Serum protein inhibition of thyrotropin binding to human thyroid tissue

    We used a modificaton of the TSH radioreceptor assay to detect TSH-binding inhibition (TBI) activity in serum and serum fractions from normal subjects and patients with Graves' disease. TBI activity is present in normal IgG prepared by DEAE-Sephadex chromatography and in normal globulins prepared by precipitation at 1.6 M ammonium sulfate. Other normal serum proteins also had TBI activity when large concentrations were tested. Gel filtration chromatography and powder block electrophoresis were used to prepare fractions of normal and Graves' disease sera. In these fractions from normal serum, TBI activity was found in both γ-globulin and α-globulin-albumin fractions electrophoretically and in both 7S and 4S peaks from gel filtration. TBI activity from Graves' disease patients' sera was similarly distributed, but relatively more TBI accompanied the electrophoretic γ-globulins. Sepharose Protein-A and anti-IgG were used as immunoabsorbents to isolate and purify IgG from normal and Graves' disease sera. TBI activity in IgG was proportional to the IgG concentration, indicating that the TBI which migrates as a γ-globulin electrophoretically is an IgG and thus may possibly be an antibody. Inhibitory activity found in normal serum globulins and in the non-IgG fractions of both normal and abnormal sera seriously interferes with attempts to use the TSH radioreceptor assay to study the hypothesized anti-TSH receptor antibody in the serum of patients with Graves' disease

  18. Effects of activation of protein kinase C (PKC) on the hormonal stimulation and inhibition of cAMP formation in intact human platelets

    Washed platelets, labelled by preincubation with [3H]adenine and [32P]P/sub i/, were studied in the presence of indomethacin, phosphocreatine and creatine phosphokinase to block thromboxane A2 formation and inhibitory effects of released ADP. Addition of phorbol 12-myristate 13-acetate (PMA) or 1,2-dioctanoyl-glycerol (diC8) decreased the initial rate of accumulation of [3H]cAMP observed with PGE1 and 3-isobutyl 1- methylxanthine. Maximal decreases of 31% (1 μM PMA) and 42% (100 μM diC8) were obtained. Also, the inhibition of [3H]cAMP formation by epinephrine (5 μM) was decreased from 68% to 16% and 31% by 1μM PMA and 100 μM diC8, respectively. The effects of increasing concentrations of PMA and diC8 on the stimulation of [3H]cAMp formation by PGE1 and on the inhibitory action of epinephrine correlated with increases in 32P incorporation into the major substrate of PKC (P47) and into two other polypeptides (P41 and P20). These results suggested that activation of PKC might explain the failure of some aggregating agents (e.g. PAF and vasopressin) to inhibit adenylate cyclase in intact platelets, although they are inhibitory with isolated membranes. However, comparison of the effects of PMA and these aggregating agents on the phosphorylation of platelet polypeptides indicated that activation of PKC by aggregating agents is inadequate to block their inhibitory effects on adenylate cyclase, when PGE1 is present

  19. Inhibition of the Unfolded Protein Response Mechanism Prevents Cardiac Fibrosis

    Jung, Joanna; Dyck, Jason R. B.; Lopaschuk, Gary D.; Agellon, Luis B.; Michalak, Marek

    2016-01-01

    Background Cardiac fibrosis attributed to excessive deposition of extracellular matrix proteins is a major cause of heart failure and death. Cardiac fibrosis is extremely difficult and challenging to treat in a clinical setting due to lack of understanding of molecular mechanisms leading to cardiac fibrosis and effective anti-fibrotic therapies. The objective in this study was to examine whether unfolded protein response (UPR) pathway mediates cardiac fibrosis and whether a pharmacological intervention to modulate UPR can prevent cardiac fibrosis and preserve heart function. Methodology/Principal Findings We demonstrate here that the mechanism leading to development of fibrosis in a mouse with increased expression of calreticulin, a model of heart failure, stems from impairment of endoplasmic reticulum (ER) homeostasis, transient activation of the unfolded protein response (UPR) pathway and stimulation of the TGFβ1/Smad2/3 signaling pathway. Remarkably, sustained pharmacologic inhibition of the UPR pathway by tauroursodeoxycholic acid (TUDCA) is sufficient to prevent cardiac fibrosis, and improved exercise tolerance. Conclusions We show that the mechanism leading to development of fibrosis in a mouse model of heart failure stems from transient activation of UPR pathway leading to persistent remodelling of cardiac tissue. Blocking the activation of the transiently activated UPR pathway by TUDCA prevented cardiac fibrosis, and improved prognosis. These findings offer a window for additional interventions that can preserve heart function. PMID:27441395

  20. Neuroprotective effects of activated protein C on intrauterine inflammation-induced neonatal white matter injury are associated with the downregulation of fibrinogen-like protein 2/fibroleukin prothrombinase and the inhibition of pro-inflammatory cytokine expression

    JIN, SHENG-JUAN; Yan LIU; DENG, SHI-HUA; LIAO, LI-HONG; LIN, TU-LIAN; Ning, Qin; LUO, XIAO-PING

    2015-01-01

    Maternal intrauterine inflammation or infection is an important risk factor for neonatal cerebral white matter injury (WMI) and future neurological deficits. Activated protein C (APC), a natural anticoagulant, has been shown to exhibit anti-inflammatory, anti-apoptotic, profibrinolytic and cytoprotective activities. Recent studies have demonstrated that the novel prothrombinase, fibrinogen-like protein 2 (fgl2), contributes to the pathogenesis of a number of inflammatory diseases through the ...

  1. MG-132 inhibits the TCDD-mediated induction of Cyp1a1 at the catalytic activity but not the mRNA or protein levels in Hepa 1c1c7 cells.

    Anwar-Mohamed, Anwar; Elbekai, Reem H; El-Kadi, Ayman O S

    2008-11-10

    Previous studies have shown that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced degradation of aryl hydrocarbon receptor (AhR) is inhibited by MG-132, a potent inhibitor of the 26S proteasome. Therefore, the current study aims to address the effect of MG-132 on the AhR-regulated gene, cytochrome P450 1a1 (Cyp1a1), using murine hepatoma Hepa 1c1c7 cells. Our results showed that MG-132 at the highest concentration tested, 10 microM significantly increased the Cyp1a1 at mRNA, protein and catalytic activity levels through a transcriptional mechanism. On the other hand, MG-132 further potentiated the TCDD-mediated induction of Cyp1a1 at mRNA but not at protein level. In contrast, MG-132 significantly inhibited the TCDD-mediated induction of Cyp1a1 catalytic activity. In addition, we showed that the decrease in Cyp1a1 catalytic activity is not Cyp specific, as MG-132 significantly inhibited Cyp2b1 and total cytochrome P450 catalytic activities. These results prompted us to examine the effect of MG-132 on total cellular heme content and heme oxygenase-1 (HO-1) mRNA, a rate limiting enzyme of heme degradation. Our results showed that MG-132 significantly induced HO-1 mRNA in a concentration-dependent manner. Furthermore, MG-132 potentiated the induction of HO-1 mRNA by TCDD in a concentration-dependent manner. The induction of HO-1 mRNA level coincided with a decrease in total cellular heme content. In conclusion, the present study demonstrates for the first time that MG-132, despite of increasing Cyp1a1 mRNA expression, it decreases its activity probably through decreasing its heme content. PMID:18835339

  2. Wild-type, but not mutant, human p53 proteins inhibit the replication activities of simian virus 40 large tumor antigen.

    Friedman, P N; Kern, S. E.; Vogelstein, B; Prives, C

    1990-01-01

    Murine p53 blocks many of the replication activities of simian virus 40 (SV40) large tumor antigen (T antigen) in vitro. As murine cells do not replicate SV40 DNA, it was of interest to determine how p53 from permissive human cells functions. Recombinant baculoviruses encoding either the wild-type form of human p53 or a mutant p53 cloned from a human tumor cell line were constructed, and p53 proteins were purified from infected insect cells. Surprisingly, we found that wild-type human p53 was...

  3. Vitamin D binding protein-derived macrophage activating factor inhibits human breast cancer cell proliferation and decreases alpha-N-acetyl galactosaminidase in breast cancer patiens.

    L. Thyer; G.Morucci; J.J.V. Branca; E. Wards; Smith, R; Noakes, D.

    2013-01-01

    Objectives: It was demonstrated that vitamin D binding protein-derived macrophage activating factor (GcMAF) decreases serum alpha-N-acetyl galactosaminidase (nagalase) in breast cancer patients; decrease of nagalase was associated with significant improvement of clinical conditions (Int J Cancer. 2008 Jan 15;122(2):461-7). Since nagalase is both an index of immune suppression and of tumour burden, we studied the direct effects of GcMAF on a human breast cancer cell line (MCF-7), and we com...

  4. Gc protein-derived macrophage-activating factor (GcMAF) stimulates cAMP formation in human mononuclear cells and inhibits angiogenesis in chick embryo chorionallantoic membrane assay

    S. Pacini; G.Morucci; T.Punzi; Gulisano, M; Ruggiero, M

    2010-01-01

    Abstract: The effects of Gc protein-derived macrophage-activating factor (GcMAF) have been studied in cancer and other conditions where angiogenesis is deregulated. In this study, we demonstrate for the first time that the mitogenic response of human peripheral blood mononuclear cells (PBMCs) to GcMAF was associated with 3'-5'-cyclic adenosine monophosphate (cAMP) formation. The effect was dose dependent, and maximal stimulation was achieved using 0.1 ng/ml. Heparin inhibited the stimulatory ...

  5. Activation and activities of the p53 tumour suppressor protein

    Bálint, É; Vousden, K H

    2001-01-01

    The p53 tumour suppressor protein inhibits malignant progression by mediating cell cycle arrest, apoptosis or repair following cellular stress. One of the major regulators of p53 function is the MDM2 protein, and multiple forms of cellular stress activate p53 by inhibiting the MDM2-mediated degradation of p53. Mutations in p53, or disruption of the pathways that allow activation of p53, seem to be a general feature of all cancers. Here we review recent advances in our understanding of the pat...

  6. Sitagliptin inhibits endothelin-1 expression in the aortic endothelium of rats with streptozotocin-induced diabetes by suppressing the nuclear factor-κB/IκBα system through the activation of AMP-activated protein kinase.

    Tang, Song-Tao; Su, Huan; Zhang, Qiu; Tang, Hai-Qin; Wang, Chang-Jiang; Zhou, Qing; Wei, Wei; Zhu, Hua-Qing; Wang, Yuan

    2016-06-01

    Emerging evidence suggests that dipeptidyl peptidase-4 (DPP-4) inhibitors, including sitagliptin, exert favourable effects on the vascular endothelium. DPP-4 inhibitors suppress the degradation of glucagon-like peptide-1 (GLP‑1), which has been reported to enhance nitric oxide (NO) production. However, the effects of DPP-4 inhibitors on endothelin-1 (ET-1) expression in the aorta, as well as the underlying mechanisms responsible for these effects, have yet to be investigated in animal models of diabetes mellitus (DM). In the present study, the rats were randomly divided into the following four groups: i) control; ii) DM; iii) DM + low‑dose sitagliptin (10 mg/kg); and iv) DM + high‑dose sitagliptin (30 mg/kg). Apart from the control group, all the rats received a high-fat diet for 8 weeks prior to the induction of diabetes with an intraperitoneal injection of streptozotocin. The treatments were then administered for 12 weeks. The serum levels of ET-1, NO, GLP-1 and insulin were measured as well as endothelial function. The expression of ET-1, AMP-activated protein kinase (AMPK) and nuclear factor (NF)-κB/IκBα were determined. After 12 weeks of treatment, the diabetic rats receiving sitagliptin showed significantly elevated serum levels of GLP-1 and NO, and reduced levels of ET-1. Moreover, sitagliptin significantly attenuated endothelial dysfunction as well as the remodeling of the aortic wall. Notably, sitagliptin inhibited ET-1 expression at the transcriptional and translational level in the aorta, which may have been mediated by the suppression of the NF-κB/IκBα system induced by AMPK activation. The majority of the above-mentioned effects were dose dependent. Taken together, the findings of the present study indicate that sitagliptin inhibits ET-1 expression in the aortic endothelium by suppressing the NF-κB/IκBα system through the activation of the AMPK pathway in diabetic rats. These findings further demonstrate some of the

  7. Inhibition of mammalian mitochondrial protein synthesis by oxazolidinones.

    McKee, E E; Ferguson, M; Bentley, A T; Marks, T A

    2006-06-01

    The effects of a variety of oxazolidinones, with different antibacterial potencies, including linezolid, on mitochondrial protein synthesis were determined in intact mitochondria isolated from rat heart and liver and rabbit heart and bone marrow. The results demonstrate that a general feature of the oxazolidinone class of antibiotics is the inhibition of mammalian mitochondrial protein synthesis. Inhibition was similar in mitochondria from all tissues studied. Further, oxazolidinones that were very potent as antibiotics were uniformly potent in inhibiting mitochondrial protein synthesis. These results were compared to the inhibitory profiles of other antibiotics that function by inhibiting bacterial protein synthesis. Of these, chloramphenicol and tetracycline were significant inhibitors of mammalian mitochondrial protein synthesis while the macrolides, lincosamides, and aminoglycosides were not. Development of future antibiotics from the oxazolidinone class will have to evaluate potential mitochondrial toxicity. PMID:16723564

  8. All-trans retinoic acid inhibits KIT activity and induces apoptosis in gastrointestinal stromal tumor GIST-T1 cell line by affecting on the expression of survivin and Bax protein

    Taguchi Takahiro

    2010-12-01

    Full Text Available Abstract Background Imatinib, a selective tyrosine kinase inhibitor, has been used as a standard first-line therapy for irresectable and metastasized gastrointestinal stromal tumor (GIST patients. Unfortunately, most patients responding to imatinib will eventually exhibit imatinib-resistance, the cause of which is not fully understood. The serious clinical problem of imatinib-resistance demands alternative therapeutic strategy. This study was conducted to investigate the effect of all-trans retinoic acid (ATRA on GIST cell lines. Methods Cell proliferation was determined by trypan blue dye exclusion test. Western blot analysis was performed to test the expression of activated KIT, its downstream proteins, and apoptosis associated proteins. The cytotoxic interactions of imatinib with ATRA were evaluated using the isobologram of Steel and Peckham. Results and conclusion In this work, for the first time we have demonstrated that ATRA affected on cell proliferation of GIST-T1 and GIST-882 cell line through inhibition of cell growth in a dose dependent manner and induced apoptosis. High dose of ATRA induced morphologic change in GIST-T1 cells, rounded-up cells, and activated the caspase-3 protein. In further examination, we found that the ATRA-induced apoptosis in GIST-T1 cells was accompanied by the down-regulated expression of survivin and up-regulated expression of Bax protein. Moreover, ATRA suppressed the activity of KIT protein in GIST-T1 cells and its downstream signal, AKT activity, but not MAPK activity. We also have demonstrated that combination of ATRA with imatinib showed additive effect by isobologram, suggesting that the combination of ATRA and imatinib may be a novel potential therapeutic option for GIST treatment. Furthermore, the scracht assay result suggested that ATRA was a potential reagent to prevent the invasion or metastasis of GIST cells.

  9. Inhibition of CDC25B Phosphatase Through Disruption of Protein-Protein Interaction

    Lund, George; Dudkin, Sergii; Borkin, Dmitry; Ni, Wendi; Grembecka, Jolanta; Cierpicki, Tomasz [Michigan

    2015-04-29

    CDC25 phosphatases are key cell cycle regulators and represent very attractive but challenging targets for anticancer drug discovery. Here, we explored whether fragment-based screening represents a valid approach to identify inhibitors of CDC25B. This resulted in identification of 2-fluoro-4-hydroxybenzonitrile, which directly binds to the catalytic domain of CDC25B. Interestingly, NMR data and the crystal structure demonstrate that this compound binds to the pocket distant from the active site and adjacent to the protein–protein interaction interface with CDK2/Cyclin A substrate. Furthermore, we developed a more potent analogue that disrupts CDC25B interaction with CDK2/Cyclin A and inhibits dephosphorylation of CDK2. Based on these studies, we provide a proof of concept that targeting CDC25 phosphatases by inhibiting their protein–protein interactions with CDK2/Cyclin A substrate represents a novel, viable opportunity to target this important class of enzymes.

  10. Kinetic evaluation of the inhibition of protein glycation during heating.

    Akıllıoğlu, H Gül; Gökmen, Vural

    2016-04-01

    This study aimed to investigate the kinetics of early stage of the Maillard reaction by a reversible bimolecular reaction mechanism and also to evaluate the compatibility of enzyme inhibition kinetics for calculating the inhibitory activity of protein anti-glycation agents. Model systems composed of ovalbumin, glucose, and anti-glycation agents (tannic acid or calcium ion) at different molar ratios were heated at 90 °C for different times in dry state or in solution. Heated samples were analysed for furosine, acid derivative of N-ε-fructoselysine (FL), to monitor the progression of the early glycation stage. Compared to a control, presence of calcium ions and tannic acid decreased FL formation significantly (p<0.05) during heating in dry state. Evaluation of the kinetic data revealed that calcium inhibited glycation of ovalbumin by a mixed non-competitive mechanism in both dry and in solution conditions; while the mode of inhibition by tannic acid was found to be purely non-competitive in the dry state. PMID:26593596

  11. Activin inhibits telomerase activity in cancer

    Katik, Indzi; Mackenzie-Kludas, Charley; Nicholls, Craig [Department of Immunology, Monash University, Melbourne (Australia); Jiang, Fang-Xu [Centre for Diabetes Research, Western Australian Institute for Medical Research and The University of Western Australia, Perth (Australia); Zhou, Shufeng [School of Health Sciences, RMIT University, Melbourne (Australia); Li, He [Department of Immunology, Monash University, Melbourne (Australia); Liu, Jun-Ping, E-mail: jun-ping.liu@med.monash.edu.au [Department of Immunology, Monash University, Melbourne (Australia)

    2009-11-27

    Activin is a pleiotropic cytokine with broad tissue distributions. Recent studies demonstrate that activin-A inhibits cancer cell proliferation with unknown mechanisms. In this report, we demonstrate that recombinant activin-A induces telomerase inhibition in cancer cells. In breast and cervical cancer cells, activin-A resulted in telomerase activity in a concentration-dependent manner. Significant inhibition was observed at 10 ng/ml of activin-A, with a near complete inhibition at 80 ng/ml. Consistently, activin-A induced repression of the telomerase reverse transcriptase (hTERT) gene, with the hTERT gene to be suppressed by 60-80% within 24 h. In addition, activin-A induced a concomitant increase in Smad3 signaling and decrease of the hTERT gene promoter activity in a concentration-dependent fashion. These data suggest that activin-A triggered telomerase inhibition by down-regulating hTERT gene expression is involved in activin-A-induced inhibition of cancer cell proliferation.

  12. Zinc-finger antiviral protein inhibits XMRV infection.

    Xinlu Wang

    Full Text Available BACKGROUND: The zinc-finger antiviral protein (ZAP is a host factor that specifically inhibits the replication of certain viruses, including Moloney murine leukemia virus (MoMLV, HIV-1, and certain alphaviruses and filoviruses. ZAP binds to specific viral mRNAs and recruits cellular mRNA degradation machinery to degrade the target RNA. The common features of ZAP-responsive RNA sequences remain elusive and thus whether a virus is susceptible to ZAP can only be determined experimentally. Xenotropic murine leukemia virus-related virus (XMRV is a recently identified γ-retrovirus that was originally thought to be involved in prostate cancer and chronic fatigue syndrome but recently proved to be a laboratory artefact. Nonetheless, XMRV as a new retrovirus has been extensively studied. Since XMRV and MoMLV share only 67.9% sequence identity in the 3'UTRs, which is the target sequence of ZAP in MoMLV, whether XMRV is susceptible to ZAP remains to be determined. FINDINGS: We constructed an XMRV-luc vector, in which the coding sequences of Gag-Pol and part of Env were replaced with luciferase-coding sequence. Overexpression of ZAP potently inhibited the expression of XMRV-luc in a ZAP expression-level-dependent manner, while downregulation of endogenous ZAP rendered cells more sensitive to infection. Furthermore, ZAP inhibited the spreading of replication-competent XMRV. Consistent with the previously reported mechanisms by which ZAP inhibits viral infection, ZAP significantly inhibited the accumulation of XMRV-luc mRNA in the cytoplasm. The ZAP-responsive element in XMRV mRNA was mapped to the 3'UTR. CONCLUSIONS: ZAP inhibits XMRV replication by preventing the accumulation of viral mRNA in the cytoplasm. Documentation of ZAP inhibiting XMRV helps to broaden the spectrum of ZAP's antiviral activity. Comparison of the target sequences of ZAP in XMRV and MoMLV helps to better understand the features of ZAP-responsive elements.

  13. Inhibition of Mammalian Mitochondrial Protein Synthesis by Oxazolidinones

    McKee, E. E.; Ferguson, M; Bentley, A T; Marks, T. A.

    2006-01-01

    The effects of a variety of oxazolidinones, with different antibacterial potencies, including linezolid, on mitochondrial protein synthesis were determined in intact mitochondria isolated from rat heart and liver and rabbit heart and bone marrow. The results demonstrate that a general feature of the oxazolidinone class of antibiotics is the inhibition of mammalian mitochondrial protein synthesis. Inhibition was similar in mitochondria from all tissues studied. Further, oxazolidinones that wer...

  14. Antiviral Protein of Momordica charantia L. Inhibits Different Subtypes of Influenza A

    Viroj Pongthanapisith; Kazuyoshi Ikuta; Pilaipan Puthavathana; Wichet Leelamanit

    2013-01-01

    The new antiviral activity of the protein extracted from Momordica charantia was determined with different subtypes of influenza A. The protein was purified from the seed of M. charantia using an anion exchanger and a Fast Protein Liquid Chromatography (FPLC) system. At the concentration of 1.401 mg/mL, the protein did not exhibit cytotoxicity in Madin-Darby canine kidney cells (MDCK) but inhibited 1 × 105 FFU influenza A/PR/8/34 H1N1 virus at 56.50%, 65.72%, and 100% inhibition by the protei...

  15. Inhibition of Heat Shock Protein 90 Prevents HIV Rebound*

    Joshi, Pheroze; Maidji, Ekaterina; Stoddart, Cheryl A.

    2016-01-01

    HIV evades eradication because transcriptionally dormant proviral genomes persist in long-lived reservoirs of resting CD4+ T cells and myeloid cells, which are the source of viral rebound after cessation of antiretroviral therapy. Dormant HIV genomes readily produce infectious virus upon cellular activation because host transcription factors activated specifically by cell stress and heat shock mediate full-length HIV transcription. The molecular chaperone heat shock protein 90 (Hsp90) is overexpressed during heat shock and activates inducible cellular transcription factors. Here we show that heat shock accelerates HIV transcription through induction of Hsp90 activity, which activates essential HIV-specific cellular transcription factors (NF-κB, NFAT, and STAT5), and that inhibition of Hsp90 greatly reduces gene expression mediated by these factors. More importantly, we show that Hsp90 controls virus transcription in vivo by specific Hsp90 inhibitors in clinical development, tanespimycin (17-(allylamino)-17-demethoxygeldanamycin) and AUY922, which durably prevented viral rebound in HIV-infected humanized NOD scid IL-2Rγ−/− bone marrow-liver-thymus mice up to 11 weeks after treatment cessation. Despite the absence of rebound viremia, we were able to recover infectious HIV from PBMC with heat shock. Replication-competent virus was detected in spleen cells from these nonviremic Hsp90 inhibitor-treated mice, indicating the presence of a tissue reservoir of persistent infection. Our novel findings provide in vivo evidence that inhibition of Hsp90 activity prevents HIV gene expression in replication-competent cellular reservoirs that would typically cause rebound in plasma viremia after antiretroviral therapy cessation. Alternating or supplementing Hsp90 inhibitors with current antiretroviral therapy regimens could conceivably suppress rebound viremia from persistent HIV reservoirs. PMID:26957545

  16. Acetaldehyde inhibition of protein synthesis in isolated rat pancreatic acini

    Exposure of isolated dispersed pancreatic acini to increasing concentrations of ethanol (5 to 500 mM) or acetaldehyde (0.5 to 100 mM) produced a progressive inhibition of [3H]leucine incorporation into both cellular (those remaining in the cell) and secretory (those released into the medium) proteins. Whereas 500 mM ethanol caused 90-95% inhibition in the synthesis of cellular and secretory proteins, the concentration of acetaldehyde needed to produce a similar inhibition was found to be 50 mM. All subsequent experiments were performed with 12.5 mM acetaldehyde, a concentration that consistently inhibited acinar protein synthesis by about 50%. The acetaldehyde-mediated inhibition of acinar protein synthesis was partially normalized when this metabolite was removed after 30 min during a 90-min incubation period. In the presence of acetaldehyde, the secretion of 3H-pulse-labeled proteins, but not amylase, trypsinogen, or chymotrypsinogen, was greatly depressed. Acetaldehyde also caused a marked reduction in [3H]uridine incorporation into acinar RNA. The entry of [3H]uridine, [3H]leucine, and [3H]aminoisobutyric acid into isolated acini was found to be slightly (15-25%) decreased by acetaldehyde. It is concluded that acetaldehyde exerts a direct toxic effect on isolated dispersed pancreatic acini as evidenced by diminution of both protein and RNA synthesis and decreased secretion of the newly synthesized proteins. This inhibitory effect of acetaldehyde could be partially reversed

  17. Hypoxia inhibits abdominal expiratory nerve activity.

    Fregosi, R F; Knuth, S L; Ward, D K; Bartlett, D

    1987-07-01

    Our purpose was to examine the influence of steady-state changes in chemical stimuli, as well as discrete peripheral chemoreceptor stimulation, on abdominal expiratory motor activity. In decerebrate, paralyzed, vagotomized, and ventilated cats that had bilateral pneumothoraces, we recorded efferent activity from a phrenic nerve and from an abdominal nerve (cranial iliohypogastric nerve, L1). All cats showed phasic expiratory abdominal nerve discharge at normocapnia [end-tidal PCO2 38 +/- 2 Torr], but small doses (2-6 mg/kg) of pentobarbital sodium markedly depressed this activity. Hyperoxic hypercapnia consistently enhanced abdominal expiratory activity and shortened the burst duration. Isocapnic hypoxia caused inhibition of abdominal nerve discharge in 11 of 13 cats. Carotid sinus nerve denervation (3 cats) exacerbated the hypoxic depression of abdominal nerve activity and depressed phrenic motor output. Stimulation of peripheral chemoreceptors with NaCN increased abdominal nerve discharge in 7 of 10 cats, although 2 cats exhibited marked inhibition. Four cats with intact neuraxis, but anesthetized with ketamine, yielded qualitatively similar results. We conclude that when cats are subjected to steady-state chemical stimuli in isolation (no interference from proprioceptive inputs), hypercapnia potentiates, but hypoxia attenuates, abdominal expiratory nerve activity. Mechanisms to explain the selective inhibition of expiratory motor activity by hypoxia are proposed, and physiological implications are discussed. PMID:3624126

  18. Linking algal growth inhibition to chemical activity

    Schmidt, Stine N.; Mayer, Philipp

    2015-01-01

    Recently, high-quality data were published on the algal growth inhibition caused by 50 non-polar narcotic compounds, of which 39 were liquid compounds with defined water solubility. In the present study, the toxicity data for these liquids were applied to challenge the chemical activity range for...

  19. Spongian diterpenoids inhibit androgen receptor activity

    Yang, Yu Chi; Labros G Meimetis; Tien, Amy H; Mawji, Nasrin R.; Carr, Gavin; Wang, Jun; Andersen, Raymond J.; Sadar, Marianne D.

    2013-01-01

    Androgen receptor (AR) is a ligand-activated transcription factor and a validated drug target for all stages of prostate cancer. Antiandrogens compete with physiological ligands for AR ligand-binding domain (LBD). High-throughput screening of a marine natural product library for small molecules that inhibit AR transcriptional activity yielded the furanoditerpenoid spongia-13(16),-14-dien-19-oic acid, designated terpene 1 (T1). Characterization of T1 and the structurally related semi-synthetic...

  20. Inhibition of Neuroinflammation in LPS-Activated Microglia by Cryptolepine.

    Olajide, Olumayokun A; Bhatia, Harsharan S; de Oliveira, Antonio C P; Wright, Colin W; Fiebich, Bernd L

    2013-01-01

    Cryptolepine, an indoloquinoline alkaloid in Cryptolepis sanguinolenta, has anti-inflammatory property. In this study, we aimed to evaluate the effects of cryptolepine on lipopolysaccharide (LPS)- induced neuroinflammation in rat microglia and its potential mechanisms. Microglial activation was induced by stimulation with LPS, and the effects of cryptolepine pretreatment on microglial activation and production of proinflammatory mediators, PGE2/COX-2, microsomal prostaglandin E2 synthase and nitric oxide/iNOS were investigated. We further elucidated the role of Nuclear Factor-kappa B (NF- κ B) and the mitogen-activated protein kinases in the antiinflammatory actions of cryptolepine in LPS-stimulated microglia. Our results showed that cryptolepine significantly inhibited LPS-induced production of tumour necrosis factor-alpha (TNF α ), interleukin-6 (IL-6), interleukin-1beta (IL-1 β ), nitric oxide, and PGE2. Protein and mRNA levels of COX-2 and iNOS were also attenuated by cryptolepine. Further experiments on intracellular signalling mechanisms show that I κ B-independent inhibition of NF- κ B nuclear translocation contributes to the anti-neuroinflammatory actions of cryptolepine. Results also show that cryptolepine inhibited LPS-induced p38 and MAPKAPK2 phosphorylation in the microglia. Cell viability experiments revealed that cryptolepine (2.5 and 5  μ M) did not produce cytotoxicity in microglia. Taken together, our results suggest that cryptolepine inhibits LPS-induced microglial inflammation by partial targeting of NF- κ B signalling and attenuation of p38/MAPKAPK2. PMID:23737832

  1. Prolonged inhibition of bacterial protein synthesis abolishes Salmonella invasion.

    MacBeth, K J; Lee, C. A.

    1993-01-01

    We have found that prolonged inhibition of bacterial protein synthesis abolishes the ability of Salmonella typhimurium to enter HEp-2 cells. Our results suggest that an essential invasion factor has a functional half-life that is seen as a gradual loss of invasiveness in the absence of protein synthesis. Therefore, Salmonella invasiveness appears to be a transient phenotype that is lost unless protein synthesis is maintained. This finding may explain why salmonellae grown to stationary phase ...

  2. Upstream and Downstream Co-inhibition of Mitogen-Activated Protein Kinase and PI3K/Akt/mTOR Pathways in Pancreatic Ductal Adenocarcinoma

    Matthew H. Wong

    2016-07-01

    Full Text Available BACKGROUND: Extensive cross talk exists between PI3K/Akt/mTOR and mitogen-activated protein kinase (MAPK pathways, and both are upregulated in pancreatic ductal adenocarcinoma (PDAC. Our previous study suggested that epidermal growth factor receptor inhibitor erlotinib which acts upstream of these pathways acts synergistically with PI3K inhibitors in PDAC. Horizontal combined blockade upstream and downstream of these two pathways is therefore explored. METHODS: Erlotinib paired with PI3K inhibitor (BYL719 was tested against erlotinib plus dual PI3K/mTOR inhibitor BEZ-235, and MEK inhibitor (PD98059 plus BEZ235, on five primary PDAC cell lines and on two pairs of parent and erlotinib-resistant (ER cell lines. A range of in vitro assays including cell proliferation, Western blotting, migration, clonogenic, cell cycle, and apopotic assays was used to test for the efficacy of combined blockade. RESULTS: Dual downstream blockade of the MAPK and PAM pathways was more effective in attenuating downstream molecular signals. Synergy was demonstrated for erlotinib and BEZ235 and for PD-98059 and BEZ-235. This resulted in a trend of increased growth cell cycle arrest, apoptosis, cell proliferation, and colony and migration suppression. This combination showed more efficacy in cell lines with acquired resistance to erlotinib. CONCLUSIONS: The additional mTOR blockade provided by BEZ235 in combined blockade resulted in increased anticancer effect. The hypersensitivity of ER cell lines to additional mTOR blockade suggested PAM pathway oncogenic dependence via mTOR. Dual downstream combined blockade of MAPK and PAM pathways with MEK and PI3K/mTOR inhibitor appeared most effective and represents an attractive therapeutic strategy against pancreatic cancer and its associated drug resistance.

  3. Interferon-Induced Transmembrane Protein-Mediated Inhibition of Host Cell Entry of Ebolaviruses.

    Wrensch, Florian; Karsten, Christina B; Gnirß, Kerstin; Hoffmann, Markus; Lu, Kai; Takada, Ayato; Winkler, Michael; Simmons, Graham; Pöhlmann, Stefan

    2015-10-01

    Ebolaviruses are highly pathogenic in humans and nonhuman primates and pose a severe threat to public health. The interferon-induced transmembrane (IFITM) proteins can restrict entry of ebolaviruses, influenza A viruses, and other enveloped viruses. However, the breadth and mechanism of the antiviral activity of IFITM proteins are incompletely understood. Here, we employed ebolavirus glycoprotein-pseudotyped vectors and ebolavirus-like particles to address this question. We show that IFITM proteins inhibit the cellular entry of diverse ebolaviruses and demonstrate that type I interferon induces IFITM protein expression in macrophages, major viral targets. Moreover, we show that IFITM proteins block entry of influenza A viruses and ebolaviruses by different mechanisms and provide evidence that antibodies and IFITM proteins can synergistically inhibit cellular entry of ebolaviruses. These results provide insights into the role of IFITM proteins in infection by ebolaviruses and suggest a mechanism by which antibodies, though poorly neutralizing in vitro, might contribute to viral control in vivo. PMID:26034199

  4. Transcriptional inhibition by the retinoblastoma protein

    Fattaey, A; Helin, K; Harlow, E

    1993-01-01

    The retinoblastoma protein, pRB, appears to play a key role in coordinating the regulation of cell cycle position and transcriptional events. pRB undergoes specific cell-cycle-dependent phosphorylation, being underphosphorylated in G1 and heavily phosphorylated in S, G2, and M. The underphosphory......The retinoblastoma protein, pRB, appears to play a key role in coordinating the regulation of cell cycle position and transcriptional events. pRB undergoes specific cell-cycle-dependent phosphorylation, being underphosphorylated in G1 and heavily phosphorylated in S, G2, and M. The......-mediated transcription would be lost by mutation in the retinoblastoma gene in human tumours, by pRB's interaction with DNA tumour virus oncoproteins, or by phosphorylation during the cell cycle....

  5. Potent Protein Glycation Inhibition of Plantagoside in Plantago major Seeds

    Nobuyasu Matsuura

    2014-01-01

    Full Text Available Plantagoside (5,7,4′,5′-tetrahydroxyflavanone-3′-O-glucoside and its aglycone (5,7,3′,4′,5′-pentahydroxyflavanone, isolated from a 50% ethanol extract of Plantago major seeds (Plantaginaceae, were established to be potent inhibitors of the Maillard reaction. These compounds also inhibited the formation of advanced glycation end products in proteins in physiological conditions and inhibited protein cross-linking glycation. These results indicate that P. major seeds have potential therapeutic applications in the prevention of diabetic complications.

  6. S-NITROSYLATED PROTEINS OF A MEDICINAL, CAM PLANT KALANCHOE PINNATA: RIBULOSE-1, 5-BISPHOSPATE CARBOXYLASE/OXYGENASE ACTIVITY TARGETED FOR INHIBITION

    Nitric oxide (NO) is a new addition to signaling molecules that affect a myriad of processes in plants. However, the mechanistic details are scanty. NO post-translationally modifies proteins by S-nitrosylation of cysteines. Soluble S-nitrosoproteome of a medicinal, crassulacean acid metabolism (CAM)...

  7. A novel mannose-binding lectin/ficolin-associated protein is highly expressed in heart and skeletal muscle tissues and inhibits complement activation

    Skjoedt, Mikkel-Ole; Hummelshoj, Tina; Palarasah, Yaseelan;

    2010-01-01

    isoforms 1 and 2, respectively) are splice variants of the MASP1 gene, whereas MASP-2 and sMAP are splice variants of the MASP2 gene. We have identified a novel serum protein of 45 kDa that is associated with MBL and the ficolins. This protein is named MBL/ficolin-associated protein 1 (MAP-1 corresponding......The human lectin complement pathway involves circulating complexes consisting of mannose-binding lectin (MBL) or three ficolins (ficolin-1, -2, and -3) in association with three MBL/ficolin-associated serine proteases (MASP) (MASP-1, -2, and -3) and a nonenzymatic sMAP. MASP-1 and MASP-3 (MASP1...... to MASP1 isoform 3). The transcript generating MAP-1 (MASP1_v3) contains exons 1-8 and a novel exon encoding an in-frame stop codon. The corresponding protein lacks the serine protease domains but contains most of the common heavy chain of MASP-1 and MASP-3. Additionally MAP-1 contains 17 unique C...

  8. Curcumin directly inhibits the transport activity of GLUT1.

    Gunnink, Leesha K; Alabi, Ola D; Kuiper, Benjamin D; Gunnink, Stephen M; Schuiteman, Sam J; Strohbehn, Lauren E; Hamilton, Kathryn E; Wrobel, Kathryn E; Louters, Larry L

    2016-06-01

    Curcumin, a major ingredient in turmeric, has a long history of medicinal applications in a wide array of maladies including treatment for diabetes and cancer. Seemingly counterintuitive to the documented hypoglycemic effects of curcumin, however, a recent report indicates that curcumin directly inhibits glucose uptake in adipocytes. The major glucose transporter in adipocytes is GLUT4. Therefore, this study investigates the effects of curcumin in cell lines where the major transporter is GLUT1. We report that curcumin has an immediate inhibitory effect on basal glucose uptake in L929 fibroblast cells with a maximum inhibition of 80% achieved at 75 μM curcumin. Curcumin also blocks activation of glucose uptake by azide, glucose deprivation, hydroxylamine, or phenylarsine oxide. Inhibition does not increase with exposure time and the inhibitory effects reverse within an hour. Inhibition does not appear to involve a reaction between curcumin and the thiol side chain of a cysteine residue since neither prior treatment of cells with iodoacetamide nor curcumin with cysteine alters curcumin's inhibitory effects. Curcumin is a mixed inhibitor reducing the Vmax of 2DG transport by about half with little effect on the Km. The inhibitory effects of curcumin are not additive to the effects of cytochalasin B and 75 μM curcumin actually reduces specific cytochalasin B binding by 80%. Taken together, the data suggest that curcumin binds directly to GLUT1 at a site that overlaps with the cytochalasin B binding site and thereby inhibits glucose transport. A direct inhibition of GLUT proteins in intestinal epithelial cells would likely reduce absorption of dietary glucose and contribute to a hypoglycemic effect of curcumin. Also, inhibition of GLUT1 activity might compromise cancer cells that overexpress GLUT1 and be another possible mechanism for the documented anticancer effects of curcumin. PMID:27039889

  9. Gc protein-derived macrophage-activating factor (GcMAF) stimulates cAMP formation in human mononuclear cells and inhibits angiogenesis in chick embryo chorionallantoic membrane assay.

    Pacini, Stefania; Morucci, Gabriele; Punzi, Tiziana; Gulisano, Massimo; Ruggiero, Marco

    2011-04-01

    The effects of Gc protein-derived macrophage-activating factor (GcMAF) have been studied in cancer and other conditions where angiogenesis is deregulated. In this study, we demonstrate for the first time that the mitogenic response of human peripheral blood mononuclear cells (PBMCs) to GcMAF was associated with 3'-5'-cyclic adenosine monophosphate (cAMP) formation. The effect was dose dependent, and maximal stimulation was achieved using 0.1 ng/ml. Heparin inhibited the stimulatory effect of GcMAF on PBMCs. In addition, we demonstrate that GcMAF (1 ng/ml) inhibited prostaglandin E(1)- and human breast cancer cell-stimulated angiogenesis in chick embryo chorionallantoic membrane (CAM) assay. Finally, we tested different GcMAF preparations on CAM, and the assay proved to be a reliable, reproducible and inexpensive method to determine the relative potencies of different preparations and their stability; we observed that storage at room temperature for 15 days decreased GcMAF potency by about 50%. These data could prove useful for upcoming clinical trials on GcMAF. PMID:21170647

  10. Androgen via p21 Inhibits Tumor Necrosis Factor α-induced JNK Activation and Apoptosis*

    Tang, Fangming; Kokontis, John; Lin, Yuting; Liao, Shutsung; Lin, Anning; Xiang, Jialing

    2009-01-01

    The male hormone androgen is a growth/survival factor for its target tissues or organs. Yet, the underlying mechanism is incompletely understood. Here, we report that androgen via p21 inhibits tumor necrosis factor α-induced JNK activation and apoptosis. Inhibition by androgen requires the transcription activity of androgen receptor (AR) and de novo protein synthesis. Androgen·AR induces expression of p21 that in turn inhibits tumor necrosis factor α-induced JNK and apoptosis. Furthermore, ge...

  11. Elongation factor 2 kinase promotes cell survival by inhibiting protein synthesis without inducing autophagy.

    Moore, Claire E J; Wang, Xuemin; Xie, Jianling; Pickford, Jo; Barron, John; Regufe da Mota, Sergio; Versele, Matthias; Proud, Christopher G

    2016-04-01

    Eukaryotic elongation factor 2 kinase (eEF2K) inhibits the elongation stage of protein synthesis by phosphorylating its only known substrate, eEF2. eEF2K is tightly regulated by nutrient-sensitive signalling pathways. For example, it is inhibited by signalling through mammalian target of rapamycin complex 1 (mTORC1). It is therefore activated under conditions of nutrient deficiency. Here we show that inhibiting eEF2K or knocking down its expression renders cancer cells sensitive to death under nutrient-starved conditions, and that this is rescued by compounds that block protein synthesis. This implies that eEF2K protects nutrient-deprived cells by inhibiting protein synthesis. Cells in which signalling through mTORC1 is highly active are very sensitive to nutrient withdrawal. Inhibiting mTORC1 protects them. Our data reveal that eEF2K makes a substantial contribution to the cytoprotective effect of mTORC1 inhibition. eEF2K is also reported to promote another potentially cytoprotective process, autophagy. We have used several approaches to test whether inhibition or loss of eEF2K affects autophagy under a variety of conditions. We find no evidence that eEF2K is involved in the activation of autophagy in the cell types we have studied. We conclude that eEF2K protects cancer cells against nutrient starvation by inhibiting protein synthesis rather than by activating autophagy. PMID:26795954

  12. Differentiation of immortal cells inhibits telomerase activity.

    Sharma, H W; Sokoloski, J A; Perez, J.R.; Maltese, J Y; Sartorelli, A C; Stein, C A; Nichols, G; Khaled, Z.; Telang, N T; Narayanan, R.

    1995-01-01

    Telomerase, a ribonucleic acid-protein complex, adds hexameric repeats of 5'-TTAGGG-3' to the ends of mammalian chromosomal DNA (telomeres) to compensate for the progressive loss that occurs with successive rounds of DNA replication. Although somatic cells do not express telomerase, germ cells and immortalized cells, including neoplastic cells, express this activity. To determine whether the phenotypic differentiation of immortalized cells is linked to the regulation of telomerase activity, t...

  13. Arabidopsis Bax Inhibitor-1 inhibits cell death induced by pokeweed antiviral protein in Saccharomyces cerevisae

    Birsen Çakır

    2015-02-01

    Full Text Available Apoptosis is an active form of programmed cell death (PCD that plays critical roles in the development, differentiation and resistance to pathogens in multicellular organisms. Ribosome inactivating proteins (RIPs are able to induce apoptotic cell death in mammalian cells. In this study, using yeast as a model system, we showed that yeast cells expressing pokeweed antiviral protein (PAP, a single-chain ribosome-inactivating protein, exhibit apoptotic-like features, such as nuclear fragmentation and ROS production. We studied the interaction between PAP and AtBI-1 (Arabidopsis thaliana Bax Inhibitor-1, a plant anti-apoptotic protein, which inhibits Bax induced cell death. Cells expressing PAP and AtBI-1 were able to survive on galactose media compared to PAP alone, indicating a reduction in the cytotoxicity of PAP in yeast. However, PAP was able to depurinate the ribosomes and to inhibit total translation in the presence of AtBI-1. A C-terminally deleted AtBI-1 was able to reduce the cytotoxicity of PAP. Since anti-apoptotic proteins form heterodimers to inhibit the biological activity of their partners, we used a co-immunoprecipitation assay to examine the binding of AtBI-1 to PAP. Both full length and C-terminal deleted AtBI-1 were capable of binding to PAP. These findings indicate that PAP induces cell death in yeast and AtBI-1 inhibits cell death induced by PAP without affecting ribosome depurination and translation inhibition.

  14. The isothiocyanate class of bioactive nutrients covalently inhibit the MEKK1 protein kinase

    Dietary isothiocyanates (ITCs) are electrophilic compounds that have diverse biological activities including induction of apoptosis and effects on cell cycle. They protect against experimental carcinogenesis in animals, an activity believed to result from the transcriptional induction of 'Phase 2' enzymes. The molecular mechanism of action of ITCs is unknown. Since ITCs are electrophiles capable of reacting with sulfhydryl groups on amino acids, we hypothesized that ITCs induce their biological effects through covalent modification of proteins, leading to changes in cell regulatory events. We previously demonstrated that stress-signaling kinase pathways are inhibited by other electrophilic compounds such as menadione. We therefore tested the effects of nutritional ITCs on MEKK1, an upstream regulator of the SAPK/JNK signal transduction pathway. The activity of MEKK1 expressed in cells was monitored using in vitro kinase assays to measure changes in catalytic activity. The activity of endogenous MEKK1, immunopurified from ITC treated and untreated LnCAP cells was also measured by in vitro kinase assay. A novel labeling and affinity reagent for detection of protein modification by ITCs was synthesized and used in competition assays to monitor direct modification of MEKK1 by ITC. Finally, immunoblots with phospho-specific antibodies were used to measure the activity of MAPK protein kinases. ITCs inhibited the MEKK1 protein kinase in a manner dependent on a specific cysteine residue in the ATP binding pocket. Inhibition of MEKK1 catalytic activity was due to direct, covalent and irreversible modification of the MEKK1 protein itself. In addition, ITCs inhibited the catalytic activity of endogenous MEKK1. This correlated with inhibition of the downstream target of MEKK1 activity, i.e. the SAPK/JNK kinase. This inhibition was specific to SAPK, as parallel MAPK pathways were unaffected. These results demonstrate that MEKK1 is directly modified and inhibited by

  15. The isothiocyanate class of bioactive nutrients covalently inhibit the MEKK1 protein kinase

    Macdonald Timothy L

    2007-09-01

    Full Text Available Abstract Background Dietary isothiocyanates (ITCs are electrophilic compounds that have diverse biological activities including induction of apoptosis and effects on cell cycle. They protect against experimental carcinogenesis in animals, an activity believed to result from the transcriptional induction of "Phase 2" enzymes. The molecular mechanism of action of ITCs is unknown. Since ITCs are electrophiles capable of reacting with sulfhydryl groups on amino acids, we hypothesized that ITCs induce their biological effects through covalent modification of proteins, leading to changes in cell regulatory events. We previously demonstrated that stress-signaling kinase pathways are inhibited by other electrophilic compounds such as menadione. We therefore tested the effects of nutritional ITCs on MEKK1, an upstream regulator of the SAPK/JNK signal transduction pathway. Methods The activity of MEKK1 expressed in cells was monitored using in vitro kinase assays to measure changes in catalytic activity. The activity of endogenous MEKK1, immunopurified from ITC treated and untreated LnCAP cells was also measured by in vitro kinase assay. A novel labeling and affinity reagent for detection of protein modification by ITCs was synthesized and used in competition assays to monitor direct modification of MEKK1 by ITC. Finally, immunoblots with phospho-specific antibodies were used to measure the activity of MAPK protein kinases. Results ITCs inhibited the MEKK1 protein kinase in a manner dependent on a specific cysteine residue in the ATP binding pocket. Inhibition of MEKK1 catalytic activity was due to direct, covalent and irreversible modification of the MEKK1 protein itself. In addition, ITCs inhibited the catalytic activity of endogenous MEKK1. This correlated with inhibition of the downstream target of MEKK1 activity, i.e. the SAPK/JNK kinase. This inhibition was specific to SAPK, as parallel MAPK pathways were unaffected. Conclusion These results

  16. Antioxidant activity of black bean (Phaseolus vulgaris L. protein hydrolysates

    Jarine Amaral do EVANGELHO

    2016-01-01

    Full Text Available Abstract The objective of this work was to study the effect of enzymatic hydrolysis of black bean protein concentrate using different enzymes. Bean proteins were extracted and hydrolyzed over a period of 120 min using the enzymes pepsin or alcalase. The protein hydrolysates’ molecular weight was assayed by electrophoresis and the antioxidant activity was evaluated by the capturing methods of free radicals ABTS●+ and DPPH. Electrophoretic results showed that the bands above 50 kDa disappeared, when the beans protein was subjected to hydrolysis with pepsin. The bean protein hydrolysate obtained by hydrolysis with alcalase enzyme, showed higher antioxidant activity for inhibition of the radical ABTS●+. However, the hydrolysates obtained by hydrolysis with pepsin had higher antioxidant activity for inhibition of the radical DPPH. The use of pepsin and alcalase enzymes, under the same reaction time, produced black bean protein hydrolysates with different molecular weight profiles and superior antioxidant activity than the native bean protein.

  17. DNA-based control of protein activity.

    Engelen, W; Janssen, B M G; Merkx, M

    2016-03-01

    DNA has emerged as a highly versatile construction material for nanometer-sized structures and sophisticated molecular machines and circuits. The successful application of nucleic acid based systems greatly relies on their ability to autonomously sense and act on their environment. In this feature article, the development of DNA-based strategies to dynamically control protein activity via oligonucleotide triggers is discussed. Depending on the desired application, protein activity can be controlled by directly conjugating them to an oligonucleotide handle, or expressing them as a fusion protein with DNA binding motifs. To control proteins without modifying them chemically or genetically, multivalent ligands and aptamers that reversibly inhibit their function provide valuable tools to regulate proteins in a noncovalent manner. The goal of this feature article is to give an overview of strategies developed to control protein activity via oligonucleotide-based triggers, as well as hurdles yet to be taken to obtain fully autonomous systems that interrogate, process and act on their environments by means of DNA-based protein control. PMID:26812623

  18. Inhibition of Dengue Virus Replication by a Class of Small-Molecule Compounds That Antagonize Dopamine Receptor D4 and Downstream Mitogen-Activated Protein Kinase Signaling

    Smith, Jessica L.; Stein, David A.; Shum, David; Fischer, Matthew A.; Radu, Constantin; Bhinder, Bhavneet; Djaballah, Hakim; Nelson, Jay A.; Früh, Klaus; Hirsch, Alec J.

    2014-01-01

    Dengue viruses (DENV) are endemic pathogens of tropical and subtropical regions that cause significant morbidity and mortality worldwide. To date, no vaccines or antiviral therapeutics have been approved for combating DENV-associated disease. In this paper, we describe a class of tricyclic small-molecule compounds—dihydrodibenzothiepines (DHBTs), identified through high-throughput screening—with potent inhibitory activity against DENV serotype 2. SKI-417616, a highly active representative of ...

  19. Inhibition of mitogen activated protein kinases increases the sensitivity of A549 lung cancer cells to the cytotoxicity induced by a kava chalcone analog

    Janel K Warmka; Solberg, Eric L.; Zeliadt, Nicholette A.; Srinivasan, Balasubramanian; Charlson, Aaron T.; Xing, Chengguo; Wattenberg, Elizabeth V.

    2012-01-01

    We are interested in investigating the biological activity of chalcones, a major class of compounds found in the beverage kava, in order to develop potent and selective chemopreventive candidates. Consumption of kava in the South Pacific Islands is inversely correlated with cancer incidence, even among smokers. Accordingly, chalcones have anti-cancer activities in animal and cell culture models. To investigate signaling pathways that affect chalcone action we studied a potent analog, (E)-3-(3...

  20. Activated protein C modulates the proinflammatory activity of dendritic cells

    Matsumoto T

    2015-05-01

    Full Text Available Takahiro Matsumoto,1,2* Yuki Matsushima,1* Masaaki Toda,1 Ziaurahman Roeen,1 Corina N D'Alessandro-Gabazza,1,5 Josephine A Hinneh,1 Etsuko Harada,1,3 Taro Yasuma,4 Yutaka Yano,4 Masahito Urawa,1,5 Tetsu Kobayashi,5 Osamu Taguchi,5 Esteban C Gabazza1 1Department of Immunology, Mie University Graduate School of Medicine, Tsu, Mie Prefecture, 2BONAC Corporation, BIO Factory 4F, Fukuoka, 3Iwade Research Institute of Mycology, 4Department of Endocrinology, Diabetes and Metabolism, 5Department of Pulmonary and Critical Care Medicine, Mie University Graduate School of Medicine, Tsu, Mie Prefecture, Japan *These authors contributed equally to this work Background: Previous studies have demonstrated the beneficial activity of activated protein C in allergic diseases including bronchial asthma and rhinitis. However, the exact mechanism of action of activated protein C in allergies is unclear. In this study, we hypothesized that pharmacological doses of activated protein C can modulate allergic inflammation by inhibiting dendritic cells. Materials and methods: Dendritic cells were prepared using murine bone marrow progenitor cells and human peripheral monocytes. Bronchial asthma was induced in mice that received intratracheal instillation of ovalbumin-pulsed dendritic cells. Results: Activated protein C significantly increased the differentiation of tolerogenic plasmacytoid dendritic cells and the secretion of type I interferons, but it significantly reduced lipopolysaccharide-mediated maturation and the secretion of inflammatory cytokines in myeloid dendritic cells. Activated protein C also inhibited maturation and the secretion of inflammatory cytokines in monocyte-derived dendritic cells. Activated protein C-treated dendritic cells were less effective when differentiating naïve CD4 T-cells from Th1 or Th2 cells, and the cellular effect of activated protein C was mediated by its receptors. Mice that received adoptive transfer of activated protein C

  1. Antiviral Protein of Momordica charantia L. Inhibits Different Subtypes of Influenza A

    Viroj Pongthanapisith

    2013-01-01

    Full Text Available The new antiviral activity of the protein extracted from Momordica charantia was determined with different subtypes of influenza A. The protein was purified from the seed of M. charantia using an anion exchanger and a Fast Protein Liquid Chromatography (FPLC system. At the concentration of 1.401 mg/mL, the protein did not exhibit cytotoxicity in Madin-Darby canine kidney cells (MDCK but inhibited FFU influenza A/PR/8/34 H1N1 virus at 56.50%, 65.72%, and 100% inhibition by the protein treated before the virus (pretreated, the protein treated alongside with the virus (simultaneously treated, and the protein treated after the virus (posttreated during incubation, respectively. Using 5, 25, and 100 TCID50 of influenza A/New Caledonia/20/99 H1N1, A/Fujian/411/01 H3N2 and A/Thailand/1(KAN-1/2004 H5N1, the IC50 was calculated to be 100, 150, and 200; 75, 175, and 300; and 40, 75, and 200 μg/mL, respectively. Our present finding indicated that the plant protein inhibited not only H1N1 and H3N2 but also H5N1 subtype. As a result of the broad spectrum of its antiviral activity, this edible plant can be developed as an effective therapeutic agent against various and even new emerging subtypes of influenza A.

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

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

  3. Identification of peptides that inhibit regulator of G protein signaling 4 function.

    Wang, Yuren; Lee, Yan; Zhang, Jie; Young, Kathleen H

    2008-01-01

    Regulators of G protein signaling (RGS) are a family of GTPase-activating proteins (GAP) that interact with heterotrimeric G proteins in the negative regulation of G-protein-coupled receptor (GPCR) signaling. RGS4, the first identified mammalian member of the RGS family, has been implicated in many GPCR signaling pathways involved in disease states. We report herein the identification of a 16-amino-acid peptide (P17) as an inhibitor of RGS4. The peptide was found by screening a random peptide library using RGS4 as 'bait' in a yeast two-hybrid system. This peptide inhibited RGS4 GAP activity on Galpha(i1)in a GTPase assay, and blocked the interaction between RGS4 and Galpha(i1)in a pull-down assay. The peptide displayed dose-dependent inhibition of RGS4 and Galpha-interacting protein (GAIP) GAP activities, yet showed no substantial effect on RGS7. Electrophysiological studies in Xenopus oocytes demonstrated that P17 attenuates RGS4 modulation of M(2) muscarinic receptor stimulation of GIRK (G-protein-mediated inwardly rectifying potassium) channels. Deletion of an arginine at the N terminus of P17 abolished its ability to inhibit RGS4 GAP activity, as did deletions of C-terminal residues. The P17 peptide showed no similarity to any known peptide sequence. Further investigation and optimization of the peptide may provide unique information for the development of RGS4 inhibitors for future therapeutic application. PMID:18547979

  4. Inhibition of Neuroinflammation in LPS-Activated Microglia by Cryptolepine

    Olumayokun A. Olajide

    2013-01-01

    Full Text Available Cryptolepine, an indoloquinoline alkaloid in Cryptolepis sanguinolenta, has anti-inflammatory property. In this study, we aimed to evaluate the effects of cryptolepine on lipopolysaccharide (LPS- induced neuroinflammation in rat microglia and its potential mechanisms. Microglial activation was induced by stimulation with LPS, and the effects of cryptolepine pretreatment on microglial activation and production of proinflammatory mediators, PGE2/COX-2, microsomal prostaglandin E2 synthase and nitric oxide/iNOS were investigated. We further elucidated the role of Nuclear Factor-kappa B (NF-κB and the mitogen-activated protein kinases in the antiinflammatory actions of cryptolepine in LPS-stimulated microglia. Our results showed that cryptolepine significantly inhibited LPS-induced production of tumour necrosis factor-alpha (TNFα, interleukin-6 (IL-6, interleukin-1beta (IL-1β, nitric oxide, and PGE2. Protein and mRNA levels of COX-2 and iNOS were also attenuated by cryptolepine. Further experiments on intracellular signalling mechanisms show that IκB-independent inhibition of NF-κB nuclear translocation contributes to the anti-neuroinflammatory actions of cryptolepine. Results also show that cryptolepine inhibited LPS-induced p38 and MAPKAPK2 phosphorylation in the microglia. Cell viability experiments revealed that cryptolepine (2.5 and 5 μM did not produce cytotoxicity in microglia. Taken together, our results suggest that cryptolepine inhibits LPS-induced microglial inflammation by partial targeting of NF-κB signalling and attenuation of p38/MAPKAPK2.

  5. Aldehyde Dehydrogenase Type 2 Activation by Adenosine and Histamine Inhibits Ischemic Norepinephrine Release in Cardiac Sympathetic Neurons: Mediation by Protein Kinase Cε

    Robador, Pablo A.; Seyedi, Nahid; Chan, Noel Yan-Ki; Koda, Kenichiro; Levi, Roberto

    2012-01-01

    During myocardial ischemia/reperfusion, lipid peroxidation leads to the formation of toxic aldehydes that contribute to ischemic dysfunction. Mitochondrial aldehyde dehydrogenase type 2 (ALDH2) alleviates ischemic heart damage and reperfusion arrhythmias via aldehyde detoxification. Because excessive norepinephrine release in the heart is a pivotal arrhythmogenic mechanism, we hypothesized that neuronal ALDH2 activation might diminish ischemic norepinephrine release. Incubation of cardiac sym...

  6. FOXP3 Inhibits HIV-1 Infection of CD4 T-cells via Inhibition of LTR Transcriptional Activity

    Selliah, Nithianandan; Zhang, Mingce; White, Sara; Zoltick, Philip; Sawaya, Bassel E.; Finkel, Terri H.; Cron, Randy Q

    2008-01-01

    FOXP3 is a necessary transcription factor for the development and function of CD4+ regulatory T-cells (Tregs). The role of Tregs in HIV-1 infection remains unclear. Here, we show expression of FOXP3 in primary human CD4 T-cells significantly inhibits HIV-1 infection. Since FOXP3 inhibits NFAT activity, and NFAT proteins contribute to HIV-1 transcription, we explore a transcriptional repressive function of HIV-1 LTR by FOXP3. Over-expression of FOXP3 in primary CD4 T-cells inhibits wild-type H...

  7. Curcumin inhibits HIV-1 by promoting Tat protein degradation

    Amjad Ali; Banerjea, Akhil C

    2016-01-01

    HIV-1 Tat is an intrinsically unfolded protein playing a pivotal role in viral replication by associating with TAR region of viral LTR. Unfolded proteins are degraded by 20S proteasome in an ubiquitin independent manner. Curcumin is known to activate 20S proteasome and promotes the degradation of intrinsically unfolded p53 tumor suppressor protein. Since HIV-1 Tat protein is largerly unfolded, we hypothesized that Tat may also be targeted through this pathway. Curcumin treated Tat transfected...

  8. Novel 2,7-Substituted (S)-1,2,3,4-Tetrahydroisoquinoline-3-carboxylic Acids: Peroxisome Proliferator-Activated Receptor γ Partial Agonists with Protein-Tyrosine Phosphatase 1B Inhibition.

    Otake, Kazuya; Azukizawa, Satoru; Takeda, Shigemitsu; Fukui, Masaki; Kawahara, Arisa; Kitao, Tatsuya; Shirahase, Hiroaki

    2015-01-01

    A novel series of 2,7-substituted 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid derivatives were synthesized and biologically evaluated. (S)-2-(2-Furylacryloyl)-7-[2-(2-methylindane-2-yl)-5-methyloxazol-4-yl]methoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid tert-butylamine salt (13jE) was identified as a potent human peroxisome proliferator-activated receptor γ (PPARγ)-selective agonist (EC50=85 nM) and human protein-tyrosine phosphatase 1B (PTP-1B) inhibitor (IC50=1.0 µM). Compound 13jE partially activated PPARγ, but not PPARα or PPARδ, and antagonized farglitazar, a full PPARγ agonist. Cmax after the oral administration of 13jE at 10 mg/kg was 28.6 µg/mL (53 µM) in male Sprague-Dawley (SD) rats. Repeated administration of 13jE and rosiglitazone for 14 d at 10 mg/kg/d decreased plasma glucose and triglyceride levels significantly in male KK-A(y) mice. Rosiglitazone, but not 13jE, significantly increased the plasma volume and liver weight. In conclusion, 13jE showed stronger hypoglycemic and hypolipidemic effects and weaker hemodilution and hepatotoxic effects than rosiglitazone, suggesting that its safer efficacy may be due to its partial PPARγ agonism and PTP-1B inhibition. PMID:26633022

  9. Inhibition of bacterial aggregation by serum- and blood-derived proteins.

    Malamud, D; Brown, C; Goldman, R

    1984-01-01

    Human and animal sera contain potent inhibitors of saliva-mediated aggregation of oral streptococci. The inhibitors consist of a high-molecular-weight heat-labile factor and a lower-molecular-weight heat-activated factor. The latter appears to be serum albumin. Analyses of purified blood-derived proteins indicated that several high-molecular-weight proteins (fibrinogen, fibronectin, and ferritin) were able to inhibit aggregation at low concentrations. These data suggest that high-molecular-we...

  10. Borna disease virus P protein inhibits nitric oxide synthase gene expression in astrocytes

    Borna disease virus (BDV) is one of the potential infectious agents involved in the development of central nervous system (CNS) diseases. Neurons and astrocytes are the main targets of BDV infection, but little is known about the roles of BDV infection in the biological effects of astrocytes. Here we reported that BDV inhibits the activation of inducible nitric oxide synthase (iNOS) in murine astrocytes induced by bacterial LPS and PMA. To determine which protein of BDV is responsible for the regulation of iNOS expression, we co-transfected murine astrocytes with reporter plasmid iNOS-luciferase and plasmid expressing individual BDV proteins. Results from analyses of reporter activities revealed that only the phosphoprotein (P) of BDV had an inhibitory effect on the activation of iNOS. In addition, P protein inhibits nitric oxide production through regulating iNOS expression. We also reported that the nuclear factor kappa B (NF-κB) binding element, AP-1 recognition site, and interferon-stimulated response element (ISRE) on the iNOS promoter were involved in the repression of iNOS gene expression regulated by the P protein. Functional analysis indicated that sequences from amino acids 134 to 174 of the P protein are necessary for the regulation of iNOS. These data suggested that BDV may suppress signal transduction pathways, which resulted in the inhibition of iNOS activation in astrocytes

  11. Inhibition of acetylcholinesterase activity by essential oil from Citrus paradisi.

    Miyazawa, M; Tougo, H; Ishihara, M

    2001-01-01

    Inhibition of acetylcholinesterase (AChE) activity by essential oils of Citrus paradisi (grapefruit pink in USA) was studied. Inhibition of AChE was measured by the colorimetric method. Nootkatone and auraptene were isolated from C. paradisi oil and showed 17-24% inhibition of AChE activity at the concentration of 1.62 microg/mL. PMID:11858553

  12. Recombinant Human Prion Protein Inhibits Prion Propagation in vitro

    Jue Yuan; Yi-An Zhan; Romany Abskharon; Xiangzhu Xiao; Manuel Camacho Martinez; Xiaochen Zhou; Geoff Kneale; Jacqueline Mikol; Sylvain Lehmann; Surewicz, Witold K.; Joaquín Castilla; Jan Steyaert; Shulin Zhang; Qingzhong Kong; Petersen, Robert B.

    2013-01-01

    Prion diseases are associated with the conformational conversion of the cellular prion protein (PrPC) into the pathological scrapie isoform (PrPSc) in the brain. Both the in vivo and in vitro conversion of PrPC into PrPSc is significantly inhibited by differences in amino acid sequence between the two molecules. Using protein misfolding cyclic amplification (PMCA), we now report that the recombinant full-length human PrP (rHuPrP23-231) (that is unglycosylated and lacks the glycophosphatidylin...

  13. Boymaw, overexpressed in brains with major psychiatric disorders, may encode a small protein to inhibit mitochondrial function and protein translation.

    Ji, Baohu; Kim, Minjung; Higa, Kerin K; Zhou, Xianjin

    2015-06-01

    The t(1,11) chromosome translocation co-segregates with major psychiatric disorders in a large Scottish family. The translocation disrupts the DISC1and Boymaw (DISC1FP1) genes on chromosomes 1 and 11, respectively. After translocation, two fusion genes are generated. Our recent studies found that the DISC1-Boymaw fusion protein is localized in mitochondria and inhibits oxidoreductase activity, rRNA expression, and protein translation. Mice carrying the DISC1-Boymaw fusion genes display intermediate behavioral phenotypes related to major psychiatric disorders. Here, we report that the Boymaw gene may encode a small protein predominantly localized in mitochondria. The Boymaw protein inhibits oxidoreductase activity, rRNA expression, and protein translation in the same way as the DISC1-Boymaw fusion protein. Interestingly, Boymaw expression is up-regulated by different stressors at RNA and/or protein translational levels. In addition, we found that Boymaw RNA expression is significantly increased in the postmortem brains of patients with major psychiatric disorders. Our studies therefore suggest that the Boymaw gene could potentially be a susceptibility gene for major psychiatric disorders in both the Scottish t(1,11) family and the general population of patients. PMID:25943690

  14. Engineered kinesin motor proteins amenable to small-molecule inhibition

    Martin F. Engelke; Winding, Michael; Yue, Yang; Shastry, Shankar; Teloni, Federico; Reddy, Sanjay(Institute for Nuclear Theory, University of Washington, Seattle, WA, United States); Blasius, T. Lynne; Soppina, Pushpanjali; Hancock, William O.; Gelfand, Vladimir I.; Verhey, Kristen J.

    2016-01-01

    The human genome encodes 45 kinesin motor proteins that drive cell division, cell motility, intracellular trafficking and ciliary function. Determining the cellular function of each kinesin would benefit from specific small-molecule inhibitors. However, screens have yielded only a few specific inhibitors. Here we present a novel chemical-genetic approach to engineer kinesin motors that can carry out the function of the wild-type motor yet can also be efficiently inhibited by small, cell-perme...

  15. Kinetics of the inhibition of calcium/calmodulin-dependent protein kinase II by pea protein-derived peptides.

    Li, Huan; Aluko, Rotimi E

    2005-11-01

    Calcium/calmodulin-dependent protein kinase II (CaMKII) catalyzes the phosphorylation of various cellular proteins and excessive activities have been implicated in the pathogenesis of various chronic diseases. We hypothesized that positively charged peptides can be produced through enzymatic hydrolysis of pea proteins; such peptides could then bind to negatively charged calmodulin (CaM) at a physiological pH level and inhibit CaMKII activity. Pea protein isolate was hydrolyzed with an alkaline protease (alcalase) and filtered through a 1000-mol wt cutoff membrane. The permeate, which contained low-molecular weight peptides, was used to isolate cationic peptides on an SP-Sepharose column by ion exchange chromatography. Separation of the permeate on the SP-Sepharose column yielded two fractions with net positive charges that were subsequently used for enzyme inhibition studies. Fraction I eluted earlier from the column and contained lower contents of lysine and arginine than Fraction II, which eluted later. Results show that both peptide fractions inhibited CaMKII activity mostly in a competitive manner, although kinetic data suggested that inhibition by Fraction II may be of the mixed type. Kinetic analysis (K(m) and K(i)) showed that affinity of peptides in Fraction II for CaM was more than that in Fraction I, which was directly correlated with the higher inhibitory properties of Fraction II against CaMKII. The results suggest that it may be possible to use pea protein-derived cationic peptides to modulate CaMKII activities. PMID:16111873

  16. Adenovirus E4 open reading frame 4-induced dephosphorylation inhibits E1A activation of the E2 promoter and E2F-1-mediated transactivation independently of the retinoblastoma tumor suppressor protein

    Mannervik, M; Fan, S; Ström, A C;

    1999-01-01

    viral E4 open reading frame 4 (E4-ORF4) protein. This effect does not to require the retinoblastoma protein that previously has been shown to regulate E2F activity. The inhibitory activity of E4-ORF4 appears to be specific because E4-ORF4 had little effect on, for example, E4-ORF6/7 transactivation of...

  17. Genetically engineered endostatin-lidamycin fusion proteins effectively inhibit tumor growth and metastasis

    Jiang, Wen-guo; Lu, Xin-an; Shang, Bo-yang; Fu, Yan; ZHANG, SHENG-HUA; Zhou, Daifu; Liang LI; Li, Yi; Luo, Yongzhang; ZHEN, YONG-SU

    2013-01-01

    Background Endostatin (ES) inhibits endothelial cell proliferation, migration, invasion, and tube formation. It also shows antiangiogenesis and antitumor activities in several animal models. Endostatin specifically targets tumor vasculature to block tumor growth. Lidamycin (LDM), which consists of an active enediyne chromophore (AE) and a non-covalently bound apo-protein (LDP), is a member of chromoprotein family of antitumor antibiotics with extremely potent cytotoxicity to cancer cells. The...

  18. Acetohydroxamate inhibition of the activity of urease from dehusked seeds of water melon (Citrullus vulgaris).

    Prakash, Om; Upadhyay, Lata Sheo Bachan

    2004-08-01

    Urease from the seeds of watermelon (Citrullus vulgaris) was purified to apparent homogeneity, using two acetone fractionation steps, heat treatment at 48 degrees C and gel filtration through Sephadex G-200. Effect of acetohydroxamic acid (AHA) on the activity of the homogeneous enzyme preparation (sp. act. 3000 +/- 550U/mg protein) was investigated. AHA exhibited a concentration-dependent inhibition both in the presence and absence of the substrate. The inhibition was uncompetitive and the Ki was 2.5 mM. Binding of AHA with the enzyme was reversible, as 63% activity could be restored by dialysis. Time-dependent inhibition revealed a monophasic inhibition of the activity. Addition of beta-mercaptoethanol (ME) gradually abolished the inhibition. Pre-treatment of native enzyme with 8.0 mM ME for 5 min at 30 degrees C exhibited protection against AHA-induced inhibition. The significance of these observations is discussed. PMID:15558957

  19. Protein kinase C regulates tonic GABAA receptor-mediated inhibition in the hippocampus and thalamus

    Bright, Damian P; Smart, Trevor G

    2013-01-01

    Tonic inhibition mediated by extrasynaptic GABAA receptors (GABAARs) is an important regulator of neuronal excitability. Phosphorylation by protein kinase C (PKC) provides a key mode of regulation for synaptic GABAARs underlying phasic inhibition; however, less attention has been focused on the plasticity of tonic inhibition and whether this can also be modulated by receptor phosphorylation. To address this issue, we used whole-cell patch clamp recording in acute murine brain slices at both room and physiological temperatures to examine the effects of PKC-mediated phosphorylation on tonic inhibition. Recordings from dentate gyrus granule cells in the hippocampus and dorsal lateral geniculate relay neurons in the thalamus demonstrated that PKC activation caused downregulation of tonic GABAAR-mediated inhibition. Conversely, inhibition of PKC resulted in an increase in tonic GABAAR activity. These findings were corroborated by experiments on human embryonic kidney 293 cells expressing recombinant α4β2δ GABAARs, which represent a key extrasynaptic GABAAR isoform in the hippocampus and thalamus. Using bath application of low GABA concentrations to mimic activation by ambient neurotransmitter, we demonstrated a similar inhibition of receptor function following PKC activation at physiological temperature. Live cell imaging revealed that this was correlated with a loss of cell surface GABAARs. The inhibitory effects of PKC activation on α4β2δ GABAAR activity appeared to be mediated by direct phosphorylation at a previously identified site on the β2 subunit, serine 410. These results indicate that PKC-mediated phosphorylation can be an important physiological regulator of tonic GABAAR-mediated inhibition. PMID:24102973

  20. Stathmin potentiates vinflunine and inhibits Paclitaxel activity.

    Soazig Malesinski

    Full Text Available Cell biology and crystallographic studies have suggested a functional link between stathmin and microtubule targeting agents (MTAs. In a previous study we showed that stathmin increases vinblastine (VLB binding to tubulin, and that conversely VLB increases stathmin binding to tubulin. This constituted the first biochemical evidence of the direct relationship between stathmin and an antimitotic drug, and revealed a new mechanism of action for VLB. The question remained if the observed interaction was specific for this drug or represented a general phenomenon for all MTAs. In the present study we investigated the binding of recombinant stathmin to purified tubulin in the presence of paclitaxel or another Vinca alkaloid, vinflunine, using Isothermal Titration Calorimetry (ITC. These experiments revealed that stathmin binding to tubulin is increased in the presence of vinflunine, whereas no signal is observed in the presence of paclitaxel. Further investigation using turbidity and co-sedimentation showed that stathmin inhibited paclitaxel microtubule-stabilizing activity. Taken together with the previous study using vinblastine, our results suggest that stathmin can be seen as a modulator of MTA activity and binding to tubulin, providing molecular explanation for multiple previous cellular and in vivo studies showing that stathmin expression level affects MTAs efficiency.

  1. Inhibition of osteoblast activity by zoledronic acid

    Fernanda Gonçalves Basso

    2013-10-01

    Full Text Available INTRODUCTION: Patients treated with nitrogen-containing bisphosphonates, such as zoledronic acid (ZA, have frequently shown oral bone exposure areas, termed osteonecrosis. In addition, these patients may also present low repair and regeneration potential, mainly after tooth extractions. These side-effects caused by bisphosphonates may be due to their inhibitory effects on oral mucosa and local bone cells. OBJECTIVE: To evaluate the effects of ZA on the mineralization capacity of cultured osteoblasts. MATERIALS AND METHODS: Human immortalized osteoblasts (SaOs-2 were grown in plain culture medium (Dulbecco's Modified Eagle Medium [DMEM] + 10% fetal bovine serum [FBS] in wells of 24-well plates. After 48-hour incubation, the plain DMEM was replaced by a solution with ZA at 5 µM which was maintained in contact with cells for seven, 14 or 21 days. After these periods, cells were evaluated regarding alkaline phosphatase (ALP activity and mineral nodule formation (alizarin red. Data were statistically analyzed by Mann-Whitney test, at 5% of significance level. RESULTS: ZA caused significant reduction on ALP activity and mineral nodules formation by cultured osteoblasts in all evaluated periods (p < 0.05. CONCLUSION: These data indicate that ZA causes inhibition on the osteogenic phenotype of cultured human osteoblasts, which, in turn, may reduce bone repair in patients subjected to ZA therapy.

  2. Inhibition of endoplasmic reticulum-associated degradation rescues native folding in loss of function protein misfolding diseases.

    Wang, Fan; Song, Wensi; Brancati, Giovanna; Segatori, Laura

    2011-12-16

    Lysosomal storage disorders are often caused by mutations that destabilize native folding and impair trafficking of secretory proteins. We demonstrate that endoplasmic reticulum (ER)-associated degradation (ERAD) prevents native folding of mutated lysosomal enzymes in patient-derived fibroblasts from two clinically distinct lysosomal storage disorders, namely Gaucher and Tay-Sachs disease. Prolonging ER retention via ERAD inhibition enhanced folding, trafficking, and activity of these unstable enzyme variants. Furthermore, combining ERAD inhibition with enhancement of the cellular folding capacity via proteostasis modulation resulted in synergistic rescue of mutated enzymes. ERAD inhibition was achieved by cell treatment with small molecules that interfere with recognition (kifunensine) or retrotranslocation (eeyarestatin I) of misfolded substrates. These different mechanisms of ERAD inhibition were shown to enhance ER retention of mutated proteins but were associated with dramatically different levels of ER stress, unfolded protein response activation, and unfolded protein response-induced apoptosis. PMID:22006919

  3. Prolonged inhibition of bacterial protein synthesis abolishes Salmonella invasion.

    MacBeth, K J; Lee, C A

    1993-01-01

    We have found that prolonged inhibition of bacterial protein synthesis abolishes the ability of Salmonella typhimurium to enter HEp-2 cells. Our results suggest that an essential invasion factor has a functional half-life that is seen as a gradual loss of invasiveness in the absence of protein synthesis. Therefore, Salmonella invasiveness appears to be a transient phenotype that is lost unless protein synthesis is maintained. This finding may explain why salmonellae grown to stationary phase lose their ability to enter cultured cells. In addition, a short-lived capacity to enter cells may be important during infection so that bacterial invasiveness is limited to certain times and host sites during pathogenesis. PMID:8454361

  4. Angiopoietin-like protein 4 inhibition of lipoprotein lipase: evidence for reversible complex formation.

    Lafferty, Michael J; Bradford, Kira C; Erie, Dorothy A; Neher, Saskia B

    2013-10-01

    Elevated triglycerides are associated with an increased risk of cardiovascular disease, and lipoprotein lipase (LPL) is the rate-limiting enzyme for the hydrolysis of triglycerides from circulating lipoproteins. The N-terminal domain of angiopoietin-like protein 4 (ANGPTL4) inhibits LPL activity. ANGPTL4 was previously described as an unfolding molecular chaperone of LPL that catalytically converts active LPL dimers into inactive monomers. Our studies show that ANGPTL4 is more accurately described as a reversible, noncompetitive inhibitor of LPL. We find that inhibited LPL is in a complex with ANGPTL4, and upon dissociation, LPL regains lipase activity. Furthermore, we have generated a variant of ANGPTL4 that is dependent on divalent cations for its ability to inhibit LPL. We show that LPL inactivation by this regulatable variant of ANGPTL4 is fully reversible after treatment with a chelator. PMID:23960078

  5. Spectroscopic studies on native proteins, glycated and inhibited nonenzymatically

    The nonenzymatic glucation is an irreversible process whose speed depends on the concentration reducer sugar in plasma. The glycated albumins is higher in diabetic people. Up to now, this has been indicated as an important mechanism in the pathology of the the secondary complications associated with diabetes and the normal aging. Recently a lot of interest has been focused on the search of certain compounds (inhibitors) to prevent the glucation and / or the formation of ending products of advanced glucation, AGE. The reaction of glucose with the human albumin and γ globulins and the effects of acid acetylsalicylic and ascorbic acid were studied. The proteins were incubated with glucose in absence and in presence of inhibitors for one month. The solutions were dialysed and then lyophilizated. The absorption spectra were taken for native proteins, glycated and inhibited (2 mg/ml) in phosphate 10 mM buffer, p H 7.3. It is observed that the spectra of the acetylate proteins and native proteins are practically same. This can be interpreted as an inhibitor effect of acid acetylsalicylic on glucation. In all the observed cases the glycated proteins absorb more than the native ones and they present a line toward the visible region. The ascorbic acid absorbs below the native proteins and it doesn't present the same characteristics. The increase and / or the decrease in the absorption picks can be associated with environmental changes affecting the groups involved in the absorption process

  6. Targeted in vivo inhibition of specific protein-protein interactions using recombinant antibodies.

    Matej Zábrady

    Full Text Available With the growing availability of genomic sequence information, there is an increasing need for gene function analysis. Antibody-mediated "silencing" represents an intriguing alternative for the precise inhibition of a particular function of biomolecules. Here, we describe a method for selecting recombinant antibodies with a specific purpose in mind, which is to inhibit intrinsic protein-protein interactions in the cytosol of plant cells. Experimental procedures were designed for conveniently evaluating desired properties of recombinant antibodies in consecutive steps. Our selection method was successfully used to develop a recombinant antibody inhibiting the interaction of ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 3 with such of its upstream interaction partners as the receiver domain of CYTOKININ INDEPENDENT HISTIDINE KINASE 1. The specific down-regulation of the cytokinin signaling pathway in vivo demonstrates the validity of our approach. This selection method can serve as a prototype for developing unique recombinant antibodies able to interfere with virtually any biomolecule in the living cell.

  7. Inhibition of mitochondrial protein synthesis results in increased endothelial cell susceptibility to nitric oxide-induced apoptosis

    Ramachandran, Anup; Moellering, Douglas R.; Ceaser, Erin; Shiva, Sruti; Xu, Jun; Darley-Usmar, Victor

    2002-01-01

    Mutations in mitochondrial DNA, affecting the activity of respiratory complexes, have been implicated in many chronic degenerative diseases. Mitochondrial proteins coded for by both the mitochondrial and nuclear genes are known to have important signaling roles in apoptosis. However, the impact of the inhibition of mitochondrial protein synthesis on apoptosis is largely unknown. This inhibition is particularly important in NO-dependent cytotoxicity, which is believed to have a significant mit...

  8. Activated AMPK inhibits PPAR-{alpha} and PPAR-{gamma} transcriptional activity in hepatoma cells.

    Sozio, Margaret S; Lu, Changyue; Zeng, Yan; Liangpunsakul, Suthat; Crabb, David W

    2011-10-01

    AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor-α (PPAR-α) are critical regulators of short-term and long-term fatty acid oxidation, respectively. We examined whether the activities of these molecules were coordinately regulated. H4IIEC3 cells were transfected with PPAR-α and PPAR-γ expression plasmids and a peroxisome-proliferator-response element (PPRE) luciferase reporter plasmid. The cells were treated with PPAR agonists (WY-14,643 and rosiglitazone), AMPK activators 5-aminoimidazole-4-carboxamide riboside (AICAR) and metformin, and the AMPK inhibitor compound C. Both AICAR and metformin decreased basal and WY-14,643-stimulated PPAR-α activity; compound C increased agonist-stimulated reporter activity and partially reversed the effect of the AMPK activators. Similar effects on PPAR-γ were seen, with both AICAR and metformin inhibiting PPRE reporter activity. Compound C increased basal PPAR-γ activity and rosiglitazone-stimulated activity. In contrast, retinoic acid receptor-α (RAR-α), another nuclear receptor that dimerizes with retinoid X receptor (RXR), was largely unaffected by the AMPK activators. Compound C modestly increased AM580 (an RAR agonist)-stimulated activity. The AMPK activators did not affect PPAR-α binding to DNA, and there was no consistent correlation between effects of the AMPK activators and inhibitor on PPAR and the nuclear localization of AMPK-α subunits. Expression of either a constitutively active or dominant negative AMPK-α inhibited basal and WY-14,643-stimulated PPAR-α activity and basal and rosiglitazone-stimulated PPAR-γ activity. We concluded that the AMPK activators AICAR and metformin inhibited transcriptional activities of PPAR-α and PPAR-γ, whereas inhibition of AMPK with compound C activated both PPARs. The effects of AMPK do not appear to be mediated through effects on RXR or on PPAR/RXR binding to DNA. These effects are independent of kinase activity and instead appear to

  9. Inhibition of hepatitis C virus production by aptamers against the core protein.

    Shi, Shali; Yu, Xiaoyan; Gao, Yimin; Xue, Binbin; Wu, Xinjiao; Wang, Xiaohong; Yang, Darong; Zhu, Haizhen

    2014-02-01

    Hepatitis C virus (HCV) core protein is essential for virus assembly. HCV core protein was expressed and purified. Aptamers against core protein were raised through the selective evolution of ligands by the exponential enrichment approach. Detection of HCV infection by core aptamers and the antiviral activities of aptamers were characterized. The mechanism of their anti-HCV activity was determined. The data showed that selected aptamers against core specifically recognize the recombinant core protein but also can detect serum samples from hepatitis C patients. Aptamers have no effect on HCV RNA replication in the infectious cell culture system. However, the aptamers inhibit the production of infectious virus particles. Beta interferon (IFN-β) and interferon-stimulated genes (ISGs) are not induced in virally infected hepatocytes by aptamers. Domains I and II of core protein are involved in the inhibition of infectious virus production by the aptamers. V31A within core is the major resistance mutation identified. Further study shows that the aptamers disrupt the localization of core with lipid droplets and NS5A and perturb the association of core protein with viral RNA. The data suggest that aptamers against HCV core protein inhibit infectious virus production by disrupting the localization of core with lipid droplets and NS5A and preventing the association of core protein with viral RNA. The aptamers for core protein may be used to understand the mechanisms of virus assembly. Core-specific aptamers may hold promise for development as early diagnostic reagents and potential therapeutic agents for chronic hepatitis C. PMID:24307579

  10. Cross inhibition improves activity selection when switching incurs time costs

    Favreau-Peigne, Angélique; Fromhage, Lutz; McNamara, John M.; Meah, Lianne F.S.; Houston, Alasdair I.

    2015-01-01

    We consider a behavioural model of an animal choosing between two activities, based on positive feedback, and examine the effect of introducing cross inhibition between the motivations for the two activities. While cross-inhibition has previously been included in models of decision making, the question of what benefit it may provide to an animal's activity selection behaviour has not previously been studied. In neuroscience and in collective behaviour cross-inhibition, and other equivalent me...

  11. Inhibition of lymphocyte activation by gold sodium thiomalate.

    Hopkins, S J; Jayson, M I; Zeil, P.

    1983-01-01

    Activation of lymphoid cells by both T and B cell mitogens was inhibited by gold sodium thiomalate (GST). The action of GST did not appear to be exerted at early stages of lymphocyte activation. Inhibition by GST was sustained throughout 4 days of culture. The inhibitory effect of GST was reduced at low serum concentrations. Sodium thiomalate and sodium chloroaurate were also able to inhibit lymphocyte activation.

  12. Inhibition of DNA gyrase by optically active ofloxacin.

    Imamura, M.; Shibamura, S; Hayakawa, I.; Osada, Y

    1987-01-01

    Inhibition of DNA gyrase activity by optically active ofloxacins was studied and compared with the inhibition of norfloxacin and ciprofloxacin. The (-)-isomer of ofloxacin inhibited the supercoiling activity of gyrase from Micrococcus luteus more effectively than did the (+)-isomer. The 50% inhibitory concentrations of (-)-, (+/-)-, and (+)-ofloxacin; norfloxacin; and ciprofloxacin for gyrase from Escherichia coli were 0.78, 0.98, 7.24, 0.78, and 1.15 microgram/ml, respectively. These values ...

  13. Hypoxia inhibits colonic ion transport via activation of AMP kinase.

    Collins, Danielle

    2012-02-01

    BACKGROUND AND AIMS: Mucosal hypoxia is a common endpoint for many pathological processes including ischemic colitis, colonic obstruction and anastomotic failure. Previous studies suggest that hypoxia modulates colonic mucosal function through inhibition of chloride secretion. However, the molecular mechanisms underlying this observation are poorly understood. AMP-activated protein kinase (AMPK) is a metabolic energy regulator found in a wide variety of cells and has been linked to cystic fibrosis transmembrane conductance regulator (CFTR) mediated chloride secretion in several different tissues. We hypothesized that AMPK mediates many of the acute effects of hypoxia on human and rat colonic electrolyte transport. METHODS: The fluorescent chloride indicator dye N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide was used to measure changes in intracellular chloride concentrations in isolated single rat colonic crypts. Ussing chamber experiments in human colonic mucosa were conducted to evaluate net epithelial ion transport. RESULTS: This study demonstrates that acute hypoxia inhibits electrogenic chloride secretion via AMPK mediated inhibition of CFTR. Pre-treatment of tissues with the AMPK inhibitor 6-[4-(2-piperidin-1-yl-ethoxy)-phenyl)]-3-pyridin-4-yl-pyyrazolo [1,5-a] pyrimidine (compound C) in part reversed the effects of acute hypoxia on chloride secretion. CONCLUSION: We therefore suggest that AMPK is a key component of the adaptive cellular response to mucosal hypoxia in the colon. Furthermore, AMPK may represent a potential therapeutic target in diseased states or in prevention of ischemic intestinal injury.

  14. Inhibition of Tomato Yellow Leaf Curl Virus (TYLCV using whey proteins

    Dawood Abdelgawad

    2010-02-01

    Full Text Available Abstract The antiviral activity of native and esterified whey proteins fractions (α-lactalbumin, β-lactoglobulin, and lactoferrin was studied to inhibit tomato yellow leaf curl virus (TYLCV on infected tomato plants. Whey proteins fractions and their esterified derivatives were sprayed into TYLCV-infected plants. Samples were collected from infected leaves before treatment, 7 and 15 days after treatment for DNA and molecular hybridization analysis. The most evident inhibition of virus replication was observed after 7 and 15 days using α-lactoferrin and α-lactalbumin, respectively. Native and esterified lactoferrin showed complete inhibition after 7 days. On the other hand, native β-lactoglobulin showed inhibition after 7 and 15 days whereas esterified β-lactoglobulin was comparatively more effective after 7 days. The relative amount of viral DNA was less affected by the esterified α-lactalbumin whereas native α-lactalbumin inhibited virus replication completely after 15 days. These results indicate that native or modified whey proteins fractions can be used for controlling the TYLCV-infected plants.

  15. The Human Antimicrobial Protein Bactericidal/Permeability-Increasing Protein (BPI) Inhibits the Infectivity of Influenza A Virus

    Pinkenburg, Olaf; Meyer, Torben; Bannert, Norbert; Norley, Steven; Bolte, Kathrin; Czudai-Matwich, Volker; Herold, Susanne; Gessner, André; Schnare, Markus

    2016-01-01

    In addition to their well-known antibacterial activity some antimicrobial peptides and proteins (AMPs) display also antiviral effects. A 27 aa peptide from the N-terminal part of human bactericidal/permeability-increasing protein (BPI) previously shown to harbour antibacterial activity inhibits the infectivity of multiple Influenza A virus strains (H1N1, H3N2 and H5N1) the causing agent of the Influenza pneumonia. In contrast, the homologous murine BPI-peptide did not show activity against Influenza A virus. In addition human BPI-peptide inhibits the activation of immune cells mediated by Influenza A virus. By changing the human BPI-peptide to the sequence of the mouse homologous peptide the antiviral activity was completely abolished. Furthermore, the human BPI-peptide also inhibited the pathogenicity of the Vesicular Stomatitis Virus but failed to interfere with HIV and measles virus. Electron microscopy indicate that the human BPI-peptide interferes with the virus envelope and at high concentrations was able to destroy the particles completely. PMID:27273104

  16. Mechanisms of HIV-1 Nucleocapsid Protein Inhibition by Lysyl-Peptidyl-Anthraquinone Conjugates.

    Sosic, Alice; Sinigaglia, Laura; Cappellini, Marta; Carli, Ilaria; Parolin, Cristina; Zagotto, Giuseppe; Sabatino, Giuseppina; Rovero, Paolo; Fabris, Dan; Gatto, Barbara

    2016-01-20

    The Nucleocapsid protein NCp7 (NC) is a nucleic acid chaperone responsible for essential steps of the HIV-1 life cycle and an attractive candidate for drug development. NC destabilizes nucleic acid structures and promotes the formation of annealed substrates for HIV-1 reverse transcription elongation. Short helical nucleic acid segments bordered by bulges and loops, such as the Trans-Activation Response element (TAR) of HIV-1 and its complementary sequence (cTAR), are nucleation elements for helix destabilization by NC and also preferred recognition sites for threading intercalators. Inspired by these observations, we have recently demonstrated that 2,6-disubstituted peptidyl-anthraquinone-conjugates inhibit the chaperone activities of recombinant NC in vitro, and that inhibition correlates with the stabilization of TAR and cTAR stem-loop structures. We describe here enhanced NC inhibitory activity by novel conjugates that exhibit longer peptidyl chains ending with a conserved N-terminal lysine. Their efficient inhibition of TAR/cTAR annealing mediated by NC originates from the combination of at least three different mechanisms, namely, their stabilizing effects on nucleic acids dynamics by threading intercalation, their ability to target TAR RNA substrate leading to a direct competition with the protein for the same binding sites on TAR, and, finally, their effective binding to the NC protein. Our results suggest that these molecules may represent the stepping-stone for the future development of NC-inhibitors capable of targeting the protein itself and its recognition site in RNA. PMID:26666402

  17. Results of a screening programme to identify plants or plant extracts that inhibit ruminal protein degradation.

    Selje, N; Hoffmann, E M; Muetzel, S; Ningrat, R; Wallace, R J; Becker, K

    2007-07-01

    One aim of the EC Framework V project, 'Rumen-up' (QLK5-CT-2001-00 992), was to find plants or plant extracts that would inhibit the nutritionally wasteful degradation of protein in the rumen. A total of 500 samples were screened in vitro using 14C-labelled casein in a 30-min incubation with ruminal digesta. Eight were selected for further investigation using a batch fermentation system and soya protein and bovine serum albumin as proteolysis substrates; proteolysis was monitored over 12 h by the disappearance of soluble protein and the production of branched SCFA and NH3. Freeze-dried, ground foliage of Peltiphyllum peltatum, Helianthemum canum, Arbutus unedo, Arctostaphylos uva-ursi and Knautia arvensis inhibited proteolysis (P Clematis vitalba and Erica arborea had little effect. Inhibition by the first four samples appeared to be caused by the formation of insoluble tannin-protein complexes. The samples were rich in phenolics and inhibition was reversed by polyethyleneglycol. In contrast, K. arvensis contained low concentrations of phenolics and no tannins, had no effect in the 30-min assay, yet inhibited the degradation rate of soluble protein (by 14 %, P < 0.0001) and the production of branched SCFA (by 17 %, P < 0.05) without precipitating protein in the 12-h batch fermentation. The effects showed some resemblance to those obtained in parallel incubations containing 3 mum-monensin, suggesting that K. arvensis may be a plant-derived feed additive that can suppress growth and activity of key proteolytic ruminal micro-organisms in a manner similar to that already well known for monensin. PMID:17445338

  18. Emerging Roles of AMP-Activated Protein Kinase

    Fritzen, Andreas Mæchel

    The cellular energy sensor AMP-activated protein kinase (AMPK) is activated, when the energy balance of the cell decreases. AMPK has been proposed to regulate multiple metabolic processes. However, much of the evidence for these general effects of AMPK relies on investigations in cell systems or...... exercise appears to inhibit pyruvate dehydrogenase (PDH) activity by an immediate up-regulation of pyruvate dehydrogenase kinase 4 (PDK4) protein content. Consequently, this may inhibit glucose oxidation and thereby generate conditions for increased FA oxidation and glycogen resynthesis in skeletal muscle...... importance for prioritising energy dissipation, inhibition of lipid storage pathways and regulation of mitochondrial and metabolic proteins, but this needs further investigations. In addition, we provide evidence that AMPK is regulating autophagic signalling in skeletal muscle. Thus, in skeletal muscle AMPK...

  19. Proteins in the Cocoon of Silkworm Inhibit the Growth of Beauveria bassiana.

    Guo, Xiaomeng; Dong, Zhaoming; Zhang, Yan; Li, Youshan; Liu, Huawei; Xia, Qingyou; Zhao, Ping

    2016-01-01

    Silk cocoons are composed of fiber proteins (fibroins) and adhesive glue proteins (sericins), which provide a physical barrier to protect the inside pupa. Moreover, other proteins were identified in the cocoon silk, many of which are immune related proteins. In this study, we extracted proteins from the silkworm cocoon by Tris-HCl buffer (pH7.5), and found that they had a strong inhibitory activity against fungal proteases and they had higher abundance in the outer cocoon layers than in the inner cocoon layers. Moreover, we found that extracted cocoon proteins can inhibit the germination of Beauveria bassiana spores. Consistent with the distribution of protease inhibitors, we found that proteins from the outer cocoon layers showed better inhibitory effects against B. bassiana spores than proteins from the inner layers. Liquid chromatography-tandem mass spectrometry was used to reveal the extracted components in the scaffold silk, the outermost cocoon layer. A total of 129 proteins were identified, 30 of which were annotated as protease inhibitors. Protease inhibitors accounted for 89.1% in abundance among extracted proteins. These protease inhibitors have many intramolecular disulfide bonds to maintain their stable structure, and remained active after being boiled. This study added a new understanding to the antimicrobial function of the cocoon. PMID:27032085

  20. Activation of an immune-regulatory macrophage response and inhibition of lung inflammation in a mouse model of COPD using heat-shock protein alpha B-crystallin-loaded PLGA microparticles

    van Noort, Johannes M.; Bsibsi, Malika; Nacken, Peter J.; Gerritsen, Wouter H.; Amor, Sandra; Holtman, Inge R.; Boddeke, Erik; van Ark, Ingrid; Leusink-Muis, Thea; Folkerts, Gert; Hennink, Wim E.; Amidi, Maryam

    2013-01-01

    As an extracellular protein, the small heat-shock protein alpha B-crystallin (HSPB5) has anti-inflammatory effects in several mouse models of inflammation. Here, we show that these effects are associated with the ability of HSPB5 to activate an immune-regulatory response in macrophages via endosomal

  1. Inhibition of Setaria cervi protein tyrosine phosphatases by Phenylarsine oxide: A proteomic and biochemical study.

    Singh, Neetu; Wadhawan, Mohit; Tiwari, Savitri; Kumar, Ranjeet; Rathaur, Sushma

    2016-07-01

    Phenylarsine oxide (PAO), a specific protein tyrosine phosphatase (PTP) inhibitor significantly decreased the motility and viability of Setaria cervi ultimately leading to its death. The PTP activity present in the cytosolic and detergent soluble fractions as well as on surface of these parasites was significantly inhibited by PAO. A marked alteration in protein spots abundance after proteomic analysis showed 14 down-regulated and 9 upregulated spots in the treated parasites as compared to the control. The PTP inhibition led to increase in the cytosolic and mitochondrial calpain activity in these parasites. PAO also blocked the ATP generation in the parasite depicted by reduced activity of phosphoglycerate kinase and expression of enolase. An increased ROS level, induced lipid peroxidation/protein carbonyl formation and decreased activity of different antioxidant enzymes like thioredoxin reductase, glutathione reductase and glutathione transferases was also observed in the PAO treated parasites. PAO, thus disturbs the overall homeostasis of the filarial parasite by inhibiting PTPs. Thereby suggesting that these molecules could be used as a good chemotherapeutic target for lymphatic filariasis. PMID:26965172

  2. Mitogen-activated protein kinases in atherosclerosis

    Dorota Bryk

    2014-01-01

    Full Text Available Intracellular signalling cascades, in which MAPK (mitogen-activated protein kinases intermediate, are responsible for a biological response of a cell to an external stimulus. MAP kinases, which include ERK1/2 (extracellular signalling-regulated kinase, JNK (c-Jun N-terminal kinase and p 38 MAPK, regulate the activity of many proteins, enzymes and transcription factors and thus have a wide spectrum of biological effects. Many basic scientific studies have defined numerous details of their pathway organization and activation. There are also more and more studies suggesting that individual MAP kinases probably play an important role in the pathogenesis of atherosclerosis. They may mediate inflammatory processes, endothelial cell activation, monocyte/macrophage recruitment and activation, smooth muscle cell proliferation and T-lymphocyte differentiation, all of which represent crucial mechanisms involved in pathogenesis of atherosclerosis. The specific inhibition of an activity of the respective MAP kinases may prove a new therapeutic approach to attenuate atherosclerotic plaque formation in the future. In this paper, we review the current state of knowledge concerning MAP kinase-dependent cellular and molecular mechanisms underlying atherosclerosis.

  3. [Mitogen-activated protein kinases in atherosclerosis].

    Bryk, Dorota; Olejarz, Wioletta; Zapolska-Downar, Danuta

    2014-01-01

    Intracellular signalling cascades, in which MAPK (mitogen-activated protein kinases) intermediate, are responsible for a biological response of a cell to an external stimulus. MAP kinases, which include ERK1/2 (extracellular signalling-regulated kinase), JNK (c-Jun N-terminal kinase) and p 38 MAPK, regulate the activity of many proteins, enzymes and transcription factors and thus have a wide spectrum of biological effects. Many basic scientific studies have defined numerous details of their pathway organization and activation. There are also more and more studies suggesting that individual MAP kinases probably play an important role in the pathogenesis of atherosclerosis. They may mediate inflammatory processes, endothelial cell activation, monocyte/macrophage recruitment and activation, smooth muscle cell proliferation and T-lymphocyte differentiation, all of which represent crucial mechanisms involved in pathogenesis of atherosclerosis. The specific inhibition of an activity of the respective MAP kinases may prove a new therapeutic approach to attenuate atherosclerotic plaque formation in the future. In this paper, we review the current state of knowledge concerning MAP kinase-dependent cellular and molecular mechanisms underlying atherosclerosis. PMID:24491891

  4. Inhibition of transcriptional activity of c-JUN by SIRT1

    c-JUN is a major component of heterodimer transcription factor AP-1 (Activator Protein-1) that activates gene transcription in cell proliferation, inflammation and stress responses. SIRT1 (Sirtuin 1) is a histone deacetylase that controls gene transcription through modification of chromatin structure. However, it is not clear if SIRT1 regulates c-JUN activity in the control of gene transcription. Here, we show that SIRT1 associated with c-JUN in co-immunoprecipitation of whole cell lysate, and inhibited the transcriptional activity of c-JUN in the mammalian two hybridization system. SIRT1 was found in the AP-1 response element in the matrix metalloproteinase-9 (MMP9) promoter DNA leading to inhibition of histone 3 acetylation as shown in a ChIP assay. The SIRT1 signal was reduced by the AP-1 activator PMA, and induced by the SIRT1 activator Resveratrol in the promoter DNA. SIRT1-mediaetd inhibition of AP-1 was demonstrated in the MMP9 gene expression at the gene promoter, mRNA and protein levels. In mouse embryonic fibroblast (MEF) with SIRT1 deficiency (SIRT1-/-), mRNA and protein of MMP9 were increased in the basal condition, and the inhibitory activity of Resveratrol was significantly attenuated. Glucose-induced MMP9 expression was also inhibited by SIRT1 in response to Resveratrol. These data consistently suggest that SIRT1 directly inhibits the transcriptional activity of AP-1 by targeting c-JUN

  5. Rapamycin Inhibits Lymphatic Endothelial Cell Tube Formation by Downregulating Vascular Endothelial Growth Factor Receptor 3 Protein Expression

    Yan Luo

    2012-03-01

    Full Text Available Mammalian target of rapamycin (mTOR controls lymphangiogenesis. However, the underlying mechanism is not clear. Here we show that rapamycin suppressed insulin-like growth factor 1 (IGF-1- or fetal bovine serum (FBS-stimulated lymphatic endothelial cell (LEC tube formation, an in vitro model of lymphangiogenesis. Expression of a rapamycin-resistant and kinase-active mTOR (S2035T, mTOR-T, but not a rapamycin-resistant and kinase-dead mTOR (S2035T/D2357E, mTOR-TE, conferred resistance to rapamycin inhibition of LEC tube formation, suggesting that rapamycin inhibition of LEC tube formation is mTOR kinase activity dependent. Also, rapamycin inhibited proliferation and motility in the LECs. Furthermore, we found that rapamycin inhibited protein expression of VEGF receptor 3 (VEGFR-3 by inhibiting protein synthesis and promoting protein degradation of VEGFR-3 in the cells. Down-regulation of VEGFR-3 mimicked the effect of rapamycin, inhibiting IGF-1- or FBS-stimulated tube formation, whereas over-expression of VEGFR-3 conferred high resistance to rapamycin inhibition of LEC tube formation. The results indicate that rapamycin inhibits LEC tube formation at least in part by downregulating VEGFR-3 protein expression.

  6. Inhibition of HIV derived lentiviral production by TAR RNA binding domain of TAT protein

    He Yukai

    2005-11-01

    Full Text Available Abstract Background A critical step in the production of new HIV virions involves the TAT protein binding to the TAR element. The TAT protein contains in close proximity its TAR RNA binding domain and protein transduction domain (PTD. The PTD domain of TAT has been identified as being instrumental in the protein's ability to cross mammalian cell and nuclear membranes. All together, this information led us to form the hypothesis that a protein containing the TAR RNA binding domain could compete with the native full length TAT protein and effectively block the TAR RNA binding site in transduced HIV infected cells. Results We synthesized a short peptide named Tat-P, which contained the TAR RNA binding and PTD domains to examine whether the peptide has the potential of inhibiting TAT dependent HIV replication. We investigated the inhibiting effects of Tat-P in vitro using a HIV derived lentiviral vector model. We found that the TAT PTD domain not only efficiently transduced test cells, but also effectively inhibited the production of lentiviral particles in a TAT dependent manner. These results were also supported by data derived from the TAT activated LTR-luciferase expression model and RNA binding assays. Conclusion Tat-P may become part of a category of anti-HIV drugs that competes with full length TAT proteins to inhibit HIV replication. In addition, this study indicates that the HIV derived lentiviral vector system is a safe and reliable screening method for anti-HIV drugs, especially for those targeting the interaction of TAT and TAR RNAs.

  7. Activating and inhibiting connections in biological network dynamics

    Knight Rob

    2008-12-01

    Full Text Available Abstract Background Many studies of biochemical networks have analyzed network topology. Such work has suggested that specific types of network wiring may increase network robustness and therefore confer a selective advantage. However, knowledge of network topology does not allow one to predict network dynamical behavior – for example, whether deleting a protein from a signaling network would maintain the network's dynamical behavior, or induce oscillations or chaos. Results Here we report that the balance between activating and inhibiting connections is important in determining whether network dynamics reach steady state or oscillate. We use a simple dynamical model of a network of interacting genes or proteins. Using the model, we study random networks, networks selected for robust dynamics, and examples of biological network topologies. The fraction of activating connections influences whether the network dynamics reach steady state or oscillate. Conclusion The activating fraction may predispose a network to oscillate or reach steady state, and neutral evolution or selection of this parameter may affect the behavior of biological networks. This principle may unify the dynamics of a wide range of cellular networks. Reviewers Reviewed by Sergei Maslov, Eugene Koonin, and Yu (Brandon Xia (nominated by Mark Gerstein. For the full reviews, please go to the Reviewers' comments section.

  8. Calmodulin binds to and inhibits the activity of phosphoglycerate kinase.

    Myre, Michael A; O'Day, Danton H

    2004-09-17

    Phosphoglycerate kinase (PGK) functions as a cytoplasmic ATP-generating glycolytic enzyme, a nuclear mediator in DNA replication and repair, a stimulator of Sendai virus transcription and an extracellular disulfide reductase in angiogenesis. Probing of a developmental expression library from Dictyostelium discoideum with radiolabelled calmodulin led to the isolation of a cDNA encoding a putative calmodulin-binding protein (DdPGK) with 68% sequence similarity to human PGK. Dictyostelium, rabbit and yeast PGKs bound to calmodulin-agarose in a calcium-dependent manner while DdPGK constructs lacking the calmodulin-binding domain (209KPFLAILGGAKVSDKIKLIE228) failed to bind. The calmodulin-binding domain shows 80% identity between diverse organisms and is situated beside the hinge and within the ATP binding domain adjacent to nine mutations associated with PGK deficiency. Calmodulin addition inhibits yeast PGK activity in vitro while the calmodulin antagonist W-7 abrogates this inhibition. Together, these data suggest that PGK activity may be negatively regulated by calcium and calmodulin signalling in eukaryotic cells. PMID:15363631

  9. Nutlin-3 down-regulates retinoblastoma protein expression and inhibits muscle cell differentiation

    The p53 tumor suppressor gene plays a critical role in regulation of proliferation, cell death and differentiation. The MDM2 oncoprotein is a major negative regulator for p53 by binding to and targeting p53 for proteasome-mediated degradation. The small molecule inhibitor, nutlin-3, disrupts MDM2-p53 interaction resulting in stabilization and activation of p53 protein. We have previously shown that nutlin-3 activates p53, leading to MDM2 accumulation as concomitant of reduced retinoblastoma (Rb) protein stability. It is well known that Rb is important in muscle development and myoblast differentiation and that rhabdomyosarcoma (RMS), or cancer of the skeletal muscle, typically harbors MDM2 amplification. In this study, we show that nutlin-3 inhibited myoblast proliferation and effectively prevented myoblast differentiation, as evidenced by lack of expression of muscle differentiation markers including myogenin and myosin heavy chain (MyHC), as well as a failure to form multinucleated myotubes, which were associated with dramatic increases in MDM2 expression and decrease in Rb protein levels. These results indicate that nutlin-3 can effectively inhibit muscle cell differentiation. - Highlights: • Nutlin-3 inhibits myoblast proliferation and prevents differentiation into myotubes. • Nutlin-3 increases MDM2 expression and down-regulates Rb protein levels. • This study has implication in nutlin-3 treatment of rhabdomyosarcomas

  10. Nutlin-3 down-regulates retinoblastoma protein expression and inhibits muscle cell differentiation

    Walsh, Erica M. [Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118 (United States); Niu, MengMeng; Bergholz, Johann [Center of Growth, Metabolism and Aging, College of Life Sciences, Sichuan University, Chengdu, 610014 China (China); Jim Xiao, Zhi-Xiong, E-mail: jxiao@bu.edu [Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118 (United States); Center of Growth, Metabolism and Aging, College of Life Sciences, Sichuan University, Chengdu, 610014 China (China)

    2015-05-29

    The p53 tumor suppressor gene plays a critical role in regulation of proliferation, cell death and differentiation. The MDM2 oncoprotein is a major negative regulator for p53 by binding to and targeting p53 for proteasome-mediated degradation. The small molecule inhibitor, nutlin-3, disrupts MDM2-p53 interaction resulting in stabilization and activation of p53 protein. We have previously shown that nutlin-3 activates p53, leading to MDM2 accumulation as concomitant of reduced retinoblastoma (Rb) protein stability. It is well known that Rb is important in muscle development and myoblast differentiation and that rhabdomyosarcoma (RMS), or cancer of the skeletal muscle, typically harbors MDM2 amplification. In this study, we show that nutlin-3 inhibited myoblast proliferation and effectively prevented myoblast differentiation, as evidenced by lack of expression of muscle differentiation markers including myogenin and myosin heavy chain (MyHC), as well as a failure to form multinucleated myotubes, which were associated with dramatic increases in MDM2 expression and decrease in Rb protein levels. These results indicate that nutlin-3 can effectively inhibit muscle cell differentiation. - Highlights: • Nutlin-3 inhibits myoblast proliferation and prevents differentiation into myotubes. • Nutlin-3 increases MDM2 expression and down-regulates Rb protein levels. • This study has implication in nutlin-3 treatment of rhabdomyosarcomas.

  11. Protein kinase B/Akt1 inhibits autophagy by down-regulating UVRAG expression

    Autophagy, or autophagocytosis, is a selective intracellular degradative process involving the cell's own lysosomal apparatus. An essential component in cell development, homeostasis, repair and resistance to stress, autophagy may result in either cell death or survival. The targeted region of the cell is sequestered within a membrane structure, the autophagosome, for regulation of the catabolic process. A key factor in both autophagosome formation and autophagosome maturation is a protein encoded by the ultraviolet irradiation resistance-associated gene (UVRAG). Conversely, the serine/threonine-specific protein kinase B (PKB, also known as Akt), which regulates survival in various cancers, inhibits autophagy through mTOR activation. We found that Akt1 may also directly inhibit autophagy by down-regulating UVRAG both in a 293T transient transfection system and breast cancer cells stably expressing Akt1. The UVRAG with mutations at putative Akt1-phosphorylation sites were still inhibited by Akt1, and dominant-negative Akt1 also inhibited UVRAG expression, suggesting that Akt1 down-regulates UVRAG by a kinase activity-independent mechanism. We showed that Akt1 overexpression in MDA-MB-231 breast cancer cells down-regulated UVRAG transcription. Cells over-expressing Akt1 were more resistant than control cells to ultraviolet light-induced autophagy and exhibited the associated reduction in cell viability. Levels of the autophagosome indicator protein LC3B-II and mRFP-GFP-LC3 were reduced in cells that over-expressing Akt1. Inhibiting Akt1 by siRNA or reintroducing UVRAG gene rescued the level of LC3B-II in UV-irradiation. Altogether, these data suggest that Akt1 may inhibit autophagy by decreasing UVRAG expression, which also sensitizes cancer cells to UV irradiation.

  12. Protein kinase B/Akt1 inhibits autophagy by down-regulating UVRAG expression

    Yang, Wonseok; Ju, Ji-hyun; Lee, Kyung-min; Nam, KeeSoo; Oh, Sunhwa [Department of Life Science, College of Natural Science, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul 133-791 (Korea, Republic of); Shin, Incheol, E-mail: incheol@hanyang.ac.kr [Department of Life Science, College of Natural Science, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul 133-791 (Korea, Republic of)

    2013-02-01

    Autophagy, or autophagocytosis, is a selective intracellular degradative process involving the cell's own lysosomal apparatus. An essential component in cell development, homeostasis, repair and resistance to stress, autophagy may result in either cell death or survival. The targeted region of the cell is sequestered within a membrane structure, the autophagosome, for regulation of the catabolic process. A key factor in both autophagosome formation and autophagosome maturation is a protein encoded by the ultraviolet irradiation resistance-associated gene (UVRAG). Conversely, the serine/threonine-specific protein kinase B (PKB, also known as Akt), which regulates survival in various cancers, inhibits autophagy through mTOR activation. We found that Akt1 may also directly inhibit autophagy by down-regulating UVRAG both in a 293T transient transfection system and breast cancer cells stably expressing Akt1. The UVRAG with mutations at putative Akt1-phosphorylation sites were still inhibited by Akt1, and dominant-negative Akt1 also inhibited UVRAG expression, suggesting that Akt1 down-regulates UVRAG by a kinase activity-independent mechanism. We showed that Akt1 overexpression in MDA-MB-231 breast cancer cells down-regulated UVRAG transcription. Cells over-expressing Akt1 were more resistant than control cells to ultraviolet light-induced autophagy and exhibited the associated reduction in cell viability. Levels of the autophagosome indicator protein LC3B-II and mRFP-GFP-LC3 were reduced in cells that over-expressing Akt1. Inhibiting Akt1 by siRNA or reintroducing UVRAG gene rescued the level of LC3B-II in UV-irradiation. Altogether, these data suggest that Akt1 may inhibit autophagy by decreasing UVRAG expression, which also sensitizes cancer cells to UV irradiation.

  13. Rhodamine 123 inhibits protein synthesis in mitochondria isolated from normal and cancer tissues

    The dye rhodamine 123 (Rho 123) is well known to specifically stain mitochrondria in living cells. Its accumulation in these organelles is associated with certain toxic effects where the dye is found to inhibit bioenergetic function in normal and cancer mitochondria. Since mitochondria appear to be a target for Rho 123 interaction, the authors investigated its effects on mitochondrial protein synthesis (PS) in rat liver, and in both erythroleukemia and chloroleukemia tumors. L-(14C) leucine incorporation into mitochondria protein was used to determine the rate of PS. While the specific activity of leucine incorporation was much higher in tumor as compared to liver mitochondria, the addition of 10 μg Rho 123/ml in all tested mitochondria resulted in 75-80% inhibition. Similar results were obtained with 10 μg/ml of chloramphenicol, the specific inhibitor for mitochondrial PS. PS inhibition in the three types of mitochondria was Rho 123 concentration-dependent being about 50% at 5 μg/ml and with total inhibition at 15-20 μg/ml. Moreover, the addition of Rho 123 to mitochondria under PS condition did not trigger any ATPase activity. If present, such activity would compete for ATP which is the energy source of PS. These results demonstrate that the mitochondrial probe Rho 123 has a potent inhibitory effect on PS in both normal and cancer mitochondria

  14. Hyperoxia Inhibits T Cell Activation in Mice

    Hughes-Fulford, M.; Meissler, J.; Aguayo, E. T.; Globus, R.; Aguado, J.; Candelario, T.

    2013-02-01

    , spleens were removed and the splenocytes were isolated and kept as individual biological samples. We have also examined transcription factors (JASPAR) and pathways of the immune system to help us understand the mechanism of regulation. Results: Our recent mouse immunology experiment aboard STS-131 suggests that the early T cell immune response was inhibited in animals that have been exposed to spaceflight, even 24 hours after return to earth. Moreover, recent experiments in hyperoxic mice show that many of the same genes involved in early T cell activation were altered. Specifically, expression of IL-2Rα, Cxcl2, TNFα, FGF2, LTA and BCL2 genes are dysregulated in mice exposed to hyperoxia. Conclusions: If these hyperoxia-induced changes of gene expression in early T cell activation are additive to the changes seen in the microgravity of spaceflight, there could be an increased infection risk to EVA astronauts, which should be addressed prior to conducting a Mars or other long-term mission.

  15. Cinnamic Acid and Its Derivatives Inhibit Fructose-Mediated Protein Glycation

    Sirintorn Yibchok-anun; Sirichai Adisakwattana; Weerachat Sompong; Sathaporn Ngamukote; Aramsri Meeprom

    2012-01-01

    Cinnamic acid and its derivatives have shown a variety of pharmacologic properties. However, little is known about the antiglycation properties of cinnamic acid and its derivatives. The present study sought to characterize the protein glycation inhibitory activity of cinnamic acid and its derivatives in a bovine serum albumin (BSA)/fructose system. The results demonstrated that cinnamic acid and its derivatives significantly inhibited the formation of advanced glycation end products (AGEs) by...

  16. Obesity and lipid stress inhibit carnitine acetyltransferase activity.

    Seiler, Sarah E; Martin, Ola J; Noland, Robert C; Slentz, Dorothy H; DeBalsi, Karen L; Ilkayeva, Olga R; An, Jie; Newgard, Christopher B; Koves, Timothy R; Muoio, Deborah M

    2014-04-01

    Carnitine acetyltransferase (CrAT) is a mitochondrial matrix enzyme that catalyzes the interconversion of acetyl-CoA and acetylcarnitine. Emerging evidence suggests that this enzyme functions as a positive regulator of total body glucose tolerance and muscle activity of pyruvate dehydrogenase (PDH), a mitochondrial enzyme complex that promotes glucose oxidation and is feedback inhibited by acetyl-CoA. Here, we used tandem mass spectrometry-based metabolic profiling to identify a negative relationship between CrAT activity and muscle content of lipid intermediates. CrAT specific activity was diminished in muscles from obese and diabetic rodents despite increased protein abundance. This reduction in enzyme activity was accompanied by muscle accumulation of long-chain acylcarnitines (LCACs) and acyl-CoAs and a decline in the acetylcarnitine/acetyl-CoA ratio. In vitro assays demonstrated that palmitoyl-CoA acts as a direct mixed-model inhibitor of CrAT. Similarly, in primary human myocytes grown in culture, nutritional and genetic manipulations that promoted mitochondrial influx of fatty acids resulted in accumulation of LCACs but a pronounced decrease of CrAT-derived short-chain acylcarnitines. These results suggest that lipid-induced antagonism of CrAT might contribute to decreased PDH activity and glucose disposal in the context of obesity and diabetes. PMID:24395925

  17. Identification and characterization of multiple TRIM proteins that inhibit hepatitis B virus transcription.

    Shijian Zhang

    Full Text Available Tripartite motif (TRIM proteins constitute a family of over 100 members that share conserved tripartite motifs and exhibit diverse biological functions. Several TRIM proteins have been shown to restrict viral infections and regulate host cellular innate immune responses. In order to identify TRIM proteins that modulate the infection of hepatitis B virus (HBV, we tested 38 human TRIMs for their effects on HBV gene expression, capsid assembly and DNA synthesis in human hepatoma cells (HepG2. The study revealed that ectopic expression of 8 TRIM proteins in HepG2 cells potently reduced the amounts of secreted HBV surface and e antigens as well as intracellular capsid and capsid DNA. Mechanistic analyses further demonstrated that the 8 TRIMs not only reduced the expression of HBV mRNAs, but also inhibited HBV enhancer I and enhancer II activities. Studies focused on TRIM41 revealed that a HBV DNA segment spanning nucleotide 1638 to nucleotide 1763 was essential for TRIM41-mediated inhibition of HBV enhancer II activity and the inhibitory effect depended on the E3 ubiquitin ligase activity of TRIM41 as well as the integrity of TRIM41 C-terminal domain. Moreover, knockdown of endogenous TRIM41 in a HepG2-derived stable cell line significantly increased the level of HBV preC/C RNA, leading to an increase in viral core protein, capsid and capsid DNA. Our studies have thus identified eight TRIM proteins that are able to inhibit HBV transcription and provided strong evidences suggesting the endogenous role of TRIM41 in regulating HBV transcription in human hepatoma cells.

  18. The feed contaminant deoxynivalenol affects the intestinal barrier permeability through inhibition of protein synthesis.

    Awad, Wageha A; Zentek, Jürgen

    2015-06-01

    Deoxynivalenol (DON) has critical health effects if the contaminated grains consumed by humans or animals. DON can have negative effects on the active transport of glucose and amino acids in the small intestine of chickens. As the underlying mechanisms are not fully elucidated, the present study was performed to delineate more precisely the effects of cycloheximide (protein synthesis inhibitor, CHX) and DON on the intestinal absorption of nutrients. This was to confirm whether DON effects on nutrient absorption are due to an inhibition of protein synthesis. Changes in ion transport and barrier function were assessed by short-circuit current (Isc) and transepithelial ion conductance (Gt) in Ussing chambers. Addition of D-glucose or L-glutamine to the luminal side of the isolated mucosa of the jejunum increased (P glutamine addition after pre-incubation of tissues with DON or CHX. Furthermore, both DON and CHX reduced Gt, indicating that the intestinal barrier is compromised and consequently induced a greater impairment of the barrier function. The remarkable similarity between the activity of CHX and DON on nutrient uptake is consistent with their common ability to inhibit protein synthesis. It can be concluded that the decreases in transport activity by CHX was evident in this study using the chicken as experimental model. Similarly, DON has negative effects on the active transport of some nutrients, and these can be explained by its influence on protein synthesis. PMID:24888376

  19. Protein C activators in snake venoms.

    Stocker, K; Fischer, H; Meier, J; Brogli, M; Svendsen, L

    1986-02-01

    Venoms of 32 snake species were tested for protein C (PC) activating potency. As measured with the chromogenic PC substrate D-Pro-L-Pro-L-Arg-pNA, eleven venoms were able to generate amidolytic activity from purified bovine PC. In five venom solutions (Bothrops moojeni, B. pradoi, Cerastes cerastes, Vipera lebetina and V. russellii) the PC activating potency was destroyed during 10 min heating at 70 degrees C at pH 3, whereas in six venom solutions (Agkistrodon contortrix contortrix, A. c. mokasen, A. c. pictigaster, Agkistrodon piscivorus, A. p. leucostoma and A. bilineatus) the PC activator was stable under these conditions. PC activator from A. c. contortrix (Protac) was purified to homogeneity and characterized as a single chain polypeptide with a molecular weight of approx. 39-42,000 Dalton. Protac does not exert proteinase activity and is not inhibited by proteinase inhibitors; PC activation with Protac seems to be a stoichiometric reaction. The use of Protac in quantitative PC determination bears significant advantages over the use of thrombin as an activator. In rabbits, i.v. injection of Protac caused a prolonged APTT and did not provoke acute toxic reactions. PMID:3755037

  20. Inhibition of cholesterol ester transfer protein CGS 25159 and changes in lipoproteins in hamsters.

    Kothari, H V; Poirier, K J; Lee, W H; Satoh, Y

    1997-01-01

    As a result of screening, several isoflavans were identified to be antagonists of cholesterol ester transfer protein (CETP) activity. The present study evaluates CGS 25159, a synthetic isoflavan, as a putative inhibitor of CETP activity of human and hamster plasma. Determined by [3]CE transfer from HDL to VLDL + LDL fraction or by fluorescent-CE transfer assay, CGS 25159 inhibited CETP in both human plasma bottom fraction (d = 1.21 g/ml) and in plasma from Golden Syrian Hamsters with an IC50 contention that pharmacological down regulation of CETP activity could result in favorable changes in lipoprotein profile. PMID:9051198

  1. Inhibition of hepatitis C virus infection by DNA aptamer against envelope protein.

    Yang, Darong; Meng, Xianghe; Yu, Qinqin; Xu, Li; Long, Ying; Liu, Bin; Fang, Xiaohong; Zhu, Haizhen

    2013-10-01

    Hepatitis C virus (HCV) envelope protein (E1E2) is essential for virus binding to host cells. Aptamers have been demonstrated to have strong promising applications in drug development. In the current study, a cDNA fragment encoding the entire E1E2 gene of HCV was cloned. E1E2 protein was expressed and purified. Aptamers for E1E2 were selected by the method of selective evolution of ligands by exponential enrichment (SELEX), and the antiviral actions of the aptamers were examined. The mechanism of their antiviral activity was investigated. The data show that selected aptamers for E1E2 specifically recognize the recombinant E1E2 protein and E1E2 protein from HCV-infected cells. CD81 protein blocks the binding of aptamer E1E2-6 to E1E2 protein. Aptamers against E1E2 inhibit HCV infection in an infectious cell culture system although they have no effect on HCV replication in a replicon cell line. Beta interferon (IFN-β) and IFN-stimulated genes (ISGs) are not induced in virus-infected hepatocytes with aptamer treatment, suggesting that E1E2-specific aptamers do not induce innate immunity. E2 protein is essential for the inhibition of HCV infection by aptamer E1E2-6, and the aptamer binding sites are located in E2. Q412R within E1E2 is the major resistance substitution identified. The data indicate that an aptamer against E1E2 exerts its antiviral effects through inhibition of virus binding to host cells. Aptamers against E1E2 can be used with envelope protein to understand the mechanisms of HCV entry and fusion. The aptamers may hold promise for development as therapeutic drugs for hepatitis C patients. PMID:23877701

  2. The Ca2+/Calmodulin-Dependent Protein Kinase Kinase, CaMKK2, Inhibits Preadipocyte Differentiation

    Lin, Fumin; Ribar, Thomas J.; Means, Anthony R.

    2011-01-01

    When fed a standard chow diet, CaMKK2 null mice have increased adiposity and larger adipocytes than do wild-type mice, whereas energy balance is unchanged. Here, we show that Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is expressed in preadipocytes, where it functions as an AMP-activated protein kinase (AMPK)α kinase. Acute inhibition or deletion of CaMKK2 in preadipocytes enhances their differentiation into mature adipocytes, which can be reversed by 5-aminoimidazole-4-carboxa...

  3. Inhibition of 5-Lipoxygenase Pathway Attenuates Acute Liver Failure by Inhibiting Macrophage Activation

    Lu Li

    2014-01-01

    Full Text Available This study aimed to investigate the role of 5-lipoxygenase (5-LO in acute liver failure (ALF and changes in macrophage activation by blocking it. ALF was induced in rats by administration of D-galactosamine (D-GalN/lipopolysaccharide (LPS. Rats were injected intraperitoneally with AA-861 (a specific 5-LO inhibitor, 24 hr before D-GalN/LPS administration. After D-GalN/LPS injection, the liver tissue was collected for assessment of histology, macrophage microstructure, macrophage counts, 5-LO mRNA formation, protein expression, and concentration of leukotrienes. Serum was collected for detecting alanine aminotransferase (ALT, aspartate transaminase (AST, total bilirubin (Tbil, and tumor necrosis factor- (TNF-α. Twenty-four hours after injection, compared with controls, ALF rats were characterized by widespread hepatocyte necrosis and elevated ALT, AST, and Tbil, and 5-LO protein expression reached a peak. Liver leukotriene B4 was also significantly elevated. However, 5-LO mRNA reached a peak 8 hr after D-GalN/LPS injection. Simultaneously, the microstructure of macrophages was changed most significantly and macrophages counts were increased significantly. Moreover, serum TNF-α was also elevated. By contrast, AA-861 pretreatment significantly decreased liver necrosis as well as all of the parameters compared with the rats without pretreatment. Macrophages, via the 5-LO pathway, play a critical role in ALF, and 5-LO inhibitor significantly alleviates ALF, possibly related to macrophage inhibition.

  4. Inhibition of protein kinase C induces differentiation in Neuro-2a cells

    1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H7), a potent inhibitor of protein kinase C, induced neuritogenesis in Neuro-2a cells, whereas N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA 1004), which inhibits more efficiently cAMP- and cGMP-dependent protein kinases, did not. The effect, noticeable after 3 hr, was maximum (13-fold increase at 500 μM H7) between 1 and 3 days and was maintained over 2 months. In controls, 90% of the cells were undifferentiated, whereas after 3 hr with 500 μM H7 only 25% of the cells remained undifferentiated. DNA synthesis decreased as the number of differentiated cells increased. Differentiation is also functional since acetylcholinesterase activity increased ∼7-fold after 48 hr with 500 μM H7. Phorbol 12-myristate 13-acetate, a specific activator of protein kinase C, prevented or reversed the induction of neuritogenesis and the inhibition of DNA synthesis by H7. There is a good correlation between the level of protein kinase C and the percentage of differentiated cells. The results indicate that protein kinase C may play a key role in the control of differentiation of neural cells. Some possible clinical implications are briefly discussed

  5. Inhibition of protein kinase C induces differentiation in Neuro-2a cells.

    Miñana, M D; Felipo, V; Grisolía, S

    1990-01-01

    1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H7), a potent inhibitor of protein kinase C, induced neuritogenesis in Neuro-2a cells, whereas N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA 1004), which inhibits more efficiently cAMP- and cGMP-dependent protein kinases, did not. The effect, noticeable after 3 hr, was maximum (13-fold increase at 500 microM H7) between 1 and 3 days and was maintained over 2 months. In controls, 90% of the cells were undifferentiated, whereas after 3 hr with 500 microM H7 only 25% of the cells remained undifferentiated. DNA synthesis decreased as the number of differentiated cells increased. Differentiation is also functional since acetylcholinesterase activity increased approximately 7-fold after 48 hr with 500 microM H7. Phorbol 12-myristate 13-acetate, a specific activator of protein kinase C, prevented or reversed the induction of neuritogenesis and the inhibition of DNA synthesis by H7. There is a good correlation between the level of protein kinase C and the percentage of differentiated cells. The results indicate that protein kinase C may play a key role in the control of differentiation of neural cells. Some possible clinical implications are briefly discussed. Images PMID:1693437

  6. Inhibition of protein kinase C induces differentiation in Neuro-2a cells

    Minana, M.D.; Felipo, V.; Grisolia, S. (Instituto de Investigaciones Citologicas de la Caja de Ahorros de Valencia (Spain))

    1990-06-01

    1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H7), a potent inhibitor of protein kinase C, induced neuritogenesis in Neuro-2a cells, whereas N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA 1004), which inhibits more efficiently cAMP- and cGMP-dependent protein kinases, did not. The effect, noticeable after 3 hr, was maximum (13-fold increase at 500 {mu}M H7) between 1 and 3 days and was maintained over 2 months. In controls, 90% of the cells were undifferentiated, whereas after 3 hr with 500 {mu}M H7 only 25% of the cells remained undifferentiated. DNA synthesis decreased as the number of differentiated cells increased. Differentiation is also functional since acetylcholinesterase activity increased {approx}7-fold after 48 hr with 500 {mu}M H7. Phorbol 12-myristate 13-acetate, a specific activator of protein kinase C, prevented or reversed the induction of neuritogenesis and the inhibition of DNA synthesis by H7. There is a good correlation between the level of protein kinase C and the percentage of differentiated cells. The results indicate that protein kinase C may play a key role in the control of differentiation of neural cells. Some possible clinical implications are briefly discussed.

  7. Inhibition of interleukin-6 expression by the V protein of parainfluenza virus 5

    The V protein of parainfluenza virus 5 (PIV5) plays an important role in the evasion of host immune responses. The V protein blocks interferon (IFN) signaling in human cells by causing degradation of the STAT1 protein, a key component of IFN signaling, and blocks IFN-β production by preventing nuclear translocation of IRF3, a key transcription factor for activating IFN-β promoter. Interleukin-6 (IL-6), along with tumor necrosis factor (TNF)-α and IL-1β, is a major proinflammatory cytokine that plays important roles in clearing virus infection through inflammatory responses. Many viruses have developed strategies to block IL-6 expression. Wild-type PIV5 infection induces little, if any, expression of cytokines such as IL-6 or TNF-α, whereas infection by a mutant PIV5 lacking the conserved C-terminal cysteine rich domain (rPIV5VΔC) induced high levels of IL-6 expression. Examination of mRNA levels of IL-6 indicated that the transcription activation of IL-6 played an important role in the increased IL-6 expression. Co-infection with wild-type PIV5 prevented the activation of IL-6 transcription by rPIV5VΔC, and a plasmid encoding the full-length PIV5 V protein prevented the activation of IL-6 promoter-driven reporter gene expression by rPIV5VΔC, indicating that the V protein played a role in inhibiting IL-6 transcription. The activation of IL-6 was independent of IFN-β even though rPIV5VΔC-infected cells produced IFN-β. Using reporter gene assays and chromatin immunoprecipitation (ChIP), it was found that NF-κB played an important role in activating expression of IL-6. We have proposed a model of activating and inhibiting IL-6 transcription by PIV5

  8. Synthesis and characterization of 18F-labeled active site inhibited factor VII (ASIS)

    Erlandsson, Maria; Nielsen, Carsten Haagen; Jeppesen, Troels Elmer;

    2015-01-01

    Activated factor VII blocked in the active site with Phe-Phe-Arg-chloromethyl ketone (active site inhibited factor VII (ASIS)) is a 50-kDa protein that binds with high affinity to its receptor, tissue factor (TF). TF is a transmembrane glycoprotein that plays an important role in, for example......, thrombosis, metastasis, tumor growth, and tumor angiogenesis. The aim of this study was to develop an 18F-labeled ASIS derivative to assess TF expression in tumors. Active site inhibited factor VII was labeled using N-succinimidyl-4-[18F]fluorobenzoate, and the [18F]ASIS was purified on a PD-10 desalting...

  9. Inhibition of host cell translation elongation by Legionella pneumophila blocks the host cell unfolded protein response

    Hempstead, Andrew D.; Isberg, Ralph R.

    2015-01-01

    Cells of the innate immune system recognize bacterial pathogens by detecting common microbial patterns as well as pathogen-specific activities. One system that responds to these stimuli is the IRE1 branch of the unfolded protein response (UPR), a sensor of endoplasmic reticulum (ER) stress. Activation of IRE1, in the context of Toll-like receptor (TLR) signaling, induces strong proinflammatory cytokine induction. We show here that Legionella pneumophila, an intravacuolar pathogen that replicates in an ER-associated compartment, blocks activation of the IRE1 pathway despite presenting pathogen products that stimulate this response. L. pneumophila TLR ligands induced the splicing of mRNA encoding XBP1s, the main target of IRE1 activity. L. pneumophila was able to inhibit both chemical and bacterial induction of XBP1 splicing via bacterial translocated proteins that interfere with host protein translation. A strain lacking five translocated translation elongation inhibitors was unable to block XBP1 splicing, but this could be rescued by expression of a single such inhibitor, consistent with limitation of the response by translation elongation inhibitors. Chemical inhibition of translation elongation blocked pattern recognition receptor-mediated XBP1 splicing, mimicking the effects of the bacterial translation inhibitors. In contrast, host cell-promoted inhibition of translation initiation in response to the pathogen was ineffective in blocking XBP1 splicing, demonstrating the need for the elongation inhibitors for protection from the UPR. The inhibition of host translation elongation may be a common strategy used by pathogens to limit the innate immune response by interfering with signaling via the UPR. PMID:26598709

  10. Methods to distinguish various types of protein phosphatase activity

    To distinguish the action of protein Tyr(P) and protein Ser(P)/Thr(P) phosphatases on 32P-labeled phosphoproteins in subcellular fractions different inhibitors and activators are utilized. Comparison of the effects of added compounds provides a convenient, indirect method to characterize dephosphorylation reactions. Protein Tyr(P) phosphatases are specifically inhibited by micromolar Zn2+ or vanadate, and show maximal activity in the presence of EDTA. The other class of cellular phosphatases, specific for protein Ser(P) and Thr(P) residues, are inhibited by fluoride and EDTA. In this class of enzymes two major functional types can be distinguished: those sensitive to inhibition by the heat-stable protein inhibitor-2 and not stimulated by polycations, and those not sensitive to inhibition and stimulated by polycations. Preparation of 32P-labeled Tyr(P) and Ser(P) phosphoproteins also is presented for the direct measurement of phosphatase activities in preparations by the release of acid-soluble [32P]phosphate

  11. Radioprotective 105 kDa protein attenuates ischemia/reperfusion-induced myocardial apoptosis and autophagy by inhibiting the activation of the TLR4/NF-κB signaling pathway in rats.

    Guo, Xin; Jiang, Hong; Yang, Jun; Chen, Jing; Yang, Jian; Ding, Jia-Wang; Li, Song; Wu, Hui; Ding, Hua-Sheng

    2016-09-01

    Toll-like receptor 4 (TLR4) serves as an important inducer of apoptotic and autophagic responses in myocardial ischemia/reperfusion (I/R) injury (MIRI). Radioprotective 105 kDa protein (RP105) is a specific inhibitor of TLR4. However, the molecular mechanisms by which RP105 represses myocardial apoptosis and autophagy through TLR4‑mediated signaling during I/R have not yet been fully elucidated. Therefore, in the present study, we aimed to examine whether adenovirus-mediated RP105 overexpression repressed myocardial apoptosis and autophagy by inhibiting the TLR4-driven mechanism in MIRI. Three days after the injection of virus or saline into the myocardium, Sprague-Dawley (SD) rats were subjected to 30 min of left anterior descending coronary artery occlusion and 6 h of reperfusion. Myocardial specimens were prepared for analysis. We performed immunohistochemichal and histopathological analysis, the measurement of cardiac biomarkers, TUNEL assay , RT-qPCR and western blot analysis. The results indicated that the overexpression of RP105 contributed to an amelioration of myocardial histological damage, decreased leakage of creatine kinase (CK) and lactate dehydrogenase (LDH), as well as a reduction in the number of TUNEL-positive cardiomyocytes. The levels of positively associated modulators of apoptosis and autophagy were also significantly downregulated by RP105, whereas Bcl-2, which plays an opposite role in inducing apoptosis and autophagy, was inversely upregulated. Furthermore, the overexpression of RP105 led to the repression of TLR4 activity and the phosphorylation of NF-κB/p65, as well as the reduced production of the cytokines interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α). Taken together, these data suggest that RP105 protects the myocardium against apoptosis and autophagy, and plays a cardioprotective role during I/R injury. This is most likely due to the inactivation of TLR4/NF-κB signaling pathway. Thus, RP105 may represent

  12. Inhibition of Methane Hydrate Formation by Ice-Structuring Proteins

    Jensen, Lars; Ramløv, Hans; Thomsen, Kaj;

    2010-01-01

    In the oil and gas industry there is ample motivation for moving toward greener kinetic inhibitors of gas hydrates as many of those used today suffer from poor biodegradability. In this work, we have investigated experimentally whether ice-structuring proteins (ISPs) found in fish and insect......, assumed biodegradable, are capable of inhibiting the growth of methane hydrate (a structure I hydrate). The ISPs investigated were type III HPLC12 (originally identified in ocean pout) and ISP type III found in meal worm (Tenebrio molitor). These were compared to polyvinylpyrrolidone (PVP) a well......-known kinetic hydrate inhibitor. The results revealed that adding ISP in sufficient amounts caused the appearance of an initial nonlinear growth period. At a certain point during the growth process the growth pattern changed to linear which is identical to the growth observed for methane hydrate in the absence...

  13. Inhibition of hepatitis C virus infection by DNA aptamer against NS2 protein.

    Gao, Yimin; Yu, Xiaoyan; Xue, Binbin; Zhou, Fei; Wang, Xiaohong; Yang, Darong; Liu, Nianli; Xu, Li; Fang, Xiaohong; Zhu, Haizhen

    2014-01-01

    NS2 protein is essential for hepatitis C virus (HCV) replication. NS2 protein was expressed and purified. Aptamers against NS2 protein were raised and antiviral effects of the aptamers were examined. The molecular mechanism through which the aptamers exert their anti-HCV activity was investigated. The data showed that aptamer NS2-3 inhibited HCV RNA replication in replicon cell line and infectious HCV cell culture system. NS2-3 and another aptamer NS2-2 were demonstrated to inhibit infectious virus production without cytotoxicity in vitro. They did not affect hepatitis B virus replication. Interferon beta (IFN-β) and interferon-stimulated genes (ISGs) were not induced by the aptamers in HCV-infected hepatocytes. Furthermore, our study showed that N-terminal region of NS2 protein is involved in the inhibition of HCV infection by NS2-2. I861T within NS2 is the major resistance mutation identified. Aptamer NS2-2 disrupts the interaction of NS2 with NS5A protein. The data suggest that NS2-2 aptamer against NS2 protein exerts its antiviral effects through binding to the N-terminal of NS2 and disrupting the interaction of NS2 with NS5A protein. NS2-specific aptamer is the first NS2 inhibitor and can be used to understand the mechanisms of virus replication and assembly. It may be served as attractive candidates for inclusion in the future HCV direct-acting antiviral combination therapies. PMID:24587329

  14. P53 and p73 differ in their ability to inhibit glucocorticoid receptor (GR transcriptional activity

    Nie Linghu

    2006-12-01

    Full Text Available Abstract Background p53 is a tumor suppressor and potent inhibitor of cell growth. P73 is highly similar to p53 at both the amino acid sequence and structural levels. Given their similarities, it is important to determine whether p53 and p73 function in similar or distinct pathways. There is abundant evidence for negative cross-talk between glucocorticoid receptor (GR and p53. Neither physical nor functional interactions between GR and p73 have been reported. In this study, we examined the ability of p53 and p73 to interact with and inhibit GR transcriptional activity. Results We show that both p53 and p73 can bind GR, and that p53 and p73-mediated transcriptional activity is inhibited by GR co-expression. Wild-type p53 efficiently inhibited GR transcriptional activity in cells expressing both proteins. Surprisingly, however, p73 was either unable to efficiently inhibit GR, or increased GR activity slightly. To examine the basis for this difference, a series of p53:p73 chimeric proteins were generated in which corresponding regions of either protein have been swapped. Replacing N- and C-terminal sequences in p53 with the corresponding sequences from p73 prevented it from inhibiting GR. In contrast, replacing p73 N- and C-terminal sequences with the corresponding sequences from p53 allowed it to efficiently inhibit GR. Differences in GR inhibition were not related to differences in transcriptional activity of the p53:p73 chimeras or their ability to bind GR. Conclusion Our results indicate that both N- and C-terminal regions of p53 and p73 contribute to their regulation of GR. The differential ability of p53 and p73 to inhibit GR is due, in part, to differences in their N-terminal and C-terminal sequences.

  15. Activation of GPR30 inhibits cardiac fibroblast proliferation.

    Wang, Hao; Zhao, Zhuo; Lin, Marina; Groban, Leanne

    2015-07-01

    The incidence of left ventricular diastolic dysfunction significantly increases in postmenopausal women suggesting the association between estrogen loss and diastolic dysfunction. The in vivo activation of G protein-coupled estrogen receptor (GPR30) attenuates the adverse effects of estrogen loss on cardiac fibrosis and diastolic dysfunction in mRen2.Lewis rats. This study was designed to address the effects of GPR30 on cardiac fibroblast proliferation in rats. The expression of GPR30 in cardiac fibroblasts isolated from adult Sprague-Dawley rats was confirmed by RT-PCR, Western blot analysis, and immunofluorescence staining. Results from BrdU incorporation assays, cell counting, carboxyfluorescein diacetate succinimidyl ester labeling in conjunction with flow cytometry, and Ki-67 staining showed that treatment with G1, a specific agonist of GPR30, inhibited cardiac fibroblast proliferation in a dose-dependent manner, which was associated with decreases in CDK1 and cyclin B1 protein expressions. In the GPR30-KO cells, BrdU incorporation, and CDK1 and cyclin B1 expressions significantly increased when compared to GPR30-intact cells. G1 had no effect on BrdU incorporation, CDK1 and cyclin B1 mRNA levels in GPR30-KO cells. In vivo studies showed increases in CDK1 and cyclin B1 mRNA levels, Ki-67-positive cells, and the immunohistochemistry staining of vimentin, a fibroblast marker, in the left ventricles from ovariectomized mRen2.Lewis rats versus hearts from ovary-intact littermates; 2 weeks of G1 treatment attenuated these adverse effects of estrogen loss. This study demonstrates that GPR30 is expressed in rat cardiac fibroblasts, and activation of GPR30 limits proliferation of these cells likely via suppression of the cell cycle proteins, cyclin B1, and CDK1. PMID:25893735

  16. Sestrins Inhibit mTORC1 Kinase Activation through the GATOR Complex

    Anita Parmigiani

    2014-11-01

    Full Text Available The mechanistic target of rapamycin complex 1 (mTORC1 kinase is a sensor of different environmental conditions and regulator of cell growth, metabolism, and autophagy. mTORC1 is activated by Rag GTPases, working as RagA:RagB and RagC:RagD heterodimers. Rags control mTORC1 activity by tethering mTORC1 to the lysosomes where it is activated by Rheb GTPase. RagA:RagB, active in its GTP-bound form, is inhibited by GATOR1 complex, a GTPase-activating protein, and GATOR1 is in turn negatively regulated by GATOR2 complex. Sestrins are stress-responsive proteins that inhibit mTORC1 via activation of AMP-activated protein kinase (AMPK and tuberous sclerosis complex. Here we report an AMPK-independent mechanism of mTORC1 inhibition by Sestrins mediated by their interaction with GATOR2. As a result of this interaction, the Sestrins suppress mTOR lysosomal localization in a Rag-dependent manner. This mechanism is potentially involved in mTORC1 regulation by amino acids, rotenone, and tunicamycin, connecting stress response with mTORC1 inhibition.

  17. Protein kinase activity associated with the nuclear lamina.

    Dessev, G; Iovcheva, C; Tasheva, B; R. Goldman

    1988-01-01

    A nuclear lamina-enriched fraction from Ehrlich ascites tumor cells contains a tightly bound protein kinase activity, which phosphorylates in vitro the nuclear lamins, a 52-kilodalton protein, and several unknown minor components. The enzyme(s) is thermolabile, independent of Ca2+ and cAMP, and inhibited by quercetin. After treatment with 4 M urea it remains bound to the nuclear lamina in an active state, but it is irreversibly inactivated in 6 M urea. The lamin proteins are phosphorylated on...

  18. Inhibition of ABC transport proteins by oil sands process affected water.

    Alharbi, Hattan A; Saunders, David M V; Al-Mousa, Ahmed; Alcorn, Jane; Pereira, Alberto S; Martin, Jonathan W; Giesy, John P; Wiseman, Steve B

    2016-01-01

    The ATP-binding cassette (ABC) superfamily of transporter proteins is important for detoxification of xenobiotics. For example, ABC transporters from the multidrug-resistance protein (MRP) subfamily are important for excretion of polycyclic aromatic hydrocarbons (PAHs) and their metabolites. Effects of chemicals in the water soluble organic fraction of relatively fresh oil sands process affected water (OSPW) from Base Mine Lake (BML-OSPW) and aged OSPW from Pond 9 (P9-OSPW) on the activity of MRP transporters were investigated in vivo by use of Japanese medaka at the fry stage of development. Activities of MRPs were monitored by use of the lipophilic dye calcein, which is transported from cells by ABC proteins, including MRPs. To begin to identify chemicals that might inhibit activity of MRPs, BML-OSPW and P9-OSPW were fractionated into acidic, basic, and neutral fractions by use of mixed-mode sorbents. Chemical compositions of fractions were determined by use of ultrahigh resolution orbitrap mass spectrometry in ESI(+) and ESI(-) mode. Greater amounts of calcein were retained in fry exposed to BML-OSPW at concentration equivalents greater than 1× (i.e., full strength). The neutral and basic fractions of BML-OSPW, but not the acidic fraction, caused greater retention of calcein. Exposure to P9-OSPW did not affect the amount of calcein in fry. Neutral and basic fractions of BML-OSPW contained relatively greater amounts of several oxygen-, sulfur, and nitrogen-containing chemical species that might inhibit MRPs, such as O(+), SO(+), and NO(+) chemical species, although secondary fractionation will be required to conclusively identify the most potent inhibitors. Naphthenic acids (O2(-)), which were dominant in the acidic fraction, did not appear to be the cause of the inhibition. This is the first study to demonstrate that chemicals in the water soluble organic fraction of OSPW inhibit activity of this important class of proteins. However, aging of OSPW attenuates

  19. Three cardiovirus Leader proteins equivalently inhibit four different nucleocytoplasmic trafficking pathways

    Ciomperlik, Jessica J. [Institute for Molecular Virology, and Department of Biochemistry, University of Wisconsin-Madison, Madison, WI (United States); Basta, Holly A. [Department of Biology, Rocky Mountain College, Billings, MT (United States); Palmenberg, Ann C., E-mail: acpalmen@wisc.edu [Institute for Molecular Virology, and Department of Biochemistry, University of Wisconsin-Madison, Madison, WI (United States)

    2015-10-15

    Cardiovirus infections inhibit nucleocytoplasmic trafficking by Leader protein-induced phosphorylation of Phe/Gly-containing nucleoporins (Nups). Recombinant Leader from encephalomyocarditis virus, Theiler's murine encephalomyelitis virus and Saffold virus target the same subset of Nups, including Nup62 and Nup98, but not Nup50. Reporter cell lines with fluorescence mCherry markers for M9, RS and classical SV40 import pathways, as well as the Crm1-mediated export pathway, all responded to transfection with the full panel of Leader proteins, showing consequent cessation of path-specific active import/export. For this to happen, the Nups had to be presented in the context of intact nuclear pores and exposed to cytoplasmic extracts. The Leader phosphorylation cascade was not effective against recombinant Nup proteins. The findings support a model of Leader-dependent Nup phosphorylation with the purpose of disrupting Nup-transportin interactions. - Highlights: • Nup98, but not Nup50 becomes phosphorylated by cardiovirus Leader protein-dependent mechanisms. • At least four independent nucleocytoplasmic trafficking pathways are inhibited by this process. • Nups must be presented in a nuclear pore context for Leader-directed phosphorylation. • Leader, by itself, does not cause activation of cellular kinases.

  20. Three cardiovirus Leader proteins equivalently inhibit four different nucleocytoplasmic trafficking pathways

    Cardiovirus infections inhibit nucleocytoplasmic trafficking by Leader protein-induced phosphorylation of Phe/Gly-containing nucleoporins (Nups). Recombinant Leader from encephalomyocarditis virus, Theiler's murine encephalomyelitis virus and Saffold virus target the same subset of Nups, including Nup62 and Nup98, but not Nup50. Reporter cell lines with fluorescence mCherry markers for M9, RS and classical SV40 import pathways, as well as the Crm1-mediated export pathway, all responded to transfection with the full panel of Leader proteins, showing consequent cessation of path-specific active import/export. For this to happen, the Nups had to be presented in the context of intact nuclear pores and exposed to cytoplasmic extracts. The Leader phosphorylation cascade was not effective against recombinant Nup proteins. The findings support a model of Leader-dependent Nup phosphorylation with the purpose of disrupting Nup-transportin interactions. - Highlights: • Nup98, but not Nup50 becomes phosphorylated by cardiovirus Leader protein-dependent mechanisms. • At least four independent nucleocytoplasmic trafficking pathways are inhibited by this process. • Nups must be presented in a nuclear pore context for Leader-directed phosphorylation. • Leader, by itself, does not cause activation of cellular kinases

  1. Immune-suppressive activity of punicalagin via inhibition of NFAT activation

    Since T cell activation is central to the development of autoimmune diseases, we screened a natural product library comprising 1400 samples of medicinal herbal extracts, to identify compounds that suppress T cell activity. Punicalagin (PCG) isolated from the fruit of Punica granatum was identified as a potent immune suppressant, based on its inhibitory action on the activation of the nuclear factor of activated T cells (NFAT). PCG downregulated the mRNA and soluble protein expression of interleukin-2 from anti-CD3/anti-CD28-stimulated murine splenic CD4+ T cells and suppressed mixed leukocytes reaction (MLR) without exhibiting cytotoxicity to the cells. In vivo, the PCG treatment inhibited phorbol 12-myristate 13-acetate (PMA)-induced chronic ear edema in mice and decreased CD3+ T cell infiltration of the inflamed tissue. These results suggest that PCG could be a potential candidate for the therapeutics of various immune pathologies

  2. Lucanthone and its derivative hycanthone inhibit apurinic endonuclease-1 (APE1) by direct protein binding

    Naidu, M.; Naidu, M.; Agarwal, R.; Pena, L.A.; Cunha, L.; Mezei, M.; Shen, M.; Wilson, D.M.; Liu, Y.; Sanchez, Z.; Chaudhary, P.; Wilson, S.H.; Waring, M.J.

    2011-09-15

    Lucanthone and hycanthone are thioxanthenone DNA intercalators used in the 1980s as antitumor agents. Lucanthone is in Phase I clinical trial, whereas hycanthone was pulled out of Phase II trials. Their potential mechanism of action includes DNA intercalation, inhibition of nucleic acid biosyntheses, and inhibition of enzymes like topoisomerases and the dual function base excision repair enzyme apurinic endonuclease 1 (APE1). Lucanthone inhibits the endonuclease activity of APE1, without affecting its redox activity. Our goal was to decipher the precise mechanism of APE1 inhibition as a prerequisite towards development of improved therapeutics that can counteract higher APE1 activity often seen in tumors. The IC{sub 50} values for inhibition of APE1 incision of depurinated plasmid DNA by lucanthone and hycanthone were 5 {mu}M and 80 nM, respectively. The K{sub D} values (affinity constants) for APE1, as determined by BIACORE binding studies, were 89 nM for lucanthone/10 nM for hycanthone. APE1 structures reveal a hydrophobic pocket where hydrophobic small molecules like thioxanthenones can bind, and our modeling studies confirmed such docking. Circular dichroism spectra uncovered change in the helical structure of APE1 in the presence of lucanthone/hycanthone, and notably, this effect was decreased (Phe266Ala or Phe266Cys or Trp280Leu) or abolished (Phe266Ala/Trp280Ala) when hydrophobic site mutants were employed. Reduced inhibition by lucanthone of the diminished endonuclease activity of hydrophobic mutant proteins (as compared to wild type APE1) supports that binding of lucanthone to the hydrophobic pocket dictates APE1 inhibition. The DNA binding capacity of APE1 was marginally inhibited by lucanthone, and not at all by hycanthone, supporting our hypothesis that thioxanthenones inhibit APE1, predominantly, by direct interaction. Finally, lucanthone-induced degradation was drastically reduced in the presence of short and long lived free radical scavengers, e

  3. Degradation of Activated Protein Kinases by Ubiquitination

    Lu, Zhimin; Hunter, Tony

    2009-01-01

    Protein kinases are important regulators of intracellular signal transduction pathways and play critical roles in diverse cellular functions. Once a protein kinase is activated, its activity is subsequently downregulated through a variety of mechanisms. Accumulating evidence indicates that the activation of protein kinases commonly initiates their downregulation via the ubiquitin/proteasome pathway. Failure to regulate protein kinase activity or expression levels can cause human diseases.

  4. Inhibition of Ly-6A antigen expression prevents T cell activation

    1990-01-01

    Antisense oligonucleotides complementary to the 5' end of the mRNA encoding the Ly-6A protein were used to block the expression of that protein. Using this approach we could inhibit the expression of Ly-6A by 60-80% in antigen-primed lymph node (LN) T cells as well as in the D10 T cell clone. Inhibition of Ly-6 expression resulted in the inability to restimulate in vitro, antigen-primed T cells. It also blocked the activation of normal spleen cells by Con A, monoclonal antibody (mAb) to CD3, ...

  5. Active inhibition of herpes simplex virus type 1-induced cell fusion

    Bzik, D.J.; Person, S.; Read, G.S.

    1982-01-01

    Previous studies have demonstrated that syn mutant-infected cells fuse less well with nonsyncytial virus-infected cells than with uninfected cells, a phenomenon defined as function inhibition. The present study characterizes the kinetics as well as the requirements for expression of fusion inhibition. Initially, the capacity of sparse syn mutant-infected cells to fuse with uninfected surrounding cells was determined throughout infection. Of seven syn mutants examined, including representatives with alterations in two different viral genes that affect cell fusion, all showed an increase in fusion capacity up to 12 hr after infection and a decrease at later times. Fusion inhibition was examined in experiments employing sparse syn20-infected cells which had been incubated to a maximum fusion capacity; it was shown that surrounding cells infected with KOS, the parent of syn20, began to inhibit fusion by the syn20-infected cells at about 4 hr after infection, and that the maximum ability to inhibit fusion was attained at about 6 hr after infection. The metabolic blocking agents actinomycin D (RNA), cycloheximide (protein), 2-deoxyglucose, and tunicamycin (glycoslyation of glycoproteins) all showed the ability to inhibit the expression of fusion inhibition by KOS-infected cells if added shortly after infection. It is concluded that fusion inhibition is an active process that requires the synthesis of RNA, proteins, and glycoproteins. 17 references, 3 figures, 2 tables.

  6. Inhibition of parathyroid hormone release by maitotoxin, a calcium channel activator

    Maitotoxin, a toxin derived from a marine dinoflagellate, is a potent activator of voltage-sensitive calcium channels. To further test the hypothesis that inhibition of PTH secretion by calcium is mediated via a calcium channel we studied the effect of maitotoxin on dispersed bovine parathyroid cells. Maitotoxin inhibited PTH release in a dose-dependent fashion, and inhibition was maximal at 1 ng/ml. Chelation of extracellular calcium by EGTA blocked the inhibition of PTH by maitotoxin. Maitotoxin enhanced the effects of the dihydropyridine calcium channel agonist (+)202-791 and increased the rate of radiocalcium uptake in parathyroid cells. Pertussis toxin, which ADP-ribosylates and inactivates a guanine nucleotide regulatory protein that interacts with calcium channels in the parathyroid cell, did not affect the inhibition of PTH secretion by maitotoxin. Maitotoxin, by its action on calcium channels allows entry of extracellular calcium and inhibits PTH release. Our results suggest that calcium channels are involved in the release of PTH. Inhibition of PTH release by maitotoxin is not sensitive to pertussis toxin, suggesting that maitotoxin may act distal to the site interacting with a guanine nucleotide regulatory protein, or maitotoxin could interact with other ions or second messengers to inhibit PTH release

  7. Cross inhibition improves activity selection when switching incurs time costs

    James A.R.MARSHALL; Angélique FAVREAU-PEIGN(E); Lutz FROMHAGE; John M.MCNAMARA; Lianne F.S.MEAH; Alasdair I.HOUSTON

    2015-01-01

    We consider a behavioural model of an animal choosing between two activities,based on positive feedback,and examine the effect of introducing cross inhibition between the motivations for the two activities.While cross-inhibition has previously been included in models of decision making,the question of what benefit it may provide to an animal's activity selection behaviour has not previously been studied.In neuroscience and in collective behaviour cross-inhibition,and other equivalent means of coupling evidence-accumulating pathways,have been shown to approximate statistically-optimal decision-making and to adaptively break deadlock,thereby improving decision performance.Switching between activities is an ongoing decision process yet here we also find that cross-inhibition robustly improves its efficiency,by reducing the frequency of costly switches between behaviours [Current Zoology 61 (2):242-250,2015].

  8. Inhibition of Gas Hydrate Nucleation and Growth: Efficacy of an Antifreeze Protein from the Longhorn BeetleRhagium mordax

    Perfeldt, Christine Malmos; Chua, Pei Cheng; Daraboina, Nagu;

    2014-01-01

    Antifreeze proteins (AFPs) are characterized by their ability to protect organisms from subfreezing temperatures by preventing tiny ice crystals in solution from growing as the solution is cooled below its freezing temperature. This inhibition of ice growth is called antifreeze activity, and in...... particular, certain insect AFPs show very high antifreeze activity. Recent studies have shown AFPs to be promising candidates as green and environmentally benign inhibitors for gas hydrate formation. Here we show that an insect antifreeze protein from the longhorn beetle, Rhagium mordax (RmAFP1), the most...... potent protein yet found for freezing inhibition, can inhibit methane hydrates as effectively as the synthetic polymeric inhibitor polyvinylpyrrolidone (PVP). In high pressure rocking cell experiments, onset hydrate nucleation temperatures and growth profiles showed repeatable results. RmAFP1 clearly...

  9. L-alpha-glycerylphosphorylcholine inhibits the transfer function of phosphatidylinositol transfer protein alpha.

    Komatsu, Hiroaki; Westerman, Jan; Snoek, Gerry T; Taraschi, Theodore F; Janes, Nathan

    2003-12-30

    Phosphatidylinositol transfer protein alpha (PITP-alpha) is a bifunctional phospholipid transfer protein that is highly selective for phosphatidylinositol (PtdIns) and phosphatidylcholine (PtdCho). Polar lipid metabolites, including L-alpha-glycerylphosphorylcholine (GroPCho), increasingly have been linked to changes in cellular function and to disease. In this study, polar lipid metabolites of PtdIns and PtdCho were tested for their ability to influence PITP-alpha activity. GroPCho inhibited the ability of PITP-alpha to transfer PtdIns or PtdCho between liposomes. The IC(50) of both processes was dependent on membrane composition. D-myo-inositol 1-phosphate and glycerylphosphorylinositol modestly enhanced PITP-alpha-mediated phospholipid transfer. Choline, phosphorylcholine (PCho), CDP-choline, glyceryl-3-phosphate, myo-inositol and D-myo-inositol 1,4,5-trisphosphate had little effect. Membrane surface charge was a strong determinant of the GroPCho inhibition with the inhibition being greatest for highly anionic membranes. GroPCho was shown to enhance the binding of PITP-alpha to anionic vesicles. In membranes of low surface charge, phosphatidylethanolamine (PtdEtn) was a determinant enabling the GroPCho inhibition. Anionic charge and PtdEtn content appeared to increase the strength of PITP-alpha-membrane interactions. The GroPCho-enhanced PITP-alpha-membrane binding was sufficient to cause inhibition, but not sufficient to account for the extent of inhibition observed. Processes associated with strengthened PITP-alpha-membrane binding in the presence of GroPCho appeared to impair the phospholipid insertion/extraction process. PMID:14729069

  10. Translation Inhibition of Capped and Uncapped Viral RNAs Mediated by Ribosome-Inactivating Proteins.

    Vivanco, Jorge M; Tumer, Nilgun E

    2003-05-01

    ABSTRACT Ribosome-inactivating proteins (RIPs) are N-glycosidases that remove specific purine residues from the sarcin/ricin (S/R) loop of the large rRNA and arrest protein synthesis at the translocation step. In addition to their enzymatic activity, RIPs have been reputed to be potent antiviral agents against many plant, animal, and human viruses. We recently showed that pokeweed antiviral protein (PAP), an RIP from pokeweed, inhibits translation in cell extracts by binding to the cap structure of eukaryotic mRNA and viral RNAs and depurinating these RNAs at multiple sites downstream of the cap structure. In this study, we examined the activity of three different RIPs against capped and uncapped viral RNAs. PAP, Mirabilis expansa RIP (ME1), and the Saponaria officinalis RIP (saporin) depurinated the capped Tobacco mosaic virus and Brome mosaic virus RNAs, but did not depurinate the uncapped luciferase RNA, indicating that other type I RIPs besides PAP can distinguish between capped and uncapped RNAs. We did not detect depurination of Alfalfa mosaic virus (AMV) RNAs at multiple sites by PAP or ME1. Because AMV RNAs are capped, these results indicate that recognition of the cap structure alone is not sufficient for depurination of the RNA at multiple sites throughout its sequence. Furthermore, PAP did not cause detectable depurination of uncapped RNAs from Tomato bushy stunt virus (TBSV), Satellite panicum mosaic virus (SPMV), and uncapped RNA containing poliovirus internal ribosome entry site (IRES). However, in vitro translation experiments showed that PAP inhibited translation of AMV, TBSV, SPMV RNAs, and poliovirus IRES dependent translation. These results demonstrate that PAP does not depurinate every capped RNA and that PAP can inhibit translation of uncapped viral RNAs in vitro without causing detectable depurination at multiple sites. Thus, the cap structure is not the only determinant for inhibition of translation by PAP. PMID:18942981

  11. A poxvirus protein that binds to and inactivates IL-18, and inhibits NK cell response.

    Born, T L; Morrison, L A; Esteban, D J; VandenBos, T; Thebeau, L G; Chen, N; Spriggs, M K; Sims, J E; Buller, R M

    2000-03-15

    IL-18 induces IFN-gamma and NK cell cytotoxicity, making it a logical target for viral antagonism of host defense. We demonstrate that the ectromelia poxvirus p13 protein, bearing homology to the mammalian IL-18 binding protein, binds IL-18, and inhibits its activity in vitro. Binding of IL-18 to the viral p13 protein was compared with binding to the cellular IL-18R. The dissociation constant of p13 for murine IL-18 is 5 nM, compared with 0.2 nM for the cellular receptor heterodimer. Mice infected with a p13 deletion mutant of ectromelia virus had elevated cytotoxicity for YAC-1 tumor cell targets compared with control animals. Additionally, the p13 deletion mutant virus exhibited decreased levels of infectivity. Our data suggest that inactivation of IL-18, and subsequent impairment of NK cell cytotoxicity, may be one mechanism by which ectromelia evades the host immune response. PMID:10706717

  12. Is Peroxiredoxin II's peroxidase activity strongly inhibited in human erythrocytes?

    Benfeitas, Rui; Selvaggio, Gianluca; Antunes, Fernando; Coelho, Pedro; Salvador, Armindo

    2014-10-01

    H2O2 elimination in human erythrocytes is mainly carried out by catalase (Cat), glutathione peroxidase (GPx1) and the more recently discovered peroxiredoxin 2 (Prx2). However, the contribution of Prx2 to H2O2 consumption is still unclear. Prx2's high reactivity with H2O2 (kPrx2=10×10(7) M(-1)s(-1), kCat =7×10(7) M(-1)s(-1), kGPx1 =4×10(7) M(-1)s(-1)) and high abundance ([Prx2]= 570µM, [Cat]= 32µM, [GPx1]= 1µM) suggest that under low H2O2 supply rates it should consume >99% of the H2O2. However, extensive evidence indicates that in intact erythrocytes Prx2 contributes no more than Cat to H2O2 consumption. In order for this to be attained, Prx2's effective rate constant with H2O2would have to be just ~10(5) M(-1)s(-1), much lower than that determined in multiple experiments with the purified proteins. Nevertheless, nearly all Prx2 is oxidized within 1min of exposing erythrocytes to a H2O2 bolus, which is inconsistent with an irreversible inhibition. A mathematical model of the H2O2 metabolism in human erythrocytes [Benfeitas et al. (2014) Free Radic. Biol. Med.] where Prx2 either has a low kPrx2 or is subject to a strong (>99%) but readily reversible inhibition achieves quantitative agreement with detailed experimental observations of the responses of the redox status of Prx2 in human erythrocytes and suggests functional advantages of this design (see companion abstract). By contrast, a variant where Prx2 is fully active with kPrx2=10(8) M(-1)s(-1) shows important qualitative discrepancies. Altogether, these results suggest that Prx2's peroxidase activity is strongly inhibited in human erythrocytes. We acknowledge fellowship SFRH/BD/51199/2010, grants PEst-C/SAU/LA0001/2013-2014, PEst-OE/QUI/UI0612/2013, PEst-OE/QUI/UI0313/2014, and FCOMP-01-0124-FEDER-020978 (PTDC/QUI-BIQ/119657/2010) co-financed by FEDER through the COMPETE program and by FCT. PMID:26461310

  13. Galectin-3 Binding Protein Secreted by Breast Cancer Cells Inhibits Monocyte-Derived Fibrocyte Differentiation.

    White, Michael J V; Roife, David; Gomer, Richard H

    2015-08-15

    To metastasize, tumor cells often need to migrate through a layer of collagen-containing scar tissue which encapsulates the tumor. A key component of scar tissue and fibrosing diseases is the monocyte-derived fibrocyte, a collagen-secreting profibrotic cell. To test the hypothesis that invasive tumor cells may block the formation of the fibrous sheath, we determined whether tumor cells secrete factors that inhibit monocyte-derived fibrocyte differentiation. We found that the human metastatic breast cancer cell line MDA-MB-231 secretes activity that inhibits human monocyte-derived fibrocyte differentiation, whereas less aggressive breast cancer cell lines secrete less of this activity. Purification indicated that Galectin-3 binding protein (LGALS3BP) is the active factor. Recombinant LGALS3BP inhibits monocyte-derived fibrocyte differentiation, and immunodepletion of LGALS3BP from MDA-MB 231 conditioned media removes the monocyte-derived fibrocyte differentiation-inhibiting activity. LGALS3BP inhibits the differentiation of monocyte-derived fibrocytes from wild-type mouse spleen cells, but not from SIGN-R1(-/-) mouse spleen cells, suggesting that CD209/SIGN-R1 is required for the LGALS3BP effect. Galectin-3 and galectin-1, binding partners of LGALS3BP, potentiate monocyte-derived fibrocyte differentiation. In breast cancer biopsies, increased levels of tumor cell-associated LGALS3BP were observed in regions of the tumor that were invading the surrounding stroma. These findings suggest LGALS3BP and galectin-3 as new targets to treat metastatic cancer and fibrosing diseases. PMID:26136428

  14. The grapevine polygalacturonase-inhibiting protein (VvPGIP1) reduces Botrytis cinerea susceptibility in transgenic tobacco and differentially inhibits fungal polygalacturonases.

    Joubert, Dirk A; Slaughter, Ana R; Kemp, Gabré; Becker, John V W; Krooshof, Geja H; Bergmann, Carl; Benen, Jacques; Pretorius, Isak S; Vivier, Melané A

    2006-12-01

    Polygalacturonase-inhibiting proteins (PGIPs) selectively inhibit polygalacturonases (PGs) secreted by invading plant pathogenic fungi. PGIPs display differential inhibition towards PGs from different fungi, also towards different isoforms of PGs originating from a specific pathogen. Recently, a PGIP-encoding gene from Vitis vinifera (Vvpgip1) was isolated and characterised. PGIP purified from grapevine was shown to inhibit crude polygalacturonase extracts from Botrytis cinerea, but this inhibitory activity has not yet been linked conclusively to the activity of the Vvpgip1 gene product. Here we use a transgenic over-expression approach to show that the PGIP encoded by the Vvpgip1 gene is active against PGs of B. cinerea and that over-expression of this gene in transgenic tobacco confers a reduced susceptibility to infection by this pathogen. A calculated reduction in disease susceptibility of 47-69% was observed for a homogeneous group of transgenic lines that was statistically clearly separated from untransformed control plants following infection with Botrytis over a 15-day-period. VvPGIP1 was subsequently purified from transgenic tobacco and used to study the specific inhibition profile of individual PGs from Botrytis and Aspergillus. The heterologously expressed and purified VvPGIP1 selectively inhibited PGs from both A. niger and B. cinerea, including BcPG1, a PG from B. cinerea that has previously been shown to be essential for virulence and symptom development. Altogether our data confirm the antifungal nature of the VvPGIP1, and the in vitro inhibition data suggest at least in part, that the VvPGIP1 contributed to the observed reduction in disease symptoms by inhibiting the macerating action of certain Botrytis PGs in planta. The ability to correlate inhibition profiles to individual PGs provides a more comprehensive analysis of PGIPs as antifungal genes with biotechnological potential, and adds to our understanding of the importance of PGIP

  15. Griffithsin binds to the glycosylated proteins (E and prM) of Japanese encephalitis virus and inhibit its infection.

    Ishag, Hassan Z A; Li, Chen; Wang, Fengjuan; Mao, Xiang

    2016-04-01

    Griffithsin (GRFT) is a broad-spectrum antiviral protein against several glycosylated viruses. In our previous publication, we have shown that GRFT exerted antiviral activity against Japanese encephalitis virus (JEV) infection. Herein, we further elucidated the mechanism by which GRFT inhibits JEV infection in BHK-21 cells. In vitro experiments using Pull-down assay and Co-immunoprecipitation (CO-IP) assay showed that GRFT binds to the JEV glycosylated viral proteins, specifically the enveloped (E) and premature (prM) glycoproteins. The binding of GRFT to the JEV was competitively inhibited by increasing concentrations of mannose; in turns abolished anti-JEV activity of GRFT. We suggested that, the binding of GRFT to the glycosylated viral proteins may contribute to its anti-JEV activity. Collectively, our data indicated a possible mechanism by which GRFT exerted its anti-JEV activity. This observation suggests GRFT's potentials in the development of therapeutics against JEV or other flavivirus infection. PMID:26820432

  16. RKIP Inhibits Local Breast Cancer Invasion by Antagonizing the Transcriptional Activation of MMP13.

    Ila Datar

    Full Text Available Raf Kinase Inhibitory Protein or RKIP was initially identified as a Raf-1 binding protein using the yeast 2-hybrid screen. RKIP inhibits the activation phosphorylation of MEK by Raf-1 by competitively inhibiting the binding of MEK to Raf-1 and thus exerting an inhibitory effect on the Raf-MEK-Erk pathway. RKIP has been identified as a metastasis suppressor gene. Expression of RKIP is low in cancer metastases. Although primary tumor growth remains unaffected, re- expression of RKIP inhibits cancer metastasis. Mechanistically, RKIP constrains metastasis by inhibiting angiogenesis, local invasion, intravasation, and colonization. The molecular mechanism of how RKIP inhibits these individual steps remains undefined. In our present study, using an unbiased PCR based screening and by analyzing DNA microarray expression datasets we observe that the expression of multiple metalloproteases (MMPs including MMP1, MMP3, MMP10 and MMP13 are negatively correlated with RKIP expression in breast cancer cell lines and clinical samples. Since expression of MMPs by cancer cells is important for cancer metastasis, we hypothesize that RKIP may mediate suppression of breast cancer metastasis by inhibiting multiple MMPs. We show that the expression signature of RKIP and MMPs is better at predicting high metastatic risk than the individual gene. Using a combination of loss- and gain-of-function approaches, we find that MMP13 is the cause of RKIP-mediated inhibition of local cancer invasion. Interestingly expression of MMP13 alone is not sufficient to reverse the inhibition of breast cancer cell metastasis to the lung due to the expression of RKIP. We find that RKIP negatively regulates MMP13 through the Erk2 signaling pathway and the repression of MMP13 by RKIP is transcription factor AP-1 independent. Together, our findings indicate that RKIP inhibits cancer cell invasion, in part, via MMP13 inhibition. These data also implicate RKIP in the regulation of MMP

  17. Anthelmintic activity of Leucaena leucocephala protein extracts on Haemonchus contortus.

    Soares, Alexandra Martins dos Santos; de Araújo, Sandra Alves; Lopes, Suzana Gomes; Costa Junior, Livio Martins

    2015-01-01

    The objective of this study was to evaluate the effects of protein extracts obtained from the plant Leucaena leucocephala on the nematode parasite Haemonchus contortus. The seeds, shell and cotyledon of L. leucocephala were separated and their proteins extracted using a sodium phosphate buffer, and named as TE (total seed extract), SE (shell extract) and CE (cotyledon extract). Soluble protein content, protease, protease inhibitory and chitinase activity assays were performed. Exsheathment inhibition of H. contortus larvae were performed at concentrations of 0.6 mg mL-1, and egg hatch assays were conducted at protein concentrations of 0.8, 0.4, 0.2, 0.1 and 0.05 mg mL-1. The effective concentration for 50% hatching inhibition (EC50) was estimated by probit. Different proportions of soluble proteins, protease and chitinase were found in TE and CE. Protease inhibitory activity was detected in all extracts. The EC50 of the CE and TE extracts were 0.48 and 0.33 mg mL-1, respectively. No ovicidal effects on H. contortus were detected in SE extracts, and none of the protein extracts demonstrated larvicidal effects on H. contortus. We therefore conclude that protein extracts of L. leucocephala had a detrimental effect on nematode eggs, which can be correlated with the high protease and chitinase activity of these extracts. PMID:26689178

  18. Human cytomegalovirus inhibits a DNA damage response by mislocalizing checkpoint proteins

    Gaspar, Miguel; Shenk, Thomas

    2006-02-01

    The DNA damage checkpoint pathway responds to DNA damage and induces a cell cycle arrest to allow time for DNA repair. Several viruses are known to activate or modulate this cellular response. Here we show that the ataxia-telangiectasia mutated checkpoint pathway, which responds to double-strand breaks in DNA, is activated in response to human cytomegalovirus DNA replication. However, this activation does not propagate through the pathway; it is blocked at the level of the effector kinase, checkpoint kinase 2 (Chk2). Late after infection, several checkpoint proteins, including ataxia-telangiectasia mutated and Chk2, are mislocalized to a cytoplasmic virus assembly zone, where they are colocalized with virion structural proteins. This colocalization was confirmed by immunoprecipitation of virion proteins with an antibody that recognizes Chk2. Virus replication was resistant to ionizing radiation, which causes double-strand breaks in DNA. We propose that human CMV DNA replication activates the checkpoint response to DNA double-strand breaks, and the virus responds by altering the localization of checkpoint proteins to the cytoplasm and thereby inhibiting the signaling pathway. ionizing radiation | ataxia-telangiectasia mutated pathway

  19. Heat Shock Protein 72 Antagonizes STAT3 Signaling to Inhibit Fibroblast Accumulation in Renal Fibrogenesis.

    Zhou, Yi; Cao, Shirong; Li, Huiyan; Peng, Xuan; Wang, Yating; Fan, Jinjin; Wang, Yihan; Zhuang, Shougang; Yu, Xueqing; Mao, Haiping

    2016-04-01

    Heat shock protein 72 (HSP72) has been shown to attenuate unilateral ureteral obstruction-induced kidney fibrosis. It remains unknown whether HSP72 has direct effects on fibroblast proliferation in the renal fibrotic evolution. Herein, we first confirmed that increased HSP72 expression occurred in fibrotic human kidneys. Using three different animal models of kidney fibrosis, pharmacological down-regulation or genetic deletion of endogenous HSP72 expression exacerbated STAT3 phosphorylation, fibroblast proliferation, and tubulointerstitial fibrosis. In contrast, treatment with geranylgeranyl acetone, a specific inducer of HSP72, reduced phosphorylated STAT3 and protected animals from kidney fibrosis. In cultured renal interstitial fibroblasts, overexpression of HSP72 blocked transforming growth factor (TGF)-β1-induced cell activation and proliferation, as evidenced by inhibiting expression of α-smooth muscle actin, fibronectin, and collagen I/III, as well as by reducing cell numbers and DNA synthesis. Mechanical studies showed that overexpressed HSP72 attenuated TGF-β-induced phosphorylation and nuclear translocation of STAT3 and its downstream protein expression. However, siRNA knockdown of HSP72 increased TGF-β-induced STAT3 activity and fibroblast proliferation. Ectopic expression of a constitutively active STAT3 conferred resistance to HSP72 inhibition of fibroblast proliferation. Thus, HSP72 blocks fibroblast activation and proliferation in renal fibrosis via targeting the STAT3 pathway and may serve as a novel therapeutic agent for chronic kidney disease regardless of the etiology. PMID:26851345

  20. The parathyroid hormone-related protein is secreted during the osteogenic differentiation of human dental follicle cells and inhibits the alkaline phosphatase activity and the expression of DLX3.

    Klingelhöffer, C; Reck, A; Ettl, T; Morsczeck, C

    2016-08-01

    The dental follicle is involved in tooth eruption and it expresses a great amount of the parathyroid hormone-related protein (PTHrP). PTHrP as an extracellular protein is required for a multitude of different regulations of enchondral bone development and differentiation of bone precursor cells and of the development of craniofacial tissues. The dental follicle contains also precursor cells (DFCs) of the periodontium. Isolated DFCs differentiate into periodontal ligament cells, alveolar osteoblast and cementoblasts. However, the role of PTHrP during the human periodontal development remains elusive. Our study evaluated the influence of PTHrP on the osteogenic differentiation of DFCs under in vitro conditions for the first time. The PTHrP protein was highly secreted after 4days of the induction of the osteogenic differentiation of DFCs with dexamethasone (2160.5pg/ml±345.7SD. in osteogenic differentiation medium vs. 315.7pg/ml±156.2SD. in standard cell culture medium; Student's t Test: pHedgehog (IHH) induces PTHrP and that PTHrP, in turn, inhibits IHH via a negative feedback loop. We showed that SUFU (Suppressor Of Fused Homolog) was not regulated during the osteogenic differentiation in DFCs. So, neither the hedgehog signaling pathway induced PTHrP nor PTHrP suppressed the hedgehog signaling pathway during the osteogenic differentiation in DFCs. In conclusion, our results suggest that PTHrP regulates independently of the hedgehog signaling pathway the osteogenic differentiated in DFCs. PMID:27368119

  1. LegC3, an effector protein from Legionella pneumophila, inhibits homotypic yeast vacuole fusion in vivo and in vitro.

    Terry L Bennett

    Full Text Available During infection, the intracellular pathogenic bacterium Legionella pneumophila causes an extensive remodeling of host membrane trafficking pathways, both in the construction of a replication-competent vacuole comprised of ER-derived vesicles and plasma membrane components, and in the inhibition of normal phagosome:endosome/lysosome fusion pathways. Here, we identify the LegC3 secreted effector protein from L. pneumophila as able to inhibit a SNARE- and Rab GTPase-dependent membrane fusion pathway in vitro, the homotypic fusion of yeast vacuoles (lysosomes. This vacuole fusion inhibition appeared to be specific, as similar secreted coiled-coiled domain containing proteins from L. pneumophila, LegC7/YlfA and LegC2/YlfB, did not inhibit vacuole fusion. The LegC3-mediated fusion inhibition was reversible by a yeast cytosolic extract, as well as by a purified soluble SNARE, Vam7p. LegC3 blocked the formation of trans-SNARE complexes during vacuole fusion, although we did not detect a direct interaction of LegC3 with the vacuolar SNARE protein complexes required for fusion. Additionally, LegC3 was incapable of inhibiting a defined synthetic model of vacuolar SNARE-driven membrane fusion, further suggesting that LegC3 does not directly inhibit the activity of vacuolar SNAREs, HOPS complex, or Sec17p/18p during membrane fusion. LegC3 is likely utilized by Legionella to modulate eukaryotic membrane fusion events during pathogenesis.

  2. Ganglioside inhibition of glutamate-mediated protein kinase C translocation in primary cultures of cerebellar neurons

    In primary cultures of cerebellar granule cells, protein kinase C (PKC) translocation and activation can be triggered by the stimulation of excitatory amino acid neurotransmitter receptors. Glutamate evokes a dose-related translocation of 4-β-[3H]phorbol 12,13-dibutyrate /[3H]-P(BtO)2/ binding sites from the cytosol to the neuronal membrane and stimulates the incorporation of 32P into a number of membrane proteins, particularly protein bands in the range of 80, 50, and 40 kDa. The glutamate-evoked PKC translocation is Mg2+ sensitive, is prevented by 2-amino-5-phosphonovalerate and phencyclidine, is not inhibited by nitrendipine (a voltage-dependent Ca2+-channel-blocker) but is abolished by the removal of Ca2+ from the incubation medium, suggesting that glutamate-mediated Ca2+ influx is operative in the redistribution of PKC. Exposure of granule cells to the gangliosides trisialosylgangliotetraglycosylceramide (GT1b) of monosialosylgangliotetraglycosylceramide (GM1) inhibits the translocation and activation of PKC evoked by glutamate. These glycosphingolipids fail to interfere with glutamate binding to its high-affinity recognition site of with the [3H]P(BtO)2 binding, nor do they affect the Ca2+ influx. These gangliosides may prevent PKC translocation by interfering with the PKC binding to the neuronal membrane phosphatidylserine

  3. Inhibition of protein synthesis in intact HeLa cells by Shigella dysenteriae 1 toxin.

    Brown, J.E.; Rothman, S W; Doctor, B P

    1980-01-01

    Shiga toxin purified to near homogeneity from cell lysates of Shigella dysenteriae 1 inhibited protein and deoxyribonucle acid syntheses in intact HeLa cells. Inhibition was dependent on toxin concentration and time of incubation. A minimal latent period of 30 min was observed with saturating doses of toxin. Ribonucleic acid synthesis, uptake of alpha-aminoisobutyric acid, and maintenance of intracellular K+ concentrations were not affected until well after maximal inhibition of protein and d...

  4. Physiological roles of mitogen-activated-protein-kinase-activated p38-regulated/activated protein kinase

    Sergiy; Kostenko; Gianina; Dumitriu; Kari; Jenssen; Lgreid; Ugo; Moens

    2011-01-01

    Mitogen-activated protein kinases(MAPKs)are a family of proteins that constitute signaling pathways involved in processes that control gene expression,cell division, cell survival,apoptosis,metabolism,differentiation and motility.The MAPK pathways can be divided into conventional and atypical MAPK pathways.The first group converts a signal into a cellular response through a relay of three consecutive phosphorylation events exerted by MAPK kinase kinases,MAPK kinase,and MAPK.Atypical MAPK pathways are not organized into this three-tiered cascade.MAPK that belongs to both conventional and atypical MAPK pathways can phosphorylate both non-protein kinase substrates and other protein kinases.The latter are referred to as MAPK-activated protein kinases.This review focuses on one such MAPK-activated protein kinase,MAPK-activated protein kinase 5(MK5)or p38-regulated/activated protein kinase(PRAK).This protein is highly conserved throughout the animal kingdom and seems to be the target of both conventional and atypical MAPK pathways.Recent findings on the regulation of the activity and subcellular localization,bona fide interaction partners and physiological roles of MK5/PRAK are discussed.

  5. Spermine inhibition of monocyte activation and inflammation.

    Zhang, M.; Borovikova, L. V.; Wang, H.; Metz, C.; Tracey, K. J.

    1999-01-01

    The innate immune system functions as a defensive front line against pathogenic invasion, but the proinflammatory products of activated monocytes and macrophages (e.g., TNF and NO) can also injure normal cells. Anti-inflammatory mediators restrain the innate immune response and prevent excessive collateral tissue damage. Spermine, a ubiquitous biogenic polyamine, specifically and reversibly suppresses the synthesis of monocyte proinflammatory cytokines. This may provide a counterregulatory me...

  6. Janus Activated Kinase inhibition in Myelofibrosis

    H Malhotra

    2012-01-01

    Full Text Available Janus Activated Kinase (JAK 2 plays an important role in the pathogenesis of myelofibrosis (MF. Ruxolitinib (INCB018424, Jakafi is a potent dual JAK1 and JAK2 inhibitor. In November 2011, it became approved by the US FDA for the treatment of intermediate or high-risk MF. This review shall outline the role of Ruxolitinib in the current management of MF and its potential future.

  7. The antipsoriatic drug, anthralin, inhibits protein kinase C--implications for its mechanism of action.

    Hegemann, L; Fruchtmann, R; van Rooijen, L A; Müller-Peddinghaus, R; Mahrle, G

    1992-01-01

    In psoriatic patients, anthralin is known to attenuate lesional inflammation, but often generates perilesional dermatitis. This phenomenon is well reflected by the contrasting action of anthralin on human leukocytes. The release of reactive oxygen species (ROS) is inhibited by anthralin in phorbol ester-activated leukocytes, whereas anthralin directly induces this cellular response in unstimulated cells. In order to elaborate further the underlying mechanisms, we compared the kinetics of anthralin and different well-characterized stimuli, including the phorbol ester, phorbol-12-myristate-13-acetate, in this test system. Compared with standard stimuli, anthralin only marginally induced the release of ROS from human leukocytes and displayed different kinetics. Protein kinase C (PKC), the major cellular phorbol ester receptor, is considered to be involved in the regulation of this cellular response. Furthermore, its involvement in the pathophysiology of psoriasis has been suggested. Therefore, we also investigated the effects of anthralin on purified PKC. Anthralin was found to inhibit the enzyme activity in a dose-dependent manner but not to display any stimulatory effects. The present results provide first evidence that the therapeutic activity of anthralin, at least in part, might be mediated by inhibition of PKC. PMID:1503504

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

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

  9. Whey protein inhibits iron overload-induced oxidative stress in rats.

    Kim, Jungmi; Paik, Hyun-Dong; Yoon, Yoh-Chang; Park, Eunju

    2013-01-01

    In this study, we evaluated the effects of whey protein on oxidative stress in rats that were subjected to oxidative stress induced by iron overload. Thirty male rats were assigned to 3 groups: the control group (regular [50 mg/kg diet] dose of iron+20% casein), iron overload group (high [2,000 mg/kg] dose of iron+20% casein, IO), and whey protein group (high dose of iron+10% casein+10% whey protein, IO+whey). After 6 wk, the IO group showed a reduction in the plasma total radical trapping antioxidant parameter and the activity of erythrocyte superoxide dismutase and an increase in lipid peroxidation (determined from the proportion of conjugated dienes). However, whey protein ameliorated the oxidative changes induced by iron overload. The concentration of erythrocyte glutathione was significantly higher in the IO+whey group than in the IO group. In addition, whey protein supplementation fully inhibited iron overload-induced DNA damage in leukocytes and colonocytes. A highly significant positive correlation was observed between plasma iron levels and DNA damage in leukocytes and colonocytes. These results show the antioxidative and antigenotoxic effects of whey protein in an in vivo model of iron overload-induced oxidative stress. PMID:23883690

  10. Vitamin k3 inhibits protein aggregation: Implication in the treatment of amyloid diseases

    Alam, Parvez; Chaturvedi, Sumit Kumar; Siddiqi, Mohammad Khursheed; Rajpoot, Ravi Kant; Ajmal, Mohd Rehan; Zaman, Masihuz; Khan, Rizwan Hasan

    2016-01-01

    Protein misfolding and aggregation have been associated with several human diseases such as Alzheimer’s, Parkinson’s and familial amyloid polyneuropathy etc. In this study, anti-fibrillation activity of vitamin k3 and its effect on the kinetics of amyloid formation of hen egg white lysozyme (HEWL) and Aβ-42 peptide were investigated. Here, in combination with Thioflavin T (ThT) fluorescence assay, circular dichroism (CD), transmission electron microscopy and cell cytotoxicity assay, we demonstrated that vitamin k3 significantly inhibits fibril formation as well as the inhibitory effect is dose dependent manner. Our experimental studies inferred that vitamin k3 exert its neuro protective effect against amyloid induced cytotoxicity through concerted pathway, modifying the aggregation formation towards formation of nontoxic aggregates. Molecular docking demonstrated that vitamin k3 mediated inhibition of HEWL and Aβ-42 fibrillogenesis may be initiated by interacting with proteolytic resistant and aggregation prone regions respectively. This work would provide an insight into the mechanism of protein aggregation inhibition by vitamin k3; pave the way for discovery of other small molecules that may exert similar effect against amyloid formation and its associated neurodegenerative diseases. PMID:27230476

  11. Inhibition of protein kinase C affects on mode of synaptic vesicle exocytosis due to cholesterol depletion

    Petrov, Alexey M., E-mail: fysio@rambler.ru; Zakyrjanova, Guzalija F., E-mail: guzik121192@mail.ru; Yakovleva, Anastasia A., E-mail: nastya1234qwer@mail.ru; Zefirov, Andrei L., E-mail: zefiroval@rambler.ru

    2015-01-02

    Highlights: • We examine the involvement of PKC in MCD induced synaptic vesicle exocytosis. • PKC inhibitor does not decrease the effect MCD on MEPP frequency. • PKC inhibitor prevents MCD induced FM1-43 unloading. • PKC activation may switch MCD induced exocytosis from kiss-and-run to a full mode. • Inhibition of phospholipase C does not lead to similar change in exocytosis. - Abstract: Previous studies demonstrated that depletion of membrane cholesterol by 10 mM methyl-beta-cyclodextrin (MCD) results in increased spontaneous exocytosis at both peripheral and central synapses. Here, we investigated the role of protein kinase C in the enhancement of spontaneous exocytosis at frog motor nerve terminals after cholesterol depletion using electrophysiological and optical methods. Inhibition of the protein kinase C by myristoylated peptide and chelerythrine chloride prevented MCD-induced increases in FM1-43 unloading, whereas the frequency of spontaneous postsynaptic events remained enhanced. The increase in FM1-43 unloading still could be observed if sulforhodamine 101 (the water soluble FM1-43 quencher that can pass through the fusion pore) was added to the extracellular solution. This suggests a possibility that exocytosis of synaptic vesicles under these conditions could occur through the kiss-and-run mechanism with the formation of a transient fusion pore. Inhibition of phospholipase C did not lead to similar change in MCD-induced exocytosis.

  12. Thyroid peroxidase activity is inhibited by amino acids

    D.P. Carvalho

    2000-03-01

    Full Text Available Normal in vitro thyroid peroxidase (TPO iodide oxidation activity was completely inhibited by a hydrolyzed TPO preparation (0.15 mg/ml or hydrolyzed bovine serum albumin (BSA, 0.2 mg/ml. A pancreatic hydrolysate of casein (trypticase peptone, 0.1 mg/ml and some amino acids (cysteine, tryptophan and methionine, 50 µM each also inhibited the TPO iodide oxidation reaction completely, whereas casamino acids (0.1 mg/ml, and tyrosine, phenylalanine and histidine (50 µM each inhibited the TPO reaction by 54% or less. A pancreatic digest of gelatin (0.1 mg/ml or any other amino acid (50 µM tested did not significantly decrease TPO activity. The amino acids that impair iodide oxidation also inhibit the TPO albumin iodination activity. The inhibitory amino acids contain side chains with either sulfur atoms (cysteine and methionine or aromatic rings (tyrosine, tryptophan, histidine and phenylalanine. Among the amino acids tested, only cysteine affected the TPO guaiacol oxidation reaction, producing a transient inhibition at 25 or 50 µM. The iodide oxidation inhibitory activity of cysteine, methionine and tryptophan was reversed by increasing iodide concentrations from 12 to 18 mM, while no such effect was observed when the cofactor (H2O2 concentration was increased. The inhibitory substances might interfere with the enzyme activity by competing with its normal substrates for their binding sites, binding to the free substrates or reducing their oxidized form.

  13. Ramiprilate inhibits functional matrix metalloproteinase activity in Crohn's disease fistulas

    Efsen, Eva; Saermark, Torben; Hansen, Alastair;

    2011-01-01

    -diamine-tetraacetic acid (EDTA), the synthetic broad-spectrum inhibitor, GM6001, the angiotensin-converting enzyme (ACE) inhibitor, ramiprilate, and the tetracycline, doxycycline. In Crohn's disease fistulas, about 50% of the total protease activity was attributable to MMP activity. The average total MMP activity was...... activity by 72%, which is comparable to the effect of GM6001 (87%). Moreover, MMP-9 activity was completely blunted by ramiprilate. Doxycycline had no effect on MMP activity. Increased functional MMP activity, notably MMP-3 and -9, is present in Crohn's fistulas and may be inhibited by ramiprilate, a...

  14. Ramiprilate inhibits functional matrix metalloproteinase activity in Crohn's disease fistulas

    Efsen, Eva; Saermark, Torben; Hansen, Alastair;

    2011-01-01

    -diamine-tetraacetic acid (EDTA), the synthetic broad-spectrum inhibitor, GM6001, the angiotensin-converting enzyme (ACE) inhibitor, ramiprilate, and the tetracycline, doxycycline. In Crohn's disease fistulas, about 50% of the total protease activity was attributable to MMP activity. The average total MMP activity...... activity by 72%, which is comparable to the effect of GM6001 (87%). Moreover, MMP-9 activity was completely blunted by ramiprilate. Doxycycline had no effect on MMP activity. Increased functional MMP activity, notably MMP-3 and -9, is present in Crohn's fistulas and may be inhibited by ramiprilate...

  15. Inhibition of intestinal disaccharidase activity by pentoses

    Halschou-Jensen, Kia

    The current health problems regarding the obesity epidemic, development of type 2 diabetes mellitus (T2D) and cardiovascular disease are a major challenge for healthcare systems worldwide.No simple or unique cure has been documented to prevent or treat this major health problem regarding T2D and...... nature of ingested food, gastric emptying, intraluminal glucose oncentration, and enzymatic activity in the brush border. The focus in this review is on evidence provided by in vitro studies, animal models and human studies on Larabinose, D-xylose and polyphenols. The focus is on their effects on...

  16. Sorafenib enhances proteasome inhibitor-mediated cytotoxicity via inhibition of unfolded protein response and keratin phosphorylation

    Hepatocellular carcinoma (HCC) is highly resistant to conventional systemic therapies and prognosis for advanced HCC patients remains poor. Recent studies of the molecular mechanisms responsible for tumor initiation and progression have identified several potential molecular targets in HCC. Sorafenib is a multi-kinase inhibitor shown to have survival benefits in advanced HCC. It acts by inhibiting the serine/threonine kinases and the receptor type tyrosine kinases. In preclinical experiments sorafenib had anti-proliferative activity in hepatoma cells and it reduced tumor angiogenesis and increased apoptosis. Here, we demonstrate for the first time that the cytotoxic mechanisms of sorafenib include its inhibitory effects on protein ubiquitination, unfolded protein response (UPR) and keratin phosphorylation in response to endoplasmic reticulum (ER) stress. Moreover, we show that combined treatment with sorafenib and proteasome inhibitors (PIs) synergistically induced a marked increase in cell death in hepatoma- and hepatocyte-derived cells. These observations may open the way to potentially interesting treatment combinations that may augment the effect of sorafenib, possibly including drugs that promote ER stress. Because sorafenib blocked the cellular defense mechanisms against hepatotoxic injury not only in hepatoma cells but also in hepatocyte-derived cells, we must be careful to avoid severe liver injury. -- Graphical abstract: Display Omitted -- Highlights: •We examined the cytotoxic mechanisms of sorafenib in hepatoma cells. •Sorafenib induces cell death via apoptotic and necrotic fashion. •Sorafenib inhibits protein ubiquitination and unfolded protein response. •Autophagy induced by sorafenib may affect its cytotoxicity. •Sorafenib inhibits keratin phosphorylation and cytoplasmic inclusion formation

  17. Sorafenib enhances proteasome inhibitor-mediated cytotoxicity via inhibition of unfolded protein response and keratin phosphorylation

    Honma, Yuichi; Harada, Masaru, E-mail: msrharada@med.uoeh-u.ac.jp

    2013-08-15

    Hepatocellular carcinoma (HCC) is highly resistant to conventional systemic therapies and prognosis for advanced HCC patients remains poor. Recent studies of the molecular mechanisms responsible for tumor initiation and progression have identified several potential molecular targets in HCC. Sorafenib is a multi-kinase inhibitor shown to have survival benefits in advanced HCC. It acts by inhibiting the serine/threonine kinases and the receptor type tyrosine kinases. In preclinical experiments sorafenib had anti-proliferative activity in hepatoma cells and it reduced tumor angiogenesis and increased apoptosis. Here, we demonstrate for the first time that the cytotoxic mechanisms of sorafenib include its inhibitory effects on protein ubiquitination, unfolded protein response (UPR) and keratin phosphorylation in response to endoplasmic reticulum (ER) stress. Moreover, we show that combined treatment with sorafenib and proteasome inhibitors (PIs) synergistically induced a marked increase in cell death in hepatoma- and hepatocyte-derived cells. These observations may open the way to potentially interesting treatment combinations that may augment the effect of sorafenib, possibly including drugs that promote ER stress. Because sorafenib blocked the cellular defense mechanisms against hepatotoxic injury not only in hepatoma cells but also in hepatocyte-derived cells, we must be careful to avoid severe liver injury. -- Graphical abstract: Display Omitted -- Highlights: •We examined the cytotoxic mechanisms of sorafenib in hepatoma cells. •Sorafenib induces cell death via apoptotic and necrotic fashion. •Sorafenib inhibits protein ubiquitination and unfolded protein response. •Autophagy induced by sorafenib may affect its cytotoxicity. •Sorafenib inhibits keratin phosphorylation and cytoplasmic inclusion formation.

  18. Phosphatidylinositol 4,5-bisphosphate competitively inhibits phorbol ester binding to protein kinase C

    Calcium phospholipid dependent protein kinase C (PKC) is activated by diacylglycerol (DG) and by phorbol esters and is recognized to be the phorbol ester receptor of cells; DG displaces phorbol ester competitively from PKC. A phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2), can also activate PKC in the presence of phosphatidylserine (PS) and Ca2+ with a KPIP2 of 0.04 mol %. Preliminary experiments have suggested a common binding site for PIP2 and DG on PKC. Here, the authors investigate the effect of PIP2 on phorbol ester binding to PKC in a mixed micellar assay. In the presence of 20 mol % PS, PIP2 inhibited specific binding of [3H]phorbol 12,13-dibutyrate (PDBu) in a dose-dependent fashion up to 85% at 1 mol %. Inhibition of binding was more pronounced with PIP2 than with DG. Scatchard analysis indicated that the decrease in binding of PDBu in the presence of PIP2 is the result of an altered affinity for the phorbol ester rather than of a change in maximal binding. The plot of apparent dissociation constants (Kd') against PIP2 concentration was linear over a range of 0.01-1 mol % with a Ki of 0.043 mol % and confirmed the competitive nature of inhibition between PDBu and PIP2. Competition between PIP2 and phorbol ester could be determined in a liposomal assay system also. These results indicate that PIP2, DG, and phorbol ester all compete for the same activator-receiving region on the regulatory moiety of protein kinase C, and they lend support to the suggestion that PIP2 is a primary activator of the enzyme

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

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

  20. Ammonium inhibition of nitrogenase activity in Herbaspirillum seropedicae

    Fu, H.; Burris, R.H. (Univ. of Wisconsin, Madison (USA))

    1989-06-01

    The effect of oxygen, ammonium ion, and amino acids on nitrogenase activity in the root-associated N{sub 2}-fixing bacterium Herbaspirillum seropedicae was investigated in comparison with Azospirillum spp. and Rhodospirillum rubrum. H. seropedicae is microaerophilic, and its optimal dissolved oxygen level is from 0.04 to 0.2 kPa for dinitrogen fixation but higher when it is supplied with fixed nitrogen. No nitrogenase activity was detected when the dissolved O{sub 2} level corresponded to 4.0 kPa. Ammonium, a product of the nitrogenase reaction, reversible inhibited nitrogenase activity when added to derepressed cell cultures. However, the inhibition of nitrogenase activity was only partial even with concentrations of ammonium chloride as high as 20 mM. Amides such as glutamine and asparagine partially inhibited nitrogenase activity, but glutamate did not. Nitrogenase in crude extracts prepared from ammonium-inhibited cells showed activity as high as in extracts from N{sub 2}-fixing cells. The pattern of the dinitrogenase and the dinitrogenase reductase revealed by the immunoblotting technique did not change upon ammonium chloride treatment of cells in vivo. No homologous sequences were detected with the draT-draG probe from Azospirillum lipoferum. There is no clear evidence that ADP-ribosylation of the dinitrogenase reductase is involved in the ammonium inhibition of H. seropedicae. The uncoupler carbonyl cyanide m-chlorophenylhydrazone decreased the intracellular ATP concentration and inhibited the nitrogenase activity of whole cells. The ATP pool was significantly disturbed when cultures were treated with ammonium in vivo.

  1. Protein-water dynamics in antifreeze protein III activity

    Xu, Yao; Bäumer, Alexander; Meister, Konrad; Bischak, Connor G.; DeVries, Arthur L.; Leitner, David M.; Havenith, Martina

    2016-03-01

    We combine Terahertz absorption spectroscopy (THz) and molecular dynamics (MD) simulations to investigate the underlying molecular mechanism for the antifreeze activity of one class of antifreeze protein, antifreeze protein type III (AFP-III) with a focus on the collective water hydrogen bond dynamics near the protein. After summarizing our previous work on AFPs, we present a new investigation of the effects of cosolutes on protein antifreeze activity by adding sodium citrate to the protein solution of AFP-III. Our results reveal that for AFP-III, unlike some other AFPs, the addition of the osmolyte sodium citrate does not affect the hydrogen bond dynamics at the protein surface significantly, as indicated by concentration dependent THz measurements. The present data, in combination with our previous THz measurements and molecular simulations, confirm that while long-range solvent perturbation is a necessary condition for the antifreeze activity of AFP-III, the local binding affinity determines the size of the hysteresis.

  2. 3,3′-Diindolylmethane, but not indole-3-carbinol, inhibits histone deacetylase activity in prostate cancer cells

    Beaver, Laura M., E-mail: beaverl@onid.orst.edu [Linus Pauling Institute, Oregon State University, 307 Linus Pauling Science Center, Corvallis, OR 97331 (United States); School of Biological and Population Health Sciences, Oregon State University, 103 Milam Hall, Corvallis, OR 97331 (United States); Yu, Tian-Wei, E-mail: david.yu@oregonstate.edu [Linus Pauling Institute, Oregon State University, 307 Linus Pauling Science Center, Corvallis, OR 97331 (United States); Sokolowski, Elizabeth I., E-mail: sokolowe@onid.orst.edu [School of Biological and Population Health Sciences, Oregon State University, 103 Milam Hall, Corvallis, OR 97331 (United States); Williams, David E., E-mail: david.williams@oregonstate.edu [Linus Pauling Institute, Oregon State University, 307 Linus Pauling Science Center, Corvallis, OR 97331 (United States); Department of Environmental and Molecular Toxicology, Oregon State University, 1007 Agriculture and Life Sciences Building, Corvallis, OR 97331 (United States); Dashwood, Roderick H., E-mail: rod.dashwood@oregonstate.edu [Linus Pauling Institute, Oregon State University, 307 Linus Pauling Science Center, Corvallis, OR 97331 (United States); Department of Environmental and Molecular Toxicology, Oregon State University, 1007 Agriculture and Life Sciences Building, Corvallis, OR 97331 (United States); Ho, Emily, E-mail: Emily.Ho@oregonstate.edu [Linus Pauling Institute, Oregon State University, 307 Linus Pauling Science Center, Corvallis, OR 97331 (United States); School of Biological and Population Health Sciences, Oregon State University, 103 Milam Hall, Corvallis, OR 97331 (United States)

    2012-09-15

    Increased consumption of cruciferous vegetables is associated with a reduced risk of developing prostate cancer. Indole-3-carbinol (I3C) and 3,3′-diindolylmethane (DIM) are phytochemicals derived from cruciferous vegetables that have shown promise in inhibiting prostate cancer in experimental models. Histone deacetylase (HDAC) inhibition is an emerging target for cancer prevention and therapy. We sought to examine the effects of I3C and DIM on HDACs in human prostate cancer cell lines: androgen insensitive PC-3 cells and androgen sensitive LNCaP cells. I3C modestly inhibited HDAC activity in LNCaP cells by 25% but no inhibition of HDAC activity was detected in PC-3 cells. In contrast, DIM significantly inhibited HDAC activity in both cell lines by as much as 66%. Decreases in HDAC activity correlated with increased expression of p21, a known target of HDAC inhibitors. DIM treatment caused a significant decrease in the expression of HDAC2 protein in both cancer cell lines but no significant change in the protein levels of HDAC1, HDAC3, HDAC4, HDAC6 or HDAC8 was detected. Taken together, these results show that inhibition of HDAC activity by DIM may contribute to the phytochemicals' anti-proliferative effects in the prostate. The ability of DIM to target aberrant epigenetic patterns, in addition to its effects on detoxification of carcinogens, may make it an effective chemopreventive agent by targeting multiple stages of prostate carcinogenesis. -- Highlights: ► DIM inhibits HDAC activity and decreases HDAC2 expression in prostate cancer cells. ► DIM is significantly more effective than I3C at inhibiting HDAC activity. ► I3C has no effect on HDAC protein expression. ► Inhibition of HDAC activity by DIM is associated with increased p21 expression. ► HDAC inhibition may be a novel epigenetic mechanism for cancer prevention with DIM.

  3. VP22 fusion protein-based dominant negative mutant can inhibit hepatitis B virus replication

    Jun Yi; Wei-Dong Gong; Ling Wang; Rui Ling; Jiang-Hao Chen; Jun Yun

    2005-01-01

    AIM: To investigate the inhibitory effect of VP22 fusion protein-based dominant negative (DN) mutant on Hepatitis Bvrus (HBV) replication.METHODS: Full-length or truncated fragment of VP22 was fused to C terminal of HBV core protein (HBc), and subcloned into pcDNA3.1 (-) vector, yielding eukaryotic expression plasmids of DN mutant. After transfection into HepG2.2.15 cells, the expression of DN mutant was identified by immunofluorescence staining. The inhibitory effect of DN mutant on HBV replication was indexed as the supernatant HBsAg concentration determined by RIA and HBV-DNA content by fluorescent quantification-PCR (FQ-PCR). Meanwhile, metabolism of HepG2.2.15 cells was evaluated by MTT colorimetry.RESULTS: VP22-based DN mutants and its truncated fragment were expressed in HepG2.2.15 cells, and had no toxic effect on host cells. DN mutants could inhibit HBV replication and the transduction ability of mutantbearing protein had a stronger inhibitory effect on HBV replication. DN mutants with full length of VP22 had the strongest inhibitory effect on HBV replication, reducing the HBsAg concentration by 81.94%, and the HBV-DNA content by 72.30%. MTT assay suggested that there were no significant differences in cell metabolic activity between the groups.CONCLUSION: VP22-based DN mutant can inhibit HBV replication effectively.

  4. Aspirin inhibits colon cancer cell and tumor growth and downregulates specificity protein (Sp transcription factors.

    Satya Pathi

    Full Text Available Acetylsalicylic acid (aspirin is highly effective for treating colon cancer patients postdiagnosis; however, the mechanisms of action of aspirin in colon cancer are not well defined. Aspirin and its major metabolite sodium salicylate induced apoptosis and decreased colon cancer cell growth and the sodium salt of aspirin also inhibited tumor growth in an athymic nude mouse xenograft model. Colon cancer cell growth inhibition was accompanied by downregulation of Sp1, Sp3 and Sp4 proteins and decreased expression of Sp-regulated gene products including bcl-2, survivin, VEGF, VEGFR1, cyclin D1, c-MET and p65 (NFκB. Moreover, we also showed by RNA interference that β-catenin, an important target of aspirin in some studies, is an Sp-regulated gene. Aspirin induced nuclear caspase-dependent cleavage of Sp1, Sp3 and Sp4 proteins and this response was related to sequestration of zinc ions since addition of zinc sulfate blocked aspirin-mediated apoptosis and repression of Sp proteins. The results demonstrate an important underlying mechanism of action of aspirin as an anticancer agent and, based on the rapid metabolism of aspirin to salicylate in humans and the high salicylate/aspirin ratios in serum, it is likely that the anticancer activity of aspirin is also due to the salicylate metabolite.

  5. DAX-1 Inhibits Hepatocellular Carcinoma Proliferation by Inhibiting β-Catenin Transcriptional Activity

    Hong-Lei Jiang

    2014-08-01

    Full Text Available Background/Aims: Hepatocellular carcinoma (HCC represents the most common type of liver cancer. DAX1 (dosage-sensitive sex reversal adrenal hypoplasia congenital critical region on X chromosome, gene 1, an atypical member of the nuclear receptor family due to lack of classical DNA-binding domains, has been known for its fundamental roles in the development, especially in the sex determination and steroidogenesis. Previous studies also showed that DAX-1 played a critical role in endocrine and sex steroid-dependent neoplasms such as adrenocortical, pituitary, endometrial, and ovarian tumors. However, its biological roles in the development of HCC remain largely unexplored. Methods: Real-time PCR and Western blot were used to detect the expression of DAX-1 in HCC tissues and cell lines. Immunoprecipitation (IP assay was used to show the interaction between DAX-1 and β-Catenin. Small interfering RNA (siRNA was used to silence the expression of DAX-1. BrdU incorporation and Cell-cycle assays were used to detect the role of DAX-1 in HCC cells proliferation. Migration and invasion assays were carried out to test the metastasis ability of DAX-1 in HCC cells. Results: In the present study, we found that mRNA and protein levels of DAX-1 were down-regulated in HCC tissues and cell lines. Furthermore, overexpression of DAX-1 could inhibit while its knockdown using small interfering RNA promoted cell proliferation in several HCC cell lines. At the molecular level, we demonstrated that DAX-1 could interact with β-Catenin and attenuate its transcriptional activity. Conclusion: Therefore, our results suggest a previously unknown DAX-1/β-Catenin molecular network controlling HCC development.

  6. DMSO inhibits human platelet activation through cyclooxygenase-1 inhibition. A novel agent for drug eluting stents?

    Asmis, L; Tanner, F C; Sudano, I; Lüscher, T F; Camici, G G

    2010-01-01

    Background: DMSO is routinely infused together with hematopoietic cells in patients undergoing myeloablative therapy and was recently found to inhibit smooth muscle cells proliferation and arterial thrombus formation in the mouse by preventing tissue factor (TF), a key activator of the coagulation cascade. This study was designed to investigate whether DMSO prevents platelet activation and thus, whether it may represent an interesting agent to be used on drug eluting stents. Methods and resul...

  7. Alzheimer's associated β-amyloid protein inhibits influenza A virus and modulates viral interactions with phagocytes.

    Mitchell R White

    Full Text Available Accumulation of β-Amyloid (βA is a key pathogenetic factor in Alzheimer's disease; however, the normal function of βA is unknown. Recent studies have shown that βA can inhibit growth of bacteria and fungi. In this paper we show that βA also inhibits replication of seasonal and pandemic strains of H3N2 and H1N1 influenza A virus (IAV in vitro. The 42 amino acid fragment of βA (βA42 had greater activity than the 40 amino acid fragment. Direct incubation of the virus with βA42 was needed to achieve optimal inhibition. Using quantitative PCR assays βA42 was shown to reduce viral uptake by epithelial cells after 45 minutes and to reduce supernatant virus at 24 hours post infection. βA42 caused aggregation of IAV particles as detected by light transmission assays and electron and confocal microscopy. βA42 did not stimulate neutrophil H2O2 production or extracellular trap formation on its own, but it increased both responses stimulated by IAV. In addition, βA42 increased uptake of IAV by neutrophils. βA42 reduced viral protein synthesis in monocytes and reduced IAV-induced interleukin-6 production by these cells. Hence, we demonstrate for the first time that βA has antiviral activity and modulates viral interactions with phagocytes.

  8. Modulation of the protein kinase activity of mTOR.

    Lawrence, J C; Lin, T A; McMahon, L P; Choi, K M

    2004-01-01

    mTOR is a founding member of a family of protein kinases having catalytic domains homologous to those in phosphatidylinositol 3-OH kinase. mTOR participates in the control by insulin of the phosphorylation of lipin, which is required for adipocyte differentiation, and the two translational regulators, p70S6K and PHAS-I. The phosphorylation of mTOR, itself, is stimulated by insulin in Ser2448, a site that is also phosphorylated by protein kinase B (PKB) in vitro and in response to activation of PKB activity in vivo. Ser2448 is located in a short stretch of amino acids not found in the two TOR proteins in yeast. A mutant mTOR lacking this stretch exhibited increased activity, and binding of the antibody, mTAb-1, to this region markedly increased mTOR activity. In contrast, rapamycin-FKBP12 inhibited mTOR activity towards both PHAS-I and p70S6K, although this complex inhibited the phosphorylation of some sites more than that of others. Mutating Ser2035 to Ile in the FKBP12-rapamycin binding domain rendered mTOR resistant to inhibition by rapamycin. Unexpectedly, this mutation markedly decreased the ability of mTOR to phosphorylate certain sites in both PHAS-I and p70S6K. The results support the hypotheses that rapamycin disrupts substrate recognition instead of directly inhibiting phosphotransferase activity and that mTOR activity in cells is controlled by the phosphorylation of an inhibitory regulatory domain containing the mTAb-1 epitope. PMID:14560959

  9. Cyclic nucleotides and mitogen-activated protein kinases: regulation of simvastatin in platelet activation

    Hou Ssu-Yu

    2010-06-01

    Full Text Available Abstract Background 3-Hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA reductase inhibitors (statins have been widely used to reduce cardiovascular risk. These statins (i.e., simvastatin may exert other effects besides from their cholesterol-lowering actions, including inhibition of platelet activation. Platelet activation is relevant to a variety of coronary heart diseases. Although the inhibitory effect of simvastatin in platelet activation has been studied; the detailed signal transductions by which simvastatin inhibit platelet activation has not yet been completely resolved. Methods The aim of this study was to systematically examine the detailed mechanisms of simvastatin in preventing platelet activation. Platelet aggregation, flow cytometric analysis, immunoblotting, and electron spin resonance studies were used to assess the antiplatelet activity of simvastatin. Results Simvastatin (20-50 μM exhibited more-potent activity of inhibiting platelet aggregation stimulated by collagen than other agonists (i.e., thrombin. Simvastatin inhibited collagen-stimulated platelet activation accompanied by [Ca2+]i mobilization, thromboxane A2 (TxA2 formation, and phospholipase C (PLCγ2, protein kinase C (PKC, and mitogen-activated protein kinases (i.e., p38 MAPK, JNKs phosphorylation in washed platelets. Simvastatin obviously increased both cyclic AMP and cyclic GMP levels. Simvastatin markedly increased NO release, vasodilator-stimulated phosphoprotein (VASP phosphorylation, and endothelial nitric oxide synthase (eNOS expression. SQ22536, an inhibitor of adenylate cyclase, markedly reversed the simvastatin-mediated inhibitory effects on platelet aggregation, PLCγ2 and p38 MAPK phosphorylation, and simvastatin-mediated stimulatory effects on VASP and eNOS phosphorylation. Conclusion The most important findings of this study demonstrate for the first time that inhibitory effect of simvastatin in platelet activation may involve activation of the cyclic AMP

  10. A viral suppressor protein inhibits host RNA silencing by hooking up with Argonautes

    Jin, Hailing

    2010-05-01

    RNA viruses are particularly vulnerable to RNAi-based defenses in the host, and thus have evolved specific proteins, known as viral suppressors of RNA silencing (VSRs), as a counterdefense. In this issue of Genes & Development, Azevedo and colleagues (pp. 904-915) discovered that P38, the VSR of Turnip crinkle virus, uses its glycine/tryptophane (GW) motifs as an ARGONAUTE (AGO) hook to attract and disarm the host\\'s essential effector of RNA silencing. Several GW motif-containing cellular proteins are known to be important partners of AGOs in RNA silencing effector complexes in yeast, plants, and animals. The GW motif appears to be a versatile and effective tool for regulating the activities of RNA silencing pathways, and the use of GW mimicry to compete for and inhibit host AGOs may be a strategy used by many pathogens to counteract host RNAi-based defenses. © 2010 by Cold Spring Harbor Laboratory Press.

  11. Platycodin D inhibits tumor growth by antiangiogenic activity via blocking VEGFR2-mediated signaling pathway

    Luan, Xin; Gao, Yun-Ge; Guan, Ying-Yun; Xu, Jian-Rong; Lu, Qin [Department of Pharmacology, Institute of Medical Sciences, Shanghai Jiao Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China); Zhao, Mei [Department of Pharmacy, Shanghai Institute of Health Sciences and Health School Attached to SJTU-SM, 279 Zhouzhu Road, Shanghai 201318 (China); Liu, Ya-Rong; Liu, Hai-Jun [Department of Pharmacology, Institute of Medical Sciences, Shanghai Jiao Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China); Fang, Chao, E-mail: fangchao100@hotmail.com [Department of Pharmacology, Institute of Medical Sciences, Shanghai Jiao Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China); Chen, Hong-Zhuan, E-mail: hongzhuan_chen@hotmail.com [Department of Pharmacology, Institute of Medical Sciences, Shanghai Jiao Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China)

    2014-11-15

    Platycodin D (PD) is an active component mainly isolated from the root of Platycodon grandiflorum. Recent studies proved that PD exhibited inhibitory effect on proliferation, migration, invasion and xenograft growth of diverse cancer cell lines. However, whether PD is suppressive for angiogenesis, an important hallmark in cancer development, remains unknown. Here, we found that PD could dose-dependently inhibit human umbilical vein endothelial cell (HUVEC) proliferation, motility, migration and tube formation. PD also significantly inhibited angiogenesis in the chick embryo chorioallantoic membrane (CAM). Moreover, the antiangiogenic activity of PD contributed to its in vivo anticancer potency shown in the decreased microvessel density and delayed growth of HCT-15 xenograft in mice with no overt toxicity. Western blot analysis indicated that PD inhibited the phosphorylation of VEGFR2 and its downstream protein kinase including PLCγ1, JAK2, FAK, Src, and Akt in endothelial cells. Molecular docking simulation showed that PD formed hydrogen bonds and hydrophobic interactions within the ATP binding pocket of VEGFR2 kinase domain. The present study firstly revealed the high antiangiogenic activity and the underlying molecular basis of PD, suggesting that PD may be a potential antiangiogenic agent for angiogenesis-related diseases. - Highlights: • Platycodin D inhibits HUVEC proliferation, motility, migration and tube formation. • Platycodin D inhibits the angiogenesis in chick embryo chorioallantoic membrane. • Platycodin D suppresses the angiogenesis and growth of HCT-15 xenograft in mice. • Platycodin D inhibits the phosphorylation of VEGFR2 and downstream kinases in HUVEC.

  12. Platycodin D inhibits tumor growth by antiangiogenic activity via blocking VEGFR2-mediated signaling pathway

    Platycodin D (PD) is an active component mainly isolated from the root of Platycodon grandiflorum. Recent studies proved that PD exhibited inhibitory effect on proliferation, migration, invasion and xenograft growth of diverse cancer cell lines. However, whether PD is suppressive for angiogenesis, an important hallmark in cancer development, remains unknown. Here, we found that PD could dose-dependently inhibit human umbilical vein endothelial cell (HUVEC) proliferation, motility, migration and tube formation. PD also significantly inhibited angiogenesis in the chick embryo chorioallantoic membrane (CAM). Moreover, the antiangiogenic activity of PD contributed to its in vivo anticancer potency shown in the decreased microvessel density and delayed growth of HCT-15 xenograft in mice with no overt toxicity. Western blot analysis indicated that PD inhibited the phosphorylation of VEGFR2 and its downstream protein kinase including PLCγ1, JAK2, FAK, Src, and Akt in endothelial cells. Molecular docking simulation showed that PD formed hydrogen bonds and hydrophobic interactions within the ATP binding pocket of VEGFR2 kinase domain. The present study firstly revealed the high antiangiogenic activity and the underlying molecular basis of PD, suggesting that PD may be a potential antiangiogenic agent for angiogenesis-related diseases. - Highlights: • Platycodin D inhibits HUVEC proliferation, motility, migration and tube formation. • Platycodin D inhibits the angiogenesis in chick embryo chorioallantoic membrane. • Platycodin D suppresses the angiogenesis and growth of HCT-15 xenograft in mice. • Platycodin D inhibits the phosphorylation of VEGFR2 and downstream kinases in HUVEC

  13. Trichinella spiralis Paramyosin Binds Human Complement C1q and Inhibits Classical Complement Activation.

    Ran Sun

    2015-12-01

    Full Text Available Trichinella spiralis expresses paramyosin (Ts-Pmy as a defense mechanism. Ts-Pmy is a functional protein with binding activity to human complement C8 and C9 and thus plays a role in evading the attack of the host's immune system. In the present study, the binding activity of Ts-Pmy to human complement C1q and its ability to inhibit classical complement activation were investigated.The binding of recombinant and natural Ts-Pmy to human C1q were determined by ELISA, Far Western blotting and immunoprecipitation, respectively. Binding of recombinant Ts-Pmy (rTs-Pmy to C1q inhibited C1q binding to IgM and consequently inhibited C3 deposition. The lysis of antibody-sensitized erythrocytes (EAs elicited by the classical complement pathway was also inhibited in the presence of rTs-Pmy. In addition to inhibiting classical complement activation, rTs-Pmy also suppressed C1q binding to THP-1-derived macrophages, thereby reducing C1q-induced macrophages migration.Our results suggest that T. spiralis paramyosin plays an important role in immune evasion by interfering with complement activation through binding to C1q in addition to C8 and C9.

  14. Genetically engineered endostatin-lidamycin fusion proteins effectively inhibit tumor growth and metastasis

    Endostatin (ES) inhibits endothelial cell proliferation, migration, invasion, and tube formation. It also shows antiangiogenesis and antitumor activities in several animal models. Endostatin specifically targets tumor vasculature to block tumor growth. Lidamycin (LDM), which consists of an active enediyne chromophore (AE) and a non-covalently bound apo-protein (LDP), is a member of chromoprotein family of antitumor antibiotics with extremely potent cytotoxicity to cancer cells. Therefore, we reasoned that endostatin-lidamycin (ES-LDM) fusion proteins upon energizing with enediyne chromophore may obtain the combined capability targeting tumor vasculature and tumor cell by respective ES and LDM moiety. In this study, we designed and obtained two new endostatin-based fusion proteins, endostatin-LDP (ES-LDP) and LDP-endostatin (LDP-ES). In vitro, the antiangiogenic effect of fusion proteins was determined by the wound healing assay and tube formation assay and the cytotoxicity of their enediyne-energized analogs was evaluated by CCK-8 assay. Tissue microarray was used to analyze the binding affinity of LDP, ES or ES-LDP with specimens of human lung tissue and lung tumor. The in vivo efficacy of the fusion proteins was evaluated with human lung carcinoma PG-BE1 xenograft and the experimental metastasis model of 4T1-luc breast cancer. ES-LDP and LDP-ES disrupted the formation of endothelial tube structures and inhibited endothelial cell migration. Evidently, ES-LDP accumulated in the tumor and suppressed tumor growth and metastasis. ES-LDP and ES show higher binding capability than LDP to lung carcinoma; in addition, ES-LDP and ES share similar binding capability. Furthermore, the enediyne-energized fusion protein ES-LDP-AE demonstrated significant efficacy against lung carcinoma xenograft in athymic mice. The ES-based fusion protein therapy provides some fundamental information for further drug development. Targeting both tumor vasculature and tumor cells by endostatin

  15. Recruitment of Perisomatic Inhibition during Spontaneous Hippocampal Activity In Vitro.

    Anna Beyeler

    Full Text Available It was recently shown that perisomatic GABAergic inhibitory postsynaptic potentials (IPSPs originating from basket and chandelier cells can be recorded as population IPSPs from the hippocampal pyramidal layer using extracellular electrodes (eIPSPs. Taking advantage of this approach, we have investigated the recruitment of perisomatic inhibition during spontaneous hippocampal activity in vitro. Combining intracellular and extracellular recordings from pyramidal cells and interneurons, we confirm that inhibitory signals generated by basket cells can be recorded extracellularly, but our results suggest that, during spontaneous activity, eIPSPs are mostly confined to the CA3 rather than CA1 region. CA3 eIPSPs produced the powerful time-locked inhibition of multi-unit activity expected from perisomatic inhibition. Analysis of the temporal dynamics of spike discharges relative to eIPSPs suggests significant but moderate recruitment of excitatory and inhibitory neurons within the CA3 network on a 10 ms time scale, within which neurons recruit each other through recurrent collaterals and trigger powerful feedback inhibition. Such quantified parameters of neuronal interactions in the hippocampal network may serve as a basis for future characterisation of pathological conditions potentially affecting the interactions between excitation and inhibition in this circuit.

  16. Mesencephalic stimulation elicits inhibition of phrenic nerve activity in cat.

    Gallman, E A; Lawing, W L; Millhorn, D E

    1991-05-01

    1. Previous work from this laboratory has indicated that the mesencephalon is the anatomical substrate for a mechanism capable of inhibiting central respiratory drive in glomectomized cats for periods of up to 1 h or more following brief exposure to systemic hypoxia; phrenic nerve activity was used as an index of central respiratory drive. 2. The present study was undertaken to further localize the region responsible for the observed post-hypoxic inhibition of respiratory drive. We studied the phrenic nerve response to stimulations of the mesencephalon in anaesthetized, paralysed peripherally chemo-denervated cats with end-expired PCO2 and body temperature servo-controlled. 3. Stimulations of two types were employed. Electrical stimulation allowed rapid determination of sites from which phrenic inhibition could be elicited. Microinjections of excitatory amino acids were used subsequently in order to confine excitation to neuronal cell bodies and not axons of passage. 4. Stimulation of discrete regions of the ventromedial aspect of the mesencephalon in the vicinity of the red nucleus produced substantial inhibition of phrenic activity which lasted up to 45 min. Stimulation of other areas of the mesencephalon either produced no phrenic inhibition or resulted in a slight stimulation of phrenic activity. 5. The results are discussed in the context of the central respiratory response to hypoxia. PMID:1676420

  17. LRRK2 kinase inhibition prevents pathological microglial phagocytosis in response to HIV-1 Tat protein

    Marker Daniel F

    2012-11-01

    Full Text Available Abstract Background Human Immunodeficiency Virus-1 (HIV-1 associated neurocognitive disorders (HANDs are accompanied by significant morbidity, which persists despite the use of combined antiretroviral therapy (cART. While activated microglia play a role in pathogenesis, changes in their immune effector functions, including phagocytosis and proinflammatory signaling pathways, are not well understood. We have identified leucine-rich repeat kinase 2 (LRRK2 as a novel regulator of microglial phagocytosis and activation in an in vitro model of HANDs, and hypothesize that LRRK2 kinase inhibition will attenuate microglial activation during HANDs. Methods We treated BV-2 immortalized mouse microglia cells with the HIV-1 trans activator of transcription (Tat protein in the absence or presence of LRRK2 kinase inhibitor (LRRK2i. We used Western blot, qRT-PCR, immunocytochemistry and latex bead engulfment assays to analyze LRRK2 protein levels, proinflammatory cytokine and phagocytosis receptor expression, LRRK2 cellular distribution and phagocytosis, respectively. Finally, we utilized ex vivo microfluidic chambers containing primary hippocampal neurons and BV-2 microglia cells to investigate microglial phagocytosis of neuronal axons. Results We found that Tat-treatment of BV-2 cells induced kinase activity associated phosphorylation of serine 935 on LRRK2 and caused the formation of cytoplasmic LRRK2 inclusions. LRRK2i decreased Tat-induced phosphorylation of serine 935 on LRRK2 and inhibited the formation of Tat-induced cytoplasmic LRRK2 inclusions. LRRK2i also decreased Tat-induced process extension in BV-2 cells. Furthermore, LRRK2i attenuated Tat-induced cytokine expression and latex bead engulfment. We examined relevant cellular targets in microfluidic chambers and found that Tat-treated BV-2 microglia cells cleared axonal arbor and engulfed neuronal elements, whereas saline treated controls did not. LRRK2i was found to protect axons in the presence

  18. Inhibition of Leukemic Cell Telomerase Activity by Antisense Phosphorothioate Oligodeoxynucleotides

    HEDongmei; ZHANGYuan

    2002-01-01

    Objective To evaluate the effect of human telomerase reverse transcriptase(hTERT) gene antisense oligodeoxynucleotide (ASON) on telomerase activity in K562 cells.Methods Telomerase activity was detemined by polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA) in K562 cells treated with ASODN and hTERTmRNA expression was detected by reverse transcriptase polymerase chain reaction (RT-PCR). Results The hTERTmRNA level was decreased,and telomerase activity was significantly inhibited when the K562 cells were treated with ASODN for 48 h. Conclusion It is suggested that hTETR ASODN might specifically inhibit telomrase activity of K562 cells at translation level,and it is further proved that hTERT gene has significant correlation with telopmerase activity.

  19. Garcinol Inhibits GCN5-Mediated Lysine Acetyltransferase Activity and Prevents Replication of the Parasite Toxoplasma gondii.

    Jeffers, Victoria; Gao, Hongyu; Checkley, Lisa A; Liu, Yunlong; Ferdig, Michael T; Sullivan, William J

    2016-04-01

    Lysine acetylation is a critical posttranslational modification that influences protein activity, stability, and binding properties. The acetylation of histone proteins in particular is a well-characterized feature of gene expression regulation. In the protozoan parasiteToxoplasma gondii, a number of lysine acetyltransferases (KATs) contribute to gene expression and are essential for parasite viability. The natural product garcinol was recently reported to inhibit enzymatic activities of GCN5 and p300 family KATs in other species. Here we show that garcinol inhibits TgGCN5b, the only nuclear GCN5 family KAT known to be required forToxoplasmatachyzoite replication. Treatment of tachyzoites with garcinol led to a reduction of global lysine acetylation, particularly on histone H3 and TgGCN5b itself. We also performed transcriptome sequencing (RNA-seq), which revealed increasing aberrant gene expression coincident with increasing concentrations of garcinol. The majority of the genes that were most significantly affected by garcinol were also associated with TgGCN5b in a previously reported chromatin immunoprecipitation assay with microarray technology (ChIP-chip) analysis. The dysregulated gene expression induced by garcinol significantly inhibitsToxoplasmatachyzoite replication, and the concentrations used exhibit no overt toxicity on human host cells. Garcinol also inhibitsPlasmodium falciparumasexual replication with a 50% inhibitory concentration (IC50) similar to that forToxoplasma Together, these data support that pharmacological inhibition of TgGCN5b leads to a catastrophic failure in gene expression control that prevents parasite replication. PMID:26810649

  20. The RecX protein interacts with the RecA protein and modulates its activity in Herbaspirillum seropedicae

    DNA repair is crucial to the survival of all organisms. The bacterial RecA protein is a central component in the SOS response and in recombinational and SOS DNA repairs. The RecX protein has been characterized as a negative modulator of RecA activity in many bacteria. The recA and recX genes of Herbaspirillum seropedicae constitute a single operon, and evidence suggests that RecX participates in SOS repair. In the present study, we show that the H. seropedicae RecX protein (RecXHs) can interact with the H. seropedicae RecA protein (RecAHs) and that RecAHs possesses ATP binding, ATP hydrolyzing and DNA strand exchange activities. RecXHs inhibited 90% of the RecAHs DNA strand exchange activity even when present in a 50-fold lower molar concentration than RecAHs. RecAHs ATP binding was not affected by the addition of RecX, but the ATPase activity was reduced. When RecXHs was present before the formation of RecA filaments (RecA-ssDNA), inhibition of ATPase activity was substantially reduced and excess ssDNA also partially suppressed this inhibition. The results suggest that the RecXHs protein negatively modulates the RecAHs activities by protein-protein interactions and also by DNA-protein interactions

  1. The RecX protein interacts with the RecA protein and modulates its activity in Herbaspirillum seropedicae

    C.W. Galvão

    2012-12-01

    Full Text Available DNA repair is crucial to the survival of all organisms. The bacterial RecA protein is a central component in the SOS response and in recombinational and SOS DNA repairs. The RecX protein has been characterized as a negative modulator of RecA activity in many bacteria. The recA and recX genes of Herbaspirillum seropedicae constitute a single operon, and evidence suggests that RecX participates in SOS repair. In the present study, we show that the H. seropedicae RecX protein (RecX Hs can interact with the H. seropedicaeRecA protein (RecA Hs and that RecA Hs possesses ATP binding, ATP hydrolyzing and DNA strand exchange activities. RecX Hs inhibited 90% of the RecA Hs DNA strand exchange activity even when present in a 50-fold lower molar concentration than RecA Hs. RecA Hs ATP binding was not affected by the addition of RecX, but the ATPase activity was reduced. When RecX Hs was present before the formation of RecA filaments (RecA-ssDNA, inhibition of ATPase activity was substantially reduced and excess ssDNA also partially suppressed this inhibition. The results suggest that the RecX Hs protein negatively modulates the RecA Hs activities by protein-protein interactions and also by DNA-protein interactions.

  2. The RecX protein interacts with the RecA protein and modulates its activity in Herbaspirillum seropedicae

    Galvão, C.W. [Departamento de Biologia Estrutural, Molecular e Genética, Universidade Estadual de Ponta Grossa, Ponta Grossa, PR (Brazil); Souza, E.M. [Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR (Brazil); Etto, R.M. [Departamento de Biologia Estrutural, Molecular e Genética, Universidade Estadual de Ponta Grossa, Ponta Grossa, PR (Brazil); Pedrosa, F.O.; Chubatsu, L.S.; Yates, M.G. [Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR (Brazil); Schumacher, J.; Buck, M. [Department of Life Sciences, Imperial College London, London (United Kingdom); Steffens, M.B.R. [Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR (Brazil)

    2012-10-15

    DNA repair is crucial to the survival of all organisms. The bacterial RecA protein is a central component in the SOS response and in recombinational and SOS DNA repairs. The RecX protein has been characterized as a negative modulator of RecA activity in many bacteria. The recA and recX genes of Herbaspirillum seropedicae constitute a single operon, and evidence suggests that RecX participates in SOS repair. In the present study, we show that the H. seropedicae RecX protein (RecX{sub Hs}) can interact with the H. seropedicae RecA protein (RecA{sub Hs}) and that RecA{sub Hs} possesses ATP binding, ATP hydrolyzing and DNA strand exchange activities. RecX{sub Hs} inhibited 90% of the RecA{sub Hs} DNA strand exchange activity even when present in a 50-fold lower molar concentration than RecA{sub Hs}. RecA{sub Hs} ATP binding was not affected by the addition of RecX, but the ATPase activity was reduced. When RecX{sub Hs} was present before the formation of RecA filaments (RecA-ssDNA), inhibition of ATPase activity was substantially reduced and excess ssDNA also partially suppressed this inhibition. The results suggest that the RecX{sub Hs} protein negatively modulates the RecA{sub Hs} activities by protein-protein interactions and also by DNA-protein interactions.

  3. Transcriptional Inhibition of Matrix Metal loproteinase 9 (MMP-9 Activity by a c-fos/Estrogen Receptor Fusion Protein is Mediated by the Proximal AP-1 Site of the MMP-9 Promoter and Correlates with Reduced Tumor Cell Invasion

    David L. Crowe

    1999-10-01

    Full Text Available Tumor cell invasion of basement membranes is one of the hallmarks of malignant transformation. Tumor cells secrete proteolytic enzymes known as matrix metalloproteinases (MMPs which degrade extracellular matrix molecules. Increased expression of MMP-9 has been associated with acquisition of invasive phenotype in many tumors. However, multiple mechanisms for regulation of MMP-9 gene expression by tumor cell lines have been proposed. A number of transcription factor binding sites have been characterized in the upstream regulatory region of the MMP-9 gene, including those for AP-1. To determine how a specific AP-1 family member, c-fos, regulates MMP-9 promoter activity through these sites, we used an expression vector containing the c-fos coding region fused to the estrogen receptor (ER ligand binding domain. This construct is activated upon binding estradiol. Stable expression of this construct in ER negative squamous cell carcinoma (SCC lines produced an estradiol dependent decrease in the number of cells that migrated through a reconstituted basement membrane. This decreased invasiveness was accompanied by estradiol dependent downregulation of MMP-9 activity as determined by gelatin zymography. Estradiol also produced transcriptional downregulation of an MMP-9 promoter construct in cells transiently transfected with the c-fosER expression vector. This downregulation was mediated by the AP-1 site at —79 by in the MMP-9 promoter. We concluded that the proximal AP-1 site mediated the transcriptional downregulation of the MMP-9 promoter by a conditionally activated c-fos fusion protein.

  4. Exosomes from myeloid derived suppressor cells carry biologically active proteins

    Burke, Meghan; Choksawangkarn, Waeowalee; Edwards, Nathan; Ostrand-Rosenberg, Suzanne; Fenselau, Catherine

    2013-01-01

    Myeloid-derived suppressor cells (MDSC) are present in most cancer patients where they inhibit natural anti-tumor immunity and are an obstacle to anti-cancer immunotherapies. They mediate immune suppression through their production of proteins and soluble mediators that prevent the activation of tumor-reactive T lymphyocytes, polarize macrophages towards a tumor-promoting phenotype, and facilitate angiogenesis. The accumulation and suppressive potency of MDSC is regulated by inflammation with...

  5. Interferon-inducible MyD88 protein inhibits hepatitis B virus replication

    Myeloid differential primary response protein (MyD88) is a critical component in the signaling cascade through Toll-like receptors (TLRs) and is induced by α interferon (IFN-α). To examine the role of MyD88 in the antiviral activity of IFN-α against hepatitis B virus (HBV), we established MyD88 stably expressing cell lines and studied HBV replication in these lines after transient transfection. The levels of HBV proteins and viral replicative intermediates were effectively reduced in MyD88-expressing cells. A significant reduction of total and cytoplasmic viral RNAs in MyD88 stably expressing cells was also observed. Using a nuclear factor-κB (NF-κB) dependent reporter assay, it was shown that activation of NF-κB was moderately increased in the presence of expression of MyD88, and further significantly increased by co-expression of HBV. These results suggest a novel mechanism for the inhibition of HBV replication by IFN-α via expression of MyD88 protein involving activation of NF-κB signaling pathway and downregulation of viral transcription

  6. Activity assay of membrane transport proteins

    Hao Xie

    2008-01-01

    Membrane transport proteins are integral membrane proteins and considered as potential drug targets. Activity assay of transport proteins is essential for developing drugs to target these proteins. Major issues related to activity assessment of transport proteins include availability of transporters,transport activity of transporters, and interactions between ligands and transporters. Researchers need to consider the physiological status of proteins (bound in lipid membranes or purified), availability and specificity of substrates, and the purpose of the activity assay (screening, identifying, or comparing substrates and inhibitors) before choosing appropriate assay strategies and techniques. Transport proteins bound in vesicular membranes can be assayed for transporting substrate across membranes by means of uptake assay or entrance counterflow assay. Alternatively, transport proteins can be assayed for interactions with ligands by using techniques such as isothermal titration calorimetry, nuclear magnetic resonance spectroscopy, or surface plasmon resonance. Other methods and techniques such as fluorometry, scintillation proximity assay, electrophysiological assay, or stopped-flow assay could also be used for activity assay of transport proteins. In this paper the major strategies and techniques for activity assessment of membrane transport proteins are reviewed.

  7. Doxycycline Indirectly Inhibits Proteolytic Activation of Tryptic Kallikrein-Related Peptidases and Activation of Cathelicidin

    Kanada, Kimberly N.; Nakatsuji, Teruaki; Richard L Gallo

    2012-01-01

    The increased abundance and activity of cathelicidin and kallikrein 5 (KLK5), a predominant trypsin-like serine protease (TLSP) in the stratum corneum, have been implicated in the pathogenesis of rosacea, a disorder treated by the use of low-dose doxycycline. Here we hypothesized that doxycycline can inhibit activation of tryptic KLKs through an indirect mechanism by inhibition of matrix metalloproteinases (MMPs) in keratinocytes. The capacity of doxycycline to directly inhibit enzyme activit...

  8. The effect of dextran to restore the activity of pulmonary surfactant inhibited by albumin.

    Lu, J J; Cheung, W W Y; Yu, L M Y; Policova, Z; Li, D; Hair, M L; Neumann, A W

    2002-04-01

    Pulmonary surfactant is crucial to maintain the proper functioning of the respiration system. Certain types of blood proteins (e.g. albumin) were found to inhibit the activity of pulmonary surfactant. Axisymmetric Drop Shape Analysis (ADSA) was used to study the effect of dextran to restore the activity of an albumin-inhibited pulmonary surfactant. It was found that dextran could effectively restore surface tension properties of the inhibited surfactant in vitro. Furthermore, dextran improved the performance of pulmonary surfactants when albumin was absent. It was found that when a surfactant film was under high compression (e.g. above 70% surface area reduction), the presence of dextran increased film stability, so that the film could sustain high surface pressures without being collapsing. PMID:12380007

  9. Oral protein kinase c β inhibition using ruboxistaurin

    Aiello, Lloyd Paul; Vignati, Louis; Sheetz, Matthew J;

    2011-01-01

    To evaluate efficacy, safety, and causes of vision loss among 813 patients (1,392 eyes) with moderately severe to very severe nonproliferative diabetic retinopathy from the Protein Kinase C β Inhibitor-Diabetic Retinopathy Study and Protein Kinase C β Inhibitor-Diabetic Retinopathy Study 2...... ruboxistaurin (RBX) protein kinase C β inhibitor trials....

  10. Irregular activity arises as a natural consequence of synaptic inhibition

    Terman, D., E-mail: terman@math.ohio-state.edu [Department of Mathematics, The Ohio State University, Columbus, Ohio 43210 (United States); Rubin, J. E., E-mail: jonrubin@pitt.edu [Department of Mathematics, University of Pittsburgh, Pittsburgh, Pennsylvania 15260 (United States); Diekman, C. O., E-mail: diekman@njit.edu [Department of Mathematical Sciences, New Jersey Institute of Technology, Newark, New Jersey 07102 (United States)

    2013-12-15

    Irregular neuronal activity is observed in a variety of brain regions and states. This work illustrates a novel mechanism by which irregular activity naturally emerges in two-cell neuronal networks featuring coupling by synaptic inhibition. We introduce a one-dimensional map that captures the irregular activity occurring in our simulations of conductance-based differential equations and mathematically analyze the instability of fixed points corresponding to synchronous and antiphase spiking for this map. We find that the irregular solutions that arise exhibit expansion, contraction, and folding in phase space, as expected in chaotic dynamics. Our analysis shows that these features are produced from the interplay of synaptic inhibition with sodium, potassium, and leak currents in a conductance-based framework and provides precise conditions on parameters that ensure that irregular activity will occur. In particular, the temporal details of spiking dynamics must be present for a model to exhibit this irregularity mechanism and must be considered analytically to capture these effects.

  11. Irregular activity arises as a natural consequence of synaptic inhibition

    Terman, D.; Rubin, J. E.; Diekman, C. O.

    2013-12-01

    Irregular neuronal activity is observed in a variety of brain regions and states. This work illustrates a novel mechanism by which irregular activity naturally emerges in two-cell neuronal networks featuring coupling by synaptic inhibition. We introduce a one-dimensional map that captures the irregular activity occurring in our simulations of conductance-based differential equations and mathematically analyze the instability of fixed points corresponding to synchronous and antiphase spiking for this map. We find that the irregular solutions that arise exhibit expansion, contraction, and folding in phase space, as expected in chaotic dynamics. Our analysis shows that these features are produced from the interplay of synaptic inhibition with sodium, potassium, and leak currents in a conductance-based framework and provides precise conditions on parameters that ensure that irregular activity will occur. In particular, the temporal details of spiking dynamics must be present for a model to exhibit this irregularity mechanism and must be considered analytically to capture these effects.

  12. Irregular activity arises as a natural consequence of synaptic inhibition

    Irregular neuronal activity is observed in a variety of brain regions and states. This work illustrates a novel mechanism by which irregular activity naturally emerges in two-cell neuronal networks featuring coupling by synaptic inhibition. We introduce a one-dimensional map that captures the irregular activity occurring in our simulations of conductance-based differential equations and mathematically analyze the instability of fixed points corresponding to synchronous and antiphase spiking for this map. We find that the irregular solutions that arise exhibit expansion, contraction, and folding in phase space, as expected in chaotic dynamics. Our analysis shows that these features are produced from the interplay of synaptic inhibition with sodium, potassium, and leak currents in a conductance-based framework and provides precise conditions on parameters that ensure that irregular activity will occur. In particular, the temporal details of spiking dynamics must be present for a model to exhibit this irregularity mechanism and must be considered analytically to capture these effects

  13. Studies of Dynamic Protein-Protein Interactions in Bacteria Using Renilla Luciferase Complementation Are Undermined by Nonspecific Enzyme Inhibition

    Hatzios, Stavroula-Artemis K.; Ringgaard, Simon; Davis, Brigid; Waldor, Matthew K.

    2012-01-01

    The luciferase protein fragment complementation assay is a powerful tool for studying protein-protein interactions. Two inactive fragments of luciferase are genetically fused to interacting proteins, and when these two proteins interact, the luciferase fragments can reversibly associate and reconstitute enzyme activity. Though this technology has been used extensively in live eukaryotic cells, split luciferase complementation has not yet been applied to studies of dynamic protein-protein inte...

  14. Minocycline and sulforaphane inhibited lipopolysaccharide-mediated retinal microglial activation

    Li-ping YANG; Zhu, Xiu-an; Tso, Mark O.M.

    2007-01-01

    Purpose To elucidate the inhibitory effect of minocycline and sulforaphane on lipopolysaccharide (LPS)-induced retinal microglial activation and the mechanisms through which they exerted their inhibitory effects. Methods Primary retinal microglial cultures were exposed to LPS with or without minocycline and sulforaphane. The mRNA expression of monocyte chemotactic protein (MCP)-1, MCP-3, macrophage inflammatory protein (MIP)-1α, MIP-1β, eotaxin, regulated upon activation normal T-cell express...

  15. Antioxidative activity of protein hydrolysates prepared from alkaline-aided channel catfish protein isolates.

    Theodore, Ann E; Raghavan, Sivakumar; Kristinsson, Hordur G

    2008-08-27

    Antioxidative activity of hydrolyzed protein prepared from alkali-solubilized catfish protein isolates was studied. The isolates were hydrolyzed to 5, 15, and 30% degree of hydrolysis using the protease enzyme, Protamex. Hydrolyzed protein was separated into hydrolysates and soluble supernatants, and both of these fractions were studied for their metal chelating ability, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging ability, ferric reducing antioxidant power (FRAP), oxygen radical absorbance capacity (ORAC), and their ability to inhibit the formation of thiobarbituric acid reactive substances (TBARS) in washed tilapia muscle containing tilapia hemolysate. Both hydrolysates and supernatants were characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results showed that DPPH radical scavenging ability and reducing power of catfish protein hydrolysates decreased, whereas the ORAC value, metal chelating ability, and ability to inhibit TBARS increased, with an increase in the degree of hydrolysis. Hydrolysate samples showed higher DPPH radical scavenging ability and Fe(3+) reducing ability, and supernatant samples had higher metal chelating ability. In general, low molecular weight (MW) peptides had high ORAC values and high metal chelating ability, and high MW peptides had a higher reducing power (FRAP) and were more effective in scavenging DPPH radicals. In a washed muscle model system, the ability of catfish protein hydrolysates and their corresponding supernatants to inhibit the formation of TBARS increased with an increase in the degree of hydrolysis. PMID:18662014

  16. Platelet Activation and Inhibition in Connection with Vascular Stents

    Christensen, Kjeld

    2007-01-01

    This thesis describes the Chandler loop, which makes it possible to conduct studies in vitro of molecular and cellular interactions between whole blood and stents. It was possible to monitor activation and inhibition of the cascades systems, leukocytes and platelets by combining different platelet inhibitors and heparin coating of stents. The clinical study was performed on patients with ACS undergoing PCI and stent implantation. In this study platelet activation markers P-selectin, and αIIb/...

  17. Transient Receptor Potential Vanilloid 4 Inhibits γ-Aminobutyric Acid-Activated Current in Hippocampal Pyramidal Neurons

    Hong, Zhiwen; Tian, Yujing; Qi, Mengwen; Li, Yingchun; Du, Yimei; Chen, Lei; Liu, Wentao; Chen, Ling

    2016-01-01

    The balance between excitatory and inhibitory neurotransmitter systems is crucial for the modulation of neuronal excitability in the central nervous system (CNS). The activation of transient receptor potential vanilloid 4 (TRPV4) is reported to enhance the response of hippocampal glutamate receptors, but whether the inhibitory neurotransmitter system can be regulated by TRPV4 remains unknown. γ-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the CNS. Here, we show that application of transient receptor potential vanilloid 4 (TRPV4) synthetic (GSK1016790A or 4α-PDD) or endogenous agonist (5,6-EET) inhibited GABA-activated current (IGABA) in hippocampal CA1 pyramidal neurons, which was blocked by specific antagonists of TRPV4 and of GABAA receptors. GSK1016790A increased the phosphorylated AMP-activated protein kinase (p-AMPK) and decreased the phosphorylated protein kinase B (p-Akt) protein levels, which was attenuated by removing extracellular calcium or by a calcium/calmodulin-dependent protein kinase kinase-β antagonist. GSK1016790A-induced decrease of p-Akt protein level was sensitive to an AMPK antagonist. GSK1016790A-inhibited IGABA was blocked by an AMPK antagonist or a phosphatidyl inositol 3 kinase (PI3K) agonist. GSK1016790A-induced inhibition of IGABA was also significantly attenuated by a protein kinase C (PKC) antagonist but was unaffected by protein kinase A or calcium/calmodulin-dependent protein kinase II antagonist. We conclude that activation of TRPV4 inhibits GABAA receptor, which may be mediated by activation of AMPK and subsequent down-regulation of PI3K/Akt signaling and activation of PKC signaling. Inhibition of GABAA receptors may account for the neuronal hyperexcitability caused by TRPV4 activation.

  18. Protein disulfide isomerase interacts with tau protein and inhibits its fibrillization.

    Li-Rong Xu

    Full Text Available BACKGROUND: Tau protein is implicated in the pathogenesis of neurodegenerative disorders such as tauopathies including Alzheimer disease, and Tau fibrillization is thought to be related to neuronal toxicity. Physiological inhibitors of Tau fibrillization hold promise for developing new strategies for treatment of Alzheimer disease. Because protein disulfide isomerase (PDI is both an enzyme and a chaperone, and implicated in neuroprotection against Alzheimer disease, we want to know whether PDI can prevent Tau fibrillization. In this study, we have investigated the interaction between PDI and Tau protein and the effect of PDI on Tau fibrillization. METHODOLOGY/PRINCIPAL FINDINGS: As evidenced by co-immunoprecipitation and confocal laser scanning microscopy, human PDI interacts and co-locates with some endogenous human Tau on the endoplasmic reticulum of undifferentiated SH-SY5Y neuroblastoma cells. The results from isothermal titration calorimetry show that one full-length human PDI binds to one full-length human Tau (or human Tau fragment Tau244-372 monomer with moderate, micromolar affinity at physiological pH and near physiological ionic strength. As revealed by thioflavin T binding assays, Sarkosyl-insoluble SDS-PAGE, and transmission electron microscopy, full-length human PDI remarkably inhibits both steps of nucleation and elongation of Tau244-372 fibrillization in a concentration-dependent manner. Furthermore, we find that two molecules of the a-domain of human PDI interact with one Tau244-372 molecule with sub-micromolar affinity, and inhibit both steps of nucleation and elongation of Tau244-372 fibrillization more strongly than full-length human PDI. CONCLUSIONS/SIGNIFICANCE: We demonstrate for the first time that human PDI binds to Tau protein mainly through its thioredoxin-like catalytic domain a, forming a 1∶1 complex and preventing Tau misfolding. Our findings suggest that PDI could act as a physiological inhibitor of Tau

  19. Inhibition of tyrosinase activity and melanine pigmentation by 2-hydroxytyrosol

    Ryuji Uchida

    2014-04-01

    Full Text Available 2-Hydroxytyrosol (2-HT, originally reported as a synthetic compound, was isolated for the first time as a fungal metabolite. 2-HT was found to inhibit mushroom tyrosinase with an IC50 value of 13.0 µmol/L. Furthermore, 2-HT dose-dependently inhibited tyrosinase activity (IC50, 32.5 µmol/L in the cell-free extract of B16 melanoma cells and α-melanocyte stimulating hormone (α-MSH-stimulated melanin formation in intact B16 melanoma cells.

  20. Knockdown of Peripheral Myelin Protein 22 Inhibits the Progression of Chronic Myeloid Leukemia.

    Liu, Hui; Cao, Hui-qin; Ta, Jin-bao; Zhang, Wen; Liu, Yu-hong

    2014-01-01

    We aimed to explore the underlying mechanism of peripheral myelin protein 22 (PMP22) in the development of chronic myeloid leukemia (CML). The level of PMP22 expression in CD34(+) cells isolated from CML patients' bone marrow samples (BMMCs) and peripheral blood samples (PBMCs) was determined by RT-PCR. In addition, PMP22-siRNA and scrambled control siRNA were transfected into human CML cell line K562 with Lipofectamine 2000 reagent. Cell viability and apoptosis were, respectively, determined by MTT assay and flow cytometry. Besides, the level of caspase 3 and Bcl-xL was then detected using Western blot. The level of PMP22 expression in CML patients' CD34(+) cells isolated from both PBMCs and BMMCs was significantly higher than the control group. PMP22 expression in K562 cells was successfully knocked down by siRNA. MTT analysis showed that knockdown of PMP22 inhibited the proliferation of CML cells. Flow cytometry showed that knockdown of PMP22 promoted the apoptosis of CML cells. Besides, Bcl-xL expression markedly decreased, while the expression of caspase 3 in CML cells significantly increased after knockdown of PMP22 expression. Our findings indicate that high expression of PMP22 may promote cell proliferation and inhibit cell apoptosis via upregulation of Bcl-xL or inhibition of caspase 3 activation, and thus may contribute to the development of CML. PMP22 may serve as a novel therapeutic target for the treatment of CML. PMID:26629937

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

    Ramón A. Lorca

    2011-01-01

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

  2. Dual modes of CLOCK:BMAL1 inhibition mediated by Cryptochrome and Period proteins in the mammalian circadian clock

    Ye, Rui; Selby, Cristopher P.; Chiou, Yi-Ying; Ozkan-Dagliyan, Irem; Gaddameedhi, Shobhan; Sancar, Aziz

    2014-01-01

    The mammalian circadian clock is based on a transcription–translation feedback loop in which CLOCK and BMAL1 proteins act as transcriptional activators of Cryptochrome and Period genes, which encode proteins that repress CLOCK–BMAL1 with a periodicity of ∼ 24 h. Ye et al. show that CRY binds to CLOCK–BMAL1 at the promoter and inhibits CLOCK–BMAL1-dependent transcription without dissociating the complex. PER alone has no effect on CLOCK–BMAL1-activated transcription, but in the presence of CRY...

  3. The VP3 structural protein of foot-and-mouth disease virus inhibits the IFN-β signaling pathway.

    Li, Dan; Yang, Wenping; Yang, Fan; Liu, Huanan; Zhu, Zixiang; Lian, Kaiqi; Lei, Caoqi; Li, Shu; Liu, Xiangtao; Zheng, Haixue; Shu, Hongbing

    2016-05-01

    Foot-and-mouth disease is a frequently occurring disease of cloven-hoofed animals that is caused by infection with the foot-and-mouth virus (FMDV). FMDV circumvents the type-I IFN response by expressing proteins that antagonize cellular innate immunity, such as leader protease and 3C protease. We identified the FMDV structural protein VP3 as a negative regulator of the virus-triggered IFN-β signaling pathway. Expression of FMDV VP3 inhibited the Sendai virus-triggered activation of IFN regulatory factor-3 and the expression of retinoic acid-inducible gene-I/melanoma differentiation-associated protein-5. Transient transfection and coimmunoprecipitation confirmed that the structural protein VP3 interacts with virus-induced signaling adapter (VISA), which is dependent on the C-terminal aa 111-220 of VP3. In addition, we found that FMDV VP3 inhibits the expression of VISA by disrupting its mRNA. Taken together, our findings reveal a novel strategy used by the structural VP3 protein of FMDV to evade host innate immunity.-Li, D., Yang, W., Yang, F., Liu, H., Zhu, Z., Lian, K., Lei, C., Li, S., Liu, X., Zheng, H., Shu, H. The VP3 structural protein of foot-and-mouth disease virus inhibits the IFN-β signaling pathway. PMID:26813975

  4. Activity-Based Protein Profiling of Microbes

    Sadler, Natalie C.; Wright, Aaron T.

    2015-02-01

    Activity-Based Protein Profiling (ABPP) in conjunction with multimodal characterization techniques has yielded impactful findings in microbiology, particularly in pathogen, bioenergy, drug discovery, and environmental research. Using small molecule chemical probes that react irreversibly with specific proteins or protein families in complex systems has provided insights in enzyme functions in central metabolic pathways, drug-protein interactions, and regulatory protein redox, for systems ranging from photoautotrophic cyanobacteria to mycobacteria, and combining live cell or cell extract ABPP with proteomics, molecular biology, modeling, and other techniques has greatly expanded our understanding of these systems. New opportunities for application of ABPP to microbial systems include: enhancing protein annotation, characterizing protein activities in myriad environments, and reveal signal transduction and regulatory mechanisms in microbial systems.

  5. Direct inhibition by nitric oxide of the transcriptional ferric uptake regulation protein via nitrosylation of the iron

    D'Autréaux, Benoît; Touati, Danièle; Bersch, Beate; Latour, Jean-Marc; Michaud-Soret, Isabelle

    2002-01-01

    Ferric uptake regulation protein (Fur) is a bacterial global regulator that uses iron as a cofactor to bind to specific DNA sequences. The function of Fur is not limited to iron homeostasis. A wide variety of genes involved in various mechanisms such as oxidative and acid stresses are under Fur control. Flavohemoglobin (Hmp) is an NO-detoxifying enzyme induced by NO and nitrosothiol compounds. Fur recently was found to regulate hmp in Salmonella typhimurium, and in Escherichia coli, the iron-chelating agent 2,2′-dipyridyl induces hmp expression. We now establish direct inhibition of E. coli Fur activity by NO. By using chromosomal Fur-regulated lacZ reporter fusion in E. coli, Fur activity is switched off by NO at micromolar concentration. In vitro Fur DNA-binding activity, as measured by protection of restriction site in aerobactin promoter, is directly sensitive to NO. NO reacts with FeII in purified FeFur protein to form a S = 1/2 low-spin FeFur–NO complex with a g = 2.03 EPR signal. Appearance of the same EPR signal in NO-treated cells links nitrosylation of the iron with Fur inhibition. The nitrosylated Fur protein is still a dimer and is stable in anaerobiosis but slowly decays in air. This inhibition probably arises from a conformational switch, leading to an inactive dimeric protein. These data establish a link between control of iron metabolism and the response to NO effects. PMID:12475930

  6. Gene activation by triplex-forming oligonucleotide coupled to the activating domain of protein VP16.

    Kuznetsova, S.; Ait-Si-Ali, S; Nagibneva, I; Troalen, F; Le Villain, J P; Harel-Bellan, A; Svinarchuk, F

    1999-01-01

    Triplex-forming oligonucleotides (TFOs) are generally designed to inhibit transcription or DNA replication but can be used for more diverse purposes. Here we have designed a chimera peptide-TFO able to activate transcription from a target gene. The designed hybrid molecule contains a triplex-forming sequence, linked through a phosphoroamidate bond to several minimal transcriptional activation domains derived from Herpes simplex virus protein 16 (VP16). We show here that this TFO-peptide chime...

  7. Transcriptional activation of the nitrogenase promoter in vitro: adenosine nucleotides are required for inhibition of NIFA activity by NIFL.

    Eydmann, T; Söderbäck, E; Jones, T; Hill, S; Austin, S; Dixon, R

    1995-03-01

    The enhancer-binding protein NIFA is required for transcriptional activation of nif promoters by the alternative holoenzyme form of RNA polymerase, which contains the sigma factor sigma 54 (sigma N). NIFA hydrolyzes nucleoside triphosphates to catalyze the isomerization of closed promoter complexes to transcriptionally competent open complexes. The activity of NIFA is antagonized by the regulatory protein NIFL in response to oxygen and fixed nitrogen in vivo. We have investigated the requirement for nucleotides in the formation and stability of open promoter complexes by NIFA and inhibition of its activity by NIFL at the Klebsiella pneumoniae nifH promoter. Open complexes formed by sigma 54-containing RNA polymerase are considerably more stable to heparin challenge in the presence of GTP than in the presence of ATP. This differential stability is most probably a consequence of GTP being the initiating nucleotide at this promoter. Adenosine nucleosides are specifically required for Azotobacter vinelandii NIFL to inhibit open complex formation by native NIFA, and the nucleoside triphosphatase activity of NIFA is strongly inhibited by NIFL under these conditions. We propose a model in which NIFL modulates the activity of NIFA via an adenosine nucleotide switch. PMID:7868590

  8. TATA binding protein associated factor 3 (TAF3 interacts with p53 and inhibits its function

    Tora Laszlo

    2008-06-01

    Full Text Available Abstract Background The tumour suppressor protein p53 is a sequence specific DNA-binding transcription regulator, which exerts its versatile roles in genome protection and apoptosis by affecting the expression of a large number of genes. In an attempt to obtain a better understanding of the mechanisms by which p53 transcription function is regulated, we studied p53 interactions. Results We identified BIP2 (Bric-à-brac interacting protein 2, the fly homolog of TAF3, a histone fold and a plant homeodomain containing subunit of TFIID, as an interacting partner of Drosophila melanogaster p53 (Dmp53. We detected physical interaction between the C terminus of Dmp53 and the central region of TAF3 both in yeast two hybrid assays and in vitro. Interestingly, DmTAF3 can also interact with human p53, and mammalian TAF3 can bind to both Dmp53 and human p53. This evolutionarily conserved interaction is functionally significant, since elevated TAF3 expression severely and selectively inhibits transcription activation by p53 in human cell lines, and it decreases the level of the p53 protein as well. Conclusion We identified TAF3 as an evolutionarily conserved negative regulator of p53 transcription activation function.

  9. Inhibition of tristetraprolin deadenylation by poly(A) binding protein

    Rowlett, Robert M.; Chrestensen, Carol A.; Schroeder, Melanie J.; Harp, Mary G.; Pelo, Jared W.; Shabanowitz, Jeffery; DeRose, Robert; Hunt, Donald F.; Sturgill, Thomas W.; Worthington, Mark T.

    2008-01-01

    Tristetraprolin (TTP) is the prototype for a family of RNA binding proteins that bind the tumor necrosis factor (TNF) messenger RNA AU-rich element (ARE), causing deadenylation of the TNF poly(A) tail, RNA decay, and silencing of TNF protein production. Using mass spectrometry sequencing we identified poly(A) binding proteins-1 and -4 (PABP1 and PABP4) in high abundance and good protein coverage from TTP immunoprecipitates. PABP1 significantly enhanced TNF ARE binding by RNA EMSA and prevente...

  10. Legionella pneumophila infection induces programmed cell death, caspase activation, and release of high-mobility group box 1 protein in A549 alveolar epithelial cells: inhibition by methyl prednisolone

    Koide Michio

    2008-05-01

    Full Text Available Abstract Background Legionella pneumophila pneumonia often exacerbates acute lung injury (ALI and acute respiratory distress syndrome (ARDS. Apoptosis of alveolar epithelial cells is considered to play an important role in the pathogenesis of ALI and ARDS. In this study, we investigated the precise mechanism by which A549 alveolar epithelial cells induced by L. pneumophila undergo apoptosis. We also studied the effect of methyl prednisolone on apoptosis in these cells. Methods Nuclear deoxyribonucleic acid (DNA fragmentation and caspase activation in L. pneumophila-infected A549 alveolar epithelial cells were assessed using the terminal deoxyribonucleotidyl transferase-mediated triphosphate (dUTP-biotin nick end labeling method (TUNEL method and colorimetric caspase activity assays. The virulent L. pneumophila strain AA100jm and the avirulent dotO mutant were used and compared in this study. In addition, we investigated whether methyl prednisolone has any influence on nuclear DNA fragmentation and caspase activation in A549 alveolar epithelial cells infected with L. pneumophila. Results The virulent strain of L. pneumophila grew within A549 alveolar epithelial cells and induced subsequent cell death in a dose-dependent manner. The avirulent strain dotO mutant showed no such effect. The virulent strains of L. pneumophila induced DNA fragmentation (shown by TUNEL staining and activation of caspases 3, 8, 9, and 1 in A549 cells, while the avirulent strain did not. High-mobility group box 1 (HMGB1 protein was released from A549 cells infected with virulent Legionella. Methyl prednisolone (53.4 μM did not influence the intracellular growth of L. pneumophila within alveolar epithelial cells, but affected DNA fragmentation and caspase activation of infected A549 cells. Conclusion Infection of A549 alveolar epithelial cells with L. pneumophila caused programmed cell death, activation of various caspases, and release of HMGB1. The dot/icm system, a

  11. GPER inhibits diabetes-mediated RhoA activation to prevent vascular endothelial dysfunction.

    Li, Zilin; Cheng, Liang; Liang, Hongliang; Duan, Weixun; Hu, Jing; Zhi, Weiwei; Yang, Jinbao; Liu, Zhenhua; Zhao, Minggao; Liu, Jincheng

    2016-02-01

    The effect of estrogen receptors on diabetes-induced vascular dysfunction is critical, but ambiguous. Individuals with diabetic vascular disease may require estrogen receptor-specific targeted therapy in the future. The G protein-coupled estrogen receptor (GPER) has beneficial effects on vascular function. However, its fundamental mechanisms are unclear. The RhoA/Rho-kinase pathway contributes to diabetic vascular complications, whereas estrogen can suppress Rho-kinase function. Thus, we assumed that GPER inhibits diabetes-mediated RhoA activation to prevent vascular dysfunction. We further investigated the underlying mechanisms involved in this process. Vascular endothelial cells and ex vivo cultured ovariectomized (OVX) C57BL/6 mouse aortae were treated with high glucose (HG) alone or in combination with GPER agonist (G1). G1 treatment was also administered to OVX db/db mice for 8 weeks. An ex-vivo isovolumic myograph was used to analyze the endothelium-dependent vasodilation and endothelium-independent contraction of mouse aortae. Apoptosis, oxidative stress, and inflammation were attenuated in G1-pretreated vascular endothelial cells. G1 significantly decreased the phosphorylation of inhibitory endothelial nitric oxide (NO) synthase residue threonine 495 (eNOS Thr495), inhibited RhoA expression, and increased NO production. Additionally, G1 rescued the impaired endothelium-dependent relaxation and inhibited RhoA activation in the thoracic aorta of OVX db/db mice and ex-vivo cultured OVX C57BL/6 mouse aortae treated with HG. Estrogens acting via GPER could protect vascular endothelium, and GPER activation might elicit ERα-independent effect to inhibit RhoA/Rho-kinase pathway. Additionally, GPER activation might reduce vascular smooth muscle contraction by inhibiting RhoA activation. Thus, the results of the present study suggest a new therapeutic paradigm for end-stage vascular dysfunction by inhibiting RhoA/Rho-kinase pathway via GPER activation. PMID:26785611

  12. Grb-IR: A SH2-Domain-Containing Protein that Binds to the Insulin Receptor and Inhibits Its Function

    Liu, Feng; Roth, Richard A.

    1995-10-01

    To identify potential signaling molecules involved in mediating insulin-induced biological responses, a yeast two-hybrid screen was performed with the cytoplasmic domain of the human insulin receptor (IR) as bait to trap high-affinity interacting proteins encoded by human liver or HeLa cDNA libraries. A SH2-domain-containing protein was identified that binds with high affinity in vitro to the autophosphorylated IR. The mRNA for this protein was found by Northern blot analyses to be highest in skeletal muscle and was also detected in fat by PCR. To study the role of this protein in insulin signaling, a full-length cDNA encoding this protein (called Grb-IR) was isolated and stably expressed in Chinese hamster ovary cells overexpressing the human IR. Insulin treatment of these cells resulted in the in situ formation of a complex of the IR and the 60-kDa Grb-IR. Although almost 75% of the Grb-IR protein was bound to the IR, it was only weakly tyrosine-phosphorylated. The formation of this complex appeared to inhibit the insulin-induced increase in tyrosine phosphorylation of two endogenous substrates, a 60-kDa GTPase-activating-protein-associated protein and, to a lesser extent, IR substrate 1. The subsequent association of this latter protein with phosphatidylinositol 3-kinase also appeared to be inhibited. These findings raise the possibility that Grb-IR is a SH2-domain-containing protein that directly complexes with the IR and serves to inhibit signaling or redirect the IR signaling pathway.

  13. Adenovirus E4orf6 protein inhibits DNA repair and radiosensitizes human tumor cells

    Full text: Double strand break repair (DSBR), although vital to normal cell survival and genomic stability, limits tumor cell kill following treatment with ionizing radiation (IR). The primary mechanism for DSBR in mammalian cells, non-homologous end joining (NHEJ), requires multiple proteins, one of which is DNA-dependent protein kinase (DNA-PK). Cells deficient in DNA-PK, although phenotypically normal, are among the most radiosensitive cells available. It has previously been shown that the E4orf6 gene product of adenovirus type 5 interacts with and inhibits the activity of DNA-PK. Therefore, we hypothesized that E4orf6, by interacting with DNA-PK, would inhibit the DSBR capacity of tumor cells and thus increase tumor cell kill upon treatment with IR. Stable clones expressing either wild type E4orf6, an E4orf6 mutant (L245P) that is defective at E1B-55K localization to the nucleus, or a neomycin control vector were established in colorectal carcinoma (RKO) cells. Based on clonogenic assays, we report a 10-fold increase in radiosensitivity of the wild type E4orf6 expressing clones at 6Gy of IR compared to both the neomycin and L245P mutant clones. Furthermore, the increase in sensitivity correlates with inhibition in DSBR based on sub-lethal damage repair assay. Preliminary data suggests that the transfected E4orf6 interacts with the endogenous DNA-PK and this results in a 20% decrease in the kinase activity of the DNA-PK compared to neomycin expressing control cells. These results indicate that E4orf6 radiosensitizes tumor cells by inhibiting their DSBR activity. We have constructed an adenoviral vector expressing E4orf6 in a tetracycline-inducible manner, which provides temporal control for E4orf6 expression. We are currently investigating the radiosensitizing properties of this expression vector. Successful use of this vector in vitro and in mouse xenografts, will set the stage for its future use in conjunction with localized radiotherapy of radioresistant

  14. Inhibition of PKB/Akt activity involved in apigenin-induced apoptosis in human gastric carcinoma cells

    YUAN LinHong; XIA Wei; ZHAO XiuJuan; ZHANG XiaoHua; ZHANG Ling; WU Kun

    2007-01-01

    Apigenin is a flavonoid widely distributed in fruits and vegetables.It possesses growth inhibitory properties against numerous cancer cell lines.However, the molecular mechanism(s) by which apigenin elicits its effects have not been fully elucidated.Here we studied whether apigenin inhibits growth and induces apoptosis in human gastric carcinoma cells.We showed that the flavonoid inhibited growth of the cells and caused apoptosis, as evidenced by DNA Ladder, cleavage of pro-caspase-3 in a time-dependent manner.Induction of apoptosis was dependent on inhibition of the PKB/Akt activity.We found that while apigenin had no effect on the expression of Akt and Bad, it inhibited specific phosphorylation of the two proteins that are associated with pro-survival mechanisms.We propose that this important flavonoid induces apoptosis in gastric cancer cells by inhibiting Akt activity.Since Akt is often activated in cancers, our findings may have clinical implications.

  15. Inhibition of ABCG2-mediated transport by protein kinase inhibitors with a bisindolylmaleimide or indolocarbazole structure.

    Robey, Robert W; Shukla, Suneet; Steadman, Kenneth; Obrzut, Tomasz; Finley, Elizabeth M; Ambudkar, Suresh V; Bates, Susan E

    2007-06-01

    ABCG2 is a transporter with potential importance in cancer drug resistance, drug oral absorption, and stem cell biology. In an effort to identify novel inhibitors of ABCG2, we examined the ability of commercially available bisindolylmaleimides (BIM) and indolocarbazole protein kinase inhibitors (PKI) to inhibit ABCG2, given the previous demonstration that the indolocarbazole PKI UCN-01 interacted with the transporter. At a concentration of 10 micromol/L, all of the compounds tested increased intracellular fluorescence of the ABCG2-specific substrate pheophorbide a in ABCG2-transfected HEK-293 cells by 1.3- to 6-fold as measured by flow cytometry; the ABCG2-specific inhibitor fumitremorgin C increased intracellular fluorescence by 6.6-fold. In 4-day cytotoxicity assays, wild-type ABCG2-transfected cells were not more than 2-fold resistant to any of the compounds, suggesting that the PKIs are not significantly transported by ABCG2. BIMs I, II, III, IV, and V, K252c, and arcyriaflavin A were also able to inhibit [(125)I]iodoarylazidoprazosin labeling of ABCG2 by 65% to 80% at 20 micromol/L, compared with a 50% to 70% reduction by 20 micromol/L fumitremorgin C. K252c and arcyriaflavin A were the most potent compounds, with IC(50) values for inhibition of [(125)I]iodoarylazidoprazosin labeling of 0.37 and 0.23 micromol/L, respectively. K252c and arcyriaflavin A did not have any effect on the ATPase activity of ABCG2. Four minimally toxic compounds--BIM IV, BIM V, arcyriaflavin A, and K252c-reduced the relative resistance of ABCG2-transfected cells to SN-38 in cytotoxicity assays. We find that indolocarbazole and BIM PKIs directly interact with the ABCG2 protein and may thus increase oral bioavailability of ABCG2 substrates. PMID:17575116

  16. Luteolin, a flavonoid, inhibits AP-1 activation by basophils

    Flavonoids including luteolin, apigenin, and fisetin are inhibitors of IL-4 synthesis and CD40 ligand expression by basophils. This study was done to search for compounds with greater inhibitory activity of IL-4 expression and to clarify the molecular mechanisms through which flavonoids inhibit their expression. Of the 37 flavonoids and related compounds examined, ayanin, luteolin, and apigenin were the strongest inhibitors of IL-4 production by purified basophils in response to anti-IgE antibody plus IL-3. Luteolin did not suppress Syk or Lyn phosphorylation in basophils, nor did suppress p54/46 SAPK/JNK, p38 MAPK, and p44/42 MAPK activation by a basophilic cell line, KU812 cells, stimulated with A23187 and PMA. However, luteolin did inhibit phosphorylation of c-Jun and DNA binding activity of AP-1 in nuclear lysates from stimulated KU812 cells. These results provide a fundamental structure of flavonoids for IL-4 inhibition and demonstrate a novel action of flavonoids that suppresses the activation of AP-1

  17. 14–3-3 Inhibits the Dictyostelium Myosin II Heavy-Chain-specific Protein Kinase C Activity by a Direct Interaction: Identification of the 14–3-3 Binding Domain

    Matto-Yelin, Meirav; Aitken, Alastair; Ravid, Shoshana

    1997-01-01

    Myosin II heavy chain (MHC) specific protein kinase C (MHC-PKC), isolated from Dictyostelium discoideum, regulates myosin II assembly and localization in response to the chemoattractant cyclic AMP. Immunoprecipitation of MHC-PKC revealed that it resides as a complex with several proteins. We show herein that one of these proteins is a homologue of the 14–3-3 protein (Dd14–3-3). This protein has recently been implicated in the regulation of intracellular signaling pathways via its interaction ...

  18. In silico prediction of inhibition of promiscuous breast cancer resistance protein (BCRP/ABCG2.

    Yi-Lung Ding

    Full Text Available Breast cancer resistant protein has an essential role in active transport of endogenous substances and xenobiotics across extracellular and intracellular membranes along with P-glycoprotein. It also plays a major role in multiple drug resistance and permeation of blood-brain barrier. Therefore, it is of great importance to derive theoretical models to predict the inhibition of both transporters in the process of drug discovery and development. Hitherto, very limited BCRP inhibition predictive models have been proposed as compared with its P-gp counterpart.An in silico BCRP inhibition model was developed in this study using the pharmacophore ensemble/support vector machine scheme to take into account the promiscuous nature of BCRP. The predictions by the PhE/SVM model were found to be in good agreement with the observed values for those molecules in the training set (n= 22, r2 =0.82, qCV2=0.73, RMSE= 0.40, s = 0.24, test set (n =97, q2=0.75-0.89, RMSE= 0.31, s= 0.21, and outlier set (n= 16, q2 =0.72-0.91, RMSE= 0.29, s=0.17. When subjected to a variety of statistical validations, the developed PhE/SVM model consistently met the most stringent criteria. A mock test by HIV protease inhibitors also asserted its predictivity.It was found that this accurate, fast, and robust PhE/SVM model can be employed to predict the BCRP inhibition of structurally diverse molecules that otherwise cannot be carried out by any other methods in a high-throughput fashion to design therapeutic agents with insignificant drug toxicity and unfavorable drug-drug interactions mediated by BCRP to enhance clinical efficacy and/or circumvent drug resistance.

  19. Serum protein inhibition of thyrotropin binding to human thyroid tissue. [/sup 125/I tracer technique

    Beall, G.N.; Chopra, I.J.; Solomon, D.H.; Kruger, S.R.

    1978-11-01

    We used a modificaton of the TSH radioreceptor assay to detect TSH-binding inhibition (TBI) activity in serum and serum fractions from normal subjects and patients with Graves' disease. TBI activity is present in normal IgG prepared by DEAE-Sephadex chromatography and in normal globulins prepared by precipitation at 1.6 M ammonium sulfate. Other normal serum proteins also had TBI activity when large concentrations were tested. Gel filtration chromatography and powder block electrophoresis were used to prepare fractions of normal and Graves' disease sera. In these fractions from normal serum, TBI activity was found in both ..gamma..-globulin and ..cap alpha..-globulin-albumin fractions electrophoretically and in both 7S and 4S peaks from gel filtration. TBI activity from Graves' disease patients' sera was similarly distributed, but relatively more TBI accompanied the electrophoretic ..gamma..-globulins. Sepharose Protein-A and anti-IgG were used as immunoabsorbents to isolate and purify IgG from normal and Graves' disease sera. TBI activity in IgG was proportional to the IgG concentration, indicating that the TBI which migrates as a ..gamma..-globulin electrophoretically is an IgG and thus may possibly be an antibody. Inhibitory activity found in normal serum globulins and in the non-IgG fractions of both normal and abnormal sera seriously interferes with attempts to use the TSH radioreceptor assay to study the hypothesized anti-TSH receptor antibody in the serum of patients with Graves' disease.

  20. Salicylate, diflunisal and their metabolites inhibit CBP/p300 and exhibit anticancer activity.

    Shirakawa, Kotaro; Wang, Lan; Man, Na; Maksimoska, Jasna; Sorum, Alexander W; Lim, Hyung W; Lee, Intelly S; Shimazu, Tadahiro; Newman, John C; Schröder, Sebastian; Ott, Melanie; Marmorstein, Ronen; Meier, Jordan; Nimer, Stephen; Verdin, Eric

    2016-01-01

    Salicylate and acetylsalicylic acid are potent and widely used anti-inflammatory drugs. They are thought to exert their therapeutic effects through multiple mechanisms, including the inhibition of cyclo-oxygenases, modulation of NF-κB activity, and direct activation of AMPK. However, the full spectrum of their activities is incompletely understood. Here we show that salicylate specifically inhibits CBP and p300 lysine acetyltransferase activity in vitro by direct competition with acetyl-Coenzyme A at the catalytic site. We used a chemical structure-similarity search to identify another anti-inflammatory drug, diflunisal, that inhibits p300 more potently than salicylate. At concentrations attainable in human plasma after oral administration, both salicylate and diflunisal blocked the acetylation of lysine residues on histone and non-histone proteins in cells. Finally, we found that diflunisal suppressed the growth of p300-dependent leukemia cell lines expressing AML1-ETO fusion protein in vitro and in vivo. These results highlight a novel epigenetic regulatory mechanism of action for salicylate and derivative drugs. PMID:27244239

  1. Inhibition of Cleavage of a Plant Viral Polyprotein by an Inhibitor Activity Present in Wheat Germ and Cowpea Embryos †

    Shih, Ding S.; Bu, Ming; Price, Mary A.; Shih, Chao-Yun T.

    1987-01-01

    In rabbit reticulocyte lysate, the bottom component RNA of cowpea mosaic virus directs the synthesis of a 200,000-molecular-weight precursor protein (200K protein) that is cleaved during synthesis by a reticulocyte enzyme to form a 32K protein and a 170K protein. Cleavage of the 200K protein was found to be effectively inhibited by inhibitor activity in wheat germ and cowpea embryo extracts. The inhibitor was nondialyzable, precipitatable by ammonium sulfate, and partially stable at high temp...

  2. Molecular Mechanism for Inhibition of G Protein-Coupled Receptor Kinase 2 by a Selective RNA Aptamer

    Tesmer, Valerie M.; Lennarz, Sabine; Mayer, Günter; Tesmer, John J.G. (Bonn); (Michigan)

    2012-08-31

    Cardiovascular homeostasis is maintained in part by the rapid desensitization of activated heptahelical receptors that have been phosphorylated by G protein-coupled receptor kinase 2 (GRK2). However, during chronic heart failure GRK2 is upregulated and believed to contribute to disease progression. We have determined crystallographic structures of GRK2 bound to an RNA aptamer that potently and selectively inhibits kinase activity. Key to the mechanism of inhibition is the positioning of an adenine nucleotide into the ATP-binding pocket and interactions with the basic {alpha}F-{alpha}G loop region of the GRK2 kinase domain. Constraints imposed on the RNA by the terminal stem of the aptamer also play a role. These results highlight how a high-affinity aptamer can be used to selectively trap a novel conformational state of a protein kinase.

  3. Oligomerization Inhibits Legionella pneumophila PlaB Phospholipase A Activity*

    Kuhle, Katja; Krausze, Joern; Curth, Ute; Rössle, Manfred; Heuner, Klaus; Lang, Christina; Flieger, Antje

    2014-01-01

    The intracellularly replicating lung pathogen Legionella pneumophila consists of an extraordinary variety of phospholipases, including at least 15 different phospholipases A (PLA). Among them, PlaB, the first characterized member of a novel lipase family, is a hemolytic virulence factor that exhibits the most prominent PLA activity in L. pneumophila. We analyzed here protein oligomerization, the importance of oligomerization for activity, addressed further essential regions for activity withi...

  4. Picomolar-affinity binding and inhibition of adenylate cyclase activity by melatonin in Syrian hamster hypothalamus

    1. The effect of melatonin on forskolin-stimulated adenylate cyclase activity was measured in homogenates of Syrian hamster hypothalamus. In addition, the saturation binding characteristics of the melatonin receptor ligand, [125I]iodomelatonin, was examined using an incubation temperature (30 degree C) similar to that used in enzyme assays. 2. At concentrations ranging from 10 pM to 1 nM, melatonin caused a significant decrease in stimulated adenylate cyclase activity with a maximum inhibition of approximately 22%. 3. Binding experiments utilizing [125I]iodomelatonin in a range of approximately 5-80 pM indicated a single class of high-affinity sites: Kd = 55 +/- 9 pM, Bmax = 1.1 +/- 0.3 fmol/mg protein. 4. The ability of picomolar concentrations of melatonin to inhibit forskolin-stimulated adenylate cyclase activity suggests that this affect is mediated by picomolar-affinity receptor binding sites for this hormone in the hypothalamus

  5. Spillover-mediated feedforward-inhibition functionally segregates interneuron activity

    Coddington, Luke T.; Rudolph, Stephanie; Lune, Patrick Vande; Overstreet-Wadiche, Linda; Wadiche, Jacques I.

    2013-01-01

    Summary Neurotransmitter spillover represents a form of neural transmission not restricted to morphologically defined synaptic connections. Communication between climbing fibers (CFs) and molecular layer interneurons (MLIs) in the cerebellum is mediated exclusively by glutamate spillover. Here, we show how CF stimulation functionally segregates MLIs based on their location relative to glutamate release. Excitation of MLIs that reside within the domain of spillover diffusion coordinates inhibition of MLIs outside the diffusion limit. CF excitation of MLIs is dependent on extrasynaptic NMDA receptors that enhance the spatial and temporal spread of CF signaling. Activity mediated by functionally segregated MLIs converges onto neighboring Purkinje cells (PCs) to generate a long-lasting biphasic change in inhibition. These data demonstrate how glutamate release from single CFs modulates excitability of neighboring PCs, thus expanding the influence of CFs on cerebellar cortical activity in a manner not predicted by anatomical connectivity. PMID:23707614

  6. Prostaglandin E2 Inhibits NLRP3 Inflammasome Activation through EP4 Receptor and Intracellular Cyclic AMP in Human Macrophages.

    Sokolowska, Milena; Chen, Li-Yuan; Liu, Yueqin; Martinez-Anton, Asuncion; Qi, Hai-Yan; Logun, Carolea; Alsaaty, Sara; Park, Yong Hwan; Kastner, Daniel L; Chae, Jae Jin; Shelhamer, James H

    2015-06-01

    PGE2 is a potent lipid mediator involved in maintaining homeostasis but also promotion of acute inflammation or immune suppression in chronic inflammation and cancer. Nucleotide-binding domain, leucine-rich repeat-containing protein (NLR)P3 inflammasome plays an important role in host defense. Uncontrolled activation of the NLRP3 inflammasome, owing to mutations in the NLRP3 gene, causes cryopyrin-associated periodic syndromes. In this study, we showed that NLRP3 inflammasome activation is inhibited by PGE2 in human primary monocyte-derived macrophages. This effect was mediated through PGE2 receptor subtype 4 (EP4) and an increase in intracellular cAMP, independently of protein kinase A or exchange protein directly activated by cAMP. A specific agonist of EP4 mimicked, whereas its antagonist or EP4 knockdown reversed, PGE2-mediated NLRP3 inhibition. PGE2 caused an increase in intracellular cAMP. Blockade of adenylate cyclase by its inhibitor reversed PGE2-mediated NLRP3 inhibition. Increase of intracellular cAMP by an activator of adenylate cyclase or an analog of cAMP, or a blockade of cAMP degradation by phosphodiesterase inhibitor decreased NLRP3 activation. Protein kinase A or exchange protein directly activated by cAMP agonists did not mimic, and their antagonists did not reverse, PGE2-mediated NLRP3 inhibition. Additionally, constitutive IL-1β secretion from LPS-primed PBMCs of cryopyrin-associated periodic fever syndromes patients was substantially reduced by high doses of PGE2. Moreover, blocking cytosolic phospholipase A2α by its inhibitor or small interfering RNA or inhibiting cyclooxygenase 2, resulting in inhibition of endogenous PGE2 production, caused an increase in NLRP3 inflammasome activation. Our results suggest that PGE2 might play a role in maintaining homeostasis during the resolution phase of inflammation and might serve as an autocrine and paracrine regulator. PMID:25917098

  7. Wogonin inhibits tumor angiogenesis via degradation of HIF-1α protein

    Wogonin, a plant-derived flavone, has been shown recently to have antitumor effects. However, the mechanisms that wogonin inhibits tumor angiogenesis are not well known. In this study, we investigated the effects of wogonin on expression of hypoxia-inducible factor-1α (HIF-1α) and secretion of vascular endothelial growth factor (VEGF) in tumor cells. We found that wogonin decreased the expression of HIF-1α by affecting its stability and reduced the secretion of VEGF, which suppressed angiogenesis in cancer. Wogonin promoted the degradation of HIF-1α by increasing its prolyl hydroxylation, which depended on prolyl hydroxylase (PHD) and the von Hippel–Lindau tumor suppressor (VHL). Intriguingly, wogonin impeded the binding between heat-shock protein 90 (Hsp90) and HIF-1α. In addition, wogonin down-regulated the Hsp90 client proteins EGFR, Cdk4 and survivin, but did not affect the level of Hsp90. Wogonin also increased ubiquitination of HIF-1α and promoted its degradation in proteasome. We also found that wogonin could inhibit nuclear translocation of HIF-1α. Electrophoresis mobility shift assay (EMSA) showed that wogonin decreased the binding activity of exogenous consensus DNA oligonucleotide with HIF-1α in nuclear extracts from MCF-7 cells. Chromatin immunoprecipitation (ChIP) assay also revealed that HIF-1α directly binded to endogenous hypoxia-responsive element (HRE) and this binding was significantly decreased in MCF-7 cells treated with wogonin. Preliminary results indicated in vivo activity of wogonin against xenograft-induced angiogenesis in nude mice. Taken together, the results suggested that wogonin was a potent inhibitor of HIF-1α and provided a new insight into the mechanisms of wogonin against cancers. - Highlights: • Wogonin is an all around inhibitor of VEGF signaling. • We firstly demonstrate that wogonin inhibits secretion of VEGF by decreasing HIF-1α. • Wogonin enhances PDH and VHL expression and inhibits Hsp90 function.

  8. Uremic toxins inhibit transport by breast cancer resistance protein and multidrug resistance protein 4 at clinically relevant concentrations.

    Henricus A M Mutsaers

    Full Text Available During chronic kidney disease (CKD, there is a progressive accumulation of toxic solutes due to inadequate renal clearance. Here, the interaction between uremic toxins and two important efflux pumps, viz. multidrug resistance protein 4 (MRP4 and breast cancer resistance protein (BCRP was investigated. Membrane vesicles isolated from MRP4- or BCRP-overexpressing human embryonic kidney cells were used to study the impact of uremic toxins on substrate specific uptake. Furthermore, the concentrations of various uremic toxins were determined in plasma of CKD patients using high performance liquid chromatography and liquid chromatography/tandem mass spectrometry. Our results show that hippuric acid, indoxyl sulfate and kynurenic acid inhibit MRP4-mediated [(3H]-methotrexate ([(3H]-MTX uptake (calculated Ki values: 2.5 mM, 1 mM, 25 µM, respectively and BCRP-mediated [(3H]-estrone sulfate ([(3H]-E1S uptake (Ki values: 4 mM, 500 µM and 50 µM, respectively, whereas indole-3-acetic acid and phenylacetic acid reduce [(3H]-MTX uptake by MRP4 only (Ki value: 2 mM and IC(50 value: 7 mM, respectively. In contrast, p-cresol, p-toluenesulfonic acid, putrescine, oxalate and quinolinic acid did not alter transport mediated by MRP4 or BCRP. In addition, our results show that hippuric acid, indole-3-acetic acid, indoxyl sulfate, kynurenic acid and phenylacetic acid accumulate in plasma of end-stage CKD patients with mean concentrations of 160 µM, 4 µM, 129 µM, 1 µM and 18 µM, respectively. Moreover, calculated Ki values are below the maximal plasma concentrations of the tested toxins. In conclusion, this study shows that several uremic toxins inhibit active transport by MRP4 and BCRP at clinically relevant concentrations.

  9. Platelet factor 4 impairs the anticoagulant activity of activated protein C.

    Preston, Roger J S

    2012-02-01

    Platelet factor 4 (PF4) is an abundant platelet alpha-granule chemokine released following platelet activation. PF4 interacts with thrombomodulin and the gamma-carboxyglutamic acid (Gla) domain of protein C, thereby enhancing activated protein C (APC) generation by the thrombin-thrombomodulin complex. However, the protein C Gla domain not only mediates protein C activation in vivo, but also plays a critical role in modulating the diverse functional properties of APC once generated. In this study we demonstrate that PF4 significantly inhibits APC anti-coagulant activity. PF4 inhibited both protein S-dependent APC anticoagulant function in plasma and protein S-dependent factor Va (FVa) proteolysis 3- to 5-fold, demonstrating that PF4 impairs protein S cofactor enhancement of APC anticoagulant function. Using recombinant factor Va variants FVa-R506Q\\/R679Q and FVa-R306Q\\/R679Q, PF4 was shown to impair APC proteolysis of FVa at position Arg(306) by 3-fold both in the presence and absence of protein S. These data suggest that PF4 contributes to the poorly understood APC resistance phenotype associated with activated platelets. Finally, despite PF4 binding to the APC Gla domain, we show that APC in the presence of PF4 retains its ability to initiate PAR-1-mediated cytoprotective signaling. In summary, we propose that PF4 acts as a critical regulator of APC generation, but also differentially targets APC toward cytoprotective, rather than anticoagulant function at sites of vascular injury with concurrent platelet activation.

  10. Platelet factor 4 impairs the anticoagulant activity of activated protein C.

    Preston, Roger J S

    2009-02-27

    Platelet factor 4 (PF4) is an abundant platelet alpha-granule chemokine released following platelet activation. PF4 interacts with thrombomodulin and the gamma-carboxyglutamic acid (Gla) domain of protein C, thereby enhancing activated protein C (APC) generation by the thrombin-thrombomodulin complex. However, the protein C Gla domain not only mediates protein C activation in vivo, but also plays a critical role in modulating the diverse functional properties of APC once generated. In this study we demonstrate that PF4 significantly inhibits APC anti-coagulant activity. PF4 inhibited both protein S-dependent APC anticoagulant function in plasma and protein S-dependent factor Va (FVa) proteolysis 3- to 5-fold, demonstrating that PF4 impairs protein S cofactor enhancement of APC anticoagulant function. Using recombinant factor Va variants FVa-R506Q\\/R679Q and FVa-R306Q\\/R679Q, PF4 was shown to impair APC proteolysis of FVa at position Arg(306) by 3-fold both in the presence and absence of protein S. These data suggest that PF4 contributes to the poorly understood APC resistance phenotype associated with activated platelets. Finally, despite PF4 binding to the APC Gla domain, we show that APC in the presence of PF4 retains its ability to initiate PAR-1-mediated cytoprotective signaling. In summary, we propose that PF4 acts as a critical regulator of APC generation, but also differentially targets APC toward cytoprotective, rather than anticoagulant function at sites of vascular injury with concurrent platelet activation.

  11. Study of hydrogenases activity inhibition by O2 and direct electrochemistry

    At the present time, a great effort of research is made on the identification and the design of enzymes insensitive or low sensitive to O2. In parallel, it seems important to understand the inhibition mechanisms in order to propose mutations able to limit this inhibition. The Protein Film Voltametry allows to obtain data which can not be observed or quantitatively obtained by other techniques. The enzyme is immobilized directly on the electrode and the electronic transfer is direct. The redox state of the enzyme depends on the potential of the electrode and the catalytic current is proportional to the enzyme activity. The data obtained for the Ni-Fe hydrogenase (Desulfovibrio fructosovorans) and for the Fe hydrogenase (Clostridium Acetobutylicum) will be compared to the data obtained for hydrogenases of other organisms, by Protein Film Voltametry as well as by other techniques. (O.M.)

  12. Inhibition of protein kinase C intracerebroventricularly attenuates sensitization

    Mrowczynski, Oliver Daniel

    2012-01-01

    Drug relapse, mediated by drug-associated memories, is a major problem associated with addiction. Protein kinase C (PKC) is a family of protein kinase enzymes that has been implicated in learning and memory with regards to addiction. This study used a PKC inhibitor, chelerythrine (10nmol), to investigate the effects of blocking PKC throughout the brain on addiction related memories. Cocaine (15mg/kg) induced locomotor sensitization, used to model the transition from casual to compulsive use, ...

  13. The Transmembrane Adaptor Protein SIT Inhibits TCR-Mediated Signaling

    Arndt, Börge; Krieger, Tina; Kalinski, Thomas; Thielitz, Anja; Reinhold, Dirk; Roessner, Albert; Schraven, Burkhart; Simeoni, Luca

    2011-01-01

    Transmembrane adaptor proteins (TRAPs) organize signaling complexes at the plasma membrane, and thus function as critical linkers and integrators of signaling cascades downstream of antigen receptors. We have previously shown that the transmembrane adaptor protein SIT regulates the threshold for thymocyte selection. Moreover, T cells from SIT-deficient mice are hyperresponsive to CD3 stimulation and undergo enhanced lymphopenia-induced homeostatic proliferation, thus indicating that SIT inhib...

  14. Inhibition of existing denitrification enzyme activity by chloramphenicol.

    Brooks, M H; Smith, R L; Macalady, D L

    1992-01-01

    Chloramphenicol completely inhibited the activity of existing denitrification enzymes in acetylene-block incubations with (i) sediments from a nitrate-contaminated aquifer and (ii) a continuous culture of denitrifying groundwater bacteria. Control flasks with no antibiotic produced significant amounts of nitrous oxide in the same time period. Amendment with chloramphenicol after nitrous oxide production had begun resulted in a significant decrease in the rate of nitrous oxide production. Chlo...

  15. Inhibition of papillomavirus protein function in cervical cancer cells by intrabody targeting.

    Griffin, Heather; Elston, Robert; Jackson, Deborah; Ansell, Keith; Coleman, Michael; Winter, Greg; Doorbar, John

    2006-01-20

    Papillomaviruses (HPVs) are a major cause of human disease, and are responsible for approximately half a million cases of cervical cancer each year. HPVs also cause genital warts, and are the most common sexually transmitted disease in many countries. Despite their importance, there are currently no specific antivirals that are active against HPVs. Papillomavirus protein function is mediated largely by protein-protein interactions, which are difficult to inhibit using conventional approaches. To circumvent these problems, we have prepared an scFv library, and have used this to isolate high-affinity binding molecules that may stearically hinder the association of E6 with p53 and prevent E6-mediated p53 degradation in cervical cancer cells. One of the molecules isolated from the library (GTE6-1), had an affinity for 16E6 of 60nM, and bound within the first zinc finger of the protein. GTE6-1 was able to associate with non-denatured E6 following expression in mammalian cells and could inhibit E6-mediated p53 degradation in in vitro assays. E6-mediated p53 degradation is essential for the continuous growth of cervical cancer cells caused by HPV16. To examine the potential of GTE6-1 as an inhibitor of E6 function in such cells, the molecule was expressed in scFv, diabody and triabody formats in a number of cell lines that are driven to proliferate by the HPV16 oncogenes E6 and E7, including the cervical cancer cell line SiHa. In contrast to small E6-binding peptides containing the ELLG E6-binding motif, GTE6-1 expression lead to changes in nuclear structure, the appearance of apoptosis markers, and an elevation in the levels of p53. No effects were seen with a control scFv molecule, or when GTE6-1 was expressed in cells that are driven to proliferate by simian virus 40 (SV40) T-antigen. Given the accessibility of HPV-associated lesions to topical therapy, our results suggest that large interfering molecules such as intrabodies may be useful inhibitors of viral protein-protein

  16. Surfactant Protein-A inhibits Aspergillus fumigatus-induced allergic T-cell responses

    Russo Scott J

    2005-08-01

    Full Text Available Abstract Background The pulmonary surfactant protein (SP-A has potent immunomodulatory activities but its role and regulation during allergic airway inflammation is unknown. Methods We studied changes in SP-A expression in the bronchoalveolar lavage (BAL using a murine model of single Aspergillus fumigatus (Af challenge of sensitized animals. Results SP-A protein levels in the BAL fluid showed a rapid, transient decline that reached the lowest values (25% of controls 12 h after intranasal Af provocation of sensitized mice. Decrease of SP-A was associated with influx of inflammatory cells and increase of IL-4 and IL-5 mRNA and protein levels. Since levels of SP-A showed a significant negative correlation with these BAL cytokines (but not with IFN-γ, we hypothesized that SP-A exerts an inhibitory effect on Th2-type immune responses. To study this hypothesis, we used an in vitro Af-rechallenge model. Af-induced lymphocyte proliferation of cells isolated from sensitized mice was inhibited in a dose-dependent manner by addition of purified human SP-A (0.1–10 μg/ml. Flow cytometric studies on Af-stimulated lymphocytes indicated that the numbers of CD4+ (but not CD8+ T cells were significantly increased in the parental population and decreased in the third and fourth generation in the presence of SP-A. Further, addition of SP-A to the tissue culture inhibited Af-induced IL-4 and IL-5 production suggesting that SP-A directly suppressed allergen-stimulated CD4+ T cell function. Conclusion We speculate that a transient lack of this lung collectin following allergen exposure of the airways may significantly contribute to the development of a T-cell dependent allergic immune response.

  17. Lactadherin inhibits secretory phospholipase A2 activity on pre-apoptotic leukemia cells.

    Steffen Nyegaard

    Full Text Available Secretory phospholipase A2 (sPLA2 is a critical component of insect and snake venoms and is secreted by mammalian leukocytes during inflammation. Elevated secretory PLA2 concentrations are associated with autoimmune diseases and septic shock. Many sPLA2's do not bind to plasma membranes of quiescent cells but bind and digest phospholipids on the membranes of stimulated or apoptotic cells. The capacity of these phospholipases to digest membranes of stimulated or apoptotic cells correlates to the exposure of phosphatidylserine. In the present study, the ability of the phosphatidyl-L-serine-binding protein, lactadherin to inhibit phospholipase enzyme activity has been assessed. Inhibition of human secretory phospholipase A2-V on phospholipid vesicles exceeded 90%, whereas inhibition of Naja mossambica sPLA2 plateaued at 50-60%. Lactadherin inhibited 45% of activity of Naja mossambica sPLA2 and >70% of human secretory phospholipase A2-V on the membranes of human NB4 leukemia cells treated with calcium ionophore A23187. The data indicate that lactadherin may decrease inflammation by inhibiting sPLA2.

  18. Mechanism of inhibition of growth hormone receptor signaling by suppressor of cytokine signaling proteins

    Hansen, J A; Lindberg, K; Hilton, D J; Nielsen, Jens Høiriis; Billestrup, N

    1999-01-01

    In this study we have investigated the role of suppressor of cytokine signaling (SOCS) proteins in GH receptor-mediated signaling. GH-induced transcription was inhibited by SOCS-1 and SOCS-3, while SOCS-2 and cytokine inducible SH2-containing protein (CIS) had no effect By using chimeric SOCS pro...

  19. Protein Kinase G-dependent Cardioprotective Mechanism of Phosphodiesterase-5 Inhibition Involves Phosphorylation of ERK and GSK3β*

    Das, Anindita; Xi, Lei; Kukreja, Rakesh C.

    2008-01-01

    Sildenafil, a potent inhibitor of phosphodiesterase-5 (PDE-5) induces powerful protection against myocardial ischemia-reperfusion injury. PDE-5 inhibition increases cGMP levels that activate cGMP-dependent protein kinase (PKG). However, the cause and effect relationship of PKG in sildenafil-induced cardioprotection and the downstream targets of PKG remain unclear. Adult ventricular myocytes were treated with sildenafil and subjected to simulated ischemia and reoxygenat...

  20. Artemisinin inhibits chloroplast electron transport activity: mode of action.

    Adyasha Bharati

    Full Text Available Artemisinin, a secondary metabolite produced in Artemisia plant species, besides having antimalarial properties is also phytotoxic. Although, the phytotoxic activity of the compound has been long recognized, no information is available on the mechanism of action of the compound on photosynthetic activity of the plant. In this report, we have evaluated the effect of artemisinin on photoelectron transport activity of chloroplast thylakoid membrane. The inhibitory effect of the compound, under in vitro condition, was pronounced in loosely and fully coupled thylakoids; being strong in the former. The extent of inhibition was drastically reduced in the presence of uncouplers like ammonium chloride or gramicidin; a characteristic feature described for energy transfer inhibitors. The compound, on the other hand, when applied to plants (in vivo, behaved as a potent inhibitor of photosynthetic electron transport. The major site of its action was identified to be the Q(B; the secondary quinone moiety of photosystemII complex. Analysis of photoreduction kinetics of para-benzoquinone and duroquinone suggest that the inhibition leads to formation of low pool of plastoquinol, which becomes limiting for electron flow through photosystemI. Further it was ascertained that the in vivo inhibitory effect appeared as a consequence of the formation of an unidentified artemisinin-metabolite rather than by the interaction of the compound per se. The putative metabolite of artemisinin is highly reactive in instituting the inhibition of photosynthetic electron flow eventually reducing the plant growth.

  1. Assessment of Novel Anti-thrombotic Fusion Proteins for Inhibition of Stenosis in a Porcine Model of Arteriovenous Graft.

    Christi M Terry

    Full Text Available Hemodialysis arteriovenous synthetic grafts (AVG provide high volumetric blood flow rates shortly after surgical placement. However, stenosis often develops at the vein-graft anastomosis contributing to thrombosis and early graft failure. Two novel fusion proteins, ANV-6L15 and TAP-ANV, inhibit the tissue factor/factor VIIa coagulation complex and the factor Xa/factor Va complex, respectively. Each inhibitor domain is fused to an annexin V domain that targets the inhibitor activity to sites of vascular injury to locally inhibit thrombosis. This study's objective was to determine if these antithrombotic proteins are safe and effective in inhibiting AVG stenosis.A bolus of either TAP-ANV or ANV-6L15 fusion protein was administered intravenously immediately prior to surgical placement of a synthetic graft between the external jugular vein and common carotid artery in a porcine model. At surgery, the vein and artery were irrigated with the anti-thrombotic fusion protein. Control animals received intravenous heparin. At 4 weeks, MRI was performed to evaluate graft patency, the pigs were then euthanized and grafts and attached vessels were explanted for histomorphometric assessment of neointimal hyperplasia at the vein-graft anastomosis. Blood was collected at surgery, immediately after surgery and at euthanasia for serum metabolic panels and coagulation chemistries.No acute thrombosis occurred in the control group or in either experimental group. No abnormal serum chemistries, activated clotting times or PT, PTT values were observed after treatment in experimental or control animals. However, at the vein-graft anastomosis, there was no difference between the control and experimental groups in cross-sectional lumen areas, as measured on MRI, and no difference in hyperplasia areas as determined by histomorphometry. These results suggest that local irrigation of TAP-ANV or ANV-6L15 intra-operatively was as effective in inhibiting acute graft thrombosis

  2. Divalent cation tolerance protein binds to β-secretase and inhibits the processing of amyloid precursor protein

    Runzhong Liu; Haibo Hou; Xuelian Yi; Shanwen Wu; Huan Zeng

    2013-01-01

    The deposition of amyloid-beta is a pathological hallmark of Alzheimer's disease. Amyloid-beta is derived from amyloid precursor protein through sequential proteolytic cleavages by β-secretase (beta-site amyloid precursor protein-cleaving enzyme 1) and γ-secretase. To further elucidate the roles of beta-site amyloid precursor protein-cleaving enzyme 1 in the development of Alzheimer's disease, a yeast two-hybrid system was used to screen a human embryonic brain cDNA library for proteins directly interacting with the intracellular domain of beta-site amyloid precursor protein-cleaving enzyme 1. A potential beta-site amyloid precursor protein-cleaving enzyme 1- interacting protein identified from the positive clones was divalent cation tolerance protein. Immunoprecipitation studies in the neuroblastoma cell line N2a showed that exogenous divalent cation tolerance protein interacts with endogenous beta-site amyloid precursor protein-cleaving enzyme 1. The overexpression of divalent cation tolerance protein did not affect beta-site amyloid precursor protein-cleaving enzyme 1 protein levels, but led to increased amyloid precursor protein levels in N2a/APP695 cells, with a concomitant reduction in the processing product amyloid precursor protein C-terminal fragment, indicating that divalent cation tolerance protein inhibits the processing of amyloid precursor protein. Our experimental findings suggest that divalent cation tolerance protein negatively regulates the function of beta-site amyloid precursor protein-cleaving enzyme 1. Thus, divalent cation tolerance protein could play a protective role in Alzheimer's disease.

  3. Dexmedetomidine inhibits vasoconstriction via activation of endothelial nitric oxide synthase

    Nong, Lidan; Ma, Jue; Zhang, Guangyan; Deng, Chunyu; Mao, Songsong; Li, Haifeng

    2016-01-01

    Despite the complex vascular effects of dexmedetomidine (DEX), its actions on human pulmonary resistance arteries remain unknown. The present study tested the hypothesis that DEX inhibits vascular tension in human pulmonary arteries through the endothelial nitric oxide synthase (eNOS) mediated production of nitric oxide (NO). Pulmonary artery segments were obtained from 62 patients who underwent lung resection. The direct effects of DEX on human pulmonary artery tension and changes in vascular tension were determined by isometric force measurements recorded on a myograph. Arterial contractions caused by increasing concentrations of serotonin with DEX in the presence or absence of L-NAME (endothelial nitric oxide synthase inhibitor), yohimbine (α2-adrenoceptor antagonist) and indomethacin (cyclooxygenase inhibitor) as antagonists were also measured. DEX had no effect on endothelium-intact pulmonary arteries, whereas at concentrations of 10–8~10–6 mol/L, it elicited contractions in endothelium-denuded pulmonary arteries. DEX (0.3, 1, or 3×10–9 mmol/L) inhibited serotonin-induced contraction in arteries with intact endothelium in a dose-dependent manner. L-NAME and yohimbine abolished DEX-induced inhibition, whereas indomethacin had no effect. No inhibitory effect was observed in endothelium-denuded pulmonary arteries. DEX-induced inhibition of vasoconstriction in human pulmonary arteries is mediated by NO production induced by the activation of endothelial α2-adrenoceptor and nitric oxide synthase. PMID:27610030

  4. Cordycepin activates AMP-activated protein kinase (AMPK) via interaction with the γ1 subunit

    Wu, Chongming; Guo, Yanshen; Su, Yan; Zhang, Xue; Luan, Hong; Zhang, Xiaopo; Zhu, Huixin; He, Huixia; Wang, Xiaoliang; Sun, Guibo; Sun, Xiaobo; Guo, Peng; Zhu, Ping

    2014-01-01

    Cordycepin is a bioactive component of the fungus Cordyceps militaris. Previously, we showed that cordycepin can alleviate hyperlipidemia through enhancing the phosphorylation of AMP-activated protein kinase (AMPK), but the mechanism of this stimulation is unknown. Here, we investigated the potential mechanisms of cordycepin-induced AMPK activation in HepG2 cells. Treatment with cordycepin largely reduced oleic acid (OA)-elicited intracellular lipid accumulation and increased AMPK activity in a dose-dependent manner. Cordycepin-induced AMPK activation was not accompanied by changes in either the intracellular levels of AMP or the AMP/ATP ratio, nor was it influenced by calmodulin-dependent protein kinase kinase (CaMKK) inhibition; however, this activation was significantly suppressed by liver kinase B1 (LKB1) knockdown. Molecular docking, fluorescent and circular dichroism measurements showed that cordycepin interacted with the γ1 subunit of AMPK. Knockdown of AMPKγ1 by siRNA substantially abolished the effects of cordycepin on AMPK activation and lipid regulation. The modulating effects of cordycepin on the mRNA levels of key lipid regulatory genes were also largely reversed when AMPKγ1 expression was inhibited. Together, these data suggest that cordycepin may inhibit intracellular lipid accumulation through activation of AMPK via interaction with the γ1 subunit. PMID:24286368

  5. Striatal-enriched protein tyrosine phosphatase modulates nociception: evidence from genetic deletion and pharmacological inhibition.

    Azkona, Garikoitz; Saavedra, Ana; Aira, Zigor; Aluja, David; Xifró, Xavier; Baguley, Tyler; Alberch, Jordi; Ellman, Jonathan A; Lombroso, Paul J; Azkue, Jon J; Pérez-Navarro, Esther

    2016-02-01

    The information from nociceptors is processed in the dorsal horn of the spinal cord by complex circuits involving excitatory and inhibitory interneurons. It is well documented that GluN2B and ERK1/2 phosphorylation contributes to central sensitization. Striatal-enriched protein tyrosine phosphatase (STEP) dephosphorylates GluN2B and ERK1/2, promoting internalization of GluN2B and inactivation of ERK1/2. The activity of STEP was modulated by genetic (STEP knockout mice) and pharmacological (recently synthesized STEP inhibitor, TC-2153) approaches. STEP(61) protein levels in the lumbar spinal cord were determined in male and female mice of different ages. Inflammatory pain was induced by complete Freund's adjuvant injection. Behavioral tests, immunoblotting, and electrophysiology were used to analyze the effect of STEP on nociception. Our results show that both genetic deletion and pharmacological inhibition of STEP induced thermal hyperalgesia and mechanical allodynia, which were accompanied by increased pGluN2B(Tyr1472) and pERK1/2(Thr202/Tyr204)levels in the lumbar spinal cord. Striatal-enriched protein tyrosine phosphatase heterozygous and knockout mice presented a similar phenotype. Furthermore, electrophysiological experiments showed that TC-2153 increased C fiber-evoked spinal field potentials. Interestingly, we found that STEP(61) protein levels in the lumbar spinal cord inversely correlated with thermal hyperalgesia associated with age and female gender in mice. Consistently, STEP knockout mice failed to show age-related thermal hyperalgesia, although gender-related differences were preserved. Moreover, in a model of inflammatory pain, hyperalgesia was associated with increased phosphorylation-mediated STEP(61) inactivation and increased pGluN2B(Tyr1472) and pERK1/2(Thr202/Tyr204)levels in the lumbar spinal cord. Collectively, the present results underscore an important role of spinal STEP activity in the modulation of nociception. PMID:26270590

  6. Pharmacological Inhibition of the Protein Kinase MRK/ZAK Radiosensitizes Medulloblastoma.

    Markowitz, Daniel; Powell, Caitlin; Tran, Nhan L; Berens, Michael E; Ryken, Timothy C; Vanan, Magimairajan; Rosen, Lisa; He, Mingzu; Sun, Shan; Symons, Marc; Al-Abed, Yousef; Ruggieri, Rosamaria

    2016-08-01

    Medulloblastoma is a cerebellar tumor and the most common pediatric brain malignancy. Radiotherapy is part of the standard care for this tumor, but its effectiveness is accompanied by significant neurocognitive sequelae due to the deleterious effects of radiation on the developing brain. We have previously shown that the protein kinase MRK/ZAK protects tumor cells from radiation-induced cell death by regulating cell-cycle arrest after ionizing radiation. Here, we show that siRNA-mediated MRK depletion sensitizes medulloblastoma primary cells to radiation. We have, therefore, designed and tested a specific small molecule inhibitor of MRK, M443, which binds to MRK in an irreversible fashion and inhibits its activity. We found that M443 strongly radiosensitizes UW228 medulloblastoma cells as well as UI226 patient-derived primary cells, whereas it does not affect the response to radiation of normal brain cells. M443 also inhibits radiation-induced activation of both p38 and Chk2, two proteins that act downstream of MRK and are involved in DNA damage-induced cell-cycle arrest. Importantly, in an animal model of medulloblastoma that employs orthotopic implantation of primary patient-derived UI226 cells in nude mice, M443 in combination with radiation achieved a synergistic increase in survival. We hypothesize that combining radiotherapy with M443 will allow us to lower the radiation dose while maintaining therapeutic efficacy, thereby minimizing radiation-induced side effects. Mol Cancer Ther; 15(8); 1799-808. ©2016 AACR. PMID:27207779

  7. SPECTROSCOPIC STUDIES OF INHIBITION OF CALMODULIN ACTIVITY BY SOME DRUGS

    Naderi

    1996-06-01

    Full Text Available The effect of four inhibitors on calmalulin (CuM were studied by a ftuorescence and ultraviolet techniques. Four compounds IN - ( 6 - aminohexyt 5-chloro - I - napthalenesulphonamide] (W-7, 1 - [ bis - (4 - chtorophenyt methyl] - 3 - [2, 4-dichloro - β - ( 2 , 4 - dichlorobenzyloxyl phenethyt] imidazolium chloride (R24571, trifluoperazine (TFP , thiodiphenylamide chloride (TDPAC showed inhibitory effect on bovine brain phosphodiesterase (PDE induced by CaM. The concentration of inhibitors producing 50% inhibition of of Ca 2+ / CaM activity activity (IC50 and the Hill coefficient were correlating closely between the methods, Ki's and thermodynamic parameters for these interactions were estimated.

  8. Chloramphenicol Inhibition of Denitrifying Enzyme Activity in Two Agricultural Soils

    Murray, Robert E.; Knowles, Roger

    1999-01-01

    Chloramphenicol, at concentrations greater than 0.1 g/liter (0.3 mM), inhibited the denitrifying enzyme activity (DEA) of slurries of humisol and sandy loam soils by disrupting the activity of existing nitrate reductase enzymes. When the concentration of chloramphenicol was increased from 0.1 to 2.0 g/liter (6.0 mM), the rate of nitrite production from nitrate decreased by 25 to 46%. The rate of NO production from nitrate decreased by 20 to 39%, and the rate of N2O production from nitrate, in...

  9. Verbascoside Inhibits Promastigote Growth and Arginase Activity of Leishmania amazonensis.

    Maquiaveli, Claudia C; Lucon-Júnior, João F; Brogi, Simone; Campiani, Giuseppe; Gemma, Sandra; Vieira, Paulo C; Silva, Edson R

    2016-05-27

    Verbascoside (1) is a phenylethanoid glycoside that has antileishmanial activity against Leishmania infantum and Leishmania donovani. In this study, we verified the activity of 1 on Leishmania amazonensis and arginase inhibition. Compound 1 showed an EC50 of 19 μM against L. amazonensis promastigotes and is a competitive arginase inhibitor (Ki = 0.7 μM). Docking studies were performed to assess the interaction of 1 with arginase at the molecular level. Arginase is an enzyme of the polyamine biosynthesis pathway that is important to parasite infectivity, and the results of our study suggest that 1 could be useful to develop new approaches for treating leishmaniasis. PMID:27096224

  10. Enzymatic and inhibiting activity in boar epididymal fluid

    Davidová, Nina; Ren, Š.; Liberda, J.; Jonáková, Věra; Maňásková-Postlerová, Pavla

    Praha: Biotechnologický ústav AVČR, v.v.i, 2014 - (Pěknicová, J.), s. 1-82 [XXth Symposium of Biology and Immunology of Reproduction with international participation. Třešť (CZ), 22.05.2014-24.05.2014] R&D Projects: GA ČR(CZ) P503/12/1834; GA ČR(CZ) P502/14/05547S; GA MŠk(CZ) CZ1.05/1.1.00/02.0109 Institutional research plan: CEZ:AV0Z50520701 Institutional support: RVO:86652036 Keywords : enzymatic activity * inhibiting activity * epididymal fluid * proteinase Subject RIV: CE - Biochemistry

  11. Trans-dominant inhibition of transcription activator LFB1.

    Nicosia, A.; Tafi, R; Monaci, P

    1992-01-01

    Liver-enriched factor LFB1 (also named HNF1) is a dimeric transcription activator which is essential for the expression of many hepatocyte-specific genes. Here we demonstrate that LFB1 mutants in the POU A-like or in the homeo domains inhibit wild-type DNA binding by forming inactive heterodimeric complexes. Co-transfection of one of these mutants with wild-type LFB1 in HeLa cells eliminated LFB1 DNA binding and transcriptional activities through a trans-dominant mechanism. Expression of the ...

  12. Menthol inhibits detrusor contractility independently of TRPM8 activation.

    Antonio Celso Saragossa Ramos-Filho

    Full Text Available Agonists such as icilin and menthol can activate the cool temperature-sensitive ion channel TRPM8. However, biological responses to menthol may occur independently of TRPM8 activation. In the rodent urinary bladder, menthol facilitates the micturition reflex but inhibits muscarinic contractions of the detrusor smooth muscle. The site(s of TRPM8 expression in the bladder are controversial. In this study we investigated the regulation of bladder contractility in vitro by menthol. Bladder strips from wild type and TRPM8 knockout male mice (25-30 g were dissected free and mounted in organ baths. Isometric contractions to carbachol (1 nM-30 µM, CaCl2 (1 µM to 100 mM and electrical field stimulation (EFS; 8, 16, 32 Hz were measured. Strips from both groups contracted similarly in response to both carbachol and EFS. Menthol (300 µM or nifedipine (1 µM inhibited carbachol and EFS-induced contractions in both wild type and TRPM8 knockout bladder strips. Incubation with the sodium channel blocker tetrodotoxin (1 µM, replacement of extracellular sodium with the impermeant cation N-Methyl-D-Glucamine, incubation with a cocktail of potassium channel inhibitors (100 nM charybdotoxin, 1 µM apamin, 10 µM glibenclamide and 1 µM tetraethylammonium or removal of the urothelium did not affect the inhibitory actions of menthol. Contraction to CaCl2 was markedly inhibited by either menthol or nifedipine. In cultured bladder smooth muscle cells, menthol or nifedipine abrogated the carbachol or KCl-induced increases in [Ca2+]i. Intravesical administration of menthol increased voiding frequency while decreasing peak voiding pressure. We conclude that menthol inhibits muscarinic bladder contractions through blockade of L-type calcium channels, independently of TRPM8 activation.

  13. Inhibition of PMN-elastase activity by semisynthetic glucan sulfates.

    Becker, Markus; Franz, Gerhard; Alban, Susanne

    2003-05-01

    Proteolysis of connective tissue by enzymes such as PMN-elastase (PMNE) is a crucial step during inflammation and metastasis. Semisynthetic sulfated carbohydrates (SC) were shown to exhibit potent antiinflammatory and antimetastatic activity in vivo. The aim of the present study was to examine whether interferences with PMN-elastase may contribute to these effects. Therefore, the interactions of these compounds with PMNE were evaluated in various test systems. Besides semisynthetic alpha-1,4/1,6- and beta-1,3-glucan sulfates, UFH, a LMWH and pentosan polysulfate (PPS) were included in the study. The inhibitory activity of SC improves not only with increasing molecular weight (MW 10 - 250 kDa: 37 - 54% inhibition at 0.25 micro g/ml) and degree of sulfation (DS 0.25 - 2.0: 16 - 50% inhibition at 0.25 micro g/ml), but depends also on their genuine polysaccharide structure (IC50 beta-1,3-glucan sulfate 0.18 / alpha-1,4/1,6-glucan sulfate 0.25 / UFH 0.5 micro g/ml). Using physiological substrate assays (collagen, elastin), beta-1,3- and alpha-1,4/1,6-glucan sulfates are more active than UFH (inhibition at 1.5 micro g/ml: 41 / 32 / 12%). According to enzyme-inhibitor binding studies, SC exhibit structure dependent affinity to the enzyme (K(d) for PMNE: beta-1,3 1,4/1,6 < UFH). Finally, SC were shown to inhibit cancer cell-mediated elastinolysis. PMID:12719790

  14. Bacterial Protein Synthesis as a Target for Antibiotic Inhibition.

    Arenz, Stefan; Wilson, Daniel N

    2016-01-01

    Protein synthesis occurs on macromolecular machines, called ribosomes. Bacterial ribosomes and the translational machinery represent one of the major targets for antibiotics in the cell. Therefore, structural and biochemical investigations into ribosome-targeting antibiotics provide not only insight into the mechanism of action and resistance of antibiotics, but also insight into the fundamental process of protein synthesis. This review summarizes the recent advances in our understanding of protein synthesis, particularly with respect to X-ray and cryoelectron microscopy (cryo-EM) structures of ribosome complexes, and highlights the different steps of translation that are targeted by the diverse array of known antibiotics. Such findings will be important for the ongoing development of novel and improved antimicrobial agents to combat the rapid emergence of multidrug resistant pathogenic bacteria. PMID:27481773

  15. Molecular mechanism by which AMP-activated protein kinase activation promotes glycogen accumulation in muscle

    Hunter, Roger W; Treebak, Jonas Thue; Wojtaszewski, Jørgen;

    2011-01-01

    OBJECTIVE During energy stress, AMP-activated protein kinase (AMPK) promotes glucose transport and glycolysis for ATP production, while it is thought to inhibit anabolic glycogen synthesis by suppressing the activity of glycogen synthase (GS) to maintain the energy balance in muscle. Paradoxically...... transgenic mice overexpressing a kinase dead (KD) AMPK were incubated with glucose tracers and the AMPK-activating compound 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) ex vivo. GS activity and glucose uptake and utilization (glycolysis and glycogen synthesis) were assessed. RESULTS Even though...

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

    Andrew Bottley

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

  17. Inhibition of p53 transcriptional activity by human cytomegalovirus UL44.

    Kwon, Yejin; Kim, Mi-Na; Young Choi, Eun; Heon Kim, Jung; Hwang, Eung-Soo; Cha, Chang-Yong

    2012-05-01

    Human cytomegalovirus (HCMV) stimulates cellular synthesis of DNA and proteins and induces transition of the cell cycle from G(1) to S and G(2) /M phase, in spite of increased amounts of p53 in the infected cells. The immediate early protein IE2-86  kDa (IE86) tethers a transcriptional repression domain to p53; however, its repression of p53 function is not enough to abrogate the G(1) checkpoint function of p53. Other HCMV proteins that suppress the activity of p53 were investigated in this study. Of the HCMV proteins that bind to p53 when assessed by immunoprecipitation and immunoblot analysis, HCMV UL44 was chosen as a candidate protein. It was found that reporter gene containing p53 consensus sequence was activated by transfection with wild type p53, but when plasmids of p53 with IE86 or UL44 were co-transfected, p53 transcriptional activity was decreased to 3-7% of the p53 control in a dose-dependent manner. When the deletion mutant of UL44 was co-transected with p53, the carboxyl one-third portion of UL44 had little effect on inhibition of p53 transcriptional activity. The amount of mRNA p21 was measured in H1299 by real time PCR after transfection of the combination of p53 and UL44 vectors and it was found that p21 transcription by p53 was inhibited dose-dependently by UL44. Increased G0/G1 and decreased S phases in p53 wild type-transfected H1299 cells were recovered to the level of p53 mutant type-transfected ones by the additional transfection of UL44 in a dose-dependent manner. In conclusion, the transcriptional activity of p53 is suppressed by UL44 as well as by IE86. PMID:22376288

  18. The grapevine polygalacturonase-inhibiting protein (VvPGIP1) reduces Botrytis cinerea susceptibility in transgenic tobacco and differentially inhibits fungal polygalacturonases

    Joubert, D.A.; Slaughter, A. R.; Kemp, G; Becker, J.V.W.; Krooshof, G. H.; Bergmann, C.; Benen, J.A.E.; Pretorius, I. S.; Vivier, M.A.

    2006-01-01

    Polygalacturonase-inhibiting proteins (PGIPs) selectively inhibit polygalacturonases (PGs) secreted by invading plant pathogenic fungi. PGIPs display differential inhibition towards PGs from different fungi, also towards different isoforms of PGs originating from a specific pathogen. Recently, a PGIP-encoding gene from Vitis vinifera (Vvpgip1) was isolated and characterised. PGIP purified from grapevine was shown to inhibit crude polygalacturonase extracts from Botrytis cinerea, but this inhi...

  19. Staphylococcal Nuclease and Tudor Domain Containing 1 (SND1 Protein) Promotes Hepatocarcinogenesis by Inhibiting Monoglyceride Lipase (MGLL).

    Rajasekaran, Devaraja; Jariwala, Nidhi; Mendoza, Rachel G; Robertson, Chadia L; Akiel, Maaged A; Dozmorov, Mikhail; Fisher, Paul B; Sarkar, Devanand

    2016-05-13

    Staphylococcal nuclease and tudor domain containing 1 (SND1) is overexpressed in multiple cancers, including hepatocellular carcinoma (HCC), and functions as an oncogene. This study was carried out to identify novel SND1-interacting proteins to better understand its molecular mechanism of action. SND1-interacting proteins were identified by a modified yeast two-hybrid assay. Protein-protein interaction was confirmed by co-immunoprecipitation analysis. Monoglyceride lipase (MGLL) expression was analyzed by quantitative RT-PCR, Western blot, and immunohistochemistry. MGLL-overexpressing clones were analyzed for cell proliferation and cell cycle analysis and in vivo tumorigenesis in nude mice. MGLL was identified as an SND1-interacting protein. Interaction of SND1 with MGLL resulted in ubiquitination and proteosomal degradation of MGLL. MGLL expression was detected in normal human hepatocytes and mouse liver, although it was undetected in human HCC cell lines. An inverse correlation between SND1 and MGLL levels was identified in a human HCC tissue microarray as well as in the TCGA database. Forced overexpression of MGLL in human HCC cells resulted in marked inhibition in cell proliferation with a significant delay in cell cycle progression and a marked decrease in tumor growth in nude mouse xenograft assays. MGLL overexpression inhibited Akt activation that is independent of enzymatic activity of MGLL and overexpression of a constitutively active Akt rescued cells from inhibition of proliferation and restored normal cell cycle progression. This study unravels a novel mechanism of SND1 function and identifies MGLL as a unique tumor suppressor for HCC. MGLL might function as a homeostatic regulator of Akt restraining its activation. PMID:26997225

  20. Soy protein diet inhibits zymosan induced monocyte migration

    Atherosclerosis has been recognized as a chronic inflammatory disease. Recently, we showed reduced atherosclerotic lesions in a hyperlipidemic mouse model fed isoflavone-free soy protein diet (SPI) compared to casein (CAS)-fed mice, despite unchanged serum lipid levels. However, the molecular mechan...

  1. Matrine Activates PTEN to Induce Growth Inhibition and Apoptosis in V600EBRAF Harboring Melanoma Cells

    Shuiying Wang

    2013-07-01

    Full Text Available Here, we report a natural chemical Matrine, which exhibits anti-melanoma potential with its PTEN activation mechanism. Matrine effectively inhibited proliferation of several carcinoma cell lines, including melanoma V600EBRAF harboring M21 cells. Flow cytometry analysis showed Matrine induced G0/G1 cell cycle arrest in M21 cells dose-dependently. Apoptosis in M21 cells induced by Matrine was identified by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL analysis and Annexin-V/FITC staining. Molecular mechanistic study suggested that Matrine upregulated both mRNA level and protein expression level of phosphatase and tensin homolog deleted on chromosome ten (PTEN, leading to inhibition of the PI3K/Akt pathway. Downregulation of phosphor-Aktser473 by Matrine activated p21 and Bax, which contributed to G0/G1 cell cycle and apoptosis. Besides, Matrine enhanced the PI3K/Akt inhibition effects to inhibit the cell proliferation with PI3K inhibitor, LY2940002. In summary, our findings suggest Matrine is a promising antitumor drug candidate with its possible PTEN activation mechanisms for treating cancer diseases, such as melanomas.

  2. Survivin inhibits anti-growth effect of p53 activated by aurora B

    Genomic instability and apoptosis evasion are hallmarks of cancer, but the molecular mechanisms governing these processes remain elusive. Here, we found that survivin, a member of the apoptosis-inhibiting gene family, and aurora B kinase, a chromosomal passenger protein, were co-overexpressed in the various glioblastoma cell lines and tumors. Notably, exogenous introduction of the aurora B in human BJ cells was shown to decrease cell growth and increase the senescence-associated β-galactosidase activity by activation of p53 tumor suppressor. However, aurora B overexpression failed to inhibit cell proliferation in BJ and U87MG cells transduced with dominant-negative p53 as well as in p53-/- mouse astrocytes. Aurora B was shown to increase centrosome amplification in the p53-/- astrocytes. Survivin was shown to induce anchorage-independent growth and inhibit anti-proliferation and drug-sensitive apoptosis caused by aurora B. Overexpression of both survivin and aurora B further accelerated the proliferation of BJ cells. Taken together, the present study indicates that survivin should accelerate tumorigenesis by inhibiting the anti-proliferative effect of p53 tumor suppressor that is activated by aurora B in normal and glioblastoma cells containing intact p53

  3. PURIFICATION AND CHARACTERIZATION OF POLYGALACTURONASE - INHIBITING PROTEIN FROM ASIAN PEAR VARIETIES

    Y.H.Han

    2015-12-01

    Full Text Available Polygalacturonase inhibitory protein (PGIP was extracted from Shinli pear tissue, purified and partially characterized. Extraction was carried out at 4oC with a high ionic strength extraction buffer. After dialysis and concentration by ultrafiltration, the extract was chromatographed on size-exclusion chromatography (S-100, and its active fractions were applied on concanavalin A-Sepharose. The PGIP activity was bound by the lectin, and then eluted using 1M α-methyl mannopyranoside, resulting in a 18-fold purification of the PGIP and demonstrating its glycoprotein nature. The following ion-exchange chromatography gave a PGIP that was 360-fold purified relative to the initial tissue extract, and having a 45kDa molecular weight, as estimated by SDS-PAGE electrophoresis. PGIP inhibitory activity was tested against A. niger, C. acutatum and B. cinerea. The radial diffusion and reducing sugar assays showed that PGIP inhibitory to three PGs was affected by pH. In vivo tests revealed that PGIP inhibited three polygalacturonase from all three fungi. Heated for 20 min at 85oC, the inhibitory activity of PGIP was reduced by 85-90%, and it was completely suppressed after being heated at 100oC for 20 min.

  4. Metformin inhibits glutaminase activity and protects against hepatic encephalopathy.

    Javier Ampuero

    Full Text Available AIM: To investigate the influence of metformin use on liver dysfunction and hepatic encephalopathy in a retrospective cohort of diabetic cirrhotic patients. To analyze the impact of metformin on glutaminase activity and ammonia production in vitro. METHODS: Eighty-two cirrhotic patients with type 2 diabetes were included. Forty-one patients were classified as insulin sensitizers experienced (metformin and 41 as controls (cirrhotic patients with type 2 diabetes mellitus without metformin treatment. Baseline analysis included: insulin, glucose, glucagon, leptin, adiponectin, TNFr2, AST, ALT. HOMA-IR was calculated. Baseline HE risk was calculated according to minimal hepatic encephalopathy, oral glutamine challenge and mutations in glutaminase gene. We performed an experimental study in vitro including an enzymatic activity assay where glutaminase inhibition was measured according to different metformin concentrations. In Caco2 cells, glutaminase activity inhibition was evaluated by ammonia production at 24, 48 and 72 hours after metformina treatment. RESULTS: Hepatic encephalopathy was diagnosed during follow-up in 23.2% (19/82: 4.9% (2/41 in patients receiving metformin and 41.5% (17/41 in patients without metformin treatment (logRank 9.81; p=0.002. In multivariate analysis, metformin use [H.R.11.4 (95% CI: 1.2-108.8; p=0.034], age at diagnosis [H.R.1.12 (95% CI: 1.04-1.2; p=0.002], female sex [H.R.10.4 (95% CI: 1.5-71.6; p=0.017] and HE risk [H.R.21.3 (95% CI: 2.8-163.4; p=0.003] were found independently associated with hepatic encephalopathy. In the enzymatic assay, glutaminase activity inhibition reached 68% with metformin 100 mM. In Caco2 cells, metformin (20 mM decreased glutaminase activity up to 24% at 72 hours post-treatment (p<0.05. CONCLUSIONS: Metformin was found independently related to overt hepatic encephalopathy in patients with type 2 diabetes mellitus and high risk of hepatic encephalopathy. Metformin inhibits glutaminase

  5. Latent membrane protein 1 inhibits apoptosis induced by 60 irradiation via Survivin triggering signal-pathway

    Objective: To investigate the anti-apoptosis mechanism of EB virus encoden latent membrane protein 1 (LMP1) via the survivin signal transduction pathway after irradiation induction. Methods: Tet-on- LMP1 HNE2 cells, as a model, were detected with morphological assay, flowcytometry and Caspase 3 assay after 60Co irradiation with LMP1 induced by doxycycline. The apoptosis in the anti-sense survivin transfected cells was tested. Results: The results showed that, with LMP1 expression, the apoptosis rates from morphological assay and flowcytometry were 32.7%±2.1% and 6.3%, which showed that they were all lower than that without LMP1 expression (66.0%±3.0% and 29.6%). When anti-sense of survivin was induced, the apoptosis rates were 59.0%±3.2% and 3.0% respectively, and caspase 3 activity was 3.78 nmol/106 cells, which were higher than that of the control (26.0%±2.6%, 8.6% and 2.79 nmol/106). Survivin restrained the cell apoptosis induced by irradiation, but anti-sense of survivin could release this inhibition of cell apoptosis triggered by LMP1 expression. Conclusion: LMP1 inhibits the irradiation-induced cell apoptosis via triggering survivin expression. Survivin may be targeted in some certain therapy

  6. MT1-MMP Inhibits the Activity of Bst-2 via Their Cytoplasmic Domains Dependent Interaction

    Fan, Long; Liu, Li; Zhu, Cuicui; Zhu, Qingyi; Lu, Shan; Liu, Ping

    2016-01-01

    Bst-2 (bone marrow stromal cell antigen 2) is a type II membrane protein, and it acts as a tetherin to inhibit virion releasing from infectious cells. Membrane type-1 matrix metalloproteinase (MT1-MMP) is a protease. It plays a pivotal role in cellular growth and migration by activating proMMP-2 into active MMP2. Our results here elaborate that MT1-MMP inhibits the tetherin activity of Bst-2 by interacting with Bst-2, and the cytoplasmic domains of both Bst-2 and MT1-MMP play critical roles within this interaction. Based on our experimental data, the assays for virion release and co-immunoprecipitation have clearly demonstrated that the activity of Bst-2 is markedly inhibited by MT1-MMP via their interaction; and both the N-terminal domain of Bst-2 and the C-terminal domain of MT1-MMP are important in the interaction. Immunostaining and Confocal Microscopy assay shows that MT1-MMP interacts with Bst-2 to form granular particles trafficking into cytoplasm from membrane and, finally, results in Bst-2 and MT1-MMP both being inhibited. In addition, mutant experiments elucidate that the N-terminal domain of Bst-2 is not only important in relating to the activity of Bst-2 itself, but is important for inhibiting the MT1-MMP/proMMP2/MMP2 pathway. These findings suggest that MT1-MMP is a novel inhibitor of Bst-2 in MT1-MMP expressed cell lines and also indicate that both the N-terminal domain of Bst-2 and the C-terminal domain of MT1-MMP are crucial in down-regulation. PMID:27240342

  7. MT1-MMP Inhibits the Activity of Bst-2 via Their Cytoplasmic Domains Dependent Interaction

    Long Fan

    2016-05-01

    Full Text Available Bst-2 (bone marrow stromal cell antigen 2 is a type II membrane protein, and it acts as a tetherin to inhibit virion releasing from infectious cells. Membrane type-1 matrix metalloproteinase (MT1-MMP is a protease. It plays a pivotal role in cellular growth and migration by activating proMMP-2 into active MMP2. Our results here elaborate that MT1-MMP inhibits the tetherin activity of Bst-2 by interacting with Bst-2, and the cytoplasmic domains of both Bst-2 and MT1-MMP play critical roles within this interaction. Based on our experimental data, the assays for virion release and co-immunoprecipitation have clearly demonstrated that the activity of Bst-2 is markedly inhibited by MT1-MMP via their interaction; and both the N-terminal domain of Bst-2 and the C-terminal domain of MT1-MMP are important in the interaction. Immunostaining and Confocal Microscopy assay shows that MT1-MMP interacts with Bst-2 to form granular particles trafficking into cytoplasm from membrane and, finally, results in Bst-2 and MT1-MMP both being inhibited. In addition, mutant experiments elucidate that the N-terminal domain of Bst-2 is not only important in relating to the activity of Bst-2 itself, but is important for inhibiting the MT1-MMP/proMMP2/MMP2 pathway. These findings suggest that MT1-MMP is a novel inhibitor of Bst-2 in MT1-MMP expressed cell lines and also indicate that both the N-terminal domain of Bst-2 and the C-terminal domain of MT1-MMP are crucial in down-regulation.

  8. Maternal Protein Restriction in the Rat Inhibits Placental Insulin, mTOR, and STAT3 Signaling and Down-Regulates Placental Amino Acid Transporters

    Rosario, Fredrick J.; Jansson, Nina; Kanai, Yoshikatsu; Prasad, Puttur D; Powell, Theresa L.; Jansson, Thomas

    2011-01-01

    The mechanisms underlying reduced fetal growth in response to maternal protein restriction are not well established. Maternal levels of insulin, IGF-I, and leptin are decreased in rats fed a low protein (LP) diet. Because these hormones stimulate placental amino acid transporters in vitro, we hypothesized that maternal protein restriction inhibits placental leptin, insulin/IGF-I, and mammalian target of rapamycin signaling and down-regulates the expression and activity of placental amino acid...

  9. GATA3 inhibits GCM1 activity and trophoblast cell invasion.

    Chiu, Yueh Ho; Chen, Hungwen

    2016-01-01

    Development of human placenta involves the invasion of trophoblast cells from anchoring villi into the maternal decidua. Placental transcription factor GCM1 regulates trophoblast cell invasion via transcriptional activation of HtrA4 gene, which encodes a serine protease enzyme. The GATA3 transcription factor regulates trophoblast cell differentiation and is highly expressed in invasive murine trophoblast giant cells. The regulation of trophoblastic invasion by GCM1 may involve novel cellular factors. Here we show that GATA3 interacts with GCM1 and inhibits its activity to suppress trophoblastic invasion. Immunohistochemistry demonstrates that GATA3 and GCM1 are coexpressed in villous cytotrophoblast cells, syncytiotrophoblast layer, and extravillous trophoblast cells of human placenta. Interestingly, GATA3 interacts with GCM1, but not the GCM2 homologue, through the DNA-binding domain and first transcriptional activation domain in GCM1 and the transcriptional activation domains and zinc finger 1 domain in GATA3. While GATA3 did not affect DNA-binding activity of GCM1, it suppressed transcriptional activity of GCM1 and therefore HtrA4 promoter activity. Correspondingly, GATA3 knockdown elevated HtrA4 expression in BeWo and JEG-3 trophoblast cell lines and enhanced the invasion activities of both lines. This study uncovered a new GATA3 function in placenta as a negative regulator of GCM1 activity and trophoblastic invasion. PMID:26899996

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

    Jarmila Vinšová

    2014-05-01

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

  11. Inhibition of Nek2 by Small Molecules Affects Proteasome Activity

    Lingyao Meng

    2014-01-01

    Full Text Available Background. Nek2 is a serine/threonine kinase localized to the centrosome. It promotes cell cycle progression from G2 to M by inducing centrosome separation. Recent studies have shown that high Nek2 expression is correlated with drug resistance in multiple myeloma patients. Materials and Methods. To investigate the role of Nek2 in bortezomib resistance, we ectopically overexpressed Nek2 in several cancer cell lines, including multiple myeloma lines. Small-molecule inhibitors of Nek2 were discovered using an in-house library of compounds. We tested the inhibitors on proteasome and cell cycle activity in several cell lines. Results. Proteasome activity was elevated in Nek2-overexpressing cell lines. The Nek2 inhibitors inhibited proteasome activity in these cancer cell lines. Treatment with these inhibitors resulted in inhibition of proteasome-mediated degradation of several cell cycle regulators in HeLa cells, leaving them arrested in G2/M. Combining these Nek2 inhibitors with bortezomib increased the efficacy of bortezomib in decreasing proteasome activity in vitro. Treatment with these novel Nek2 inhibitors successfully mitigated drug resistance in bortezomib-resistant multiple myeloma. Conclusion. Nek2 plays a central role in proteasome-mediated cell cycle regulation and in conferring resistance to bortezomib in cancer cells. Taken together, our results introduce Nek2 as a therapeutic target in bortezomib-resistant multiple myeloma.

  12. Structural basis of metallo-β-lactamase, serine-β-lactamase and penicillin-binding protein inhibition by cyclic boronates.

    Brem, Jürgen; Cain, Ricky; Cahill, Samuel; McDonough, Michael A; Clifton, Ian J; Jiménez-Castellanos, Juan-Carlos; Avison, Matthew B; Spencer, James; Fishwick, Colin W G; Schofield, Christopher J

    2016-01-01

    β-Lactamases enable resistance to almost all β-lactam antibiotics. Pioneering work revealed that acyclic boronic acids can act as 'transition state analogue' inhibitors of nucleophilic serine enzymes, including serine-β-lactamases. Here we report biochemical and biophysical analyses revealing that cyclic boronates potently inhibit both nucleophilic serine and zinc-dependent β-lactamases by a mechanism involving mimicking of the common tetrahedral intermediate. Cyclic boronates also potently inhibit the non-essential penicillin-binding protein PBP 5 by the same mechanism of action. The results open the way for development of dual action inhibitors effective against both serine- and metallo-β-lactamases, and which could also have antimicrobial activity through inhibition of PBPs. PMID:27499424

  13. Structural basis of metallo-β-lactamase, serine-β-lactamase and penicillin-binding protein inhibition by cyclic boronates

    Brem, Jürgen; Cain, Ricky; Cahill, Samuel; McDonough, Michael A.; Clifton, Ian J.; Jiménez-Castellanos, Juan-Carlos; Avison, Matthew B.; Spencer, James; Fishwick, Colin W. G.; Schofield, Christopher J.

    2016-08-01

    β-Lactamases enable resistance to almost all β-lactam antibiotics. Pioneering work revealed that acyclic boronic acids can act as `transition state analogue' inhibitors of nucleophilic serine enzymes, including serine-β-lactamases. Here we report biochemical and biophysical analyses revealing that cyclic boronates potently inhibit both nucleophilic serine and zinc-dependent β-lactamases by a mechanism involving mimicking of the common tetrahedral intermediate. Cyclic boronates also potently inhibit the non-essential penicillin-binding protein PBP 5 by the same mechanism of action. The results open the way for development of dual action inhibitors effective against both serine- and metallo-β-lactamases, and which could also have antimicrobial activity through inhibition of PBPs.

  14. Omega-3 free fatty acids suppress macrophage inflammasome activation by inhibiting NF-κB activation and enhancing autophagy.

    Yolanda Williams-Bey

    Full Text Available The omega-3 (ω3 fatty acid docosahexaenoic acid (DHA can suppress inflammation, specifically IL-1β production through poorly understood molecular mechanisms. Here, we show that DHA reduces macrophage IL-1β production by limiting inflammasome activation. Exposure to DHA reduced IL-1β production by ligands that stimulate the NLRP3, AIM2, and NAIP5/NLRC4 inflammasomes. The inhibition required Free Fatty Acid Receptor (FFAR 4 (also known as GPR120, a G-protein coupled receptor (GPR known to bind DHA. The exposure of cells to DHA recruited the adapter protein β-arrestin1/2 to FFAR4, but not to a related lipid receptor. DHA treatment reduced the initial inflammasome priming step by suppressing the nuclear translocation of NF-κB. DHA also reduced IL-1β levels by enhancing autophagy in the cells. As a consequence macrophages derived from mice lacking the essential autophagy protein ATG7 were partially resistant to suppressive effects of DHA. Thus, DHA suppresses inflammasome activation by two distinct mechanisms, inhibiting the initial priming step and by augmenting autophagy, which limits inflammasome activity.

  15. Modulation of mitogen-activated protein kinase-activated protein kinase 3 by hepatitis C virus core protein

    Ngo, HT; Pham, Long; Kim, JW;

    2013-01-01

    , approximately 100 cellular proteins were identified as HCV core-interacting partners. Of these candidates, mitogen-activated protein kinase-activated protein kinase 3 (MAPKAPK3) was selected for further characterization. MAPKAPK3 is a serine/threonine protein kinase that is activated by stress and growth...... inducers. Binding of HCV core to MAPKAPK3 was confirmed by in vitro pulldown assay and further verified by coimmunoprecipitation assay. HCV core protein interacted with MAPKAPK3 through amino acid residues 41 to 75 of core and the N-terminal half of kinase domain of MAPKAPK3. In addition, both RNA and......Hepatitis C virus (HCV) is highly dependent on cellular proteins for its own propagation. In order to identify the cellular factors involved in HCV propagation, we performed protein microarray assays using the HCV core protein as a probe. Of ~9,000 host proteins immobilized in a microarray...

  16. Prion protein inhibits microtubule assembly by inducing tubulin oligomerization

    A growing body of evidence points to an association of prion protein (PrP) with microtubular cytoskeleton. Recently, direct binding of PrP to tubulin has also been found. In this work, using standard light scattering measurements, sedimentation experiments, and electron microscopy, we show for First time the effect of a direct interaction between these proteins on tubulin polymerization. We demonstrate that full-length recombinant PrP induces a rapid increase in the turbidity of tubulin diluted below the critical concentration for microtubule assembly. This effect requires magnesium ions and is weakened by NaCl. Moreover, the PrP-induced light scattering structures of tubulin are cold-stable. In preparations of diluted tubulin incubated with PrP, electron microscopy revealed the presence of ∼50 nm disc-shaped structures not reported so far. These unique tubulin oligomers may form large aggregates. The effect of PrP is more pronounced under the conditions promoting microtubule formation. In these tubulin samples, PrP induces formation of the above oligomers associated with short protofilaments and sheets of protofilaments into aggregates. Noticeably, this is accompanied by a significant reduction of the number and length of microtubules. Hence, we postulate that prion protein may act as an inhibitor of microtubule assembly by inducing formation of stable tubulin oligomers

  17. The chromosomal protein HMGBI inhibits DNA replication in vitro. The role of post-synthetic acetylation

    The effect of HMGB1 protein on the replication of closed circular plasmid DNA in cell free extract have been studied using parental form of the protein, post-synthetically acetylated HMGB1 and HMGB1 lacking its acidic C-terminal tail. We have shown that HMGB1 protein inhibits DNA replication and that this effect is eliminated upon either acetylation of the protein or removal of the acidic C-terminal domain. An explanation of these findings suggests interactions of HMGB1 with a protein(s) of the replication complex resulting in reduction of its functional efficiency. Acetylation of HMGB1 affects these interactions in a way that restores the initial replication capacity of the system. The eventual protein-protein interactions are supposed to proceed via the C-terminal domain of HMGB1. (authors)

  18. CYP24A1 Inhibition Enhances the Antitumor Activity of Calcitriol

    Muindi, Josephia R.; Yu, Wei-Dong; Ma, Yingyu; Engler, Kristie L.; Kong, Rui-Xian; Trump, Donald L.; Johnson, Candace S.

    2010-01-01

    High systemic exposures to calcitriol are necessary for optimal antitumor effects. Human prostate cancer PC3 cells are insensitive to calcitriol treatment. Therefore, we investigated whether the inhibition of 24-hydroxylase (CYP24A1), the major calcitriol inactivating enzyme, by ketoconazole (KTZ) or RC2204 modulates calcitriol serum pharmacokinetics and biologic effects. Dexamethasone (Dex) was added to minimize calcitriol-induced hypercalcemia and as a steroid replacement for the KTZ inhibition of steroid biosynthesis cytochrome P450 enzymes. KTZ effectively inhibited time-dependent calcitriol-inducible CYP24A1 protein expression and enzyme activity in PC3 cells and C3H/HeJ mouse kidney tissues. Systemic calcitriol exposure area under the curve was higher in mice treated with a combination of calcitriol and KTZ than with calcitriol alone. KTZ and Dex synergistically potentiated calcitriol-mediated antiproliferative effects in PC3 cells in vitro; this effect was associated with enhanced apoptosis. After treatment with calcitriol and KTZ/Dex, although caspase-9 and caspase-3 were not activated and cytochrome c was not released by mitochondria, caspase-8 was activated and the truncated Bid protein level was increased. Translocation of apoptosis-inducing factor to the nucleus was observed, indicating a role of the apoptosis-inducing factor-mediated and caspase-independent apoptotic pathways. Calcitriol and KTZ/Dex combination suppressed the clonogenic survival and enhanced the growth inhibition observed with calcitriol alone in PC3 human prostate cancer xenograft mouse model. Our results show that the administration of calcitriol in combination with CYP24A1 inhibitor enhances antiproliferative effects, increases systemic calcitriol exposure, and promotes the activation of caspase-independent apoptosis pathway. PMID:20591973

  19. Synergistic inhibition of T-cell activation by a cell-permeable ZAP-70 mutant and ctCTLA-4

    Kim, Kyun-Do [Department of Biotechnology, Yonsei University, Seodaemun-Gu, Shinchon-Dong 134, Seoul 120-749 (Korea, Republic of); Choi, Je-Min; Chae, Wook-Jin [Department of Immunobiology, Yale University School of Medicine, New Haven CT 06520 (United States); Lee, Sang-Kyou, E-mail: sjrlee@yonsei.ac.kr [Department of Biotechnology, Yonsei University, Seodaemun-Gu, Shinchon-Dong 134, Seoul 120-749 (Korea, Republic of); ForHumanTech Co., Ltd., Kowoon Institute of Technology Innovation, Bldg. 706, Suwon (Korea, Republic of)

    2009-04-10

    T-cell activation requires TcR-mediated and co-stimulatory signals. ZAP-70 participates in the initial step of TcR signal transduction, while a co-receptor, CTLA-4, inhibits T-cell activation. In previous studies, the overexpression of a ZAP-70 mutant (ZAP-70-Y319F) inhibited the TcR-induced activation of NFAT and IL-2 production, while Hph-1-ctCTLA-4 prevented allergic inflammation. To develop an effective immunosuppressive protein drug that blocks both TcR-mediated and co-stimulatory signaling pathways, a fusion protein of ZAP-70-Y319F and the Hph-1 protein transduction domain was generated. Hph-1-ZAP-70-Y319F inhibited the phosphorylation of ZAP-70-Tyr{sup 319}, LAT-Tyr{sup 191}, and p44/42 MAPK induced by TcR stimulation, NFAT- and AP-1-mediated gene transcription, and the induction of CD69 expression and IL-2 secretion. Hph-1-ZAP-70-Y319F and Hph-1-ctCTLA-4 synergistically inhibited signaling events during T-cell activation. This is the first report to demonstrate the synergistic inhibition of signals transmitted via TcR and its co-stimulatory receptor by cell-permeable forms of intracellular signal mediators.

  20. Evaluation of Jatropha curcas Linn. leaf extracts for its cytotoxicity and potential to inhibit hemagglutinin protein of influenza virus.

    Patil, Deepak; Roy, Soumen; Dahake, Ritwik; Rajopadhye, Shreewardhan; Kothari, Sweta; Deshmukh, Ranjana; Chowdhary, Abhay

    2013-09-01

    Influenza is a serious respiratory illness which can be debilitating and cause complications that lead to hospitalization and death. Although influenza vaccine can prevent influenza virus infection, the only therapeutic options to treat influenza virus infection are antiviral agents. Given temporal and geographic changes and the shifts in antiviral drug resistance among influenza viruses, it is time to consider natural antiviral agents against influenza virus. Jatropha curcas is known for various medicinal uses. Its antimicrobial, anti-cancer and anti-HIV activity has been well recognized. Because of its broad-spectrum activity, we investigated aqueous and methanol leaf extracts for cytotoxicity and its potential to inhibit hemagglutinin protein of influenza virus. The bioactive compounds from leaf extracts were characterized by high-performance thinlayer chromatography which revealed the presence of major phytochemicals including flavonoids, saponins and tannins. The cytotoxic concentration 50 for aqueous and methanol extracts were determined using trypan blue dye exclusion assay. Inhibition of hemagglutinin protein was assessed using minimal cytotoxic concentrations of the extracts and 10(2.5) TCID50 (64 HA titre) of the Influenza A (H1N1) virus with different exposure studies using hemagglutination assay. Aqueous and methanol extracts were found to be non toxic to Madin darby canine kidney cells below concentration of 15.57 and 33.62 mg/mL for respectively. Inhibition of hemagglutinin was studied using reducing hemagglutination titre which confirmed that the J. curcas extracts have direct effect on the process of virus adsorption leading to its inhibition. Our results provide the information which shows the potential of Jatropha extracts in the treatment of influenza A (H1N1) virus infection. With an established reduced toxicity and prevention of infection by inhibiting hemagglutinin protein, these extracts and its derivatives may be further developed as broad

  1. [6]-Shogaol inhibits melanogenesis in B16 mouse melanoma cells through activation of the ERK pathway

    Cheng YAO; Jang-hee OH; Inn Gyung OH; Chi-hyun PARK; Jin Ho CHUNG

    2013-01-01

    Aim: To investigate the effect of [6]-shogaol,an active ingredient in ginger,on melanogenesis and the underlying mechanisms.Methods: B16F10 mouse melanoma cells were tested.Cell viability was determined with the MTT assay.Melanin content and tyrosinase activity were analyzed with a spectrophotometer.The protein expression of tyrosinase and microphthalmia associated transcription factor (MITF),as well as phosphorylated or total ERK1/2 and Akt were measured using Western blot.Results: Treatment of the cells with [6]-shogaol (1,5,10 μmol/L) reduced the melanin content in a concentration-dependent manner.[6]-Shogaol (5 and 10 μmol/L) significantly decreased the intracellular tyrosinase activity,and markedly suppressed the expression levels of tyrosinase and MITF proteins in the cells.Furthermore,[6]-shogaol (10 μmol/L) activated ERK,which was known to negatively regulate melanin synthesis in these cells.Pretreatment with the specific ERK pathway inhibitor PD98059 (20 μmol/L) greatly attenuated the inhibition of melanin synthesis by [6]-shogaol (10 μmol/L).Conclusion: The results demonstrate that [6]-shogaol inhibits melanogenesis in B16F10 mouse melanoma cells via activating the ERK pathway.

  2. DMSO inhibits human platelet activation through cyclooxygenase-1 inhibition. A novel agent for drug eluting stents?

    Background: DMSO is routinely infused together with hematopoietic cells in patients undergoing myeloablative therapy and was recently found to inhibit smooth muscle cells proliferation and arterial thrombus formation in the mouse by preventing tissue factor (TF), a key activator of the coagulation cascade. This study was designed to investigate whether DMSO prevents platelet activation and thus, whether it may represent an interesting agent to be used on drug eluting stents. Methods and results: Human venous blood from healthy volunteers was collected in citrated tubes and platelet activation was studied by cone and platelet analyzer (CPA) and rapid-platelet-function-assay (RPFA). CPA analysis showed that DMSO-treated platelets exhibit a lower adherence in response to shear stress (-15.54 ± 0.9427%, n = 5, P < 0.0001 versus control). Additionally, aggregometry studies revealed that DMSO-treated, arachidonate-stimulated platelets had an increased lag phase (18.0% ± 4.031, n = 9, P = 0.0004 versus control) as well as a decreased maximal aggregation (-6.388 ± 2.212%, n = 6, P = 0.0162 versus control). Inhibitory action of DMSO could be rescued by exogenous thromboxane A2 and was mediated, at least in part, by COX-1 inhibition. Conclusions: Clinically relevant concentrations of DMSO impair platelet activation by a thromboxane A2-dependent, COX-1-mediated effect. This finding may be crucial for the previously reported anti-thrombotic property displayed by DMSO. Our findings support a role for DMSO as a novel drug to prevent not only proliferation, but also thrombotic complications of drug eluting stents.

  3. DMSO inhibits human platelet activation through cyclooxygenase-1 inhibition. A novel agent for drug eluting stents?

    Asmis, Lars [Institute for Clinical Hematology, University Hospital Zuerich, Zuerich (Switzerland); Tanner, Felix C. [Cardiovascular Research, Physiology Institute, University of Zuerich, Zuerich (Switzerland); Center for Integrative Human Physiology, University of Zuerich, Zuerich (Switzerland); Cardiology, Cardiovascular Center, University Hospital Zuerich, Zuerich (Switzerland); Sudano, Isabella [Cardiology, Cardiovascular Center, University Hospital Zuerich, Zuerich (Switzerland); Luescher, Thomas F. [Cardiovascular Research, Physiology Institute, University of Zuerich, Zuerich (Switzerland); Center for Integrative Human Physiology, University of Zuerich, Zuerich (Switzerland); Cardiology, Cardiovascular Center, University Hospital Zuerich, Zuerich (Switzerland); Camici, Giovanni G., E-mail: giovannic@access.uzh.ch [Cardiovascular Research, Physiology Institute, University of Zuerich, Zuerich (Switzerland); Center for Integrative Human Physiology, University of Zuerich, Zuerich (Switzerland)

    2010-01-22

    Background: DMSO is routinely infused together with hematopoietic cells in patients undergoing myeloablative therapy and was recently found to inhibit smooth muscle cells proliferation and arterial thrombus formation in the mouse by preventing tissue factor (TF), a key activator of the coagulation cascade. This study was designed to investigate whether DMSO prevents platelet activation and thus, whether it may represent an interesting agent to be used on drug eluting stents. Methods and results: Human venous blood from healthy volunteers was collected in citrated tubes and platelet activation was studied by cone and platelet analyzer (CPA) and rapid-platelet-function-assay (RPFA). CPA analysis showed that DMSO-treated platelets exhibit a lower adherence in response to shear stress (-15.54 {+-} 0.9427%, n = 5, P < 0.0001 versus control). Additionally, aggregometry studies revealed that DMSO-treated, arachidonate-stimulated platelets had an increased lag phase (18.0% {+-} 4.031, n = 9, P = 0.0004 versus control) as well as a decreased maximal aggregation (-6.388 {+-} 2.212%, n = 6, P = 0.0162 versus control). Inhibitory action of DMSO could be rescued by exogenous thromboxane A2 and was mediated, at least in part, by COX-1 inhibition. Conclusions: Clinically relevant concentrations of DMSO impair platelet activation by a thromboxane A2-dependent, COX-1-mediated effect. This finding may be crucial for the previously reported anti-thrombotic property displayed by DMSO. Our findings support a role for DMSO as a novel drug to prevent not only proliferation, but also thrombotic complications of drug eluting stents.

  4. Inhibition of neuraminidase inhibitor-resistant influenza virus by DAS181, a novel sialidase fusion protein.

    Gallen B Triana-Baltzer

    Full Text Available Antiviral drug resistance for influenza therapies remains a concern due to the high prevalence of H1N1 2009 seasonal influenza isolates which display H274Y associated oseltamivir-resistance. Furthermore, the emergence of novel H1N1 raises the potential that additional reassortments can occur, resulting in drug resistant virus. Thus, additional antiviral approaches are urgently needed. DAS181 (Fludase, a sialidase fusion protein, has been shown to have inhibitory activity against a large number of seasonal influenza strains and a highly pathogenic avian influenza (HPAI strain (H5N1. Here, we examine the in vitro activity of DAS181 against a panel of 2009 oseltamivir-resistant seasonal H1N1 clinical isolates. The activity of DAS181 against nine 2009, two 2007, and two 2004 clinical isolates of seasonal IFV H1N1 was examined using plaque number reduction assay on MDCK cells. DAS181 strongly inhibited all tested isolates. EC50 values remained constant against isolates from 2004, 2007, and 2009, suggesting that there was no change in DAS181 sensitivity over time. As expected, all 2007 and 2009 isolates were resistant to oseltamivir, consistent with the identification of the H274Y mutation in the NA gene of all these isolates. Interestingly, several of the 2007 and 2009 isolates also exhibited reduced sensitivity to zanamivir, and accompanying HA mutations near the sialic acid binding site were observed. DAS181 inhibits IFV that is resistant to NAIs. Thus, DAS181 may offer an alternative therapeutic option for seasonal or pandemic IFVs that become resistant to currently available antiviral drugs.

  5. AMP-Activated Protein Kinase alpha1 Dependent Signaling in Renal Tissue Fibrosis

    Mia, Sobuj

    2016-01-01

    Tubulointerstitial fibrosis is a common hallmark of chronic kidney disease caused by diabetes, hypertension, ischemia, renal injury and obstructive uropathy. The hetereotrimeric AMP-activated protein kinase (AMPK) consists of three subunits (α, β, γ) and is a master sensor of cellular energy status. Activation of AMPK contributes to monocyte-fibroblast transition and production of matrix protein even though accumulating evidence suggests that activated AMPK inhibits tissue fibrosis, which may...

  6. Antioxidant Activity and Acetylcholinesterase Inhibition of Grape Skin Anthocyanin (GSA

    Mehnaz Pervin

    2014-07-01

    Full Text Available We aimed to investigate the antioxidant and acetylcholinesterase inhibitory activities of the anthocyanin rich extract of grape skin. Grape skin anthocyanin (GSA neutralized free radicals in different test systems, such as 2,-2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS and 2,2-diphenyl-1-picrylhydrazyl (DPPH assays, to form complexes with Fe2+ preventing 2,2'-azobis(2-amidinopropane dihydrochloride (AAPH-induced erythrocyte hemolysis and oxidative DNA damage. Moreover, GSA decreased reactive oxygen species (ROS generation in isolated mitochondria thus inhibiting 2',-7'-dichlorofluorescin (DCFH oxidation. In an in vivo study, female BALB/c mice were administered GSA, at 12.5, 25, and 50 mg per kg per day orally for 30 consecutive days. Herein, we demonstrate that GSA administration significantly elevated the level of antioxidant enzymes in mice sera, livers, and brains. Furthermore, GSA inhibited acetylcholinesterase (AChE in the in vitro assay with an IC50 value of 363.61 µg/mL. Therefore, GSA could be an excellent source of antioxidants and its inhibition of cholinesterase is of interest with regard to neurodegenerative disorders such as Alzheimer’s disease.

  7. Protective effect of nectandrin B, a potent AMPK activator on neointima formation: inhibition of Pin1 expression through AMPK activation

    Ki, Sung Hwan; Lee, Jung-Woon; Lim, Sung Chul; Hien, Tran Thi; Im, Ji Hye; Oh, Won Keun; Lee, Moo Yeol; Ji, Young Hyun; Kim, Yoon Gyoon; Kang, Keon Wook

    2013-01-01

    Background and Purpose Neointima is considered a critical event in the development of vascular occlusive disease. Nectandrin B from nutmeg functions as a potent AMP-activated protein kinase (AMPK) activators. The present study addressed whether nectandrin B inhibits intimal hyperplasia in guide wire-injured arteries and examined its molecular mechanism. Experimental Approach Neointima was induced by guide wire injury in mouse femoral arteries. Cell proliferation and mechanism studies were performed in rat vascular smooth muscle cells (VSMC) culture model. Key Results Nectandrin B increased AMPK activity in VSMC. Nectandrin B inhibited the cell proliferation induced by PDGF and DNA synthesis. Moreover, treatment of nectandrin B suppressed neointima formation in femoral artery after guide wire injury. We have recently shown that Pin1 plays a critical role in VSMC proliferation and neointima formation. Nectandrin B potently blocked PDGF-induced Pin1 and cyclin D1 expression and nectandrin B‘s anti-proliferation effect was diminished in Pin1 overexpressed VSMC. PDGF-induced phosphorylation of ERK and Akt was marginally affected by nectandrin B. However, nectandrin B increased the levels of p53 and its downstream target p21 and, also reversibly decreased the expression of E2F1 and phosphorylated Rb in PDGF-treated VSMC. AMPK inhibition by dominant mutant form of adenovirus rescued nectandrin B-mediated down-regulation of Pin1 and E2F1. Conclusions and Implications Nectandrin B inhibited VSMC proliferation and neointima formation via inhibition of E2F1-dependent Pin1 gene transcription, which is mediated through the activation of an AMPK/p53-triggered pathway. PMID:23004677

  8. Radicicol, a heat shock protein 90 inhibitor, inhibits differentiation and adipogenesis in 3T3-L1 preadipocytes

    He, Yonghan [Department of Nutrition and Food Hygiene, Public Health College, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China); Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3 (Canada); State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223 (China); Li, Ying [Department of Nutrition and Food Hygiene, Public Health College, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China); Zhang, Shuocheng [Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3 (Canada); Perry, Ben [Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3 (Canada); Department of Biomedical Sciences, University of Prince Edward Island, 550 University Avenue, Charlottetown, PE, Canada C1A 4P3 (Canada); Zhao, Tiantian [Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3 (Canada); Department of Psychology, University of Toronto, 1265 Military Trail, Toronto, ON, Canada M1C 1A4 (Canada); Wang, Yanwen, E-mail: yanwen.wang@nrc.ca [Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3 (Canada); Department of Biomedical Sciences, University of Prince Edward Island, 550 University Avenue, Charlottetown, PE, Canada C1A 4P3 (Canada); Sun, Changhao, E-mail: sun2002changhao@yahoo.com [Department of Nutrition and Food Hygiene, Public Health College, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China)

    2013-06-28

    Highlights: •Radicicol suppressed intracellular fat accumulation in 3T3-L1 adipocytes. •Radicicol inhibited the expression of FAS and FABP4. •Radicicol blocked cell cycle at the G1-S phase during cell differentiation. •Radicicol inhibited the PDK1/Akt pathway in adipocyte differentiation. -- Abstract: Heat shock protein 90 (Hsp90) is involved in various cellular processes, such as cell proliferation, differentiation and apoptosis. As adipocyte differentiation plays a critical role in obesity development, the present study investigated the effect of an Hsp90 inhibitor radicicol on the differentiation of 3T3-L1 preadipocytes and potential mechanisms. The cells were treated with different concentrations of radicicol during the first 8 days of cell differentiation. Adipogenesis, the expression of adipogenic transcriptional factors, differentiation makers and cell cycle were determined. It was found that radicicol dose-dependently decreased intracellular fat accumulation through down-regulating the expression of peroxisome proliferator-activated receptor γ (PPAR{sub γ}) and CCAAT element binding protein α (C/EBP{sub α}), fatty acid synthase (FAS) and fatty acid-binding protein 4 (FABP4). Flow cytometry analysis revealed that radicicol blocked cell cycle at G1-S phase. Radicicol redcued the phosphorylation of Akt while showing no effect on β-catenin expression. Radicicol decreased the phosphorylation of phosphoinositide-dependent kinase 1 (PDK1). The results suggest that radicicol inhibited 3T3-L1 preadipocyte differentiation through affecting the PDK1/Akt pathway and subsequent inhibition of mitotic clonal expansion and the expression/activity of adipogenic transcriptional factors and their downstream adipogenic proteins.

  9. Radicicol, a heat shock protein 90 inhibitor, inhibits differentiation and adipogenesis in 3T3-L1 preadipocytes

    Highlights: •Radicicol suppressed intracellular fat accumulation in 3T3-L1 adipocytes. •Radicicol inhibited the expression of FAS and FABP4. •Radicicol blocked cell cycle at the G1-S phase during cell differentiation. •Radicicol inhibited the PDK1/Akt pathway in adipocyte differentiation. -- Abstract: Heat shock protein 90 (Hsp90) is involved in various cellular processes, such as cell proliferation, differentiation and apoptosis. As adipocyte differentiation plays a critical role in obesity development, the present study investigated the effect of an Hsp90 inhibitor radicicol on the differentiation of 3T3-L1 preadipocytes and potential mechanisms. The cells were treated with different concentrations of radicicol during the first 8 days of cell differentiation. Adipogenesis, the expression of adipogenic transcriptional factors, differentiation makers and cell cycle were determined. It was found that radicicol dose-dependently decreased intracellular fat accumulation through down-regulating the expression of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT element binding protein α (C/EBPα), fatty acid synthase (FAS) and fatty acid-binding protein 4 (FABP4). Flow cytometry analysis revealed that radicicol blocked cell cycle at G1-S phase. Radicicol redcued the phosphorylation of Akt while showing no effect on β-catenin expression. Radicicol decreased the phosphorylation of phosphoinositide-dependent kinase 1 (PDK1). The results suggest that radicicol inhibited 3T3-L1 preadipocyte differentiation through affecting the PDK1/Akt pathway and subsequent inhibition of mitotic clonal expansion and the expression/activity of adipogenic transcriptional factors and their downstream adipogenic proteins

  10. Copper oxide nanoparticles inhibit the metabolic activity of Saccharomyces cerevisiae.

    Mashock, Michael J; Kappell, Anthony D; Hallaj, Nadia; Hristova, Krassimira R

    2016-01-01

    Copper oxide nanoparticles (CuO NPs) are used increasingly in industrial applications and consumer products and thus may pose risk to human and environmental health. The interaction of CuO NPs with complex media and the impact on cell metabolism when exposed to sublethal concentrations are largely unknown. In the present study, the short-term effects of 2 different sized manufactured CuO NPs on metabolic activity of Saccharomyces cerevisiae were studied. The role of released Cu(2+) during dissolution of NPs in the growth media and the CuO nanostructure were considered. Characterization showed that the 28 nm and 64 nm CuO NPs used in the present study have different primary diameter, similar hydrodynamic diameter, and significantly different concentrations of dissolved Cu(2+) ions in the growth media released from the same initial NP mass. Exposures to CuO NPs or the released Cu(2+) fraction, at doses that do not have impact on cell viability, showed significant inhibition on S. cerevisiae cellular metabolic activity. A greater CuO NP effect on the metabolic activity of S. cerevisiae growth under respiring conditions was observed. Under the tested conditions the observed metabolic inhibition from the NPs was not explained fully by the released Cu ions from the dissolving NPs. PMID:26178758

  11. An Impermeant Ganetespib Analog Inhibits Extracellular Hsp90-Mediated Cancer Cell Migration that Involves Lysyl Oxidase 2-like Protein

    McCready, Jessica [Department of Natural Sciences, Assumption College, Worcester, MA 01609 (United States); Wong, Daniel S. [Department of Developmental Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111 (United States); Cell and Molecular Physiology Program, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, MA 02111 (United States); Burlison, Joseph A.; Ying, Weiwen [Synta Pharmaceuticals, Lexington, MA 02421 (United States); Jay, Daniel G., E-mail: daniel.jay@tufts.edu [Department of Developmental Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111 (United States); Cell and Molecular Physiology Program, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, MA 02111 (United States)

    2014-04-30

    Extracellular Hsp90 (eHsp90) activates a number of client proteins outside of cancer cells required for migration and invasion. Therefore, eHsp90 may serve as a novel target for anti-metastatic drugs as its inhibition using impermeant Hsp90 inhibitors would not affect the numerous vital intracellular Hsp90 functions in normal cells. While some eHsp90 clients are known, it is important to establish other proteins that act outside the cell to validate eHsp90 as a drug target to limit cancer spread. Using mass spectrometry we identified two precursor proteins Galectin 3 binding protein (G3BP) and Lysyl oxidase 2-like protein (LOXL2) that associate with eHsp90 in MDA-MB231 breast cancer cell conditioned media and confirmed that LOXL2 binds to eHsp90 in immunoprecipitates. We introduce a novel impermeant Hsp90 inhibitor STA-12-7191 derived from ganetespib and show that it is markedly less toxic to cells and can inhibit cancer cell migration in a dose dependent manner. We used STA-12-7191 to test if LOXL2 and G3BP are potential eHsp90 clients. We showed that while LOXL2 can increase wound healing and compensate for STA-12-7191-mediated inhibition of wound closure, addition of G3BP had no affect on this assay. These findings support of role for LOXL2 in eHsp90 stimulated cancer cell migration and provide preliminary evidence for the use of STA-12-7191 to inhibit eHsp90 to limit cancer invasion.

  12. An Impermeant Ganetespib Analog Inhibits Extracellular Hsp90-Mediated Cancer Cell Migration that Involves Lysyl Oxidase 2-like Protein

    Extracellular Hsp90 (eHsp90) activates a number of client proteins outside of cancer cells required for migration and invasion. Therefore, eHsp90 may serve as a novel target for anti-metastatic drugs as its inhibition using impermeant Hsp90 inhibitors would not affect the numerous vital intracellular Hsp90 functions in normal cells. While some eHsp90 clients are known, it is important to establish other proteins that act outside the cell to validate eHsp90 as a drug target to limit cancer spread. Using mass spectrometry we identified two precursor proteins Galectin 3 binding protein (G3BP) and Lysyl oxidase 2-like protein (LOXL2) that associate with eHsp90 in MDA-MB231 breast cancer cell conditioned media and confirmed that LOXL2 binds to eHsp90 in immunoprecipitates. We introduce a novel impermeant Hsp90 inhibitor STA-12-7191 derived from ganetespib and show that it is markedly less toxic to cells and can inhibit cancer cell migration in a dose dependent manner. We used STA-12-7191 to test if LOXL2 and G3BP are potential eHsp90 clients. We showed that while LOXL2 can increase wound healing and compensate for STA-12-7191-mediated inhibition of wound closure, addition of G3BP had no affect on this assay. These findings support of role for LOXL2 in eHsp90 stimulated cancer cell migration and provide preliminary evidence for the use of STA-12-7191 to inhibit eHsp90 to limit cancer invasion

  13. Protein kinase domain of twitchin has protein kinase activity and an autoinhibitory region.

    Lei, J; Tang, X; Chambers, T C; Pohl, J; Benian, G M

    1994-08-19

    Twitchin is a 753-kDa polypeptide located in the muscle A-bands of the nematode, Caenorhabditis elegans. It consists of multiple copies of both fibronectin III and immunoglobulin C2 domains and, near the C terminus, a protein kinase domain with greatest homology to the catalytic domains of myosin light chain kinases. We have expressed and purified from Escherichia coli twitchin's protein kinase catalytic core and flanking sequences that do not include fibronectin III and immunoglobulin C2 domains. The protein was shown to phosphorylate a model substrate and to undergo autophosphorylation. The autophosphorylation occurs at a slow rate, attaining a maximum at 3 h with a stoichiometry of about 1.0 mol of phosphate/mol of protein, probably through an intramolecular mechanism. Sequence analysis of proteolytically derived phosphopeptides revealed that autophosphorylation occurred N-terminal to the catalytic core, predominantly at Thr-5910, with possible minor sites at Ser5912 and/or Ser-5913. This portion of twitchin (residues 5890-6268) was also phosphorylated in vitro by protein kinase C in the absence of calcium and phosphotidylserine, but not by cAMP-dependent protein kinase. By comparing the activities of three twitchin segments, the enzyme appears to be inhibited by the 60-amino acid residues lying just C-terminal to the kinase catalytic core. Thus, like a number of other protein kinases including myosin light chain kinases, the twitchin kinase appears to be autoregulated. PMID:8063727

  14. R-Ras Inhibits VEGF-Induced p38MAPK Activation and HSP27 Phosphorylation in Endothelial Cells.

    Sawada, Junko; Li, Fangfei; Komatsu, Masanobu

    2015-01-01

    R-Ras is a Ras family small GTPase that is highly expressed in mature functional blood vessels in normal tissues. It inhibits pathological angiogenesis and promotes vessel maturation and stabilization. Previous studies suggest that R-Ras affects cellular signaling in endothelial cells, pericytes and smooth-muscle cells to regulate vessel formation and remodeling in adult tissues. R-Ras suppresses VEGF-induced endothelial permeability and vessel sprouting while promoting normalization of pathologically developing vessels in mice. It attenuates VEGF receptor-2 (VEGFR2) activation by inhibiting internalization of the receptor upon VEGF ligand binding, leading to significant reduction of VEGFR2 autophosphorylation. Here, we show that R-Ras strongly suppresses the VEGF-dependent activation of stress-activated protein kinase-2/p38 mitogen-activated protein kinase (SAPK2/p38MAPK) and the phosphorylation of downstream heat-shock protein 27 (HSP27), a regulator of actin cytoskeleton organization, in endothelial cells. The suppression of p38MAPK activation and HSP27 phosphorylation by R-Ras concurred with altered actin cytoskeleton architecture, reduced membrane protrusion and inhibition of endothelial cell migration toward VEGF. Silencing of endogenous R-Ras by RNA interference increased membrane protrusion and cell migration stimulated by VEGF, and these effects were offset by p38MAPK inhibitor SB203580. These results suggest that R-Ras regulates angiogenic activities of endothelial cells in part via inhibition of the p38MAPK-HSP27 axis of VEGF signaling. PMID:27029009

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

    Pawin Pongkorpsakol

    2014-09-01

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

  16. Activated FXR Inhibits Leptin Signaling and Counteracts Tumor-promoting Activities of Cancer-Associated Fibroblasts in Breast Malignancy.

    Giordano, Cinzia; Barone, Ines; Vircillo, Valentina; Panza, Salvatore; Malivindi, Rocco; Gelsomino, Luca; Pellegrino, Michele; Rago, Vittoria; Mauro, Loredana; Lanzino, Marilena; Panno, Maria Luisa; Bonofiglio, Daniela; Catalano, Stefania; Andò, Sebastiano

    2016-01-01

    Cancer-associated fibroblasts (CAFs), the principal components of the tumor stroma, play a central role in cancer development and progression. As an important regulator of the crosstalk between breast cancer cells and CAFs, the cytokine leptin has been associated to breast carcinogenesis. The nuclear Farnesoid X Receptor-(FXR) seems to exert an oncosuppressive role in different tumors, including breast cancer. Herein, we demonstrated, for the first time, that the synthetic FXR agonist GW4064, inhibiting leptin signaling, affects the tumor-promoting activities of CAFs in breast malignancy. GW4064 inhibited growth, motility and invasiveness induced by leptin as well as by CAF-conditioned media in different breast cancer cell lines. These effects rely on the ability of activated FXR to increase the expression of the suppressor of the cytokine signaling 3 (SOCS3) leading to inhibition of leptin-activated signaling and downregulation of leptin-target genes. In vivo xenograft studies, using MCF-7 cells alone or co-injected with CAFs, showed that GW4064 administration markedly reduced tumor growth. Interestingly, GW4064-treated tumors exhibited decreased levels of leptin-regulated proteins along with a strong staining intensity for SOCS3. Thus, FXR ligands might represent an emerging potential anti-cancer therapy able to block the tumor supportive role of activated fibroblasts within the breast microenvironment. PMID:26899873

  17. Silybin-mediated inhibition of Notch signaling exerts antitumor activity in human hepatocellular carcinoma cells.

    Song Zhang

    Full Text Available Hepatocellular carcinoma (HCC is a global health burden that is associated with limited treatment options and poor patient prognoses. Silybin (SIL, an antioxidant derived from the milk thistle plant (Silybum marianum, has been reported to exert hepatoprotective and antitumorigenic effects both in vitro and in vivo. While SIL has been shown to have potent antitumor activity against various types of cancer, including HCC, the molecular mechanisms underlying the effects of SIL remain largely unknown. The Notch signaling pathway plays crucial roles in tumorigenesis and immune development. In the present study, we assessed the antitumor activity of SIL in human HCC HepG2 cells in vitro and in vivo and explored the roles of the Notch pathway and of the apoptosis-related signaling pathway on the activity of SIL. SIL treatment resulted in a dose- and time-dependent inhibition of HCC cell viability. Additionally, SIL exhibited strong antitumor activity, as evidenced not only by reductions in tumor cell adhesion, migration, intracellular glutathione (GSH levels and total antioxidant capability (T-AOC but also by increases in the apoptotic index, caspase3 activity, and reactive oxygen species (ROS. Furthermore, SIL treatment decreased the expression of the Notch1 intracellular domain (NICD, RBP-Jκ, and Hes1 proteins, upregulated the apoptosis pathway-related protein Bax, and downregulated Bcl2, survivin, and cyclin D1. Notch1 siRNA (in vitro or DAPT (a known Notch1 inhibitor, in vivo further enhanced the antitumor activity of SIL, and recombinant Jagged1 protein (a known Notch ligand in vitro attenuated the antitumor activity of SIL. Taken together, these data indicate that SIL is a potent inhibitor of HCC cell growth that targets the Notch signaling pathway and suggest that the inhibition of Notch signaling may be a novel therapeutic intervention for HCC.

  18. Lamprey buccal gland secretory protein-2 (BGSP-2 inhibits human T lymphocyte proliferation

    Jing SUN, Shuiyan YU, Zhuang XUE, Cenjie LIU, Yu WU, Xin LIU, Qingwei LI

    2010-04-01

    Full Text Available Lamprey is a representative of the agnathans, the most ancient class of vertebrates. Parasitic lampreys secrete anticoagulant from their buccal glands and prevent blood coagulation of host fishes. We identified a buccal gland secretory protein-2 (BGSP-2 from a buccal gland cDNA library of Lampetra japonica. The full-length BGSP-2 gene was cloned and the recombinant BGSP-2 protein was generated. The role of BGSP-2 on lymphocyte proliferation was studied by examining its effects on human T lymphocytes. We found that lamprey BGSP-2 was able to effectively block the proliferation of T cells in vitro by inducing G1/S cell cycle arrest. Furthermore, it inhibited the proliferation of human T lymphocytes stimulated by phytohemagglutinin (PHA at a minimum concentration of 0.1μg/ml. Our data suggest that lamprey BGSP-2 is able to block the mitosis of human T lymphocytes at the G1/S point, and has the potential of anti-proliferative effect on PHA-activated T lymphocytes [Current Zoology 56 (2: 252–258, 2010].

  19. Cichorium intybus Linn. Extract Prevents Type 2 Diabetes Through Inhibition of NLRP3 Inflammasome Activation.

    Shim, Do-Wan; Han, Ji-Won; Ji, Young-Eun; Shin, Woo-Young; Koppula, Sushruta; Kim, Myong-Ki; Kim, Tae-Kweon; Park, Pyo-Jam; Kang, Tae-Bong; Lee, Kwang-Ho

    2016-03-01

    This study provides the scientific basis for the inhibitory effect of the aerial parts of Cichorium intybus Linn. (C. intybus) on the activation of the NLRP3 inflammasome in vitro and on high-fat diet (HFD)-induced type-2 diabetes (T2D). Lipopolysaccharide (LPS)-primed bone marrow-derived macrophages were used to study the effects methanolic extract of C. intybus leaf (CI) on inflammasome activation. An insulin resistance model (mice fed a HFD) was used to study the in vivo effect of CI on T2D. CI attenuated interleukin-1β (IL-1β) secretion by inhibiting the activation of the NLRP3 inflammasome in mouse bone marrow macrophages. The CI treatment attenuated the intracellular movement of NLRP3 in Triton X-100 insoluble fraction, without affecting the expression of other NLRP3 inflammasome-related proteins. Attenuated IL-1β secretion may improve glucose metabolism in the HFD-fed insulin resistance mouse model. CI also attenuated the infiltration of M1 macrophages and increased the M2 macrophage population in white adipose tissue. Collectively, our data showed that CI inhibits IL-1β secretion through attenuation of NLRP3 inflammasome activation, leading to an antidiabetic effect by improving glucose metabolism and inhibiting metainflammation. PMID:26987023

  20. Inhibition of the mitochondrial unfolded protein response by acetylcholine alleviated hypoxia/reoxygenation-induced apoptosis of endothelial cells.

    Xu, Man; Bi, Xueyuan; He, Xi; Yu, Xiaojiang; Zhao, Ming; Zang, Weijin

    2016-05-18

    The mitochondrial unfolded protein response (UPR(mt)) is involved in numerous diseases that have the common feature of mitochondrial dysfunction. However, its pathophysiological relevance in the context of hypoxia/reoxygenation (H/R) in endothelial cells remains elusive. Previous studies have demonstrated that acetylcholine (ACh) protects against cardiomyocyte injury by suppressing generation of mitochondrial reactive oxygen species (mtROS). This study aimed to explore the role of UPR(mt) in endothelial cells during H/R and to clarify the beneficial effects of ACh. Our results demonstrated that H/R triggered UPR(mt) in endothelial cells, as evidenced by the elevation of heat shock protein 60 and LON protease 1 protein levels, and resulted in release of mitochondrial pro-apoptotic proteins, including cytochrome C, Omi/high temperature requirement protein A 2 and second mitochondrial activator of caspases/direct inhibitor of apoptosis-binding protein with low PI, from the mitochondria to cytosol. ACh administration markedly decreased UPR(mt) by inhibiting mtROS and alleviating the mitonuclear protein imbalance. Consequently, ACh alleviated the release of pro-apoptotic proteins and restored mitochondrial ultrastructure and function, thereby reducing the number of terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL)-positive cells. Intriguingly, 4-diphenylacetoxy-N-methylpiperidine methiodide, a type-3 muscarinic ACh receptor (M3AChR) inhibitor, abolished the ACh-elicited attenuation of UPR(mt) and TUNEL positive cells, indicating that the salutary effects of ACh were likely mediated by M3AChR in endothelial cells. In conclusion, our studies demonstrated that UPR(mt) might be essential for triggering the mitochondrion-associated apoptotic pathway during H/R. ACh markedly suppressed UPR(mt) by inhibiting mtROS and alleviating the mitonuclear protein imbalance, presumably through M3AChR. PMID:27111378

  1. Smooth Muscle-Alpha Actin Inhibits Vascular Smooth Muscle Cell Proliferation and Migration by Inhibiting Rac1 Activity

    Chen, Lihua; DeWispelaere, Allison; Dastvan, Frank; Osborne, William R. A.; Blechner, Christine; Windhorst, Sabine; Daum, Guenter

    2016-01-01

    Smooth muscle alpha-actin (SMA) is a marker for the contractile, non-proliferative phenotype of adult smooth muscle cells (SMCs). Upon arterial injury, expression of SMA and other structural proteins decreases and SMCs acquire a pro-migratory and proliferative phenotype. To what extent SMA regulates migration and proliferation of SMCs is unclear and putative signaling pathways involved remain to be elucidated. Here, we used lentiviral-mediated gene transfer and siRNA technology to manipulate expression of SMA in carotid mouse SMCs and studied effects of SMA. Overexpression of SMA results in decreased proliferation and migration and blunts serum-induced activation of the small GTPase Rac, but not RhoA. All inhibitory effects of SMA are rescued by expression of a constitutively active Rac1 mutant (V12rac1). Moreover, reduction of SMA expression by siRNA technology results in an increased activation of Rac. Taken together, this study identifies Rac1 as a downstream target for SMA to inhibit SMC proliferation and migration. PMID:27176050

  2. Colchicine prevents NSAID-induced small intestinal injury by inhibiting activation of the NLRP3 inflammasome.

    Otani, Koji; Watanabe, Toshio; Shimada, Sunao; Takeda, Shogo; Itani, Shigehiro; Higashimori, Akira; Nadatani, Yuji; Nagami, Yasuaki; Tanaka, Fumio; Kamata, Noriko; Yamagami, Hirokazu; Tanigawa, Tetsuya; Shiba, Masatsugu; Tominaga, Kazunari; Fujiwara, Yasuhiro; Arakawa, Tetsuo

    2016-01-01

    The inflammasome is a large, multiprotein complex that consists of a nucleotide-binding oligomerization domain-like receptor (NLR), an apoptosis-associated speck-like protein containing a caspase recruitment domain, and pro-caspase-1. Activation of the inflammasome results in cleavage of pro-caspase-1 into cleaved caspase-1, which promotes the processing of pro-interleukin (IL)-1β into mature IL-1β. We investigated the effects of colchicine on non-steroidal anti-inflammatory drug (NSAID)-induced small intestinal injury and activation of the NLR family pyrin domain-containing 3 (NLRP3) inflammasome. Colchicine treatment inhibited indomethacin-induced small intestinal injury by 86% (1 mg/kg) and 94% (3 mg/kg) as indicated by the lesion index 24 h after indomethacin administration. Colchicine inhibited the protein expression of cleaved caspase-1 and mature IL-1β, without affecting the mRNA expression of NLRP3 and IL-1β. Although treatment with recombinant IL-1β (0.1 μg/kg) did not change the severity of small intestinal damage, the preventive effects of colchicine were abolished by supplementation with the same dose of recombinant IL-1β. Indomethacin-induced small intestinal damage was reduced by 77%, as determined by the lesion index in NLRP3(-/-) mice, and colchicine treatment failed to inhibit small intestinal damage in NLRP3(-/-) mice. These results demonstrate that colchicine prevents NSAID-induced small intestinal injury by inhibiting activation of the NLRP3 inflammasome. PMID:27585971

  3. Eriodictyol Inhibits RANKL-Induced Osteoclast Formation and Function Via Inhibition of NFATc1 Activity.

    Song, Fangming; Zhou, Lin; Zhao, Jinmin; Liu, Qian; Yang, Mingli; Tan, Renxiang; Xu, Jun; Zhang, Ge; Quinn, Julian M W; Tickner, Jennifer; Huang, Yuanjiao; Xu, Jiake

    2016-09-01

    Receptor activator of nuclear factor kappa-B ligand (RANKL) induces differentiation and function of osteoclasts through triggering multiple signaling cascades, including NF-κB, MAPK, and Ca(2+) -dependent signals, which induce and activate critical transcription factor NFATc1. Targeting these signaling cascades may serve as an effective therapy against osteoclast-related diseases. Here, by screening a panel of natural plant extracts with known anti-inflammatory, anti-tumor, or anti-oxidant properties for possible anti-osteoclastogenic activities we identified Eriodictyol. This flavanone potently suppressed RANKL-induced osteoclastogenesis and bone resorption in a dose-dependent manner without detectable cytotoxicity, suppressing RANKL-induced NF-κB, MAPK, and Ca(2+) signaling pathways. Eriodictyol also strongly inhibited RANKL-induction of c-Fos levels (a critical component of AP-1 transcription factor required by osteoclasts) and subsequent activation of NFATc1, concomitant with reduced expression of osteoclast specific genes including cathepsin K (Ctsk), V-ATPase-d2 subunit, and tartrate resistant acid phosphatase (TRAcP/Acp5). Taken together, these data provide evidence that Eriodictyol could be useful for the prevention and treatment of osteolytic disorders associated with abnormally increased osteoclast formation and function. J. Cell. Physiol. 231: 1983-1993, 2016. © 2016 Wiley Periodicals, Inc. PMID:26754483

  4. Pharmacological inhibition of dynamin II reduces constitutive protein secretion from primary human macrophages.

    Maaike Kockx

    Full Text Available Dynamins are fission proteins that mediate endocytic and exocytic membrane events and are pharmacological therapeutic targets. These studies investigate whether dynamin II regulates constitutive protein secretion and show for the first time that pharmacological inhibition of dynamin decreases secretion of apolipoprotein E (apoE and several other proteins constitutively secreted from primary human macrophages. Inhibitors that target recruitment of dynamin to membranes (MiTMABs or directly target the GTPase domain (Dyngo or Dynole series, dose- and time- dependently reduced the secretion of apoE. SiRNA oligo's targeting all isoforms of dynamin II confirmed the involvement of dynamin II in apoE secretion. Inhibition of secretion was not mediated via effects on mRNA or protein synthesis. 2D-gel electrophoresis showed that inhibition occurred after apoE was processed and glycosylated in the Golgi and live cell imaging showed that inhibited secretion was associated with reduced post-Golgi movement of apoE-GFP-containing vesicles. The effect was not restricted to macrophages, and was not mediated by the effects of the inhibitors on microtubules. Inhibition of dynamin also altered the constitutive secretion of other proteins, decreasing the secretion of fibronectin, matrix metalloproteinase 9, Chitinase-3-like protein 1 and lysozyme but unexpectedly increasing the secretion of the inflammatory mediator cyclophilin A. We conclude that pharmacological inhibitors of dynamin II modulate the constitutive secretion of macrophage apoE as a class effect, and that their capacity to modulate protein secretion may affect a range of biological processes.

  5. A novel role of sesamol in inhibiting NF-κB-mediated signaling in platelet activation

    Chang Chao-Chien

    2011-12-01

    Full Text Available Abstract Background Platelet activation is relevant to a variety of coronary heart diseases. Our previous studies revealed that sesamol possesses potent antiplatelet activity through increasing cyclic AMP formation. Although platelets are anucleated cells, they also express the transcription factor, NF-κB, that may exert non-genomic functions in platelet activation. Therefore, we further investigated the inhibitory roles of sesamol in NF-κB-mediated platelet function. Methods Platelet aggregation, Fura 2-AM fluorescence, and immunoblotting analysis were used in this study. Results NF-κB signaling events, including IKKβ phosphorylation, IκBα degradation, and p65 phosphorylation, were markedly activated by collagen (1 μg/ml in washed human platelets, and these signaling events were attenuated by sesamol (2.5~25 μM. Furthermore, SQ22536 and ODQ, inhibitors of adenylate cyclase and guanylate cyclase, respectively, strongly reversed the sesamol (25 μM-mediated inhibitory effects of IKKβ phosphorylation, IκBα degradation, and p65 phosphorylation stimulated by collagen. The protein kinase A (PKA inhibitor, H89, also reversed sesamol-mediated inhibition of IκBα degradation. Moreover, BAY11-7082, an NF-κB inhibitor, abolished IκBα degradation, phospholipase C (PLCγ2 phosphorylation, protein kinase C (PKC activation, [Ca2+]i mobilization, and platelet aggregation stimulated by collagen. Preincubation of platelets with the inhibitors, SQ22536 and H89, both strongly reversed sesamol-mediated inhibition of platelet aggregation and [Ca2+]i mobilization. Conclusions Sesamol activates cAMP-PKA signaling, followed by inhibition of the NF-κB-PLC-PKC cascade, thereby leading to inhibition of [Ca2+]i mobilization and platelet aggregation. Because platelet activation is not only linked to hemostasis, but also has a relevant role in inflammation and metastasis, our data demonstrating that inhibition of NF-κB interferes with platelet function may

  6. Amiloride inhibits rat mucosal ornithine decarboxylase activity and DNA synthesis

    Refeeding fasted rats induces a dramatic trophic response in gastrointestinal mucosa and is associated with elevations in both rate of DNA synthesis and ornithine decarboxylase (ODC) activity. The signal for these increases is unknown. Amiloride prevents cell alkalinization by blocking Na+-H+ exchange at apical epithelial cell membranes. In study 1, rats were fasted 48 h, treated with amiloride (0.5 to 500 mg/kg), and refed for 4 h. Refeeding increased ODC activities in the jejunal mucosa (X8) and liver (X19) but not in the oxyntic gland mucosa. In the jejunum, but not the liver, the activation of ODC was completely abolished by 100 mg/kg amiloride. In study 2, the rate of DNA synthesis was determine by measuring the rate of [3H]thymidine incorporation 16 h after refeeding. Refeeding resulted in significantly increased rates of DNA synthesis over fasted levels, and amiloride at 100 mg/kg significantly reduced the elevations in the jejenum and liver. In conclusion, amiloride inhibits the postprandial increases in jejunal ODC activity and DNA synthesis in the jejunum and liver. The results indicate that (1) the Na+-H+ antiport is essential to the increased ODC activity in the jejunum and liver after a meal and (2) increases in DNA synthesis and their suppression by amiloride are not necessary linked to ODC activity

  7. Restoration of Brain Acid Soluble Protein 1 Inhibits Proliferation and Migration of Thyroid Cancer Cells

    Guo, Run-Sheng; Yu, Yue; Chen, Jun; Chen, Yue-Yu; Shen, Na; Qiu, Ming

    2016-01-01

    Background: Brain acid soluble protein 1 (BASP1) is identified as a novel potential tumor suppressor in several cancers. However, its role in thyroid cancer has not been investigated yet. In the present study, the antitumor activities of BASP1 against the growth and migration of thyroid cancer cells were evaluated. Methods: BASP1 expression in thyroid cancer tissues and normal tissues were examined by immunohistochemical staining and the association between its expression and prognosis was analyzed. pcDNA-BASP1 carrying full length of BASP1 cDNA was constructed to restore the expression of BASP1 in thyroid cancer cell lines (BHT-101 and KMH-2). The cell proliferation in vitro and in vivo was evaluated by WST-1 assay and xenograft tumor models, respectively. Cell cycle distribution after transfection was analyzed using flow cytometry. Cell apoptosis after transfection was examined by annexin V/propidium iodide assay. The migration was examined using transwell assay. Results: BASP1 expression was abundant in normal tissues while it is significantly decreased in cancer tissues (P = 0.000). pcDNA-BASP1 restored the expression of BASP1 and significantly inhibited the growth of BHT-101 and KMH-2 cells as well as xenograft tumors in nude mice (P = 0.000). pcDNA-BASP1 induced G1 arrest and apoptosis in BHT-101 and KMH-2 cells. In addition, pcDNA-BASP1 significantly inhibited the cell migration. Conclusions: Downregulation of BASP1 expression may play a role in the tumorigenesis of thyroid cancer. Restoration of BASP1 expression exerted extensive antitumor activities against growth and migration of thyroid cancer cells, which suggested that BASP1 gene might act as a potential therapeutic agent for the treatment of thyroid cancer. PMID:27270539

  8. Behavioral Inhibition and Activation Systems: Differences in Substance Use Expectancy Organization and Activation in Memory

    Simons, Jeffrey S.; Dvorak, Robert D.; Lau-Barraco, Cathy

    2009-01-01

    We used multidimensional scaling to model the semantic network of alcohol and marijuana expectancies (N = 897). Preference mapping was used to estimate vectors representing patterns of activation through the network as a function of levels of behavioral inhibition (BIS) and behavioral activation (BAS). Individuals with low BIS combined with high BAS levels exhibited patterns of activation emphasizing behavioral activation similar to heavier drug users in previous research. High BIS, low BAS i...

  9. Fast inhibition of glutamate-activated currents by caffeine.

    Nicholas P Vyleta

    Full Text Available BACKGROUND: Caffeine stimulates calcium-induced calcium release (CICR in many cell types. In neurons, caffeine stimulates CICR presynaptically and thus modu