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Sample records for aggregation induces phosphorylation

  1. Effects of platelet inhibitors on propyl gallate-induced platelet aggregation, protein tyrosine phosphorylation, and platelet factor 3 activation.

    Science.gov (United States)

    Xiao, Hongyan; Kovics, Richard; Jackson, Van; Remick, Daniel G

    2004-04-01

    Propyl gallate (PG) is a platelet agonist characterized by inducing platelet aggregation, protein tyrosine phosphorylation, and platelet factor 3 activity. The mechanisms of platelet activation following PG stimulation were examined by pre-incubating platelets with well-defined platelet inhibitors using platelet aggregation, protein tyrosine phosphorylation, activated plasma clotting time, and annexin V binding by flow cytometry. PG-induced platelet aggregation and tyrosine phosphorylation of multiple proteins were substantially abolished by aspirin, apyrase, and abciximab (c7E3), suggesting that PG is associated with activation of platelet cyclooxygenase 1, adenosine phosphate receptors, and glycoprotein IIb/IIIa, respectively. The phosphorylation of the cytoskeletal enzyme pp60(c-src) increased following PG stimulation, but was blunted by pre-incubation of platelets with aspirin, apyrase, and c7E3, suggesting that tyrosine kinase is important for the signal transduction of platelet aggregation. Propyl gallate also activates platelet factor 3 by decreasing the platelet coagulation time and increasing platelet annexin V binding. Platelet incubation with aspirin, apyrase, and c7E3 did not alter PG-induced platelet coagulation and annexin V binding. The results suggest that platelet factor 3 activation and membrane phosphotidylserine expression were not involved with activation of platelet cyclooxygenase, adenosine phosphate receptors, and glycoprotein IIb/IIIa. PG is unique in its ability to stimulate platelet aggregation and coagulation simultaneously, and platelet inhibitors in this study affect only platelet aggregation but not platelet coagulation. PMID:15060414

  2. Covalent modifications of ribosomal proteins in growing and aggregation-competent dictyostelium discoideum: phosphorylation and methylation.

    Science.gov (United States)

    Ramagopal, S

    1991-04-01

    Phosphorylated and methylated ribosomal proteins were identified in vegetatively growing amoebae and in the starvation-induced, aggregation-competent cells of Dictyostelium discoideum. Of the 15 developmentally regulated cell-specific ribosomal proteins reported earlier, protein A and the acidic proteins A1, A2, and A3 were identified as phosphoproteins, and S5, S6, S10, and D were identified as methylated proteins. Three other ribosomal proteins were phosphorylated and 19 others methylated. S19, L13, A1, A2, and A3 were the predominant phosphoproteins in growing amoebae, whereas S20 and A were the predominant ones in the aggregation-competent cells. Among the methylated proteins, eight (S6, S10, S13, S30, D, L1, L2, and L31) were modified only during growth phase, six (S5, S7, S8, S24, S31, and L36) were altered only during aggregation-competent phase, and nine (S9, S27, S28, S29, S34, L7, L35, L41, and L42) were modified under both phases. Five proteins (S6, S24, L7, L41, and L42) were heavily methylated and of these, the large subunit proteins were present in both growing amoebae and aggregation-competent cells. These findings demonstrate that covalent modification of specific ribosomal proteins is regulated during cell differentiation in D. discoideum.

  3. Gap junction communication between cells aggregated on a cellulose-coated polystyrene: influence of connexin 43 phosphorylation.

    Science.gov (United States)

    Faucheux, N; Zahm, J M; Bonnet, N; Legeay, G; Nagel, M D

    2004-06-01

    The appropriate functioning of tissues and organ systems depends on intercellular communication such as gap junctions formed by connexin (Cx) protein channels between adjacent cells. We have previously shown that Swiss 3T3 cells aggregated on hydrophilic cellulose substratum Cuprophan (CU) establish short linear gap junctions composed of Cx 43 in cell surface plaques. This phenomenon seems to depend on the high intracellular cyclic AMP (cAMP) concentration triggered by attachment of the cells to CU. We have now used a cellulose-coated polystyrene inducing the same cell behaviour to analyse the gap junction communication between aggregated cells. The transfer of the dye Lucifer Yellow (LY) between cells showed that cells aggregated on cellulose substratum rapidly (within 90 min) establish functional gap junctions. Inhibitors of cAMP protein kinase (PKI) or protein kinase C (GF109203X) both inhibited the diffusion of LY between neighbouring cells. Western blot analysis showed that this change in permeability was correlated with a decrease in Cx 43 phosphorylation. Thus, cellulose substrata seem to induce cell-cell communication through Cx 43 phosphorylation modulated by PKA and PKC. To understand the mechanisms by which a substratum regulates gap junctional communication is critically important for the emerging fields of tissue engineering and biohybrid devices. PMID:14751734

  4. Aluminum induces lipid peroxidation and aggregation of human blood platelets

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    Neiva T.J.C.

    1997-01-01

    Full Text Available Aluminum (Al3+ intoxication is thought to play a major role in the development of Alzheimer's disease and in certain pathologic manifestations arising from long-term hemodialysis. Although the metal does not present redox capacity, it can stimulate tissue lipid peroxidation in animal models. Furthermore, in vitro studies have revealed that the fluoroaluminate complex induces diacylglycerol formation, 43-kDa protein phosphorylation and aggregation. Based on these observations, we postulated that Al3+-induced blood platelet aggregation was mediated by lipid peroxidation. Using chemiluminescence (CL of luminol as an index of total lipid peroxidation capacity, we established a correlation between lipid peroxidation capacity and platelet aggregation. Al3+ (20-100 µM stimulated CL production by human blood platelets as well as their aggregation. Incubation of the platelets with the antioxidants nor-dihydroguaiaretic acid (NDGA (100 µM and n-propyl gallate (NPG (100 µM, inhibitors of the lipoxygenase pathway, completely prevented CL and platelet aggregation. Acetyl salicylic acid (ASA (100 µM, an inhibitor of the cyclooxygenase pathway, was a weaker inhibitor of both events. These findings suggest that Al3+ stimulates lipid peroxidation and the lipoxygenase pathway in human blood platelets thereby causing their aggregation

  5. Neuroinflammation is not a prerequisite for diabetes-induced tau phosphorylation

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    Judith M Van Der Harg

    2015-11-01

    Full Text Available Abnormal phosphorylation and aggregation of tau is a key hallmark of Alzheimer's disease (AD. AD is a multifactorial neurodegenerative disorder for which Diabetes Mellitus (DM is a risk factor. In animal models for DM, the phosphorylation and aggregation of tau is induced or exacerbated, however the underlying mechanism is unknown. In addition to the metabolic dysfunction, DM is characterized by chronic low-grade inflammation. This was reported to be associated with a neuroinflammatory response in the hypothalamus of DM animal models. Neuroinflammation is also implicated in the development and progression of AD. It is unknown whether DM also induces neuroinflammation in brain areas affected in AD, the cortex and hippocampus. Here we investigated whether neuroinflammation could be the mechanistic trigger to induce tau phosphorylation in the brain of DM animals. Two distinct diabetic animal models were used; rats on free-choice high-fat high-sugar (fcHFHS diet that are insulin resistant and streptozotocin-treated rats that are insulin deficient. The streptozotocin-treated animals demonstrated increased tau phosphorylation in the brain as expected, whereas the fcHFHS diet fed animals did not. Remarkably, neither of the diabetic animal models showed reactive microglia or increased GFAP and COX-2 levels in the cortex or hippocampus. From this, we conclude: 1. DM does not induce neuroinflammation in brain regions affected in AD, and 2. Neuroinflammation is not a prerequisite for tau phosphorylation. Neuroinflammation is therefore not the mechanism that explains the close connection between DM and AD.

  6. Cisplatinum and Taxol Induce Different Patterns of p53 Phosphorylation

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    Giovanna Damia

    2001-01-01

    Full Text Available Posttranslational modifications of p53 induced by two widely used anticancer agents, cisplatinum (DDP and taxol were investigated in two human cancer cell lines. Although both drugs were able to induce phosphorylation at serine 20 (Ser20, only DDP treatment induced p53 phosphorylation at serine 15 (Ser15. Moreover, both drug treatments were able to increase p53 levels and consequently the transcription of waf1 and mdm-2 genes, although DDP treatment resulted in a stronger inducer of both genes. Using two ataxia telangiectasia mutated (ATM cell lines, the role of ATM in druginduced p53 phosphorylations was investigated. No differences in drug-induced p53 phosphorylation could be observed, indicating that ATM is not the kinase involved in these phosphorylation events. In addition, inhibition of DNA-dependent protein kinase activity by wortmannin did not abolish p53 phosphorylation at Ser15 and Ser20, again indicating that DNA-PK is unlikely to be the kinase involved. After both taxol and DDP treatments, an activation of hCHK2 was found and this is likely to be responsible for phosphorylation at Ser20. In contrast, only DDP was able to activate ATR, which is the candidate kinase for phosphorylation of Ser15 by this drug. This data clearly suggests that differential mechanisms are involved in phosphorylation and activation of p53 depending on the drug type.

  7. Control of aggregation-induced emission by DNA hybridization

    OpenAIRE

    Li, Shaoguang; Langenegger, Simon Matthias; Häner, Robert

    2013-01-01

    Aggregation-induced emission (AIE) was studied by hybridization of dialkynyl-tetraphenylethylene (DATPE) modified DNA strands. Molecular aggregation and fluorescence of DATPEs are controlled by duplex formation.

  8. Cyanogen induced phosphorylation of D-fructose. [prebiotic modeling

    Science.gov (United States)

    Degani, CH.; Kawatsuji, M.; Halmann, M.

    1975-01-01

    It has been demonstrated that a phosphorylated sugar, identified as alpha-D-fructopyranose, can be formed as the result of cyanogen-induced phosphorylation of D-fructose at pH 8.8. The product was isolated from barium and cyclohexylammonium salts and identified on the basis of its chromatographic and electrophoretic properties, its lability to hydrolysis by alkaline phosphatase, the rate of its acid-catalyzed hydrolysis, and the results of periodate oxidation and optical rotatory measurements. These results support the suggestion that the cyanogen-induced phosphorylation of free sugars could be a possible process for formation of sugar phosphates under prebiotic conditions (Halman et al., 1969).

  9. Histone deacetylase inhibitors block IFNγ-induced STAT1 phosphorylation.

    Science.gov (United States)

    Ginter, Torsten; Bier, Carolin; Knauer, Shirley K; Sughra, Kalsoom; Hildebrand, Dagmar; Münz, Tobias; Liebe, Theresa; Heller, Regine; Henke, Andreas; Stauber, Roland H; Reichardt, Werner; Schmid, Johannes A; Kubatzky, Katharina F; Heinzel, Thorsten; Krämer, Oliver H

    2012-07-01

    Signal transducer and activator of transcription 1 (STAT1) is important for innate and adaptive immunity. Histone deacetylase inhibitors (HDACi) antagonize unbalanced immune functions causing chronic inflammation and cancer. Phosphorylation and acetylation regulate STAT1 and different IFNs induce phosphorylated STAT1 homo-/heterodimers, e.g. IFNα activates several STATs whereas IFNγ only induces phosphorylated STAT1 homodimers. In transformed cells HDACi trigger STAT1 acetylation linked to dephosphorylation by the phosphatase TCP45. It is unclear whether acetylation differentially affects STAT1 activated by IFNα or IFNγ, and if cellular responses to both cytokines depend on a phosphatase-dependent inactivation of acetylated STAT1. Here, we report that HDACi counteract IFN-induced phosphorylation of a critical tyrosine residue in the STAT1 C-terminus in primary cells and hematopoietic cells. STAT1 mutants mimicking a functionally inactive DNA binding domain (DBD) reveal that the number of acetylation-mimicking sites in STAT1 determines whether STAT1 is recruited to response elements after stimulation with IFNγ. Furthermore, we show that IFNα-induced STAT1 heterodimers carrying STAT1 molecules mimicking acetylation bind cognate DNA and provide innate anti-viral immunity. IFNγ-induced acetylated STAT1 homodimers are though inactive, suggesting that heterodimerization and complex formation can rescue STAT1 lacking a functional DBD. Apparently, the type of cytokine determines how acetylation affects the nuclear entry and DNA binding of STAT1. Our data contribute to a better understanding of STAT1 regulation by acetylation. PMID:22425562

  10. Potent anticholinesterasic and neuroprotective pyranotacrines as inhibitors of beta-amyloid aggregation, oxidative stress and tau-phosphorylation for Alzheimer's disease.

    Science.gov (United States)

    García-Font, Nuria; Hayour, Hasna; Belfaitah, Ali; Pedraz, Jorge; Moraleda, Ignacio; Iriepa, Isabel; Bouraiou, Abdelmalek; Chioua, Mourad; Marco-Contelles, José; Oset-Gasque, María Jesús

    2016-08-01

    Herein we describe the synthesis and in vitro biological evaluation of thirteen new, racemic, diversely functionalized 2-chloroquinolin-3-yl substituted PyranoTacrines (PTs) as multipotent tacrine analogues for Alzheimer's disease (AD) therapy. Among these compounds, 1-(5-amino-4-(2-chloro-7-methoxyquinolin-3-yl)-2-methyl-6,7,8,9-tetrahydro-4H-pyrano [2,3-b]quinolin-3-yl)éthanone (9) and ethyl 5-amino-4-(2-chloroquinolin-3-yl)-2-methyl-6,7,8,9-tetrahydro-4H-pyrano[2,3-b]quinoline-3-carboxylate (4) were found to be non-neurotoxic agents in human neuroblastoma SHSY5Y cells. Compounds 9 (IC50 = 0.47 ± 0.13 μM) and 4 (IC50 = 0.48 ± 0.05 μM) are potent, mixed-type (9: Ki = 0.0142 ± 0.003 μM), and selective EeAChE inhibitors, binding at the both catalytic and peripheral anionic site of the enzyme. Compounds 9 and 4 are neuroprotective agents at low μM concentrations upon decreased viability of SHSY5Y cells induced by oxidative stress, and stimulators of GSK3β-dependent tau phosphorylation. In addition, molecules 9 and 4 effectively counteract Aβ-aggregation on exposure to Aβ1-40, as well as Aβ1-40 aggregation-dependent tau-oligomerization and phosphorylation in (396)Ser, which could be ascribed to the anti-aggregating properties shown in vitro. Thus, a new family of tacrine analogues, whose potent AChEI activity is linked to both their Aβ-aggregating and tau-phosphorylation inhibitory capacities, has been discovered for the potential treatment of AD. PMID:27128182

  11. Non-aggregating tau phosphorylation by cyclin-dependent kinase 5 contributes to motor neuron degeneration in spinal muscular atrophy.

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    Miller, Nimrod; Feng, Zhihua; Edens, Brittany M; Yang, Ben; Shi, Han; Sze, Christie C; Hong, Benjamin Taige; Su, Susan C; Cantu, Jorge A; Topczewski, Jacek; Crawford, Thomas O; Ko, Chien-Ping; Sumner, Charlotte J; Ma, Long; Ma, Yong-Chao

    2015-04-15

    Mechanisms underlying motor neuron degeneration in spinal muscular atrophy (SMA), the leading inherited cause of infant mortality, remain largely unknown. Many studies have established the importance of hyperphosphorylation of the microtubule-associated protein tau in various neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. However, tau phosphorylation in SMA pathogenesis has yet to be investigated. Here we show that tau phosphorylation on serine 202 (S202) and threonine 205 (T205) is increased significantly in SMA motor neurons using two SMA mouse models and human SMA patient spinal cord samples. Interestingly, phosphorylated tau does not form aggregates in motor neurons or neuromuscular junctions (NMJs), even at late stages of SMA disease, distinguishing it from other tauopathies. Hyperphosphorylation of tau on S202 and T205 is mediated by cyclin-dependent kinase 5 (Cdk5) in SMA disease condition, because tau phosphorylation at these sites is significantly reduced in Cdk5 knock-out mice; genetic knock-out of Cdk5 activating subunit p35 in an SMA mouse model also leads to reduced tau phosphorylation on S202 and T205 in the SMA;p35(-/-) compound mutant mice. In addition, expression of the phosphorylation-deficient tauS202A,T205A mutant alleviates motor neuron defects in a zebrafish SMA model in vivo and mouse motor neuron degeneration in culture, whereas expression of phosphorylation-mimetic tauS202E,T205E promotes motor neuron defects. More importantly, genetic knock-out of tau in SMA mice rescues synapse stripping on motor neurons, NMJ denervation, and motor neuron degeneration in vivo. Altogether, our findings suggest a novel mechanism for SMA pathogenesis in which hyperphosphorylation of non-aggregating tau by Cdk5 contributes to motor neuron degeneration.

  12. Cognitive defects are reversible in inducible mice expressing pro-aggregant full-length human Tau

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    Sydow, Astrid; Hofmann, Anne; Wu, Dan; Messing, Lars; Balschun, Detlef; D'Hooge, Rudi; Mandelkow, Eva-Maria

    2016-01-01

    Neurofibrillary lesions of abnormal Tau are hallmarks of Alzheimer´s disease and frontotemporal dementias. Our regulatable (Tet-OFF) mouse models of tauopathy express variants of human full-length Tau in the forebrain (CaMKIIα promoter) either with mutation ΔK280 (pro-aggregant) or ΔK280/I277P/I308P (anti-aggregant). Co-expression of luciferase enables in vivo quantification of gene expression by bioluminescence imaging. Pro-aggregant mice develop synapse loss and Tau pathology including missorting, phosphorylation and early pretangle formation, whereas anti-aggregant mice do not. We correlated hippocampal Tau pathology with learning/memory performance and synaptic plasticity. Pro-aggregant mice at 16 months of gene expression exhibited severe cognitive deficits in Morris water-maze and in passive-avoidance paradigms, whereas anti-aggregant mice were comparable to controls. Cognitive impairment of pro-aggregant mice was accompanied by loss of hippocampal LTP in CA1 and CA3 areas and by a reduction of synaptic proteins and dendritic spines, although no neuronal loss was observed. Remarkably, memory and LTP recovered when pro-aggregant Tau was switched-OFF for ∼4 months, Tau phosphorylation and missorting were reversed, and synapses recovered. Moreover soluble and insoluble pro-aggregant hTau40 disappeared while insoluble mouse Tau was still present. This study links early Tau pathology without neurofibrillary tangles and neuronal death to cognitive decline and synaptic dysfunction. It demonstrates that Tau-induced impairments are reversible after switching-OFF pro-aggregant Tau. Therefore our mouse model may mimic an early phase of AD when the hippocampus does not yet suffer from irreversible cell death but cognitive deficits are already striking. It offers potential to evaluate drugs with regard to learning and memory performance. PMID:22532069

  13. Phosphorylation by Cdk1 induces Plk1-mediated vimentin phosphorylation during mitosis

    NARCIS (Netherlands)

    Yamaguchi, Tomoya; Goto, Hidemasa; Yokoyama, Tomoya; Silljé, Herman; Hanisch, Anja; Uldschmid, Andreas; Takai, Yasushi; Oguri, Takashi; Nigg, Erich A; Inagaki, Masaki

    2005-01-01

    Several kinases phosphorylate vimentin, the most common intermediate filament protein, in mitosis. Aurora-B and Rho-kinase regulate vimentin filament separation through the cleavage furrow-specific vimentin phosphorylation. Cdk1 also phosphorylates vimentin from prometaphase to metaphase, but its si

  14. Adriamycin induces H2AX phosphorylation in human spermatozoa

    Institute of Scientific and Technical Information of China (English)

    Zhong-Xiang Li; Ting-Ting Wang; Yan-Ting Wu; Chen-Ming Xu; Min-Yue Dong; Jian-Zhong Sheng; He-Feng Huang

    2008-01-01

    Aim: To investigate whether adriamycin induces DNA damage and the formation of γH2AX (the phosphorylated form of histone H2AX) foci in mature spermatozoa. Methods: Human spermatozoa were treated with adriamycin at different concentrations. γH2AX was analyzed by immunofluorescent staining and flow cytometry and double- strand breaks (DSB) were detected by the comet assay. Results: The neutral comet assay revealed that the treatment with adriamycin at 2 μg/mL for different times (0.5, 2, 8 and 24 h), or for 8 h at different concentrations (0.4, 2 and 10 μg/mL), induced significant DSB in spermatozoa. Immunofluorent staining and flow cytometry showed that the expression of γH2AX was increased in a dose-dependent and time-dependant manner after the treatment of adriamycin. Adriamycin also induced the concurrent appearance of DNA maintenance/repair proteins RAD50 and 53BP1 with γH2AX in spermatozoa. Wortmannin, an inhibitor of the phosphatidylinositol 3-kinase (PI3K) family, abolished the co-appearance of these two proteins with γH2AX. Conclusion: Human mature spermatozoa have the same response to DSB-induced H2AX phosphorylation and subsequent recruitment of DNA maintenance/repair proteins as somatic cells.

  15. Rapid changes in protein phosphorylation associated with light-induced gravity perception in corn roots

    Science.gov (United States)

    McFadden, J. J.; Poovaiah, B. W.

    1988-01-01

    The effect of light and calcium depletion on in vivo protein phosphorylation was tested using dark-grown roots of Merit corn. Light caused rapid and specific promotion of phosphorylation of three polypeptides. Pretreatment of roots with ethylene glycol bis N,N,N',N' tetraacetic acid and A23187 prevented light-induced changes in protein phosphorylation. We postulate that these changes in protein phosphorylation are involved in the light-induced gravity response.

  16. Salt-induced aggregation of stiff polyelectrolytes

    International Nuclear Information System (INIS)

    Molecular dynamics simulation techniques are used to study the process of aggregation of highly charged stiff polyelectrolytes due to the presence of multivalent salt. The dominant kinetic mode of aggregation is found to be the case of one end of one polyelectrolyte meeting others at right angles, and the kinetic pathway to bundle formation is found to be similar to that of flocculation dynamics of colloids as described by Smoluchowski. The aggregation process is found to favor the formation of finite bundles of 10-11 filaments at long times. Comparing the distribution of the cluster sizes with the Smoluchowski formula suggests that the energy barrier for the aggregation process is negligible. Also, the formation of long-lived metastable structures with similarities to the raft-like structures of actin filaments is observed within a range of salt concentration.

  17. Effects of Suilysin on Streptococcus suis-Induced Platelet Aggregation

    Science.gov (United States)

    Zhang, Shengwei; Wang, Junping; Chen, Shaolong; Yin, Jiye; Pan, Zhiyuan; Liu, Keke; Li, Lin; Zheng, Yuling; Yuan, Yuan; Jiang, Yongqiang

    2016-01-01

    Blood platelets play important roles during pathological thrombocytopenia in streptococcal toxic shock syndrome (STSS). Streptococcus suis (S. suis) an emerging human pathogen, can cause STSS similarly to S. pyogenes. However, S. suis interactions with platelets are poorly understood. Here, we found that suilysin (SLY), different from other bacterial cholesterol-dependent cytolysins (CDCs), was the sole stimulus that induced platelet aggregation. Furthermore, the inside-out activation of GPIIb/IIIa of platelets mediated SLY-induced platelet aggregation. This process was triggered by Ca2+ influx that depend on the pore forming on platelets by SLY. Additionally, although SLY induced α-granule release occurred via the MLCK-dependent pathway, PLC-β-IP3/DAG-MLCK and Rho-ROCK-MLCK signaling were not involved in SLY-induced platelet aggregation. Interestingly, the pore dependent Ca2+ influx was also found to participate in the induction of platelet aggregation with pneumolysin (PLY) and streptolysin O (SLO), two other CDCs. It is possible that the CDC-mediated platelet aggregation we observed in S. suis is a similar response mechanism to that used by a wide range of bacteria. These findings might lead to the discovery of potential therapeutic targets for S. suis-associated STSS. PMID:27800304

  18. Aggregation in charged nanoparticles solutions induced by different interactions

    Science.gov (United States)

    Abbas, S.; Kumar, Sugam; Aswal, V. K.; Kohlbrecher, J.

    2016-05-01

    Small-angle neutron scattering (SANS) has been used to study the aggregation of anionic silica nanoparticles as induced through different interactions. The nanoparticle aggregation is induced by addition of salt (NaCl), cationic protein (lysozyme) and non-ionic surfactant (C12E10) employing different kind of interactions. The results show that the interaction in presence of salt can be explained using DLVO theory whereas non-DLVO forces play important role for interaction of nanoparticles with protein and surfactant. The presence of salt screens the repulsion between charged nanoparticles giving rise to a net attraction in the DLVO potential. On the other hand, strong electrostatic attraction between nanoparticle and oppositely charged protein leads to protein-mediated nanoparticle aggregation. In case of non-ionic surfactant, the relatively long-range attractive depletion interaction is found to be responsible for the particle aggregation. Interestingly, the completely different interactions lead to similar kind of aggregate morphology. The nanoparticle aggregates formed are found to have mass fractal nature having a fractal dimension (~2.5) consistent with diffusion limited type of fractal morphology in all three cases.

  19. The Stimulus-induced Tyrosine Phosphorylation of Munc18c Facilitates Vesicle Exocytosis*

    OpenAIRE

    Oh, Eunjin; Thurmond, Debbie C.

    2006-01-01

    Stimulus-induced tyrosine phosphorylation of Munc18c was investigated as a potential regulatory mechanism by which the Munc18c-Syntaxin 4 complex can be dissociated in response to divergent stimuli in multiple cell types. Use of [32P]orthophosphate incorporation, pervanadate treatment, and phosphotyrosine-specific antibodies demonstrated that Munc18c underwent tyrosine phosphorylation. Phosphorylation was apparent under basal conditions, but levels were significantly increased within 5 min of...

  20. Formaldehyde-induced histone H3 phosphorylation via JNK and the expression of proto-oncogenes

    Energy Technology Data Exchange (ETDEWEB)

    Yoshida, Ikuma; Ibuki, Yuko, E-mail: ibuki@u-shizuoka-ken.ac.jp

    2014-12-15

    Graphical abstract: - Highlights: • Formaldehyde modified histones. • The phosphorylation of H3S10 was increased at the promoter regions of proto-oncogenes. • The phosphorylation of H2AXS139 was attributed to FA-induced DNA damage. • The FA-induced initiation and promotion of cancer could be judged by these modifications. - Abstract: Formaldehyde (FA) is a very reactive compound that forms DNA adducts and DNA-protein crosslinks, which are known to contribute to FA-induced mutations and carcinogenesis. Post-translational modifications to histones have recently attracted attention due to their link with cancer. In the present study, we examined histone modifications following a treatment with FA. FA significantly phosphorylated histone H3 at serine 10 (H3S10), and at serine 28 (H3S28), the time-course of which was similar to the phosphorylation of H2AX at serine 139 (γ-H2AX), a marker of DNA double strand breaks. The temporal deacetylation of H3 was observed due to the reaction of FA with the lysine residues of histones. The phosphorylation mechanism was then analyzed by focusing on H3S10. The nuclear distribution of the phosphorylation of H3S10 and γ-H2AX did not overlap, and the phosphorylation of H3S10 could not be suppressed with an inhibitor of ATM/ATR, suggesting that the phosphorylation of H3S10 was independent of the DNA damage response. ERK and JNK in the MAPK pathways were phosphorylated by the treatment with FA, in which the JNK pathway was the main target for phosphorylation. The phosphorylation of H3S10 increased at the promoter regions of c-fos and c-jun, indicating a relationship between FA-induced tumor promotion activity and phosphorylation of H3S10. These results suggested that FA both initiates and promotes cancer, as judged by an analysis of histone modifications.

  1. Tumor suppressor PTEN affects tau phosphorylation: deficiency in the phosphatase activity of PTEN increases aggregation of an FTDP-17 mutant Tau

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    Zhang Xue

    2006-07-01

    Full Text Available Abstract Background Aberrant hyperphosphorylation of tau protein has been implicated in a variety of neurodegenerative disorders. Although a number of protein kinases have been shown to phosphorylate tau in vitro and in vivo, the molecular mechanisms by which tau phosphorylation is regulated pathophysiologically are largely unknown. Recently, a growing body of evidence suggests a link between tau phosphorylation and PI3K signaling. In this study, phosphorylation, aggregation and binding to the microtubule of a mutant frontal temporal dementia and parkinsonism linked to chromosome 17 (FTDP-17 tau in the presence of tumor suppressor PTEN, a major regulatory component in PI3K signaling, were investigated. Results Phosphorylation of the human mutant FTDP-17 tau, T40RW, was evaluated using different phospho-tau specific antibodies in the presence of human wild-type or phosphatase activity null mutant PTEN. Among the evaluated phosphorylation sites, the levels of Ser214 and Thr212 phospho-tau proteins were significantly decreased in the presence of wild-type PTEN, and significantly increased when the phosphatase activity null mutant PTEN was ectopically expressed. Fractionation of the mutant tau transfected cells revealed a significantly increased level of soluble tau in cytosol when wild-type PTEN was expressed, and an elevated level of SDS-soluble tau aggregates in the presence of the mutant PTEN. In addition, the filter/trap assays detected more SDS-insoluble mutant tau aggregates in the cells overexpressing the mutant PTEN compared to those in the cells overexpressing wild-type PTEN and control DNA. This notion was confirmed by the immunocytochemical experiment which demonstrated that the overexpression of the phosphatase activity null mutant PTEN caused the mutant tau to form aggregates in the COS-7 cells. Conclusion Tumor suppressor PTEN can alleviate the phosporylation of the mutant FTDP-17 tau at specific sites, and the phosphatase activity

  2. Accurate modelling of flow induced stresses in rigid colloidal aggregates

    Science.gov (United States)

    Vanni, Marco

    2015-07-01

    A method has been developed to estimate the motion and the internal stresses induced by a fluid flow on a rigid aggregate. The approach couples Stokesian dynamics and structural mechanics in order to take into account accurately the effect of the complex geometry of the aggregates on hydrodynamic forces and the internal redistribution of stresses. The intrinsic error of the method, due to the low-order truncation of the multipole expansion of the Stokes solution, has been assessed by comparison with the analytical solution for the case of a doublet in a shear flow. In addition, it has been shown that the error becomes smaller as the number of primary particles in the aggregate increases and hence it is expected to be negligible for realistic reproductions of large aggregates. The evaluation of internal forces is performed by an adaptation of the matrix methods of structural mechanics to the geometric features of the aggregates and to the particular stress-strain relationship that occurs at intermonomer contacts. A preliminary investigation on the stress distribution in rigid aggregates and their mode of breakup has been performed by studying the response to an elongational flow of both realistic reproductions of colloidal aggregates (made of several hundreds monomers) and highly simplified structures. A very different behaviour has been evidenced between low-density aggregates with isostatic or weakly hyperstatic structures and compact aggregates with highly hyperstatic configuration. In low-density clusters breakup is caused directly by the failure of the most stressed intermonomer contact, which is typically located in the inner region of the aggregate and hence originates the birth of fragments of similar size. On the contrary, breakup of compact and highly cross-linked clusters is seldom caused by the failure of a single bond. When this happens, it proceeds through the removal of a tiny fragment from the external part of the structure. More commonly, however

  3. Assembly of naphthalenediimide conjugated peptides: aggregation induced changes in fluorescence.

    Science.gov (United States)

    Basak, Shibaji; Nanda, Jayanta; Banerjee, Arindam

    2013-08-01

    Naphthalenediimide appended peptide based self-assembly was studied. Interestingly, an aggregation induced drastic change in the fluorescence property and gel formation were observed depending on the solvent composition (chloroform : methylcyclohexane) at a fixed concentration of 1.6 mM at room temperature. PMID:23799544

  4. α- and γ-mangostin cause shape changes, inhibit aggregation and induce cytolysis of rat platelets.

    Science.gov (United States)

    Liu, Yingqiu; Park, Jung-Min; Chang, Kyung-Hwa; Chin, Young-Won; Lee, Moo-Yeol

    2015-10-01

    α- and γ-mangostin are natural xanthones isolated from mangosteen (Garcinia mangostana) and the major constituents responsible for the plant's diverse biological activities. In this study, the effects of α- and γ-mangostin on platelets were investigated based on their possible antiplatelet activity. Treatment of isolated platelets with α-mangostin resulted in attenuation of platelet aggregatory response to collagen, thrombin or ADP. Such antiaggregatory effects were concentration-dependent in ranges of 1-10 μM. Interestingly, α-mangostin alone induced shape changes in platelets at the same concentration, and higher levels, 25 and 50 μM caused platelet lysis. Similarly, γ-mangostin induced shape changes and inhibited aggregation at 2.5-25 μM, while 50 and 100 μM γ-mangostin exhibited cytotoxicity. Platelet shape change induced by α- and γ-mangostin was accompanied by increases in myosin light chain (MLC) phosphorylation. MLC phosphorylation and subsequent shape changes were prevented by pretreatment with Rho kinase (ROCK) inhibitor Y-27632, but not by the intracellular Ca(2+) chelating with BAPTA-AM and extracellular Ca(2+) removal. Cytolysis by both α- and γ-mangostin was abolished in the absence of extracellular Ca(2+). Taken together, α- and γ-mangostin have differential effects on platelets depending on their concentration, which includes inducing shape change, inhibiting aggregation and causing cytolysis. Platelet shape change is attributed to stimulation of the Rho/ROCK signaling pathway, while platelet lysis is presumably mediated by extracellular Ca(2+) influx. These results suggest that mangosteen consumption may have potential platelet effects, although the in vivo or clinical consequences have yet to be assessed. PMID:26343955

  5. EBP50 inhibits EGF-induced breast cancer cell proliferation by blocking EGFR phosphorylation.

    Science.gov (United States)

    Yao, Wenfang; Feng, Duiping; Bian, Weihua; Yang, Longyan; Li, Yang; Yang, Zhiyu; Xiong, Ying; Zheng, Junfang; Zhai, Renyou; He, Junqi

    2012-11-01

    Ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50) suppresses breast cancer cell proliferation, potentially through its regulatory effect on epidermal growth factor receptor (EGFR) signaling, although the mechanism by which this occurs remains unknown. Thus in our studies, we aimed to determine the effect of EBP50 expression on EGF-induced cell proliferation and activation of EGFR signaling in the breast cancer cell lines, MDA-MB-231 and MCF-7. In MDA-MB-231 cells, which express low levels of EBP50, EBP50 overexpression inhibited EGF-induced cell proliferation, ERK1/2 and AKT phosphorylation. In MCF-7 cells, which express high levels of EBP50, EBP50 knockdown promoted EGF-induced cell proliferation, ERK1/2 and AKT phosphorylation. Knockdown of EBP50 in EBP50-overexpressed MDA-MB-231 cells abrogated the inhibitory effect of EBP50 on EGF-stimulated ERK1/2 phosphorylation and restoration of EBP50 expression in EBP50-knockdown MCF-7 cells rescued the inhibition of EBP50 on EGF-stimulated ERK1/2 phosphorylation, further confirming that the activation of EGF-induced downstream molecules could be specifically inhibited by EBP50 expression. Since EGFR signaling was triggered by EGF ligands via EGFR phosphorylation, we further detected the phosphorylation status of EGFR in the presence or absence of EBP50 expression. Overexpression of EBP50 in MDA-MB-231 cells inhibited EGF-stimulated EGFR phosphorylation, whereas knockdown of EBP50 in MCF-7 cells enhanced EGF-stimulated EGFR phosphorylation. Meanwhile, total expression levels of EGFR were unaffected during EGF stimulation. Taken together, our data shows that EBP50 can suppress EGF-induced proliferation of breast cancer cells by inhibiting EGFR phosphorylation and blocking EGFR downstream signaling in breast cancer cells. These results provide further insight into the molecular mechanism by which EBP50 regulates the development and progression of breast cancer.

  6. Rubrene analogues with the aggregation-induced emission enhancement behaviour

    DEFF Research Database (Denmark)

    Zhang, Xiaoxu; Sørensen, Jakob Kryger; Fu, Xiaowei;

    2014-01-01

    In the light of the principle of aggregation-induced emission enhancement (AIEE), the rubrene analogue with orange light-emitting properties is designed and synthesized by substituting the phenyl side groups of rubrene with thienyl groups. To the best of our knowledge, this is the first report...... on the synthesis of rubrene with AIEE behaviour, thus paving the way for the development of light-emitting rubrene derivatives. This journal is © 2014 The Royal Society of Chemistry....

  7. Formaldehyde-induced histone H3 phosphorylation via JNK and the expression of proto-oncogenes.

    Science.gov (United States)

    Yoshida, Ikuma; Ibuki, Yuko

    2014-12-01

    Formaldehyde (FA) is a very reactive compound that forms DNA adducts and DNA-protein crosslinks, which are known to contribute to FA-induced mutations and carcinogenesis. Post-translational modifications to histones have recently attracted attention due to their link with cancer. In the present study, we examined histone modifications following a treatment with FA. FA significantly phosphorylated histone H3 at serine 10 (H3S10), and at serine 28 (H3S28), the time-course of which was similar to the phosphorylation of H2AX at serine 139 (γ-H2AX), a marker of DNA double strand breaks. The temporal deacetylation of H3 was observed due to the reaction of FA with the lysine residues of histones. The phosphorylation mechanism was then analyzed by focusing on H3S10. The nuclear distribution of the phosphorylation of H3S10 and γ-H2AX did not overlap, and the phosphorylation of H3S10 could not be suppressed with an inhibitor of ATM/ATR, suggesting that the phosphorylation of H3S10 was independent of the DNA damage response. ERK and JNK in the MAPK pathways were phosphorylated by the treatment with FA, in which the JNK pathway was the main target for phosphorylation. The phosphorylation of H3S10 increased at the promoter regions of c-fos and c-jun, indicating a relationship between FA-induced tumor promotion activity and phosphorylation of H3S10. These results suggested that FA both initiates and promotes cancer, as judged by an analysis of histone modifications.

  8. Dexmedetomidine-Induced Contraction Involves CPI-17 Phosphorylation in Isolated Rat Aortas

    Science.gov (United States)

    Ok, Seong-Ho; Kwon, Seong-Chun; Baik, Jiseok; Hong, Jeong-Min; Oh, Jiah; Han, Jeong Yeol; Sohn, Ju-Tae

    2016-01-01

    Dexmedetomidine, a highly selective α-2 adrenoceptor agonist, produces vasoconstriction, which leads to transiently increased blood pressure. The goal of this study was to investigate specific protein kinases and the associated cellular signal pathways responsible for the increased calcium sensitization induced by dexmedetomidine in isolated rat aortas, with a particular focus on phosphorylation-dependent inhibitory protein of myosin phosphatase (CPI-17). The effect of Y-27632 and chelerythrine on the dexmedetomidine-induced intracellular calcium concentration ([Ca2+]i) and tension were assessed using fura-2-loaded aortic strips. The effects of rauwolscine, Y-27632, chelerythrine, and ML-7 hydrochloride on the dexmedetomidine-induced phosphorylation of CPI-17 or of the 20-kDa regulatory light chain of myosin (MLC20) were investigated in rat aortic vascular smooth muscle cells. The effects of rauwolscine, Y-27632, and chelerythrine on the membrane translocation of Rho-kinase and protein kinase C (PKC) phosphorylation induced by dexmedetomidine were assessed. Y-27632 and chelerythrine each reduced the slopes of the [Ca2+]i-tension curves of dexmedetomidine-induced contraction, and Y-27632 more strongly reduced these slopes than did chelerythrine. Rauwolscine, Y-27632, chelerythrine, and ML-7 hydrochloride attenuated the dexmedetomidine-induced phosphorylation of CPI-17 and MLC20. Taken together, these results suggest that dexmedetomidine-induced contraction involves calcium sensitization, which appears to be mediated by CPI-17 phosphorylation via Rho-kinase or PKC. PMID:27706026

  9. Flagellin-induced NLRC4 phosphorylation primes the inflammasome for activation by NAIP5.

    Science.gov (United States)

    Matusiak, Magdalena; Van Opdenbosch, Nina; Vande Walle, Lieselotte; Sirard, Jean-Claude; Kanneganti, Thirumala-Devi; Lamkanfi, Mohamed

    2015-02-01

    The Nlrc4 inflammasome contributes to immunity against intracellular pathogens that express flagellin and type III secretion systems, and activating mutations in NLRC4 cause autoinflammation in patients. Both Naip5 and phosphorylation of Nlrc4 at Ser533 are required for flagellin-induced inflammasome activation, but how these events converge upon inflammasome activation is not known. Here, we showed that Nlrc4 phosphorylation occurs independently of Naip5 detection of flagellin because Naip5 deletion in macrophages abolished caspase-1 activation, interleukin (IL)-1β secretion, and pyroptosis, but not Nlrc4 phosphorylation by cytosolic flagellin of Salmonella Typhimurium and Yersinia enterocolitica. ASC speck formation and caspase-1 expression also were dispensable for Nlrc4 phosphorylation. Interestingly, Helicobacter pylori flagellin triggered robust Nlrc4 phosphorylation, but failed to elicit caspase-1 maturation, IL-1β secretion, and pyroptosis, suggesting that it retained Nlrc4 Ser533 phosphorylating-activity despite escaping Naip5 detection. In agreement, the flagellin D0 domain was required and sufficient for Nlrc4 phosphorylation, whereas deletion of the S. Typhimurium flagellin carboxy-terminus prevented caspase-1 maturation only. Collectively, this work suggests a biphasic activation mechanism for the Nlrc4 inflammasome in which Ser533 phosphorylation prepares Nlrc4 for subsequent activation by the flagellin sensor Naip5. PMID:25605939

  10. Fluorescence Aggregation-Caused Quenching versus Aggregation-Induced Emission: A Visual Teaching Technology for Undergraduate Chemistry Students

    Science.gov (United States)

    Ma, Xiaofeng; Sun, Rui; Cheng, Jinghui; Liu, Jiaoyan; Gou, Fei; Xiang, Haifeng; Zhou, Xiangge

    2016-01-01

    A laboratory experiment visually exploring two opposite basic principles of fluorescence of aggregation-caused quenching (ACQ) and aggregation-induced emission (AIE) is demonstrated. The students would prepared two salicylaldehyde-based Schiff bases through a simple one-pot condensation reaction of one equiv of 1,2-diamine with 2 equiv of…

  11. Dexmedetomidine-induced Contraction Involves Phosphorylation of Caldesmon by JNK in Endothelium-denuded Rat Aortas

    OpenAIRE

    Baik, Jiseok; Ok, Seong-Ho; Cho, Hyunhoo; Yu, Jongsun; Kim, Woochan; Nam, In-Koo; Choi, Mun-Jeoung; Lee, Heon-Keun; Sohn, Ju-Tae

    2014-01-01

    Caldesmon, an inhibitory actin binding protein, binds to actin and inhibits actin-myosin interactions, whereas caldesmon phosphorylation reverses the inhibitory effect of caldesmon on actin-myosin interactions, potentially leading to enhanced contraction. The goal of this study was to investigate the cellular signaling pathway responsible for caldesmon phosphorylation, which is involved in the regulation of the contraction induced by dexmedetomidine (DMT), an alpha-2 adrenoceptor agonist, in ...

  12. Alzheimer—like phosphorylation of tau and neurofilament induced by cocaine in vivo

    Institute of Scientific and Technical Information of China (English)

    LIUShi-Jie; FANGZheng-Yu; YANGYing; DENGHeng-Mei; WANGJian-Zhi

    2003-01-01

    AIM:To explore the relationship between cocaine-induced cyclin-dependent kinase-5(CDK5) overexpression or overactivation and Alzheimer-like hyperphosphorylation of cytoskeletal protein. METHODS: Cocaine was injected (ip,20mg·kg-1·d-1) into rats and the phosphorylation of neuronal cytoskeletal proteins was measured by Western blotting.RESULTS:The levels of phosphorylated tau at PHF-1 epitope and phosphorylated neurofilament determined by SMI31 were elevated in rat brain hippocampus, cortex, and caudatoputamen on d 8 and d 16 after the injection of cocaine, when compared with saline control rat at the same brain regions. On the other hand, the levels of tau non-phosphorylated at tau-1 site and non-phosphorylated neurofilament determined by SIM32 were decreased in same brain regions at the same time points examined. No significant difference of phosphorylated tau and neurofilament at those epitopes was seen on d 4. Although cocaine injection could induce significant hyperphosphorylation of neuronal cytoskeletal proteins, the overexpression of CDK5 and p35 was not detected. CONCLUSION:Peritoneal injection of cocaine induces Alzheimer-like hyperphosphorylation of tau and neurofilament in rat brain, and the effect may be not relevant to an increase in overexpression or overactivation of CDK5.

  13. Aggregation-induced emission—fluorophores and applications

    Science.gov (United States)

    Hong, Yuning

    2016-06-01

    Aggregation-induced emission (AIE) is a novel photophysical phenomenon found in a group of luminogens that are not fluorescent in solution but are highly emissive in the aggregate or solid state. Since the first publication of AIE luminogens in 2001, AIE has become a hot research area in which the number of research papers regarding new AIE molecules and their applications has been increasing in an exponential manner. Thomson Reuters Essential Science Indicators ranked AIE no.3 among the Top 100 Research Frontiers in the field of Chemistry and Materials Science in 2013. In this review, I will give a general introduction of the AIE phenomenon, discuss the structure-property relationship of the AIE lumingens and summarize the recent progress in the applications including as light-emitting materials in optoelectronics, as chemosensors and bioprobes, and for bioimaging (total 69 references cited).

  14. N-Terminus of the Protein Kinase CLK1 Induces SR Protein Hyper-Phosphorylation

    Science.gov (United States)

    Aubol, Brandon E.; Plocinik, Ryan M.; Keshwani, Malik M.; McGlone, Maria L.; Hagopian, Jonathan C.; Ghosh, Gourisankar; Fu, Xiang-Dong; Adams, Joseph A.

    2016-01-01

    SR proteins are essential splicing factors that are regulated through multisite phosphorylation of their RS (arginine-serine-rich) domains by two major families of protein kinases. The SRPKs efficiently phosphorylate the arginine-serine dipeptides in the RS domain using a conserved docking groove in the kinase domain. In contrast, CLKs lack a docking groove and phosphorylate both arginine-serine and serine-proline dipeptides, modifications that generate a hyper-phosphorylated state important for unique SR protein-dependent splicing activities. All CLKs contain long, flexible N-terminal extensions (140-300 residues) that resemble the RS domains present in their substrate SR proteins. We showed that the N-terminus in CLK1 contacts both the kinase domain and the RS domain of the SR protein SRSF1. This interaction not only is essential for facilitating hyper-phosphorylation but also induces cooperative binding of SRSF1 to RNA. The N-terminus of CLK1 enhances the total phosphoryl contents of a panel of physiological substrates including SRSF1, SRSF2, SRSF5 and Tra2β1 by 2–3-fold. These findings suggest that CLK1-dependent hyper-phosphorylation is the result of a general mechanism in which the N-terminus acts as a bridge connecting the kinase domain and the RS domain of the SR protein. PMID:24869919

  15. Doubly Phosphorylated Peptide Vaccines to Protect Transgenic P301S Mice against Alzheimer’s Disease Like Tau Aggregation

    Directory of Open Access Journals (Sweden)

    Monique Richter

    2014-07-01

    Full Text Available Intracellular neurofibrillary tangles and extracellular senile plaques are potential targets for active and passive immunotherapies. In this study we used the transgenic mouse model P301S for active immunizations with peptide vaccines composed of a double phosphorylated tau neoepitope (pSer202/pThr205, pThr212/pSer214, pThr231/pSer235 and an immunomodulatory T cell epitope from the tetanus toxin or tuberculosis antigen Ag85B. Importantly, the designed vaccine combining Alzheimer’s disease (AD specific B cell epitopes with foreign (bacterial T cell epitopes induced fast immune responses with high IgG1 titers after prophylactic immunization that subsequently decreased over the observation period. The effectiveness of the immunization was surveyed by evaluating the animal behavior, as well as the pathology in the brain by biochemical and histochemical techniques. Immunized mice clearly lived longer with reduced paralysis than placebo-treated mice. Additionally, they performed significantly better in rotarod and beam walk tests at the age of 20 weeks, indicating that the disease development was slowed down. Forty-eight weeks old vaccinated mice passed the beam walk test significantly better than control animals, which together with the increased survival rates undoubtedly prove the treatment effect. In conclusion, the data provide strong evidence that active immune therapies can reduce toxic effects of deposits formed in AD.

  16. Interleukin-2 induces tyrosine phosphorylation and nuclear translocation of stat3 in human T lymphocytes

    DEFF Research Database (Denmark)

    Nielsen, M; Svejgaard, A; Skov, S;

    1994-01-01

    stimulation through the IL-2R induced tyrosine phosphorylation and subsequent nuclear translocation of stat3, a newly identified member of the signal transducers and activators of transcription (STAT) family of proteins. In contrast, stat1 proteins were not tyrosine phosphorylated after IL-2 ligation, whereas...... an apparent molecular mass of 84 kDa and was not recognized by stat3 or stat1 mAb or antisera. Since IL-2 induced nuclear translocation of the 84 kDa protein and stat3 followed identical kinetics, p84 is a candidate for a new, yet undefined, member of the STAT family. Taken together, we report that...... IL-2 induces tyrosine phosphorylation and subsequent nuclear translocation of stat3 and an as yet undefined 84-kDa protein in antigen-specific human T cell lines....

  17. Induced expression of nucleolin phosphorylation-deficient mutant confers dominant-negative effect on cell proliferation.

    Directory of Open Access Journals (Sweden)

    Shu Xiao

    Full Text Available Nucleolin (NCL is a major nucleolar phosphoprotein that has pleiotropic effects on cell proliferation and is elevated in a variety of tumors. NCL is highly phosphorylated at the N-terminus by two major kinases: interphase casein kinase 2 (CK2 and mitotic cyclin-dependent kinase 1 (CDK1. Earlier we demonstrated that a NCL-mutant that is partly defective in undergoing phosphorylation by CK2 inhibits chromosomal replication through its interactions with Replication Protein A, mimicking the cellular response to DNA damage. We further delineated that the N-terminus of NCL associates with Hdm2, the most common E3 ubiquitin ligase of p53. We reported that NCL antagonizes Hdm2 to stabilize p53 and stimulates p53 transcriptional activity. Although NCL-phosphorylation by CK2 and ribosomal DNA transcription are closely coordinated during interphase, the role of NCL phosphorylation in regulating cell proliferation remains unexplored. We have therefore engineered unique human cells that specifically induce expression of NCL-wild type (WT or a phosphorylation-deficient NCL-mutant, 6/S*A where all the six CK2 consensus serine sites residing in the N-terminus NCL were mutated to alanine. Here we show that this NCL-mutant is defective in undergoing phosphorylation by CK2. We also demonstrate that NCL-phosphorylation by CK2 is required through the S-phase progression in cell cycle and hence proliferation. Induced expression of NCL with mutated CK2 phosphorylation sites stabilizes p53, results in higher expression of Bcl2 (B-cell lymphoma 2 homology 3 (BH3-only apoptotic markers and causes a dominant-negative effect on cell viability. Our unique cellular system thus provides the first evidential support to delineate phospho-specific functions of NCL on cell proliferation.

  18. Aggregation of Ribosomal Protein S6 at Nucleolus Is Cell Cycle-Controlled and Its Function in Pre-rRNA Processing Is Phosphorylation Dependent.

    Science.gov (United States)

    Zhang, Duo; Chen, Hui-Peng; Duan, Hai-Feng; Gao, Li-Hua; Shao, Yong; Chen, Ke-Yan; Wang, You-Liang; Lan, Feng-Hua; Hu, Xian-Wen

    2016-07-01

    Ribosomal protein S6 (rpS6) has long been regarded as one of the primary r-proteins that functions in the early stage of 40S subunit assembly, but its actual role is still obscure. The correct forming of 18S rRNA is a key step in the nuclear synthesis of 40S subunit. In this study, we demonstrate that rpS6 participates in the processing of 30S pre-rRNA to 18S rRNA only when its C-terminal five serines are phosphorylated, however, the process of entering the nucleus and then targeting the nucleolus does not dependent its phosphorylation. Remarkably, we also find that the aggregation of rpS6 at the nucleolus correlates to the phasing of cell cycle, beginning to concentrate in the nucleolus at later S phase and disaggregate at M phase. J. Cell. Biochem. 117: 1649-1657, 2016. © 2015 Wiley Periodicals, Inc.

  19. Methylglyoxal-induced modification causes aggregation of myoglobin

    Science.gov (United States)

    Banerjee, Sauradipta; Maity, Subhajit; Chakraborti, Abhay Sankar

    2016-02-01

    Post-translational modification of proteins by Maillard reaction, known as glycation, is thought to be the root cause of different complications, particularly in diabetes mellitus and age-related disorders. Methylglyoxal (MG), a reactive α-oxoaldehyde, increases in diabetic condition and reacts with proteins to form advanced glycation end products (AGEs) following Maillard-like reaction. We have investigated the in vitro effect of MG (200 μM) on the monomeric heme protein myoglobin (Mb) (100 μM) in a time-dependent manner (7 to 18 days incubation at 25 °C). MG induces significant structural alterations of the heme protein, including heme loss, changes in tryptophan fluorescence, decrease of α-helicity with increased β-sheet content etc. These changes occur gradually with increased period of incubation. Incubation of Mb with MG for 7 days results in formation of the AGE adducts: carboxyethyllysine at Lys-16, carboxymethyllysine at Lys-87 and carboxyethyllysine or pyrraline-carboxymethyllysine at Lys-133. On increasing the period of incubation up to 14 days, additional AGEs namely, carboxyethyllysine at Lys-42 and hydroimidazolone or argpyrimidine at Arg-31 and Arg-139 have been detected. MG also induces aggregation of Mb, which is clearly evident with longer period of incubation (18 days), and appears to have amyloid nature. MG-derived AGEs may thus have an important role as the precursors of protein aggregation, which, in turn, may be associated with physiological complications.

  20. Temperature Induced Aggregation and Clouding in Humic Acid Solutions

    Directory of Open Access Journals (Sweden)

    Leah Shaffer

    2015-01-01

    Full Text Available Humic acids in aqueous solution demonstrate inverse temperature-solubility relationships when solution conditions are manipulated to reduce coulombic repulsion among the humic polyanions. These effects were followed by dynamic light scattering (DLS measurements of the resulting aggregates, as well as the addition of a polarity sensitive fluorescent probe (pyrene. The humic solutions could be primed for temperature induced clouding by carefully lowering the pH to a point where hydration effects became dominant. The exact value of the cloud point (CP was a function of both pH and humate concentration. The CPs mostly lay in the range 50–90°C, but DLS showed that temperature induced aggregation proceeded from approximately 30°C onward. Similar effects could be achieved by adding multivalent cations at concentrations below those which cause spontaneous precipitation. The declouding of clouded humate solutions could be affected by lowering the temperature combined with mechanical agitation to disentangle the humic polymers.

  1. Nrf2 reduces levels of phosphorylated tau protein by inducing autophagy adaptor protein NDP52

    Science.gov (United States)

    Jo, Chulman; Gundemir, Soner; Pritchard, Susanne; Jin, Youngnam N.; Rahman, Irfan; Johnson, Gail V. W.

    2014-03-01

    Nuclear factor erythroid 2-related factor 2 (Nrf2) is a pivotal transcription factor in the defence against oxidative stress. Here we provide evidence that activation of the Nrf2 pathway reduces the levels of phosphorylated tau by induction of an autophagy adaptor protein NDP52 (also known as CALCOCO2) in neurons. The expression of NDP52, which we show has three antioxidant response elements (AREs) in its promoter region, is strongly induced by Nrf2, and its overexpression facilitates clearance of phosphorylated tau in the presence of an autophagy stimulator. In Nrf2-knockout mice, phosphorylated and sarkosyl-insoluble tau accumulates in the brains concurrent with decreased levels of NDP52. Moreover, NDP52 associates with phosphorylated tau from brain cortical samples of Alzheimer disease cases, and the amount of phosphorylated tau in sarkosyl-insoluble fractions is inversely proportional to that of NDP52. These results suggest that NDP52 plays a key role in autophagy-mediated degradation of phosphorylated tau in vivo.

  2. Structural basis of how stress-induced MDMX phosphorylation activates p53.

    Science.gov (United States)

    Chen, X; Gohain, N; Zhan, C; Lu, W-Y; Pazgier, M; Lu, W

    2016-04-14

    The tumor-suppressor protein p53 is tightly controlled in normal cells by its two negative regulators--the E3 ubiquitin ligase MDM2 and its homolog MDMX. Under stressed conditions such as DNA damage, p53 escapes MDM2- and MDMX-mediated functional inhibition and degradation, acting to prevent damaged cells from proliferating through induction of cell cycle arrest, DNA repair, senescence or apoptosis. Ample evidence suggests that stress signals induce phosphorylation of MDM2 and MDMX, leading to p53 activation. However, the structural basis of stress-induced p53 activation remains poorly understood because of the paucity of technical means to produce site-specifically phosphorylated MDM2 and MDMX proteins for biochemical and biophysical studies. Herein, we report total chemical synthesis, via native chemical ligation, and functional characterization of (24-108)MDMX and its Tyr99-phosphorylated analog with respect to their ability to interact with a panel of p53-derived peptide ligands and PMI, a p53-mimicking but more potent peptide antagonist of MDMX, using FP and surface plasmon resonance techniques. Phosphorylation of MDMX at Tyr99 weakens peptide binding by approximately two orders of magnitude. Comparative X-ray crystallographic analyses of MDMX and of pTyr99 MDMX in complex with PMI as well as modeling studies reveal that the phosphate group of pTyr99 imposes extensive steric clashes with the C-terminus of PMI or p53 peptide and induces a significant lateral shift of the peptide ligand, contributing to the dramatic decrease in the binding affinity of MDMX for p53. Because DNA damage activates c-Abl tyrosine kinase that phosphorylates MDMX at Tyr99, our findings afford a rare glimpse at the structural level of how stress-induced MDMX phosphorylation dislodges p53 from the inhibitory complex and activates it in response to DNA damage. PMID:26148237

  3. Phosphorylation dynamics of radixin in hypoxia-induced hepatocyte injury.

    Science.gov (United States)

    Suda, Jo; Rockey, Don C; Karvar, Serhan

    2015-02-15

    The most prominent ezrin-radixin-moesin protein in hepatocytes is radixin, which is localized primarily at the canalicular microvilli and appears to be important in regulation of cell polarity and in localizing the multidrug resistance-associated protein 2 (Mrp-2) function. Our aim was to investigate how hypoxia affects radixin distribution and Mrp-2 function. We created wild-type and mutant constructs (in adenoviral vectors), which were expressed in WIF-B cells. The cellular distribution of Mrp-2 and radixin was visualized by fluorescence microscopy, and a 5-chloromethylfluorescein diacetate (CMFDA) assay was used to measure Mrp-2 function. Under usual conditions, cells infected with wild-type radixin, nonphosphorylatable radixin-T564A, and radixin-T564D (active phospho-mimicking mutant) were found to be heavily expressed in canalicular membrane compartment vacuoles, typically colocalizing with Mrp-2. In contrast, after hypoxia for 24 h, both endogenous and overexpressed wild-type radixin and the radixin-T564A mutant were found to be translocated to the cytoplasmic space. However, distribution of the radixin-T564D mutant, which mimics constant phosphorylation, was remarkably different, being associated with canalicular membranes even in hypoxic conditions. This dominant-active construct also prevented dissociation of radixin from the plasma membrane. Hypoxia also led to Mrp-2 mislocalization and caused Mrp-2 to be dissociated from radixin; the radixin phospho-mimicking mutant (T564D) abrogated this effect of hypoxia. Finally, hypoxia diminished the secretory response (measured using the CMFDA assay) in WIF-B cells, and the dominant-active construct (radixin-T567D) rescued this phenotype. Taken collectively, these findings suggest that radixin regulates Mrp-2 localization and function in hepatocytes and is important in hypoxic liver injury. PMID:25501552

  4. Phorbol ester induced phosphorylation of the estrogen receptor in intact MCF-7 human breast cancer cells

    International Nuclear Information System (INIS)

    Recent studies with a variety of cellular receptors have shown that phorbol ester induced phosphorylation modulates ligand binding and function. In this study the authors present direct evidence that the estrogen receptor in MCF-7 human breast cancer cells is a phosphoprotein whose phosphorylation state can be enhanced specifically by phorbol-12-myristate-13-acetate (PMA). Cells were cultured to 6h in the presence of [32P]-orthophosphate. Whole cell extracts were immunoprecipitated with a monoclonal antibody (D58) against the estrogen receptor and subjected to SDS-polyacrylamide electrophoresis. Autoradiography showed a specific band in the region of 60-62 kDa which was significantly increased in preparations from PMA treated cells. Phospho-amino acid analysis demonstrated specific phosphorylation of serine and threonine residues. Cholera toxin or forskolin did not change the phosphorylation state of this protein. In a parallel binding analysis PMA led to a rapid decrease of estrogen binding sites. The estrogen induction of both progesterone receptors and growth in semisolid medium was blocked by PMA, whereas the estrogen induction of the 8kDa protein corresponding to the ps2 gene product and of the 52 kDa protein was not affected. In conclusion, phorbol esters can induce phosphorylation of the estrogen receptor. This process may be associated with the inactivation of certain receptor functions

  5. p53 Aggregates penetrate cells and induce the co-aggregation of intracellular p53.

    Directory of Open Access Journals (Sweden)

    Karolyn J Forget

    Full Text Available Prion diseases are unique pathologies in which the infectious particles are prions, a protein aggregate. The prion protein has many particular features, such as spontaneous aggregation, conformation transmission to other native PrP proteins and transmission from an individual to another. Protein aggregation is now frequently associated to many human diseases, for example Alzheimer's disease, Parkinson's disease or type 2 diabetes. A few proteins associated to these conformational diseases are part of a new category of proteins, called prionoids: proteins that share some, but not all, of the characteristics associated with prions. The p53 protein, a transcription factor that plays a major role in cancer, has recently been suggested to be a possible prionoid. The protein has been shown to accumulate in multiple cancer cell types, and its aggregation has also been reproduced in vitro by many independent groups. These observations suggest a role for p53 aggregates in cancer development. This study aims to test the «prion-like» features of p53. Our results show in vitro aggregation of the full length and N-terminally truncated protein (p53C, and penetration of these aggregates into cells. According to our findings, the aggregates enter cells using macropinocytosis, a non-specific pathway of entry. Lastly, we also show that once internalized by the cell, p53C aggregates can co-aggregate with endogenous p53 protein. Together, these findings suggest prion-like characteristics for p53 protein, based on the fact that p53 can spontaneously aggregate, these aggregates can penetrate cells and co-aggregate with cellular p53.

  6. Tyrosine 402 phosphorylation of Pyk2 is involved in ionomycin-induced neurotransmitter release.

    Directory of Open Access Journals (Sweden)

    Zhao Zhang

    Full Text Available Protein tyrosine kinases, which are highly expressed in the central nervous system, are implicated in many neural processes. However, the relationship between protein tyrosine kinases and neurotransmitter release remains unknown. In this study, we found that ionomycin, a Ca²⁺ ionophore, concurrently induced asynchronous neurotransmitter release and phosphorylation of a non-receptor protein tyrosine kinase, proline-rich tyrosine kinase 2 (Pyk2, in clonal rat pheochromocytoma PC12 cells and cerebellar granule cells, whereas introduction of Pyk2 siRNA dramatically suppressed ionomycin-induced neurotransmitter release. Further study indicated that Tyr-402 (Y402 in Pyk2, instead of other tyrosine sites, underwent rapid phosphorylation after ionomycin induction in 1 min to 2 min. We demonstrated that the mutant of Pyk2 Y402 could abolish ionomycin-induced dopamine (DA release by transfecting cells with recombinant Pyk2 and its mutants (Y402F, Y579F, Y580F, and Y881F. In addition, Src inhibition could prolong phosphorylation of Pyk2 Y402 and increase DA release. These findings suggested that Pyk2 was involved in ionomycin-induced neurotransmitter release through phosphorylation of Y402.

  7. Mineral trioxide aggregate induces osteoblastogenesis via Atf6

    Directory of Open Access Journals (Sweden)

    Toyonobu Maeda

    2015-06-01

    Full Text Available Mineral trioxide aggregate (MTA has been recommended for various uses in endodontics. To understand the effects of MTA on alveolar bone, we examined whether MTA induces osteoblastic differentiation using MC3T3-E1 cells. MTA enhanced mineralization concomitant with alkaline phosphatase activity in a dose- and time-dependent manner. MTA increased production of collagens (Type I and Type III and matrix metalloproteinases (MMP-9 and MMP-13, suggesting that MTA affects bone matrix remodeling. MTA also induced Bglap (osteocalcin but not Bmp2 (bone morphogenetic protein-2 mRNA expression. We observed induction of Atf6 (activating transcription factor 6, an endoplasmic reticulum (ER stress response transcription factor mRNA expression and activation of Atf6 by MTA treatment. Forced expression of p50Atf6 (active form of Atf6 markedly enhanced Bglap mRNA expression. Chromatin immunoprecipitation assay was performed to investigate the increase in p50Atf6 binding to the Bglap promoter region by MTA treatment. Furthermore, knockdown of Atf6 gene expression by introduction of Tet-on Atf6 shRNA expression vector abrogated MTA-induced mineralization. These results suggest that MTA induces in vitro osteoblastogenesis through the Atf6–osteocalcin axis as ER stress signaling. Therefore, MTA in endodontic treatment may affect alveolar bone healing in the resorbed region caused by pulpal infection.

  8. Protein kinase C {alpha} activity is important for contraction-induced FXYD1 phosphorylation in skeletal muscle

    DEFF Research Database (Denmark)

    Thomassen, Martin; Rose, Adam John; Jensen, Thomas Elbenhardt;

    2011-01-01

    Exercise induced phosphorylation of FXYD1 is a potential important regulator of Na(+), K(+) pump activity. It was investigated if skeletal muscle contractions induce phosphorylation of FXYD1 and if Protein Kinase C a (PKCa) activity is a prerequisite for this possible mechanism. In part 1, human...

  9. Fe65 Is Phosphorylated on Ser289 after UV-Induced DNA Damage.

    Directory of Open Access Journals (Sweden)

    Hannah Langlands

    Full Text Available Fe65 undergoes a phosphatase-sensitive gel mobility shift after DNA damage, consistent with protein phosphorylation. A recent study identified Ser228 as a specific site of phosphorylation, targeted by the ATM and ATR protein kinases, with phosphorylation inhibiting the Fe65-dependent transcriptional activity of the amyloid precursor protein (APP. The direct binding of Fe65 to APP not only regulates target gene expression, but also contributes to secretase-mediated processing of APP, producing cytoactive proteolytic fragments including the APP intracellular domain (AICD and cytotoxic amyloid β (Aβ peptides. Given that the accumulation of Aβ peptides in neural plaques is a pathological feature of Alzheimer's disease (AD, it is essential to understand the mechanisms controlling Aβ production. This will aid in the development of potential therapeutic agents that act to limit the deleterious production of Aβ peptides. The Fe65-APP complex has transcriptional activity and the complex is regulated by multiple post-translational modifications and other protein binding partners. In the present study, we have identified Ser289 as a novel site of UV-induced phosphorylation. Interestingly, this phosphorylation was mediated by ATM, rather than ATR, and occurred independently of APP. Neither phosphorylation nor mutation of Ser289 affected the Fe65-APP interaction, though this was markedly decreased after UV treatment, with a concomitant decrease in the protein levels of APP in cells. Using mutagenesis, we demonstrated that Fe65 Ser289 phosphorylation did not affect the transcriptional activity of the Fe65-APP complex, in contrast to the previously described Ser228 site.

  10. Hypoxia activates Akt and induces phosphorylation of GSK-3 in PC12 cells.

    Science.gov (United States)

    Beitner-Johnson, D; Rust, R T; Hsieh, T C; Millhorn, D E

    2001-01-01

    Akt is a serine/threonine kinase that has been shown to play a central role in promoting cell survival and opposing apoptosis. We evaluated the effect of hypoxia on Akt in rat pheochromocytoma (PC12) cells. PC12 cells were exposed to varying levels of hypoxia, including 21%, 15%, 10%, 5%, and 1% O(2). Hypoxia dramatically increased phosphorylation of Akt (Ser(473)). This effect peaked after 6 h exposure to hypoxia, but persisted strongly for up to 24 h. Phosphorylation of Akt was paralleled with a progressive increase in phosphorylation of glycogen synthase kinase-3 (GSK-3), one of its downstream substrates. The effect of hypoxia on phosphorylation of Akt was completely blocked by pretreatment of the cells with wortmannin (100 nM), indicating that this effect is mediated by phosphatidylinositol 3-kinase (P13K). In contrast, whereas hypoxia also strongly induced phosphorylation of the transcription factors CREB and EPAS1, these effects persisted in the presence of wortmannin. Thus, hypoxia regulates both P13K-dependent and P13K-independent signaling pathways. Furthermore, activation of the P13K and Akt signaling pathways may be one mechanism by which cells adapt and survive under conditions of hypoxia. PMID:11257444

  11. Reactive oxygen species contribute to arsenic-induced EZH2 phosphorylation in human bronchial epithelial cells and lung cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Li, Lingzhi; Qiu, Ping; Chen, Bailing; Lu, Yongju; Wu, Kai; Thakur, Chitra; Chang, Qingshan; Sun, Jiaying; Chen, Fei, E-mail: fchen@wayne.edu

    2014-05-01

    Our previous studies suggested that arsenic is able to induce serine 21 phosphorylation of the EZH2 protein through activation of JNK, STAT3, and Akt signaling pathways in the bronchial epithelial cell line, BEAS-2B. In the present report, we further demonstrated that reactive oxygen species (ROS) were involved in the arsenic-induced protein kinase activation that leads to EZH2 phosphorylation. Several lines of evidence supported this notion. First, the pretreatment of the cells with N-acetyl-L-cysteine (NAC), a potent antioxidant, abolishes arsenic-induced EZH2 phosphorylation along with the inhibition of JNK, STAT3, and Akt. Second, H{sub 2}O{sub 2}, the most important form of ROS in the cells in response to extracellular stress signals, can induce phosphorylation of the EZH2 protein and the activation of JNK, STAT3, and Akt. By ectopic expression of the myc-tagged EZH2, we additionally identified direct interaction and phosphorylation of the EZH2 protein by Akt in response to arsenic and H{sub 2}O{sub 2}. Furthermore, both arsenic and H{sub 2}O{sub 2} were able to induce the translocation of ectopically expressed or endogenous EZH2 from nucleus to cytoplasm. In summary, the data presented in this report indicate that oxidative stress due to ROS generation plays an important role in the arsenic-induced EZH2 phosphorylation. - Highlights:: • Arsenic (As{sup 3+}) induces EZH phosphorylation. • JNK, STAT3, and Akt contribute to EZH2 phosphorylation. • Oxidative stress is involved in As{sup 3+}-induced EZH2 phosphorylation. • As{sup 3+} induces direct interaction of Akt and EZH2. • Phosphorylated EZH2 localized in cytoplasm.

  12. Phosphorylation of AMPA receptors is required for sensory deprivation-induced homeostatic synaptic plasticity.

    Science.gov (United States)

    Goel, Anubhuti; Xu, Linda W; Snyder, Kevin P; Song, Lihua; Goenaga-Vazquez, Yamila; Megill, Andrea; Takamiya, Kogo; Huganir, Richard L; Lee, Hey-Kyoung

    2011-01-01

    Sensory experience, and the lack thereof, can alter the function of excitatory synapses in the primary sensory cortices. Recent evidence suggests that changes in sensory experience can regulate the synaptic level of Ca(2+)-permeable AMPA receptors (CP-AMPARs). However, the molecular mechanisms underlying such a process have not been determined. We found that binocular visual deprivation, which is a well-established in vivo model to produce multiplicative synaptic scaling in visual cortex of juvenile rodents, is accompanied by an increase in the phosphorylation of AMPAR GluR1 (or GluA1) subunit at the serine 845 (S845) site and the appearance of CP-AMPARs at synapses. To address the role of GluR1-S845 in visual deprivation-induced homeostatic synaptic plasticity, we used mice lacking key phosphorylation sites on the GluR1 subunit. We found that mice specifically lacking the GluR1-S845 site (GluR1-S845A mutants), which is a substrate of cAMP-dependent kinase (PKA), show abnormal basal excitatory synaptic transmission and lack visual deprivation-induced homeostatic synaptic plasticity. We also found evidence that increasing GluR1-S845 phosphorylation alone is not sufficient to produce normal multiplicative synaptic scaling. Our study provides concrete evidence that a GluR1 dependent mechanism, especially S845 phosphorylation, is a necessary pre-requisite step for in vivo homeostatic synaptic plasticity.

  13. Phosphorylation of AMPA receptors is required for sensory deprivation-induced homeostatic synaptic plasticity.

    Directory of Open Access Journals (Sweden)

    Anubhuti Goel

    Full Text Available Sensory experience, and the lack thereof, can alter the function of excitatory synapses in the primary sensory cortices. Recent evidence suggests that changes in sensory experience can regulate the synaptic level of Ca(2+-permeable AMPA receptors (CP-AMPARs. However, the molecular mechanisms underlying such a process have not been determined. We found that binocular visual deprivation, which is a well-established in vivo model to produce multiplicative synaptic scaling in visual cortex of juvenile rodents, is accompanied by an increase in the phosphorylation of AMPAR GluR1 (or GluA1 subunit at the serine 845 (S845 site and the appearance of CP-AMPARs at synapses. To address the role of GluR1-S845 in visual deprivation-induced homeostatic synaptic plasticity, we used mice lacking key phosphorylation sites on the GluR1 subunit. We found that mice specifically lacking the GluR1-S845 site (GluR1-S845A mutants, which is a substrate of cAMP-dependent kinase (PKA, show abnormal basal excitatory synaptic transmission and lack visual deprivation-induced homeostatic synaptic plasticity. We also found evidence that increasing GluR1-S845 phosphorylation alone is not sufficient to produce normal multiplicative synaptic scaling. Our study provides concrete evidence that a GluR1 dependent mechanism, especially S845 phosphorylation, is a necessary pre-requisite step for in vivo homeostatic synaptic plasticity.

  14. Involvement of Protein Phosphorylation in Water Stress-induced Antioxidant Defense in Maize Leaves

    Institute of Scientific and Technical Information of China (English)

    Shu-cheng Xu; Hai-dong Ding; Feng-xia Su; A-ying Zhang; Ming-yi Jiang

    2009-01-01

    Using pharmacological and biochemical approaches, the role of protein phosphorylation and the interrelationship between water stress-enhanced kinase activity, antioxidant enzyme activity, hydrogen peroxide (H2O2) accumulation and endogenous abscisic acid in maize (Zea mays L.) leaves were investigated. Water-stress upregulated the activities of total protein phosphorylation and Ca2+ -dependent protein kinase, and the upregulation was blocked in abscisic acid-deficient vp5 mutant. Furthermore, pretreatments with a nicotinamide adenine dinucleotide phosphate oxidase inhibitor and a scavenger of H2O2 significantly reduced the increased activities of total protein kinase and Ca2+-dependent protein kinase in maize leaves exposed to water stress. Pretreatments with different protein kinase inhibitors also reduced the water stress-induced H2O2 production and the water stress-enhanced activities of antioxidant enzymes such as superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase. The data suggest that protein phosphorylation and H2O2 generation are required for water stress-induced antioxidant defense in maize leaves and that crosstalk between protein phosphorylation and H2O2 generation may occur.

  15. The role of eNOS phosphorylation in causing drug-induced vascular injury.

    Science.gov (United States)

    Tobin, Grainne A McMahon; Zhang, Jun; Goodwin, David; Stewart, Sharron; Xu, Lin; Knapton, Alan; González, Carlos; Bancos, Simona; Zhang, Leshuai; Lawton, Michael P; Enerson, Bradley E; Weaver, James L

    2014-06-01

    Previously we found that regulation of eNOS is an important part of the pathogenic process of Drug-induced vascular injury (DIVI) for PDE4i. The aims of the current study were to examine the phosphorylation of eNOS in mesentery versus aorta at known regulatory sites across DIVI-inducing drug classes and to compare changes across species. We found that phosphorylation at S615 in rats was elevated 35-fold 2 hr after the last dose of CI-1044 in mesentery versus 3-fold in aorta. Immunoprecipitation studies revealed that many of the upstream regulators of eNOS activation were associated with eNOS in 1 or more signalosome complexes. Next rats were treated with drugs from 4 other classes known to cause DIVI. Each drug was given alone and in combination with SIN-1 (NO donor) or L-NAME (eNOS inhibitor), and the level of eNOS phosphorylation in mesentery and aorta tissue was correlated with the extent of vascular injury and measured serum nitrite. Drugs or combinations produced altered serum nitrite levels as well as vascular injury score in the mesentery. The results suggested that phosphorylation of S615 may be associated with DIVI activity. Studies with the species-specific A2A adenosine agonist CI-947 in rats versus primates showed a similar pattern.

  16. Human lymphocyte damage and phosphorylation of H2AX and ATM induced by γ-rays

    International Nuclear Information System (INIS)

    Objective: To investigate 60Co γ-ray induced damage in lymphocytes and the relationship between doses of 60Co γ-ray irradiation and the levels of phosphorylated H2AX and ATM. Methods: Cells were irradiated with 60Co γ-rays in the range of 0-8 Gy. The levels of phosphorylated H2AX and ATM were detected by Western blot and FACScan,respectively. The micronucleus(MN)was analyzed by CB method to evaluate DNA damage. Results: FACScan results showed the dose-effect relationship of γ-H2AX expression were linear.square at 0.5 h post-irradiation to different doses, and the fitting curve was shown as Y=3.96+11.29D-0.45D2. The level of phosphorylated ATM (p-ATM) was not changed significantly by using the same method. Western blot showed that p-ATM protein expression was significantly increased after irradiation compared with sham, irradiated group. The MN assay which represented DNA damage was sensitive to different doses. Conclusions: γ-ray irradiation could induce the phosphorylation of H2AX and ATM, which may play an important role in indicating DNA damage. Both of H2AX and ATM have the potential as sensitive biomarker and biodosimeter for radiation damage. (authors)

  17. Phosphorylated endothelial nitric oxide synthase mediates vascular endothelial growth factor-induced penile erection.

    Science.gov (United States)

    Musicki, Biljana; Palese, Michael A; Crone, Julie K; Burnett, Arthur L

    2004-02-01

    The objective of the present study was to evaluate whether vascular endothelial growth factor (VEGF)-induced penile erection is mediated by activation of endothelial nitric oxide synthase (eNOS) through its phosphorylation. We assessed the role of constitutively activated eNOS in VEGF-induced penile erection using wild-type (WT) and eNOS-knockout (eNOS(-/-)) mice with and without vasculogenic erectile dysfunction. Adult WT and eNOS(-/-) mice were subjected to sham operation or bilateral castration to induce vasculogenic erectile dysfunction. At the time of surgery, animals were injected intracavernosally with a replication-deficient adenovirus expressing human VEGF145 (10(9) particle units) or with empty virus (Ad.Null). After 7 days, erectile function was assessed in response to cavernous nerve electrical stimulation. Total and phosphorylated protein kinase B (Akt) as well as total and phosphorylated eNOS were quantitatively assessed in mice penes using Western immunoblot and immunohistochemistry. In intact WT mice, VEGF145 significantly increased erectile responses, and in WT mice after castration, it completely recovered penile erection. However, VEGF145 failed to increase erectile responses in intact eNOS(-/-) mice and only partially recovered erectile function in castrated eNOS(-/-) mice. In addition, VEGF145 significantly increased phosphorylation of eNOS at Serine 1177 by approximately 2-fold in penes of both intact and castrated WT mice. The data provide a molecular explanation for VEGF stimulatory effect on penile erection, which involves phosphorylated eNOS (Serine 1177) mediation. PMID:14522830

  18. Reversible NaCl-induced aggregation of a monoclonal antibody at low pH: Characterization of aggregates and factors affecting aggregation.

    Science.gov (United States)

    Bickel, Fabian; Herold, Eva Maria; Signes, Alba; Romeijn, Stefan; Jiskoot, Wim; Kiefer, Hans

    2016-10-01

    We investigated the influence of pH and sodium chloride concentration on aggregation kinetics of a monoclonal antibody. Aggregation was induced by sodium chloride addition at low pH. Protein conformation before and after salt addition was determined as well as the reversibility of aggregation. Aggregation was monitored at pH values between 2 and 7 with NaCl up to 1.5M by turbidity measurement and size-exclusion chromatography. Particle size distribution was assessed by using size-exclusion chromatography as well as nanoparticle tracking analysis and flow imaging microscopy. Structural changes were monitored by circular dichroism, Fourier transform infrared and fluorescence spectroscopy. Thermal stability was measured by differential scanning fluorimetry. Aggregation propensity was maximal at low pH and high ionic strength. While thermal stability decreased with pH, the secondary structure remained unchanged down to pH 3.5 and up to 1.5M NaCl. Precipitated protein could be largely reverted to monomers by dilution into salt-free buffer. The re-solubilized antibody was indistinguishable in structure, solubility and monodispersity from the unstressed protein. Also, binding to Protein A was steady. Aggregation could be reduced in the presence of trehalose. The results suggest a reversible aggregation mechanism characterized by a limited change in tertiary structure at low pH and a subsequent loss of colloidal stability resulting from electrostatic repulsion once salt is added to the sample. The experimental setup is robust and allows high-throughput quantification of the effect of additives on aggregation kinetics.

  19. First selective dual inhibitors of tau phosphorylation and Beta-amyloid aggregation, two major pathogenic mechanisms in Alzheimer's disease.

    Science.gov (United States)

    Mariano, Marica; Schmitt, Christian; Miralinaghi, Parisa; Catto, Marco; Hartmann, Rolf W; Carotti, Angelo; Engel, Matthias

    2014-12-17

    In Alzheimer's disease (AD), multiple factors account for the accumulation of neurocellular changes, which may begin several years before symptoms appear. The most important pathogenic brain changes that are contributing to the development of AD are the formation of the cytotoxic β-amyloid aggregates and of the neurofibrillary tangles, which originate from amyloid-β peptides and hyperphosphorylated tau protein, respectively. New therapeutic agents that target both major pathogenic mechanisms may be particularly efficient. In this study, we introduce bis(hydroxyphenyl)-substituted thiophenes as a novel class of selective, dual inhibitors of the tau kinase Dyrk1A and of the amyloid-β aggregation. PMID:25247807

  20. Suppressive effect of CORM-2 on LPS-induced platelet activation by glycoprotein mediated HS1 phosphorylation interference.

    Directory of Open Access Journals (Sweden)

    Dadong Liu

    Full Text Available In recent years, it has been discovered that septic patients display coagulation abnormalities. Platelets play a major role in the coagulation system. Studies have confirmed that carbon monoxide (CO has important cytoprotective and anti-inflammatory function. However, whether CO could alter abnormal activation of platelets and coagulation and thereby reduce the incidence of mortality during sepsis has not been defined. In this report, we have used CO-releasing molecules (CORM-2 to determine whether CO inhibits LPS-induced abnormal activation of platelets and have explored the potential mechanisms. LPS was used to induce activation of platelets in vitro, which were purified from the peripheral venous blood of healthy adult donors. CORM-2 was applied as a potential therapeutic agent. CORM-2 preconditioning and delayed treatment were also studied. We found that in the LPS groups, the function of platelets such as spreading, aggregation, and release were enhanced abnormally. By contrast, the platelets in the CORM-2 group were gently activated. Further studies showed that the expression of platelet membrane glycoproteins increased in the LPS group. Coincidently, both hematopoietic lineage cell-specific protein 1 and its phosphorylated form also increased dramatically. These phenomena were less dramatically seen in the CORM-2 groups. Taken together, we conclude that during LPS stimulation, platelets were abnormally activated, and this functional state may be associated with the signal that is transmitted between membrane glycoproteins and HS1. CORM-released CO suppresses the abnormal activation of platelets by interfering with glycoprotein-mediated HS1 phosphorylation.

  1. Phytochelatins inhibit the metal-induced aggregation of alpha-crystallin.

    Science.gov (United States)

    Hori, Yasuhisa; Yoshikawa, Tomoaki; Tsuji, Naoki; Bamba, Takeshi; Aso, Yoshikazu; Kudou, Motonori; Uchida, Yoshiki; Takagi, Masahiro; Harada, Kazuo; Hirata, Kazumasa

    2009-02-01

    Phytochelatins (PCs) are heavy-metal-binding peptides found in some eukaryotes. This study investigates the use of plant-derived PCs for the inhibition of metal-induced protein aggregation. The results of this study show that PCs inhibit zinc-induced alpha-crystallin aggregation, and suggest that PCs might be useful as anti-cataract agents.

  2. Tyrosine 402 Phosphorylation of Pyk2 Is Involved in Ionomycin-Induced Neurotransmitter Release

    OpenAIRE

    Zhao Zhang; Yun Zhang; Zheng Mou; Shifeng Chu; Xiaoyu Chen; Wenbin He; Xiaofeng Guo; Yuhe Yuan; Masami Takahashi; Naihong Chen

    2014-01-01

    Protein tyrosine kinases, which are highly expressed in the central nervous system, are implicated in many neural processes. However, the relationship between protein tyrosine kinases and neurotransmitter release remains unknown. In this study, we found that ionomycin, a Ca²⁺ ionophore, concurrently induced asynchronous neurotransmitter release and phosphorylation of a non-receptor protein tyrosine kinase, proline-rich tyrosine kinase 2 (Pyk2), in clonal rat pheochromocytoma PC12 cells and ce...

  3. Engagement of CD81 induces ezrin tyrosine phosphorylation and its cellular redistribution with filamentous actin

    Energy Technology Data Exchange (ETDEWEB)

    Coffey, Greg P.; Rajapaksa, Ranjani; Liu, Raymond; Sharpe, Orr; Kuo, Chiung-Chi; Wald Krauss, Sharon; Sagi, Yael; Davis, R. Eric; Staudt, Louis M.; Sharman, Jeff P.; Robinson, William H.; Levy, Shoshana

    2009-06-09

    CD81 is a tetraspanin family member involved in diverse cellular interactions in the immune and nervous systems and in cell fusion events. However, the mechanism of action of CD81 and of other tetraspanins has not been defined. We reasoned that identifying signaling molecules downstream of CD81 would provide mechanistic clues. We engaged CD81 on the surface of Blymphocytes and identified the induced tyrosine-phosphorylated proteins by mass spectrometry. This analysis showed that the most prominent tyrosine phosphorylated protein was ezrin, an actin binding protein and a member of the ezrin-radixin-moesin family. We also found that CD81 engagement induces spleen tyrosine kinase (Syk) and that Syk was involved in tyrosine phosphorylation of ezrin. Ezrin colocalized with CD81 and F-actin upon stimulation and this association was disrupted when Syk activation was blocked. Taken together, these studies suggest a model in which CD81 interfaces between the plasma membrane and the cytoskeleton by activating Syk, mobilizing ezrin, and recruiting F-actin to facilitate cytoskeletal reorganization and cell signaling. This may be a mechanism explaining the pleiotropic effects induced in response to stimulating cells by anti-CD81 antibodies or by the hepatitis C virus, which uses this molecule as its key receptor.

  4. Activation of TORC1 transcriptional coactivator through MEKK1-induced phosphorylation.

    Science.gov (United States)

    Siu, Yeung-Tung; Ching, Yick-Pang; Jin, Dong-Yan

    2008-11-01

    CREB is a prototypic bZIP transcription factor and a master regulator of glucose metabolism, synaptic plasticity, cell growth, apoptosis, and tumorigenesis. Transducers of regulated CREB activity (TORCs) are essential transcriptional coactivators of CREB and an important point of regulation on which various signals converge. In this study, we report on the activation of TORC1 through MEKK1-mediated phosphorylation. MEKK1 potently activated TORC1, and this activation was independent of downstream effectors MEK1/MEK2, ERK2, JNK, p38, protein kinase A, and calcineurin. MEKK1 induced phosphorylation of TORC1 both in vivo and in vitro. Expression of the catalytic domain of MEKK1 alone in cultured mammalian cells sufficiently caused phosphorylation and subsequent activation of TORC1. MEKK1 physically interacted with TORC1 and stimulated its nuclear translocation. An activation domain responsive to MEKK1 stimulation was mapped to amino acids 431-650 of TORC1. As a physiological activator of CREB, interleukin 1alpha triggered MEKK1-dependent phosphorylation of TORC1 and its consequent recruitment to the cAMP response elements in the interleukin 8 promoter. Taken together, our findings suggest a new mechanism for regulated activation of TORC1 transcriptional coactivator and CREB signaling.

  5. Induced Interval-Valued Intuitionistic Fuzzy Hybrid Aggregation Operators with TOPSIS Order-Inducing Variables

    Directory of Open Access Journals (Sweden)

    Jun-Ling Zhang

    2012-01-01

    Full Text Available Two induced aggregation operators with novelly designed TOPSIS order-inducing variables are proposed: Induced Interval-valued Intuitionistic Fuzzy Hybrid Averaging (I-IIFHA operator and Induced Interval-valued Intuitionistic Fuzzy Hybrid Geometric (I-IIFHG operator. The merit of two aggregation operators is that they can consider additional preference information of decision maker’s attitudinal characteristics besides argument-dependent information and argument-independent information. Some desirable properties of I-IIFHA and I-IIFHG are studied and theoretical analysis also shows that they can include a wide range of aggregation operators as special cases. Further, we extend these operators to form a novel group decision-making method for selecting the most desirable alternative in multiple attribute multi-interest group decision-making problems with attribute values and decision maker’s interest values taking the form of interval-valued intuitionistic fuzzy numbers, and application research to real estate purchase selection shows its practicality.

  6. Inhibition of phosphorylated tyrosine hydroxylase attenuates ethanol-induced hyperactivity in adult zebrafish (Danio rerio).

    Science.gov (United States)

    Nowicki, Magda; Tran, Steven; Chatterjee, Diptendu; Gerlai, Robert

    2015-11-01

    Zebrafish have been successfully employed in the study of the behavioural and biological effects of ethanol. Like in mammals, low to moderate doses of ethanol induce motor hyperactivity in zebrafish, an effect that has been attributed to the activation of the dopaminergic system. Acute ethanol exposure increases dopamine (DA) in the zebrafish brain, and it has been suggested that tyrosine hydroxylase, the rate-limiting enzyme of DA synthesis, may be activated in response to ethanol via phosphorylation. The current study employed tetrahydropapaveroline (THP), a selective inhibitor of phosphorylated tyrosine hydroxylase, for the first time, in zebrafish. We treated zebrafish with a THP dose that did not alter baseline motor responses to examine whether it can attenuate or abolish the effects of acute exposure to alcohol (ethanol) on motor activity, on levels of DA, and on levels of dopamine's metabolite 3,4-dihydroxyphenylacetic acid (DOPAC). We found that 60-minute exposure to 1% alcohol induced motor hyperactivity and an increase in brain DA. Both of these effects were attenuated by pre-treatment with THP. However, no differences in DOPAC levels were found among the treatment groups. These findings suggest that tyrosine hydroxylase is activated via phosphorylation to increase DA synthesis during alcohol exposure in zebrafish, and this partially mediates alcohol's locomotor stimulant effects. Future studies will investigate other potential candidates in the molecular pathway to further decipher the neurobiological mechanism that underlies the stimulatory properties of this popular psychoactive drug.

  7. EMT phenotype is induced by increased Src kinase activity via Src-mediated caspase-8 phosphorylation.

    Science.gov (United States)

    Zhao, Yang; Li, XiaoJun; Sun, XiangFei; Zhang, YunFeng; Ren, Hong

    2012-01-01

    Caspase-8 governs multiple cell responses to the microenvironmental cues. However, its integration of "death-life" signalings remains elusive. In our study, the role of caspase-8-Src is well-established as a promoter for migration or metastasis in Casp8(+)Src(+) A549/H226 cells in vivo and in vitro. In particular for nude mice models, mice implanted with Casp8(+)Src(+) A459/H226 cells remarkably increased spontaneous tumor metastatic burden with a significant survival disadvantage. Additionally, we detect that Src-mediated caspase-8 phosphorylation stimulates Src phosphorylation at Tyr-416 via the linkage of Src SH2 domain with phosph-Tyr-380 site of caspase-8. In turn, activated Src can efficiently induce epithelial-mesenchymal transition (EMT) phenotypic features to promote tumor cells metastasis. Surprisingly, RXDLL motif deletion in the DEDa of caspase-8 attenuates tumor cell migration or metastasis via impairing the recruitment of caspase-8 into the cellular periphery where activated Src is subject to caspase-8 phosphorylation. Together, a simple model is that the peripherization of caspase-8 is well-poised to facilitate Src-mediated caspase-8 phosphrylation at Tyr-380, then binding of phospho-Tyr380 of caspase-8 to Src SH2 domain may maintain Src in an active conformation to induce EMT phenotype, a key step toward cancer metastasis. PMID:22508042

  8. Phosphorylation-induced mechanical regulation of intrinsically disordered neurofilament protein assemblies

    CERN Document Server

    Malka-Gibor, Eti; Laser-Azogui, Adi; Doron, Ofer; Zingerman-Koladko, Irena; Medalia, Ohad; Beck, Roy

    2016-01-01

    The biological function of protein assemblies was conventionally equated with a unique three-dimensional protein structure and protein-specific interactions. However, in the past 20 years it was found that some assemblies contain long flexible regions that adopt multiple structural conformations. These include neurofilament (NF) proteins that constitute the stress-responsive supportive network of neurons. Herein, we show that NF networks macroscopic properties are tuned by enzymatic regulation of the charge found on the flexible protein regions. The results reveal an enzymatic (phosphorylation) regulation of macroscopic properties such as orientation, stress-response and expansion in flexible protein assemblies. Together with a model explaining the attractive electrostatic interactions induced by enzymatically added charges, we demonstrate that phosphorylation-regulation is far richer and versatile than previously considered.

  9. Multivalent scaffolds induce galectin-3 aggregation into nanoparticles

    Directory of Open Access Journals (Sweden)

    Candace K. Goodman

    2014-07-01

    Full Text Available Galectin-3 meditates cell surface glycoprotein clustering, cross linking, and lattice formation. In cancer biology, galectin-3 has been reported to play a role in aggregation processes that lead to tumor embolization and survival. Here, we show that lactose-functionalized dendrimers interact with galectin-3 in a multivalent fashion to form aggregates. The glycodendrimer–galectin aggregates were characterized by dynamic light scattering and fluorescence microscopy methodologies and were found to be discrete particles that increased in size as the dendrimer generation was increased. These results show that nucleated aggregation of galectin-3 can be regulated by the nucleating polymer and provide insights that improve the general understanding of the binding and function of sugar-binding proteins.

  10. Ralstonia insidiosa induces cell aggregation by Listeria monocytogenes

    Science.gov (United States)

    Biofilm formation is an important strategy for foodborne bacterial pathogens to survive in stressful environments such as fresh produce processing facilities. Bacterial cell aggregation strongly promotes the initiation of microcolonies and the formation of biofilms on abiological surfaces. We previ...

  11. MAPKs and Mst1/Caspase-3 pathways contribute to H2B phosphorylation during UVB-induced apoptosis

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Apoptosis is a highly coordinated or programmed cell suicide mechanism in eukaryotes.Histone modification is associated with nuclear events in apoptotic cells.Specifically H2B phosphorylation at serine 14 (Ser14) catalyzed by Mst1 kinase has been linked to chromatin condensation during apoptosis.We report that activation of MAPKs (ERK1/2,JNK1/2 and p38) together with Mst1 and caspase-3 is required for phosphorylation of H2B (Ser14) during ultraviolet B light (UVB)-induced apoptosis.UVB can trigger activation of MAPKs and induce H2B phosphorylation at Ser14 but not acetylation in a time-dependent manner.Inhibition of ERK1/2,JNK1/2 or p38 activity blocked H2B phosphorylation (Ser14).Furthermore,caspase-3 was activated by UVB to regulate Mst1 activity,which phosphorylates H2B at Ser14,leading to chromatin condensation.Full inhibition of caspase-3 activity reduced Mst1 activation and partially inhibited H2B phosphorylation (Ser14),but ERK1/2,JNK1/2 and p38 activities were not affected.Taken together,these data revealed that H2B phosphorylation is regulated by both MAPKs and caspase-3/Mst1 pathways during UVB-induced apoptosis.

  12. SP600125 Induces Src and Type I IGF Receptor Phosphorylation Independent of JNK

    Directory of Open Access Journals (Sweden)

    Qingbin Kong

    2014-09-01

    Full Text Available c-Jun N-terminal kinases (JNK are members of the mitogen-activated protein kinase (MAPK family that have important roles in signal transduction. The small molecule SP600125 is widely used in biochemical studies as a JNK inhibitor. However, recent studies indicate that SP600125 may also act independent of JNK. Here, we report that SP600125 can induce Src, type I insulin-like growth factor receptor (IGF-IR, Akt and Erk1/2 phosphorylation. Notably, these effects are independent of its inhibition of JNK. Inhibition of Src abrogates the stimulation of IGF-IR, Akt and Erk1/2 phosphorylation. IGF-IR knockdown blunts the induction of both Akt and Erk1/2 phosphorylation by SP600125. Moreover, combination of SP600125 and the Src inhibitor saracatinib synergistically inhibits cell proliferation. We conclude that SP600125 can activate Src-IGF-IR-Akt/Erk1/2 signaling pathways independent of JNK.

  13. CaMKII induces permeability transition through Drp1 phosphorylation during chronic β-AR stimulation

    Science.gov (United States)

    Xu, Shangcheng; Wang, Pei; Zhang, Huiliang; Gong, Guohua; Gutierrez Cortes, Nicolas; Zhu, Weizhong; Yoon, Yisang; Tian, Rong; Wang, Wang

    2016-01-01

    Mitochondrial permeability transition pore (mPTP) is involved in cardiac dysfunction during chronic β-adrenergic receptor (β-AR) stimulation. The mechanism by which chronic β-AR stimulation leads to mPTP openings is elusive. Here, we show that chronic administration of isoproterenol (ISO) persistently increases the frequency of mPTP openings followed by mitochondrial damage and cardiac dysfunction. Mechanistically, this effect is mediated by phosphorylation of mitochondrial fission protein, dynamin-related protein 1 (Drp1), by Ca2+/calmodulin-dependent kinase II (CaMKII) at a serine 616 (S616) site. Mutating this phosphorylation site or inhibiting Drp1 activity blocks CaMKII- or ISO-induced mPTP opening and myocyte death in vitro and rescues heart hypertrophy in vivo. In human failing hearts, Drp1 phosphorylation at S616 is increased. These results uncover a pathway downstream of chronic β-AR stimulation that links CaMKII, Drp1 and mPTP to bridge cytosolic stress signal with mitochondrial dysfunction in the heart. PMID:27739424

  14. Chlorogenic acid ameliorates endotoxin-induced liver injury by promoting mitochondrial oxidative phosphorylation.

    Science.gov (United States)

    Zhou, Yan; Ruan, Zheng; Zhou, Lili; Shu, Xugang; Sun, Xiaohong; Mi, Shumei; Yang, Yuhui; Yin, Yulong

    2016-01-22

    Acute or chronic hepatic injury is a common pathology worldwide. Mitochondrial dysfunction and the depletion of adenosine triphosphate (ATP) play important roles in liver injury. Chlorogenic acids (CGA) are some of the most abundant phenolic acids in human diet. This study was designed to test the hypothesis that CGA may protect against chronic lipopolysaccharide (LPS)-induced liver injury by modulating mitochondrial energy generation. CGA decreased the activities of serum alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase. The contents of ATP and adenosine monophosphate (AMP), as well as the ratio of AMP/ATP, were increased after CGA supplementation. The activities of enzymes that are involved in glycolysis were reduced, while those of enzymes involved in oxidative phosphorylation were increased. Moreover, phosphorylated AMP-activated protein kinase (AMPK), and mRNA levels of AMPK-α, peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α), nuclear respiratory factor 1, and mitochondrial DNA transcription factor A were increased after CGA supplementation. Collectively, these findings suggest that the hepatoprotective effect of CGA might be associated with enhanced ATP production, the stimulation of mitochondrial oxidative phosphorylation and the inhibition of glycolysis. PMID:26740181

  15. Glutamate-induced protein phosphorylation in cerebellar granule cells: role of protein kinase C.

    Science.gov (United States)

    Eboli, M L; Mercanti, D; Ciotti, M T; Aquino, A; Castellani, L

    1994-10-01

    Protein phosphorylation in response to toxic doses of glutamate has been investigated in cerebellar granule cells. 32P-labelled cells have been stimulated with 100 microM glutamate for up to 20 min and analysed by one and two dimensional gel electrophoresis. A progressive incorporation of label is observed in two molecular species of about 80 and 43 kDa (PP80 and PP43) and acidic isoelectric point. Glutamate-stimulated phosphorylation is greatly reduced by antagonists of NMDA and non-NMDA glutamate receptors. The effect of glutamate is mimicked by phorbol esters and is markedly reduced by inhibitors of protein kinase C (PKC) such as staurosporine and calphostin C. PP80 has been identified by Western blot analysis as the PKC substrate MARCKS (myristoylated alanine-rich C kinase substrate), while antibody to GAP-43 (growth associated protein-43), the nervous tissue-specific substrate of PKC, failed to recognize PP43. Our results suggest that PKC is responsible for the early phosphorylative events induced by toxic doses of glutamate in cerebellar granule cells. PMID:7891841

  16. Antimicrobial preservatives induce aggregation of interferon alpha-2a: the order in which preservatives induce protein aggregation is independent of the protein.

    Science.gov (United States)

    Bis, Regina L; Mallela, Krishna M G

    2014-09-10

    Antimicrobial preservatives (APs) are included in liquid multi-dose protein formulations to combat the growth of microbes and bacteria. These compounds have been shown to cause protein aggregation, which leads to serious immunogenic and toxic side-effects in patients. Our earlier work on a model protein cytochrome c (Cyt c) demonstrated that APs cause protein aggregation in a specific manner. The aim of this study is to validate the conclusions obtained from our model protein studies on a pharmaceutical protein. Interferon α-2a (IFNA2) is available as a therapeutic treatment for numerous immune-compromised disorders including leukemia and hepatitis C, and APs have been used in its multi-dose formulation. Similar to Cyt c, APs induced IFNA2 aggregation, demonstrated by the loss of soluble monomer and increase in solution turbidity. The extent of IFNA2 aggregation increased with the increase in AP concentration. IFNA2 aggregation also depended on the nature of AP, and followed the order m-cresol>phenol>benzyl alcohol>phenoxyethanol. This specific order exactly matched with that observed for the model protein Cyt c. These and previously published results on antibodies and other recombinant proteins suggest that the general mechanism by which APs induce protein aggregation may be independent of the protein.

  17. Rapid Oligo-Galacturonide Induced Changes in Protein Phosphorylation in Arabidopsis.

    Science.gov (United States)

    Kohorn, Bruce D; Hoon, Divya; Minkoff, Benjamin B; Sussman, Michael R; Kohorn, Susan L

    2016-04-01

    The wall-associated kinases (WAKs)(1)are receptor protein kinases that bind to long polymers of cross-linked pectin in the cell wall. These plasma-membrane-associated protein kinases also bind soluble pectin fragments called oligo-galacturonides (OGs) released from the wall after pathogen attack and damage. WAKs are required for cell expansion during development but bind water soluble OGs generated from walls with a higher affinity than the wall-associated polysaccharides. OGs activate a WAK-dependent, distinct stress-like response pathway to help plants resist pathogen attack. In this report, a quantitative mass-spectrometric-based phosphoproteomic analysis was used to identify Arabidopsis cellular events rapidly induced by OGsin planta Using N(14/)N(15)isotopicin vivometabolic labeling, we screened 1,000 phosphoproteins for rapid OG-induced changes and found 50 proteins with increased phosphorylation, while there were none that decreased significantly. Seven of the phosphosites within these proteins overlap with those altered by another signaling molecule plants use to indicate the presence of pathogens (the bacterial "elicitor" peptide Flg22), indicating distinct but overlapping pathways activated by these two types of chemicals. Genetic analysis of genes encoding 10 OG-specific and two Flg22/OG-induced phosphoproteins reveals that null mutations in eight proteins compromise the OG response. These phosphorylated proteins with genetic evidence supporting their role in the OG response include two cytoplasmic kinases, two membrane-associated scaffold proteins, a phospholipase C, a CDPK, an unknown cadmium response protein, and a motor protein. Null mutants in two proteins, the putative scaffold protein REM1.3, and a cytoplasmic receptor like kinase ROG2, enhance and suppress, respectively, a dominantWAKallele. Altogether, the results of these chemical and genetic experiments reveal the identity of several phosphorylated proteins involved in the kinase

  18. Prolactin-induced Jak2 phosphorylation of RUSH: a key element in Jak/RUSH signaling

    OpenAIRE

    Helmer, Rebecca A.; Panchoo, Marlyn; Dertien, Janet S.; Bhakta, Suhani M.; Hewetson, Aveline; Beverly S Chilton

    2010-01-01

    Jak2/Stat-mediated prolactin signaling culminates in Stat5a-DNA-binding. However, not all Jak2-dependent genes have Stat5 sites. Western analysis with inhibitors showed Jak2 is a proximal intermediate in prolactin-induced RUSH phosphorylation. Transfection assays with HRE-H9 cells showed the RUSH-binding site mediated the ability of prolactin to augment progesterone-dependent transcription of the RUSH gene. Jak2 inhibitors or targeted RUSH-site mutation blocked the prolactin effect. RUSH co-i...

  19. The Down syndrome-related protein kinase DYRK1A phosphorylates p27(Kip1) and Cyclin D1 and induces cell cycle exit and neuronal differentiation.

    Science.gov (United States)

    Soppa, Ulf; Schumacher, Julian; Florencio Ortiz, Victoria; Pasqualon, Tobias; Tejedor, Francisco J; Becker, Walter

    2014-01-01

    A fundamental question in neurobiology is how the balance between proliferation and differentiation of neuronal precursors is maintained to ensure that the proper number of brain neurons is generated. Substantial evidence implicates DYRK1A (dual specificity tyrosine-phosphorylation-regulated kinase 1A) as a candidate gene responsible for altered neuronal development and brain abnormalities in Down syndrome. Recent findings support the hypothesis that DYRK1A is involved in cell cycle control. Nonetheless, how DYRK1A contributes to neuronal cell cycle regulation and thereby affects neurogenesis remains poorly understood. In the present study we have investigated the mechanisms by which DYRK1A affects cell cycle regulation and neuronal differentiation in a human cell model, mouse neurons, and mouse brain. Dependent on its kinase activity and correlated with the dosage of overexpression, DYRK1A blocked proliferation of SH-SY5Y neuroblastoma cells within 24 h and arrested the cells in G₁ phase. Sustained overexpression of DYRK1A induced G₀ cell cycle exit and neuronal differentiation. Furthermore, we provide evidence that DYRK1A modulated protein stability of cell cycle-regulatory proteins. DYRK1A reduced cellular Cyclin D1 levels by phosphorylation on Thr286, which is known to induce proteasomal degradation. In addition, DYRK1A phosphorylated p27(Kip1) on Ser10, resulting in protein stabilization. Inhibition of DYRK1A kinase activity reduced p27(Kip1) Ser10 phosphorylation in cultured hippocampal neurons and in embryonic mouse brain. In aggregate, these results suggest a novel mechanism by which overexpression of DYRK1A may promote premature neuronal differentiation and contribute to altered brain development in Down syndrome.

  20. Molecular Mechanisms in the Pathogenesis of Alzheimer’s disease and Tauopathies-Prion-Like Seeded Aggregation and Phosphorylation

    Science.gov (United States)

    Hasegawa, Masato

    2016-01-01

    Neurofibrillary tau pathology (tangles and threads) and extracellular amyloid-β (Aβ) pathology are defining features of Alzheimer’s disease. For 25 years, most research has focused on the amyloid hypothesis of AD pathogenesis and progression. But, because of failures in clinical trials of Aβ-targeted therapies and the new concept of prion-like propagation of intracellular abnormal proteins, tau has come back into the spotlight as a candidate therapeutic target in AD. Tau pathologies are found in a range of neurodegenerative disorders, but extensive analyses of pathological tau in diseased brains has demonstrated that the abnormal tau protein in each disease is structurally distinct, supporting the idea that progression of the diverse but characteristic tau pathologies occurs through prion-like seed-dependent aggregation. Therefore, intervention in the conversion of normal tau to abnormal forms and in cell-to-cell transmission of tau may be the key to development of disease-modifying therapies for AD and other dementing disorders. PMID:27136595

  1. Composite alginate hydrogel microparticulate delivery system of zidovudine hydrochloride based on counter ion induced aggregation

    OpenAIRE

    Roy, Harekrishna; Rao, P. Venkateswar; Panda, Sanjay Kumar; Biswal, Asim Kumar; Parida, Kirti Ranjan; Dash, Jharana

    2014-01-01

    Aim: The present study deals with preparation of zidovudine loaded microparticle by counter ion induced aggregation method. During this study effect of polyacrylates and hypromellose polymers on release study were investigated. Materials and Methods: The ion induced aggregated alginate based microparticles were characterized for surface morphology, particle size analysis, drug entrapment study, in-vitro study, Fourier-transform infrared (FTIR) spectroscopy, and differential scanning calorimet...

  2. Global protein phosphorylation dynamics during deoxynivalenol-induced ribotoxic stress response in the macrophage

    Energy Technology Data Exchange (ETDEWEB)

    Pan, Xiao [Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824 (United States); Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824 (United States); Whitten, Douglas A. [Research Technology Support Facility, Proteomics Core, Michigan State University, East Lansing, MI 48824 (United States); Wu, Ming [Department of Computer Science and Engineering, Michigan State University, East Lansing, MI 48824 (United States); Chan, Christina [Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824 (United States); Department of Computer Science and Engineering, Michigan State University, East Lansing, MI 48824 (United States); Wilkerson, Curtis G. [Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824 (United States); Research Technology Support Facility, Proteomics Core, Michigan State University, East Lansing, MI 48824 (United States); Pestka, James J., E-mail: pestka@msu.edu [Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824 (United States); Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824 (United States); Department of Food Science and Human Nutrition, Michigan State University, East Lansing, MI 48824 (United States)

    2013-04-15

    Deoxynivalenol (DON), a trichothecene mycotoxin produced by Fusarium that commonly contaminates food, is capable of activating mononuclear phagocytes of the innate immune system via a process termed the ribotoxic stress response (RSR). To encapture global signaling events mediating RSR, we quantified the early temporal (≤ 30 min) phosphoproteome changes that occurred in RAW 264.7 murine macrophage during exposure to a toxicologically relevant concentration of DON (250 ng/mL). Large-scale phosphoproteomic analysis employing stable isotope labeling of amino acids in cell culture (SILAC) in conjunction with titanium dioxide chromatography revealed that DON significantly upregulated or downregulated phosphorylation of 188 proteins at both known and yet-to-be functionally characterized phosphosites. DON-induced RSR is extremely complex and goes far beyond its prior known capacity to inhibit translation and activate MAPKs. Transcriptional regulation was the main target during early DON-induced RSR, covering over 20% of the altered phosphoproteins as indicated by Gene Ontology annotation and including transcription factors/cofactors and epigenetic modulators. Other biological processes impacted included cell cycle, RNA processing, translation, ribosome biogenesis, monocyte differentiation and cytoskeleton organization. Some of these processes could be mediated by signaling networks involving MAPK-, NFκB-, AKT- and AMPK-linked pathways. Fuzzy c-means clustering revealed that DON-regulated phosphosites could be discretely classified with regard to the kinetics of phosphorylation/dephosphorylation. The cellular response networks identified provide a template for further exploration of the mechanisms of trichothecenemycotoxins and other ribotoxins, and ultimately, could contribute to improved mechanism-based human health risk assessment. - Highlights: ► Mycotoxin deoxynivalenol (DON) induces immunotoxicity via ribotoxic stress response. ► SILAC phosphoproteomics using

  3. Blocking rpS6 Phosphorylation Exacerbates Tsc1 Deletion-Induced Kidney Growth.

    Science.gov (United States)

    Wu, Huijuan; Chen, Jianchun; Xu, Jinxian; Dong, Zheng; Meyuhas, Oded; Chen, Jian-Kang

    2016-04-01

    The molecular mechanisms underlying renal growth and renal growth-induced nephron damage remain poorly understood. Here, we report that in murine models, deletion of the tuberous sclerosis complex protein 1 (Tsc1) in renal proximal tubules induced strikingly enlarged kidneys, with minimal cystogenesis and occasional microscopic tumorigenesis. Signaling studies revealed hyperphosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and increased phosphorylation of ribosomal protein S6 (rpS6) in activated renal tubules. Notably, knockin of a nonphosphorylatable rpS6 in theseTsc1-mutant mice exacerbated cystogenesis and caused drastic nephron damage and renal fibrosis, leading to kidney failure and a premature death rate of 67% by 9 weeks of age. In contrast,Tsc1single-mutant mice were all alive and had far fewer renal cysts at this age. Mechanistic studies revealed persistent activation of mammalian target of rapamycin complex 1 (mTORC1) signaling causing hyperphosphorylation and consequent accumulation of 4E-BP1, along with greater cell proliferation, in the renal tubules ofTsc1andrpS6double-mutant mice. Furthermore, pharmacologic treatment ofTsc1single-mutant mice with rapamycin reduced hyperphosphorylation and accumulation of 4E-BP1 but also inhibited phosphorylation of rpS6. Rapamycin also exacerbated cystic and fibrotic lesions and impaired kidney function in these mice, consequently leading to a premature death rate of 40% within 2 weeks of treatment, despite destroying tumors and decreasing kidney size. These findings indicate that Tsc1 prevents aberrant renal growth and tumorigenesis by inhibiting mTORC1 signaling, whereas phosphorylated rpS6 suppresses cystogenesis and fibrosis inTsc1-deleted kidneys. PMID:26296742

  4. Does ocean acidification induce an upward flux of marine aggregates?

    Directory of Open Access Journals (Sweden)

    X. Mari

    2008-07-01

    Full Text Available The absorption of anthropogenic atmospheric carbon dioxide (CO2 by the ocean provokes its acidification. This acidification may alter several oceanic processes, including the export of biogenic carbon from the upper layer of the ocean, hence providing a feedback on rising atmospheric carbon concentrations. The effect of seawater acidification on transparent exopolymeric particles (TEP driven aggregation and sedimentation processes were investigated by studying the interactions between latex beads and TEP precursors collected in the lagoon of New Caledonia. A suspension of TEP and beads was prepared and the formation of mixed aggregates was monitored as a function of pH under increasing turbulence intensities. The pH was controlled by addition of sulfuric acid. Aggregation and sedimentation processes driven by TEP were drastically reduced when the pH of seawater decreases within the expected limits imposed by increased anthropogenic CO2 emissions. In addition to the diminution of TEP sticking properties, the diminution of seawater pH led to a significant increase of the TEP pool, most likely due to swollen structures. A diminution of seawater pH by 0.2 units or more led to a stop or a reversal of the downward flux of particles. If applicable to oceanic conditions, the sedimentation of marine aggregates may slow down or even stop as the pH decreases, and the vertical flux of organic carbon may reverse. This would enhance both rising atmospheric carbon and ocean acidification.

  5. Does ocean acidification induce an upward flux of marine aggregates?

    Directory of Open Access Journals (Sweden)

    X. Mari

    2008-04-01

    Full Text Available The adsorption of anthropogenic atmospheric carbon dioxide (CO2 by the ocean provokes its acidification. This acidification may alter several oceanic processes, including the export of biogenic carbon from the upper layer of the ocean, hence providing a feedback on rising atmospheric carbon concentrations. The effect of seawater acidification on transparent exopolymeric particles (TEP driven aggregation and sedimentation processes were investigated by studying the interactions between latex beads and TEP precursors collected in the lagoon of New Caledonia. A suspension of TEP and beads was prepared and the formation of mixed aggregates was monitored as a function of pH under increasing turbulence intensities. The pH was controlled by addition of sulfuric acid. Aggregation and sedimentation processes driven by TEP were drastically reduced when the pH of seawater decreases within the expected limits imposed by increased anthropogenic CO2 emissions. In addition to the diminution of TEP sticking properties, the diminution of seawater pH led to a significant increase of the TEP pool, most likely due to swollen structures. A diminution of seawater pH by 0.2 units or more led to a stop or a reversal of the downward flux of particles. If applicable to oceanic conditions, the sedimentation of marine aggregates may slow down or even stop as the pH decreases, and the vertical flux of organic carbon may reverse. This would enhance both rising atmospheric carbon and ocean acidification.

  6. Dendritic copper phthalocyanine with aggregation induced blue emission and solid-state fluorescence

    Science.gov (United States)

    Wang, Jiayi; Pan, Lin; Zhou, Xuefei; Jia, Kun; Liu, Xiaobo

    2016-09-01

    In this work, dendritic copper phthalocyanine (CuPc) showing obvious aggregation induced emission (AIE) and strong solid-state fluorescence was synthesized. It was found that synthesized CuPc can be easily solubilized in polar aprotic solvent, where no fluorescence signal was detected. Interestingly, both the CuPc aggregates in solution and solid-state powder exhibited strong fluorescence emission around 480 nm, which should be attributed to the restriction of intramolecular rotation as rationalized in aggregation induced emission framework. Meanwhile the obvious crystalline enhanced solid-state fluorescent emission is observed for CuPc powder.

  7. Towards meso -Ester BODIPYs with Aggregation-Induced Emission Properties: The Effect of Substitution Positions

    KAUST Repository

    Chua, Ming Hui

    2015-06-17

    Three meso-ester boron dipyrromethene (BODIPY) dyes have been synthesized and functionalized with aggregation-induced emission (AIE)-active tetraphenylethene or triphenylethene moieties. It was found that functionalizing at the different positions of the BODIPY core resulted in the final dye having different emission properties in response to aggregation: from aggregation-induced quenching (ACQ) to being AIE active. X-ray crystallographic analysis was thus performed to provide an explanation for these differences. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. PKC-Mediated ZYG1 Phosphorylation Induces Fusion of Myoblasts as well as of Dictyostelium Cells

    Directory of Open Access Journals (Sweden)

    Aiko Amagai

    2012-01-01

    Full Text Available We have previously demonstrated that a novel protein ZYG1 induces sexual cell fusion (zygote formation of Dictyostelium cells. In the process of cell fusion, involvements of signal transduction pathways via Ca2+ and PKC (protein kinase C have been suggested because zygote formation is greatly enhanced by PKC activators. In fact, there are several deduced sites phosphorylated by PKC in ZYG1 protein. Thereupon, we designed the present work to examine whether or not ZYG1 is actually phosphorylated by PKC and localized at the regions of cell-cell contacts where cell fusion occurs. These were ascertained, suggesting that ZYG1 might be the target protein for PKC. A humanized version of zyg1 cDNA (mzyg1 was introduced into myoblasts to know if ZYG1 is also effective in cell fusion of myoblasts. Quite interestingly, enforced expression of ZYG1 in myoblasts was found to induce markedly their cell fusion, thus strongly suggesting the existence of a common signaling pathway for cell fusion beyond the difference of species.

  9. Light-induced phosphorylation of a membrane protein plays an early role in signal transduction for phototropism in Arabidopsis thaliana

    Science.gov (United States)

    Reymond, P.; Short, T. W.; Briggs, W. R.; Poff, K. L.

    1992-01-01

    Blue light is known to cause rapid phosphorylation of a membrane protein in etiolated seedlings of several plant species, a protein that, at least in etiolated pea seedlings and maize coleoptiles, has been shown to be associated with the plasma membrane. The light-driven phosphorylation has been proposed on the basis of correlative evidence to be an early step in the signal transduction chain for phototropism. In the Arabidopsis thaliana mutant JK224, the sensitivity to blue light for induction of first positive phototropism is known to be 20- to 30-fold lower than in wild type, whereas second positive curvature appears to be normal. While light-induced phosphorylation can be demonstrated in crude membrane preparations from shoots of the mutant, the level of phosphorylation is dramatically lower than in wild type, as is the sensitivity to blue light. Another A. thaliana mutant, JK218, that completely lacks any phototropic responses to up to 2 h of irradiation, shows a normal level of light-induced phosphorylation at saturation. Since its gravitropic sensitivity is normal, it is presumably blocked in some step between photoreception and the confluence of the signal transduction pathways for phototropism and gravitropism. We conclude from mutant JK224 that light-induced phosphorylation plays an early role in the signal transduction chain for phototropism in higher plants.

  10. Pervanadate-induced adhesion of CD4+ T cell to fibronectin is associated with tyrosine phosphorylation of paxillin.

    Science.gov (United States)

    Miron, S; Kachalsky, S G; Hershkoviz, R; Lider, O

    1997-09-01

    The initial stages of T cell activation involve tyrosine protein kinase-mediated intracellular signaling events. Integrin-mediated adhesion of CD4+ T lymphocytes to extracellular matrix glycoproteins, such as fibronectin, is an activation-dependent process. The involvement of tyrosine protein kinases in the adhesion of CD4+ T cells to fibronectin was examined using pervanadate, a protein-tyrosine phosphatase inhibitor. Pervanadate induced the adhesion of human CD4+ T cells to immobilized fibronectin in a beta1 integrin-mediated fashion, and adhesion was associated with an increase of protein tyrosine phosphorylation. Tyrosine protein kinase inhibitors abrogated both T cell adhesion and intracellular protein tyrosine phosphorylation. Participation of cytoskeletal proteins in the pervanadate-induced T cell adhesion was indicated because cytoskeleton disruption by cytochalasin B inhibited cell adhesion to fibronectin. We demonstrate that the cytoskeletal protein paxillin underwent time-dependent tyrosine phosphorylation simultaneously with pervanadate-induced T cell adhesion to fibronectin. Tyrosine phosphorylation of paxillin was related to cell adhesion, since pretreatment of T cells with cytochalasin B abrogated both adhesion and phosphorylation. This study demonstrates a correlation between activation of protein tyrosine kinases, tyrosine phosphorylation of paxillin, and integrin-mediated T cell adhesion to extracellular matrix glycoproteins. PMID:9307082

  11. 14-3-3ζ Mediates Tau Aggregation in Human Neuroblastoma M17 Cells.

    Science.gov (United States)

    Li, Tong; Paudel, Hemant K

    2016-01-01

    Microtubule-associated protein tau is the major component of paired helical filaments (PHFs) associated with the neuropathology of Alzheimer's disease (AD). Tau in the normal brain binds and stabilizes microtubules. Tau isolated from PHFs is hyperphosphorylated, which prevents it from binding to microtubules. Tau phosphorylation has been suggested to be involved in the development of NFT pathology in the AD brain. Recently, we showed that 14-3-3ζ is bound to tau in the PHFs and when incubated in vitro with 14-3-3ζ, tau formed amorphous aggregates, single-stranded straight filaments, double stranded ribbon-like filaments and PHF-like filaments that displayed close resemblance with corresponding ultrastructures of AD brain. Surprisingly however, phosphorylated and non-phosphorylated tau aggregated in a similar manner, indicating that tau phosphorylation does not affect in vitro tau aggregation (Qureshi et al (2013) Biochemistry 52, 6445-6455). In this study, we have examined the role of tau phosphorylation in tau aggregation in cellular level. We have found that in human M17 neuroblastoma cells, tau phosphorylation by GSK3β or PKA does not cause tau aggregation, but promotes 14-3-3ζ-induced tau aggregation by destabilizing microtubules. Microtubule disrupting drugs also promoted 14-3-3ζ-induced tau aggregation without changing tau phosphorylation in M17 cell. In vitro, when incubated with 14-3-3ζ and microtubules, nonphosphorylated tau bound to microtubules and did not aggregate. Phosphorylated tau on the other hand did not bind to microtubules and aggregated. Our data indicate that microtubule-bound tau is resistant to 14-3-3ζ-induced tau aggregation and suggest that tau phosphorylation promotes tau aggregation in the brain by detaching tau from microtubules and thus making it accessible to 14-3-3ζ. PMID:27548710

  12. Spermine-induced aggregation of DNA, nucleosome, and chromatin.

    OpenAIRE

    Raspaud, E.; Chaperon, I; Leforestier, A; Livolant, F

    1999-01-01

    We have analyzed the conditions of aggregation or precipitation of DNA in four different states: double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), mononucleosome core particles (NCP), and H1-depleted chromatin fragments (ChF) in the presence of the multivalent cation spermine (4+). In an intermediate regime of DNA concentration, these conditions are identical for the four states. This result demonstrates that the mechanism involved is general from flexible chains to rigid rods and qua...

  13. High LET - induced H2AX phosphorylation at sites of DNA double strand breaks

    Science.gov (United States)

    Desai, N.; Cucinotta, F.; Wu, H.

    Within cell nuclei, traversing charged heavy ion particles lead to the accumulation of proteins related to DNA lesions and repair along the ion trajectories. Irradiation using a standard geometric setup with the beam path perpendicular to the cell monolayer generates discrete foci of several proteins known to localize at sites of DNA double strand breaks (DSBs). One such molecule is the histone protein H2AX (gamma-H2AX), which gets rapidly phosphorylated in response to ionizing radiation. Here we present data obtained with a modified irradiation geometry characterized by a beam path parallel to a monolayer of human fibroblast cells. This new irradiation geometry leads to the formation of gamma-H2AX aggregates in the shape of streaks stretching over several micrometers in the x/y plane, thus enabling the analysis of the fluorescence distributions along the particle trajectories. Qualitative analysis of these distributions presented insights into the DNA repair kinetics along the primary track structure and visualization of possible chromatin movement. We also present evidence of colocalization of gamma-H2AX with several other proteins in responses to ionizing radiation exposure. Analysis of gamma-H2AX has the potential to provide useful information on human cell responses to high LET radiation after exposure to space-like radiation.

  14. Mechanism of neem limonoids-induced cell death in cancer: Role of oxidative phosphorylation.

    Science.gov (United States)

    Yadav, Neelu; Kumar, Sandeep; Kumar, Rahul; Srivastava, Pragya; Sun, Leimin; Rapali, Peter; Marlowe, Timothy; Schneider, Andrea; Inigo, Joseph R; O'Malley, Jordan; Londonkar, Ramesh; Gogada, Raghu; Chaudhary, Ajay K; Yadava, Nagendra; Chandra, Dhyan

    2016-01-01

    We have previously reported that neem limonoids (neem) induce multiple cancer cell death pathways. Here we dissect the underlying mechanisms of neem-induced apoptotic cell death in cancer. We observed that neem-induced caspase activation does not require Bax/Bak channel-mediated mitochondrial outer membrane permeabilization, permeability transition pore, and mitochondrial fragmentation. Neem enhanced mitochondrial DNA and mitochondrial biomass. While oxidative phosphorylation (OXPHOS) Complex-I activity was decreased, the activities of other OXPHOS complexes including Complex-II and -IV were unaltered. Increased reactive oxygen species (ROS) levels were associated with an increase in mitochondrial biomass and apoptosis upon neem exposure. Complex-I deficiency due to the loss of Ndufa1-encoded MWFE protein inhibited neem-induced caspase activation and apoptosis, but cell death induction was enhanced. Complex II-deficiency due to the loss of succinate dehydrogenase complex subunit C (SDHC) robustly decreased caspase activation, apoptosis, and cell death. Additionally, the ablation of Complexes-I, -III, -IV, and -V together did not inhibit caspase activation. Together, we demonstrate that neem limonoids target OXPHOS system to induce cancer cell death, which does not require upregulation or activation of proapoptotic Bcl-2 family proteins.

  15. Aggregation induced enhanced emission of 2-(2'-hydroxyphenyl)benzimidazole.

    Science.gov (United States)

    Malakar, Ashim; Kumar, Manishekhar; Reddy, Anki; Biswal, Himadree T; Mandal, Biman B; Krishnamoorthy, G

    2016-07-01

    In this study, the aggregation induced emission enhancement (AIEE) of 2-(2'-hydroxyphenyl)benzimidazole (HPBI) is reported. To investigate the AIEE process of HPBI, absorption/fluorescence spectroscopy, fluorescence imaging and field emission scanning electron microscopy were employed. A comparative study with 2-phenylbenzimidazole (PBI) divulges the significance of the hydroxyl group in the AIEE process. Further, molecular dynamics simulations have been carried out with explicit solvent molecules to follow the aggregation process of HPBI with time. The obtained molecular dynamics simulation results not only predicted the formation of aggregates but also provided detailed insight and information on the molecular interactions. The cellular studies showed aggregates yield higher fluorescence in the visible region inside HeLa cells in comparison to monomeric compounds which failed to exhibit any visible fluorescence inside the cell. The obtained aggregates were further found to be biocompatible and therefore can be used for bio-imaging applications. PMID:27334264

  16. Evaluation of butyrate-induced production of a mannose-6-phosphorylated therapeutic enzyme using parallel bioreactors.

    Science.gov (United States)

    Madhavarao, Chikkathur N; Agarabi, Cyrus D; Wong, Lily; Müller-Loennies, Sven; Braulke, Thomas; Khan, Mansoor; Anderson, Howard; Johnson, Gibbes R

    2014-01-01

    Bioreactor process changes can have a profound effect on the yield and quality of biotechnology products. Mannose-6-phosphate (M6P) glycan content and the enzymatic catalytic kinetic parameters are critical quality attributes (CQAs) of many therapeutic enzymes used to treat lysosomal storage diseases (LSDs). Here, we have evaluated the effect of adding butyrate to bioreactor production cultures of human recombinant β-glucuronidase produced from CHO-K1 cells, with an emphasis on CQAs. The β-glucuronidase produced in parallel bioreactors was quantified by capillary electrophoresis, the catalytic kinetic parameters were measured using steady-state analysis, and mannose-6-phosphorylation status was assessed using an M6P-specific single-chain antibody fragment. Using this approach, we found that butyrate treatment increased β-glucuronidase production up to approximately threefold without significantly affecting the catalytic properties of the enzyme. However, M6P content in β-glucuronidase was inversely correlated with the increased enzyme production induced by butyrate treatment. This assessment demonstrated that although butyrate dramatically increased β-glucuronidase production in bioreactors, it adversely impacted the mannose-6-phosphorylation of this LSD therapeutic enzyme. This strategy may have utility in evaluating manufacturing process changes to improve therapeutic enzyme yields and CQAs.

  17. Mobile phone base station-emitted radiation does not induce phosphorylation of Hsp27.

    Science.gov (United States)

    Hirose, H; Sakuma, N; Kaji, N; Nakayama, K; Inoue, K; Sekijima, M; Nojima, T; Miyakoshi, J

    2007-02-01

    An in vitro study focusing on the effects of low-level radiofrequency (RF) fields from mobile radio base stations employing the International Mobile Telecommunication 2000 (IMT-2000) cellular system was conducted to test the hypothesis that modulated RF fields act to induce phosphorylation and overexpression of heat shock protein hsp27. First, we evaluated the responses of human cells to microwave exposure at a specific absorption rate (SAR) of 80 mW/kg, which corresponds to the limit of the average whole-body SAR for general public exposure defined as a basic restriction in the International Commission on Non-Ionizing Radiation Protection (ICNIRP) guidelines. Second, we investigated whether continuous wave (CW) and Wideband Code Division Multiple Access (W-CDMA) modulated signal RF fields at 2.1425 GHz induced activation or gene expression of hsp27 and other heat shock proteins (hsps). Human glioblastoma A172 cells were exposed to W-CDMA radiation at SARs of 80 and 800 mW/kg for 2-48 h, and CW radiation at 80 mW/kg for 24 h. Human IMR-90 fibroblasts from fetal lungs were exposed to W-CDMA at 80 and 800 mW/kg for 2 or 28 h, and CW at 80 mW/kg for 28 h. Under the RF field exposure conditions described above, no significant differences in the expression levels of phosphorylated hsp27 at serine 82 (hsp27[pS82]) were observed between the test groups exposed to W-CDMA or CW signal and the sham-exposed negative controls, as evaluated immediately after the exposure periods by bead-based multiplex assays. Moreover, no noticeable differences in the gene expression of hsps were observed between the test groups and the negative controls by DNA Chip analysis. Our results confirm that exposure to low-level RF field up to 800 mW/kg does not induce phosphorylation of hsp27 or expression of hsp gene family. PMID:17004241

  18. Bio-/Chemosensors and Imaging with Aggregation-Induced Emission Luminogens.

    Science.gov (United States)

    Zhan, Chi; You, Xue; Zhang, Guanxin; Zhang, Deqing

    2016-08-01

    Aggregation-induced emission (AIE) luminogens show abnormal fluorescent behavior; they are non-emissive in solution, but they become strongly emissive after aggregation. Sensing and imaging are the major applications of AIE luminogens. By properly manipulating the aggregation and deaggregation of AIE molecules, various bio-/chemosensors have been developed. Moreover, AIE molecules with targeting groups have been devised for imaging of organelles and cancer cells. In this account, we report our recent work on the application of AIE luminogens for the construction of bio-/chemosensors and imaging. PMID:27427427

  19. Primary WWOX phosphorylation and JNK activation during etoposide induces cytotoxicity in HEK293 cells

    Directory of Open Access Journals (Sweden)

    M Jamshidiha

    2010-06-01

    Full Text Available "n  "nBackground and the purpose of the study: Etoposide is an antineoplastic agent used in multiple cancers. It is known that etoposide induce cell death via interaction with topoisomerase II; however, the etopoisde cellular response is poorly understood. Upon etoposide induced DNA damage, many stress signaling pathways including JNK are activated. In response to DNA damage, it has been shown that WWOX, a recently introduced tumor suppressor, can be activated. In this study the activation of WWOX and JNK and their interaction following etoposide treatment were evaluated. "nMaterials and Methods:HEK293 cells treated with etoposide were lysed in a time course manner. The whole cell lysates were used to evaluate JNK and WWOX activation pattern using Phospho specific antibodies on western blots. The viability of cells treated with etoposide, JNK specific inhibitor and their combination was examined using MTT assay. "nResults:Findings of this study indicate that WWOX and JNK are activated in a simultaneous way in response to DNA damage. Moreover, JNK inhibition enhances etoposide induced cytotoxicity in HEK293. "nConclusion:Taken together, our results indicate that etoposide induces cytotoxicity and WWOX phosphorylation and the cytotoxicty is augmented by blocking JNK pathway.

  20. Detection of Gold Nanoparticles Aggregation Growth Induced by Nucleic Acid through Laser Scanning Confocal Microscopy

    OpenAIRE

    Ramla Gary; Giovani Carbone; Gia Petriashvili; Maria Penelope De Santo; Riccardo Barberi

    2016-01-01

    The gold nanoparticle (GNP) aggregation growth induced by deoxyribonucleic acid (DNA) is studied by laser scanning confocal and environmental scanning electron microscopies. As in the investigated case the direct light scattering analysis is not suitable, we observe the behavior of the fluorescence produced by a dye and we detect the aggregation by the shift and the broadening of the fluorescence peak. Results of laser scanning confocal microscopy images and the fluorescence emission spectra ...

  1. Break-induced replication requires DNA damage-induced phosphorylation of Pif1 and leads to telomere lengthening.

    Directory of Open Access Journals (Sweden)

    Yulia Vasianovich

    2014-10-01

    Full Text Available Broken replication forks result in DNA breaks that are normally repaired via homologous recombination or break induced replication (BIR. Mild insufficiency in the replicative ligase Cdc9 in budding yeast Saccharomyces cerevisiae resulted in a population of cells with persistent DNA damage, most likely due to broken replication forks, constitutive activation of the DNA damage checkpoint and longer telomeres. This telomere lengthening required functional telomerase, the core DNA damage signaling cascade Mec1-Rad9-Rad53, and the components of the BIR repair pathway - Rad51, Rad52, Pol32, and Pif1. The Mec1-Rad53 induced phosphorylation of Pif1, previously found necessary for inhibition of telomerase at double strand breaks, was also important for the role of Pif1 in BIR and telomere elongation in cdc9-1 cells. Two other mutants with impaired DNA replication, cdc44-5 and rrm3Δ, were similar to cdc9-1: their long telomere phenotype was dependent on the Pif1 phosphorylation locus. We propose a model whereby the passage of BIR forks through telomeres promotes telomerase activity and leads to telomere lengthening.

  2. Stress-induced ceramide generation and apoptosis via the phosphorylation and activation of nSMase1 by JNK signaling.

    Science.gov (United States)

    Yabu, T; Shiba, H; Shibasaki, Y; Nakanishi, T; Imamura, S; Touhata, K; Yamashita, M

    2015-02-01

    Neutral sphingomyelinase (nSMase) activation in response to environmental stress or inflammatory cytokine stimuli generates the second messenger ceramide, which mediates the stress-induced apoptosis. However, the signaling pathways and activation mechanism underlying this process have yet to be elucidated. Here we show that the phosphorylation of nSMase1 (sphingomyelin phosphodiesterase 2, SMPD2) by c-Jun N-terminal kinase (JNK) signaling stimulates ceramide generation and apoptosis and provide evidence for a signaling mechanism that integrates stress- and cytokine-activated apoptosis in vertebrate cells. An nSMase1 was identified as a JNK substrate, and the phosphorylation site responsible for its effects on stress and cytokine induction was Ser-270. In zebrafish cells, the substitution of Ser-270 for alanine blocked the phosphorylation and activation of nSMase1, whereas the substitution of Ser-270 for negatively charged glutamic acid mimicked the effect of phosphorylation. The JNK inhibitor SP600125 blocked the phosphorylation and activation of nSMase1, which in turn blocked ceramide signaling and apoptosis. A variety of stress conditions, including heat shock, UV exposure, hydrogen peroxide treatment, and anti-Fas antibody stimulation, led to the phosphorylation of nSMase1, activated nSMase1, and induced ceramide generation and apoptosis in zebrafish embryonic ZE and human Jurkat T cells. In addition, the depletion of MAPK8/9 or SMPD2 by RNAi knockdown decreased ceramide generation and stress- and cytokine-induced apoptosis in Jurkat cells. Therefore the phosphorylation of nSMase1 is a pivotal step in JNK signaling, which leads to ceramide generation and apoptosis under stress conditions and in response to cytokine stimulation. nSMase1 has a common central role in ceramide signaling during the stress and cytokine responses and apoptosis.

  3. Stabilization of Microtubule-Unbound Tau via Tau Phosphorylation at Ser262/356 by Par-1/MARK Contributes to Augmentation of AD-Related Phosphorylation and Aβ42-Induced Tau Toxicity.

    Directory of Open Access Journals (Sweden)

    Kanae Ando

    2016-03-01

    Full Text Available Abnormal accumulation of the microtubule-interacting protein tau is associated with neurodegenerative diseases including Alzheimer's disease (AD. β-amyloid (Aβ lies upstream of abnormal tau behavior, including detachment from microtubules, phosphorylation at several disease-specific sites, and self-aggregation into toxic tau species in AD brains. To prevent the cascade of events leading to neurodegeneration in AD, it is essential to elucidate the mechanisms underlying the initial events of tau mismetabolism. Currently, however, these mechanisms remain unclear. In this study, using transgenic Drosophila co-expressing human tau and Aβ, we found that tau phosphorylation at AD-related Ser262/356 stabilized microtubule-unbound tau in the early phase of tau mismetabolism, leading to neurodegeneration. Aβ increased the level of tau detached from microtubules, independent of the phosphorylation status at GSK3-targeted SP/TP sites. Such mislocalized tau proteins, especially the less phosphorylated species, were stabilized by phosphorylation at Ser262/356 via PAR-1/MARK. Levels of Ser262 phosphorylation were increased by Aβ42, and blocking this stabilization of tau suppressed Aβ42-mediated augmentation of tau toxicity and an increase in the levels of tau phosphorylation at the SP/TP site Thr231, suggesting that this process may be involved in AD pathogenesis. In contrast to PAR-1/MARK, blocking tau phosphorylation at SP/TP sites by knockdown of Sgg/GSK3 did not reduce tau levels, suppress tau mislocalization to the cytosol, or diminish Aβ-mediated augmentation of tau toxicity. These results suggest that stabilization of microtubule-unbound tau by phosphorylation at Ser262/356 via the PAR-1/MARK may act in the initial steps of tau mismetabolism in AD pathogenesis, and that such tau species may represent a potential therapeutic target for AD.

  4. HDAC Inhibitor-Mediated Beta-Cell Protection Against Cytokine-Induced Toxicity Is STAT1 Tyr701 Phosphorylation Independent

    DEFF Research Database (Denmark)

    Dahllöf, Mattias Salling; Christensen, Dan P; Harving, Mette;

    2015-01-01

    regulates IFN-γ signaling at the level of STAT1 Tyr701 phosphorylation. Using different small molecule HDAC inhibitors with varying class selectivity, INS-1E wild type and stable HDAC1-3 knockdown pancreatic INS-1 cell lines, we show that IFN-γ-induced Cxcl9 and iNos expression as well as Cxcl9 and GAS...

  5. Hypoxia induces phosphorylation of the cyclic AMP response element-binding protein by a novel signaling mechanism.

    Science.gov (United States)

    Beitner-Johnson, D; Millhorn, D E

    1998-07-31

    To investigate signaling mechanisms by which hypoxia regulates gene expression, we examined the effect of hypoxia on the cyclic AMP response element-binding protein (CREB) in PC12 cells. Exposure to physiological levels of hypoxia (5% O2, approximately 50 mm Hg) rapidly induced a persistent phosphorylation of CREB on Ser133, an event that is required for CREB-mediated transcriptional activation. Hypoxia-induced phosphorylation of CREB was more robust than that induced by any other stimulus tested, including forskolin, depolarization, and osmotic stress. Furthermore, this effect was not mediated by any of the previously known signaling pathways that lead to phosphorylation of CREB, including protein kinase A, calcium/calmodulin-dependent protein kinase, protein kinase C, ribosomal S6 kinase-2, and mitogen-activated protein kinase-activated protein kinase-2. Hypoxic activation of a CRE-containing reporter (derived from the 5'-flanking region of the tyrosine hydroxylase gene) was attenuated markedly by mutation of the CRE. Thus, a physiological reduction in O2 levels induces a functional phosphorylation of CREB at Ser133 via a novel signaling pathway. PMID:9677418

  6. PROLACTIN-INDUCED TYROSINE PHOSPHORYLATION, ACTIVATION AND RECEPTOR ASSOCIATION OF FOCAL ADHESION KINASE (FAK) IN MAMMARY EPITHELIAL CELLS

    Science.gov (United States)

    Prolactin-Induced Tyrosine Phosphorylation, Activation and ReceptorAssociation of Focal Adhesion Kinase (FAK) in Mammary Epithelial Cells. Suzanne E. Fenton1 and Lewis G. Sheffield2. 1U.S. Environmental ProtectionAgency, MD-72, Research Triangle Park, NC 27711, and

  7. Modulation of curcumin-induced Akt phosphorylation and apoptosis by PI3K inhibitor in MCF-7 cells

    International Nuclear Information System (INIS)

    Curcumin has been shown to induce apoptosis in various malignant cancer cell lines. One mechanism of curcumin-induced apoptosis is through the PI3K/Akt signaling pathway. Akt, also known as protein kinase B (PKB), is a member of the family of phosphatidylinositol 3-OH-kinase regulated Ser/Thr kinases. The active Akt regulates cell survival and proliferation; and inhibits apoptosis. In this study we found that curcumin induces apoptotic cell death in MCF-7 cells, as assessed by MTT assay, DNA ladder formation, PARP cleavage, p53 and Bax induction. At apoptotic inducing concentration, curcumin induces a dramatic Akt phosphorylation, accompanied by an increased phosphorylation of glycogen synthase kinase 3β (GSK3β), which has been considered to be a pro-growth signaling molecule. Combining curcumin with PI3K inhibitor, LY290042, synergizes the apoptotic effect of curcumin. The inhibitor LY290042 was capable of attenuating curcumin-induced Akt phosphorylation and activation of GSK3β. All together, our data suggest that blocking the PI3K/Akt survival pathway sensitizes the curcumin-induced apoptosis in MCF-7 cells.

  8. Modulation of curcumin-induced Akt phosphorylation and apoptosis by PI3K inhibitor in MCF-7 cells

    Energy Technology Data Exchange (ETDEWEB)

    Kizhakkayil, Jaleel; Thayyullathil, Faisal; Chathoth, Shahanas; Hago, Abdulkader; Patel, Mahendra [Cell Signaling Laboratory, Department of Biochemistry, Faculty of Medicine and Health Sciences, UAE University, P.O. Box 17666, Al Ain (United Arab Emirates); Galadari, Sehamuddin, E-mail: sehamuddin@uaeu.ac.ae [Cell Signaling Laboratory, Department of Biochemistry, Faculty of Medicine and Health Sciences, UAE University, P.O. Box 17666, Al Ain (United Arab Emirates)

    2010-04-09

    Curcumin has been shown to induce apoptosis in various malignant cancer cell lines. One mechanism of curcumin-induced apoptosis is through the PI3K/Akt signaling pathway. Akt, also known as protein kinase B (PKB), is a member of the family of phosphatidylinositol 3-OH-kinase regulated Ser/Thr kinases. The active Akt regulates cell survival and proliferation; and inhibits apoptosis. In this study we found that curcumin induces apoptotic cell death in MCF-7 cells, as assessed by MTT assay, DNA ladder formation, PARP cleavage, p53 and Bax induction. At apoptotic inducing concentration, curcumin induces a dramatic Akt phosphorylation, accompanied by an increased phosphorylation of glycogen synthase kinase 3{beta} (GSK3{beta}), which has been considered to be a pro-growth signaling molecule. Combining curcumin with PI3K inhibitor, LY290042, synergizes the apoptotic effect of curcumin. The inhibitor LY290042 was capable of attenuating curcumin-induced Akt phosphorylation and activation of GSK3{beta}. All together, our data suggest that blocking the PI3K/Akt survival pathway sensitizes the curcumin-induced apoptosis in MCF-7 cells.

  9. Dynamics of a shear-induced aggregation process by a combined Monte Carlo-Stokesian Dynamics approach

    OpenAIRE

    Frungieri, Graziano; Vanni, Marco

    2016-01-01

    In the present work we investigated the collision efficiency of colloidal aggregates suspended in a shear flow. A Discrete Element Method (DEM), built in the framework of Stokesian Dynamics, was developed to model hydrodynamic and colloidal interactions acting on each primary particle composing the aggregates. Aggregates with complex geometries were generated by means of a combined DEM-Monte Carlo algorithm able to reproduce a shear-induced aggregation process occurring in a dilute colloidal ...

  10. Platelet-derived growth factor-induced Akt phosphorylation requires mTOR/Rictor and phospholipase C-γ1, whereas S6 phosphorylation depends on mTOR/Raptor and phospholipase D

    Directory of Open Access Journals (Sweden)

    Razmara Masoud

    2013-01-01

    Full Text Available Abstract Mammalian target of rapamycin (mTOR can be found in two multi-protein complexes, i.e. mTORC1 (containing Raptor and mTORC2 (containing Rictor. Here, we investigated the mechanisms by which mTORC1 and mTORC2 are activated and their downstream targets in response to platelet-derived growth factor (PDGF-BB treatment. Inhibition of phosphatidylinositol 3-kinase (PI3K inhibited PDGF-BB activation of both mTORC1 and mTORC2. We found that in Rictor-null mouse embryonic fibroblasts, or after prolonged rapamycin treatment of NIH3T3 cells, PDGF-BB was not able to promote phosphorylation of Ser473 in the serine/threonine kinase Akt, whereas Thr308 phosphorylation was less affected, suggesting that Ser473 in Akt is phosphorylated in an mTORC2-dependent manner. This reduction in Akt phosphorylation did not influence the phosphorylation of the S6 protein, a well established protein downstream of mTORC1. Consistently, triciribine, an inhibitor of the Akt pathway, suppressed PDGF-BB-induced Akt phosphorylation without having any effect on S6 phosphorylation. Thus, mTORC2 does not appear to be upstream of mTORC1. We could also demonstrate that in Rictor-null cells the phosphorylation of phospholipase Cγ1 (PLCγ1 and protein kinase C (PKC was impaired, and the PKCα protein levels strongly reduced. Furthermore, interfering with the PLCγ/Ca2+/PKC pathway inhibited PDGF-BB-induced Akt phosphorylation. In addition, PDGF-BB-induced activation of mTORC1, as measured by phosphorylation of the downstream S6 protein, was dependent on phospholipase D (PLD. It has been shown that Erk1/2 MAP-kinase directly phosphorylates and activates mTORC1; in partial agreement with this finding, we found that a Mek1/2 inhibitor delayed S6 phosphorylation in response to PDGF-BB, but it did not block it. Thus, whereas both mTORC1 and mTORC2 are activated in a PI3K-dependent manner, different additional signaling pathways are needed. mTORC1 is activated in a PLD-dependent manner

  11. Effect of polymer aggregation on the open circuit voltage in organic photovoltaic cells: aggregation-induced conjugated polymer gel and its application for preventing open circuit voltage drop.

    Science.gov (United States)

    Kim, Bong-Gi; Jeong, Eun Jeong; Park, Hui Joon; Bilby, David; Guo, L Jay; Kim, Jinsang

    2011-03-01

    To investigate the structure-dependent aggregation behavior of conjugated polymers and the effect of aggregation on the device performance of conjugated polymer photovoltaic cells, new conjugated polymers (PVTT and CN-PVTT) having the same regioregularity but different intermolecular packing were prepared and characterized by means of UV-vis spectroscopy and atomic force microscopy (AFM). Photovoltaic devices were prepared with these polymers under different polymer-aggregate conditions. Polymer aggregation induced by thermal annealing increases the short circuit current but provides no advantage in the overall power conversion efficiency because of a decrease in the open circuit voltage. The device fabricated from a pre-aggregated polymer suspension, acquired from ultrasonic agitation of a conjugated polymer gel, showed enhanced performance because of better phase separation and reduced recombination between polymer/PCBM.

  12. Nitric Oxide and Brassinosteroids Mediated Fungal Endophyte-Induced Volatile Oil Production Through Protein Phosphorylation Pathways in Atractylodes lancea Plantlets

    Institute of Scientific and Technical Information of China (English)

    Cheng-Gang Ren; Chuan-Chao Dai

    2013-01-01

    Fungal endophytes have been isolated from almost every plant, infecting their hosts without causing visible disease symptoms, and yet have still proved to be involved in plant secondary metabolites accumulation. To decipher the possible physiological mechanisms of the endophytic fungus-host interaction, the role of protein phosphorylation and the relationship between endophytic fungus-induced kinase activity and nitric oxide (NO) and brassinolide (BL) in endophyte-enhanced volatile oil accumulation in Atractylodes lancea plantlets were investigated using pharmacological and biochemical approaches. Inoculation with the endophytic fungus Gilmaniella sp. AL12 enhanced the activities of total protein phosphorylation, Ca2þ-dependent protein kinase, and volatile oil accumulation in A. lancea plantlets. The upregulation of protein kinase activity could be blocked by the BL inhibitor brassinazole. Furthermore, pretreatments with the NO-specific scavenger cPTIO significantly reduced the increased activities of protein kinases in A. lancea plantlets inoculated with endophytic fungus. Pretreatments with different protein kinase inhibitors also reduced fungus-induced NO production and volatile oil accumulation, but had barely no effect on the BL level. These data suggest that protein phosphorylation is required for endophyte-induced volatile oil production in A. lancea plantlets, and that crosstalk between protein phosphorylation and the NO pathway may occur and act as a downstream signaling event of the BL pathway.

  13. Molecular dynamics simulation of phosphorylation-induced conformational transitions in the mycobacterium tuberculosis response regulator PrrA

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Guo [Los Alamos National Laboratory; Mcmahon, Benjamin H [Los Alamos National Laboratory; Tung, Chang - Shung [Los Alamos National Laboratory

    2008-01-01

    Phosphorylation-activated modulation of response regulators (RR) is predominantly used by bacteria as a strategy in regulating their two-component signaling (TCS) systems, the underlying molecular mechanisms are however far from fully understood. In this work we have conducted a molecular dynamics (MD) simulation of the phosphorylation-induced conformational transitions of RRs with the Mycobacterium Tuberculosis PrrA as a particular example. Starting from the full-length inactive structure of PrrA we introduced a local disturbance by phosphorylating the conserved aspartic acid residue, Asp-58, in the regulatory domain. A Go-model-type algorithm packaged with AMBER force fields was then applied to simulate the dynamics upon phosphorylation. The MD simulation shows that the phosphorylation of Asp-58 facilitates PrrA, whose inactive state has a compact conformation with a closed interdomain interface, to open up with its interdomain separation being increased by an average of about 1.5 {angstrom} for a simulation of 20 ns. The trans-activation loop, which is completely buried within the interdomain interface in the inactive PrrA, is found to become more exposed with the phosphorylated structure as well. These results provide more structural details of how the phosphorylation of a local aspartate activates PrrA to undergo a global conformational rearrangement toward its extended active state. This work also indicates that MD simulations can serve as a fast tool to unravel the regulation mechanisms of all RRs, which is especially valuable when the structures of full-length active RRs are currently unavailable.

  14. Spontaneous motion of the oil-water interface induced by the generation of surfactant aggregates. In-situ measurement of aggregate formation with SAXS and SANS

    International Nuclear Information System (INIS)

    Motion of an oil-water interface induced by the formation of surfactant aggregates is reported. An oil-water system that generates surfactant aggregates at the oil-water interface is constructed with biomimetic motivation. We applied a system composed of ionic surfactant, co-surfactant and water, which is known to generate lamellar structure with low surfactant ratio (∼ a few wt.%). In our system, a cationic surfactant is dissolved in water, whereas co-surfactant is dissolved in hydrocarbon solvent. Setting the oil and the water in contact with each other, the aggregates were formed at the oil-water interface. Accompanied with the generation of surfactant aggregates, the oil-water interface showed extension and retraction of circular extrude within a quasi 2-dimensional cell. Furthermore, the aggregates formed pillar-like structure due to the interfacial motion. By in situ measurement with small angel X-ray scattering and small angle neutron scattering, we revealed the aggregates have the lamellar structure where their interlayer distance was 28 nm to 30 nm. The internal structure of the aggregate pillar is also revealed. SANS measurement further revealed that the aggregates do not contain large amount of the organic solvent. (author)

  15. Fractal aggregates induced by liposome-liposome interaction in the presence of Ca2+.

    Science.gov (United States)

    Sabín, J; Prieto, G; Ruso, J M; Sarmiento, F

    2007-10-01

    We present a study of the fractal dimension of clusters of large unilamellar vesicles (LUVs) formed by egg yolk phosphatidylcholine (EYPC), dimyristoylphosphocholine (DMPC) and dipalmitoylphosphocholine (DPPC) induced by Ca2+ . Fractal dimensions were calculated by application of two methods, measuring the angular dependency of the light scattered by the clusters and following the evolution of the cluster size. In all cases, the fractal dimensions fell in the range from 2.1 to 1.8, corresponding to two regimes: diffusion-limited cluster aggregation (DLCA) and reaction-limited cluster aggregation (RLCA). Whereas DMPC clusters showed a typical transition from the RLCA to the DLCA aggregation, EYPC exhibited an unusual behaviour, since the aggregation was limited for a higher concentration than the critical aggregation concentration. The behaviour of DPPC was intermediate, with a transition from the RLCA to the DLCA regimes with cluster sizes depending on Ca2+ concentration. Studies on the reversibility of the aggregates show that EYPC and DPPC clusters can be re-dispersed by dilution with water. DMPC does not present reversibility. Reversibility is evidence of the existence of secondary minima in the DLVO potential between two liposomes. To predict these secondary minima, a correction of the DLVO model was necessary taking into account a repulsive force of hydration.

  16. Enzyme-induced aggregation of whey proteins with Bacillus licheniformis protease

    NARCIS (Netherlands)

    Creusot, N.P.

    2006-01-01

    Whey proteins are commonly used as ingredient in food. In relation with the gelation properties of whey proteins, this thesis deals with understanding the mechanism of peptide-induced aggregation of whey protein hydrolysates made with Bacillus licheniformis protease (BLP). The results show that BLP

  17. Aggregation-induced emission molecules in layered matrices for two-color luminescence films.

    Science.gov (United States)

    Guan, Weijiang; Lu, Jun; Zhou, Wenjuan; Lu, Chao

    2014-10-14

    We fabricated two-color luminescence ultrathin films (UTFs) composed of the layered double hydroxide host-aggregation-induced emission guests by LBL assembly. The fabricated UTFs were simple, tunable, controllable and highly luminescent. Moreover, reversible thermochromic luminescence further exhibited their potential in practical applications. PMID:25154856

  18. Vasopressin induces phosphorylation of the thiazide-sensitive sodium chloride cotransporter in the distal convoluted tubule

    DEFF Research Database (Denmark)

    Pedersen, Nis Borbye; Hofmeister, Marlene Vind; Rosenbaek, Lena L;

    2010-01-01

    The thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC) is important for renal electrolyte balance and its phosphorylation causes an increase in its transport activity and cellular localization. Here, we generated phospho-specific antibodies against two conserved N-terminal phosphorylation sites...

  19. Halogenated salicylaldehyde azines: The heavy atom effect on aggregation-induced emission enhancement properties

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Xiao-tong, E-mail: chenxiaotong@tsinghua.edu.cn [Institute of Nuclear and New Energy Technology, Tsinghua University, Beijing 100084 (China); Tong, Ai-jun [Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing 100084 (China)

    2014-01-15

    This study investigates the heavy-atom effect (HAE) on aggregation-induced emission enhancement (AIEE) properties of salicylaldehyde azines. For this purpose, a series of halogenated salicylaldehyde azine derivatives, namely, chloro-salicylaldehyde azine (1), bromo-salicylaldehyde azine (2) and iodo-salicylaldehyde azine (3) are synthesized. 1 and 2 display typical AIEE characteristics of salicylaldehyde azine compounds; whereas for the iodo-substituent in 3, is found to be effective “external” heavy atom quenchers to salicylaldehyde azine fluorescence in aggregated state. Based on its weak fluorescence in aggregated state and relative strong fluorescence in dispersed state, 3 can also be applied as a turn-on fluorescence probe for egg albumin detection attributed to hydrophobic interaction. -- Highlights: • This study investigates the heavy-atom effect (HAE) on aggregation-induced emission enhancement (AIEE) properties of salicylaldehyde azines. • Chloro- and bromo-salicylaldehyde display typical AIEE properties of salicylaldehyde azine, whereas the iodo-substitute quenches AIEE in aggregated state. • Iodo-salicylaldehyde can be applied as a turn-on fluorescence probe for egg albumin detection attributed to hydrophobic interaction.

  20. Synthesis, crystal Structures and aggregation-induced emission enhancement of aryl-substituted cyclopentadiene derivatives

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Xiangdong [State Key Laboratory of Fine Chemicals and School of Chemical Engineering, Dalian University of Technology, 2 Linggong Road, Dalian 116012 (China); State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000 (China); Ye, Junwei, E-mail: junweiye@dlut.edu.cn [State Key Laboratory of Fine Chemicals and School of Chemical Engineering, Dalian University of Technology, 2 Linggong Road, Dalian 116012 (China); Xu, Lifeng; Yang, Lijian; Deng, Dai [State Key Laboratory of Fine Chemicals and School of Chemical Engineering, Dalian University of Technology, 2 Linggong Road, Dalian 116012 (China); Ning, Guiling, E-mail: ninggl@dlut.edu.cn [State Key Laboratory of Fine Chemicals and School of Chemical Engineering, Dalian University of Technology, 2 Linggong Road, Dalian 116012 (China)

    2013-07-15

    A series of cyclopentadiene derivates, namely, 1,2,4-triphenylcyclopenta-1,3-diene (1), 1,2-diphenyl-4-(p-methoxyphenyl)cyclopenta-1,3-diene (2) and 1,2-di(p-methoxyphenyl)-4-phenylcyclopenta-1,3-diene (3), were synthesized and their photoluminescence properties in solutions and aggregation state were investigated. Different photoluminescence properties of 1–3 were tuned by changing types and replace position of substituent groups. These cyclopentadiene derivates display solvent-dependent fluorescence emissions and typical aggregation-induced emission enhancement (AIEE) characteristics. The crystal structure analysis reveals that intermolecular C–H…π interactions and X-aggregation molecule stacking have a dramatic effect on the photoluminescent properties of 1–3. Additionally, the DFT calculations of these compounds were investigated. -- Graphical abstract: A series of aryl-substituted cyclopentadiene derivatives with AIEE properties were synthesized and their crystal structures and photoluminescence properties in solution and aggregation state were studied. Highlights: ► A series of cyclopentadiene derivates were synthesized via aldol condensation followed cyclizing and dehydrating reaction. ► The effect of solvents on fluorescence properties of these compounds were investigated in seven organic solvents. ► These compounds show typical aggregation-induced emission enhancement properties.

  1. Serum- and glucocorticoid-induced protein kinase 1 (SGK1) increases the cystic fibrosis transmembrane conductance regulator (CFTR) in airway epithelial cells by phosphorylating Shank2E protein.

    Science.gov (United States)

    Koeppen, Katja; Coutermarsh, Bonita A; Madden, Dean R; Stanton, Bruce A

    2014-06-13

    The glucocorticoid dexamethasone increases cystic fibrosis transmembrane conductance regulator (CFTR) abundance in human airway epithelial cells by a mechanism that requires serum- and glucocorticoid-induced protein kinase 1 (SGK1) activity. The goal of this study was to determine whether SGK1 increases CFTR abundance by phosphorylating Shank2E, a PDZ domain protein that contains two SGK1 phosphorylation consensus sites. We found that SGK1 phosphorylates Shank2E as well as a peptide containing the first SGK1 consensus motif of Shank2E. The dexamethasone-induced increase in CFTR abundance was diminished by overexpression of a dominant-negative Shank2E in which the SGK1 phosphorylation sites had been mutated. siRNA-mediated reduction of Shank2E also reduced the dexamethasone-induced increase in CFTR abundance. Taken together, these data demonstrate that the glucocorticoid-induced increase in CFTR abundance requires phosphorylation of Shank2E at an SGK1 consensus site.

  2. Mitochondrial Complex I Inhibitors and Forced Oxidative Phosphorylation Synergize in Inducing Cancer Cell Death

    Directory of Open Access Journals (Sweden)

    Roberta Palorini

    2013-01-01

    Full Text Available Cancer cells generally rely mostly on glycolysis rather than oxidative phosphorylation (OXPHOS for ATP production. In fact, they are particularly sensitive to glycolysis inhibition and glucose depletion. On the other hand mitochondrial dysfunctions, involved in the onset of the Warburg effect, are sometimes also associated with the resistance to apoptosis that characterizes cancer cells. Therefore, combined treatments targeting both glycolysis and mitochondria function, exploiting peculiar tumor features, might be lethal for cancer cells. In this study, we show that glucose deprivation and mitochondrial Complex I inhibitors synergize in inducing cancer cell death. In particular, our results reveal that low doses of Complex I inhibitors, ineffective on immortalized cells and in high glucose growth, become specifically cytotoxic on cancer cells deprived of glucose. Importantly, the cytotoxic effect of the inhibitors on cancer cells is strongly enhanced by forskolin, a PKA pathway activator, that we have previously shown to stimulate OXPHOS. Taken together, we demonstrate that induction in cancer cells of a switch from a glycolytic to a more respirative metabolism, obtained by glucose depletion or mitochondrial activity stimulation, strongly increases their sensitivity to low doses of mitochondrial Complex I inhibitors. Our findings might be a valuable approach to eradicate cancer cells.

  3. VE-cadherin cleavage by ovarian cancer microparticles induces β-catenin phosphorylation in endothelial cells.

    Science.gov (United States)

    Al Thawadi, Hamda; Abu-Kaoud, Nadine; Al Farsi, Haleema; Hoarau-Véchot, Jessica; Rafii, Shahin; Rafii, Arash; Pasquier, Jennifer

    2016-02-01

    Microparticles (MPs) are increasingly recognized as important mediators of cell-cell communication in tumour growth and metastasis by facilitating angiogenesis-related processes. While the effects of the MPs on recipient cells are usually well described in the literature, the leading process remains unclear. Here we isolated MPs from ovarian cancer cells and investigated their effect on endothelial cells. First, we demonstrated that ovarian cancer MPs trigger β-catenin activation in endothelial cells, inducing the upregulation of Wnt/β-catenin target genes and an increase of angiogenic properties. We showed that this MPs mediated activation of β-catenin in ECs was Wnt/Frizzled independent; but dependent on VE-cadherin localization disruption, αVβ3 integrin activation and MMP activity. Finally, we revealed that Rac1 and AKT were responsible for β-catenin phosphorylation and translocation to the nucleus. Overall, our results indicate that MPs released from cancer cells could play a major role in neo-angiogenesis through activation of beta catenin pathway in endothelial cells. PMID:26700621

  4. The mitosis-regulating and protein-protein interaction activities of astrin are controlled by aurora-A-induced phosphorylation.

    Science.gov (United States)

    Chiu, Shao-Chih; Chen, Jo-Mei Maureen; Wei, Tong-You Wade; Cheng, Tai-Shan; Wang, Ya-Hui Candice; Ku, Chia-Feng; Lian, Chiao-Hsuan; Liu, Chun-Chih Jared; Kuo, Yi-Chun; Yu, Chang-Tze Ricky

    2014-09-01

    Cells display dramatic morphological changes in mitosis, where numerous factors form regulatory networks to orchestrate the complicated process, resulting in extreme fidelity of the segregation of duplicated chromosomes into two daughter cells. Astrin regulates several aspects of mitosis, such as maintaining the cohesion of sister chromatids by inactivating Separase and stabilizing spindle, aligning and segregating chromosomes, and silencing spindle assembly checkpoint by interacting with Src kinase-associated phosphoprotein (SKAP) and cytoplasmic linker-associated protein-1α (CLASP-1α). To understand how Astrin is regulated in mitosis, we report here that Astrin acts as a mitotic phosphoprotein, and Aurora-A phosphorylates Astrin at Ser(115). The phosphorylation-deficient mutant Astrin S115A abnormally activates spindle assembly checkpoint and delays mitosis progression, decreases spindle stability, and induces chromosome misalignment. Mechanistic analyses reveal that Astrin phosphorylation mimicking mutant S115D, instead of S115A, binds and induces ubiquitination and degradation of securin, which sequentially activates Separase, an enzyme required for the separation of sister chromatids. Moreover, S115A fails to bind mitosis regulators, including SKAP and CLASP-1α, which results in the mitotic defects observed in Astrin S115A-transfected cells. In conclusion, Aurora-A phosphorylates Astrin and guides the binding of Astrin to its cellular partners, which ensures proper progression of mitosis.

  5. Disruption of dopamine D1 receptor phosphorylation at serine 421 attenuates cocaine-induced behaviors in mice.

    Science.gov (United States)

    Zhang, Ying; Wang, Ning; Su, Ping; Lu, Jie; Wang, Yun

    2014-12-01

    Dopamine D1 receptors (D1Rs) play a key role in cocaine addiction, and multiple protein kinases such as GRKs, PKA, and PKC are involved in their phosphorylation. Recently, we reported that protein kinase D1 phosphorylates the D1R at S421 and promotes its membrane localization. Moreover, this phosphorylation of S421 is required for cocaineinduced behaviors in rats. In the present study, we generated transgenic mice over-expressing S421A-D1R in the forebrain. These transgenic mice showed reduced phospho-D1R (S421) and its membrane localization, and reduced downstream ERK1/2 activation in the striatum. Importantly, acute and chronic cocaine-induced locomotor hyperactivity and conditioned place preference were significantly attenuated in these mice. These findings provide in vivo evidence for the critical role of S421 phosphorylation of the D1R in its membrane localization and in cocaine-induced behaviors. Thus, S421 on the D1R represents a potential pharmacotherapeutic target for cocaine addiction and other drug-abuse disorders. PMID:25304015

  6. Theoretical study on electromagnetically induced transparency in molecular aggregate models using quantum Liouville equation method

    Energy Technology Data Exchange (ETDEWEB)

    Minami, Takuya; Nakano, Masayoshi [Department of Materials Engineering Science, Graduate School of Engineering Science, Osaka University, Toyonaka, Osaka 560-8531 (Japan)

    2015-01-22

    Electromagnetically induced transparency (EIT), which is known as an efficient control method of optical absorption property, is investigated using the polarizability spectra and population dynamics obtained by solving the quantum Liouville equation. In order to clarify the intermolecular interaction effect on EIT, we examine several molecular aggregate models composed of three-state monomers with the dipole-dipole coupling. On the basis of the present results, we discuss the applicability of EIT in molecular aggregate systems to a new type of optical switch.

  7. Ultrasensitive detection of target analyte-induced aggregation of gold nanoparticles using laser-induced nanoparticle Rayleigh scattering.

    Science.gov (United States)

    Lin, Jia-Hui; Tseng, Wei-Lung

    2015-01-01

    Detection of salt- and analyte-induced aggregation of gold nanoparticles (AuNPs) mostly relies on costly and bulky analytical instruments. To response this drawback, a portable, miniaturized, sensitive, and cost-effective detection technique is urgently required for rapid field detection and monitoring of target analyte via the use of AuNP-based sensor. This study combined a miniaturized spectrometer with a 532-nm laser to develop a laser-induced Rayleigh scattering technique, allowing the sensitive and selective detection of Rayleigh scattering from the aggregated AuNPs. Three AuNP-based sensing systems, including salt-, thiol- and metal ion-induced aggregation of the AuNPs, were performed to examine the sensitivity of laser-induced Rayleigh scattering technique. Salt-, thiol-, and metal ion-promoted NP aggregation were exemplified by the use of aptamer-adsorbed, fluorosurfactant-stabilized, and gallic acid-capped AuNPs for probing K(+), S-adenosylhomocysteine hydrolase-induced hydrolysis of S-adenosylhomocysteine, and Pb(2+), in sequence. Compared to the reported methods for monitoring the aggregated AuNPs, the proposed system provided distinct advantages of sensitivity. Laser-induced Rayleigh scattering technique was improved to be convenient, cheap, and portable by replacing a diode laser and a miniaturized spectrometer with a laser pointer and a smart-phone. Using this smart-phone-based detection platform, we can determine whether or not the Pb(2+) concentration exceed the maximum allowable level of Pb(2+) in drinking water. PMID:25476277

  8. PPARγ ligands decrease hydrostatic pressure-induced platelet aggregation and proinflammatory activity.

    Directory of Open Access Journals (Sweden)

    Fang Rao

    Full Text Available Hypertension is known to be associated with platelet overactivity, but the direct effects of hydrostatic pressure on platelet function remain unclear. The present study sought to investigate whether elevated hydrostatic pressure is responsible for platelet activation and to address the potential role of peroxisome proliferator-activated receptor-γ (PPARγ. We observed that hypertensive patients had significantly higher platelet volume and rate of ADP-induced platelets aggregation compared to the controls. In vitro, Primary human platelets were cultured under standard (0 mmHg or increased (120, 180, 240 mmHg hydrostatic pressure for 18 h. Exposure to elevated pressure was associated with morphological changes in platelets. Platelet aggregation and PAC-1 (the active confirmation of GPIIb/IIIa binding were increased, CD40L was translocated from cytoplasm to the surface of platelet and soluble CD40L (sCD40L was released into the medium in response to elevated hydrostatic pressure (180 and 240 mmHg. The PPARγ activity was up-regulated as the pressure was increased from 120 mmHg to 180 mmHg. Pressure-induced platelet aggregation, PAC-1 binding, and translocation and release of CD40L were all attenuated by the PPARγ agonist Thiazolidinediones (TZDs. These results demonstrate that platelet activation and aggregation are increased by exposure to elevated pressure and that PPARγ may modulate platelet activation induced by high hydrostatic pressure.

  9. Ginkgo Biloba Extract Ameliorates Oxidative Phosphorylation Performance and Rescues Aβ-Induced Failure

    Science.gov (United States)

    Rhein, Virginie; Giese, Maria; Baysang, Ginette; Meier, Fides; Rao, Stefania; Schulz, Kathrin L.; Hamburger, Matthias; Eckert, Anne

    2010-01-01

    Background Energy deficiency and mitochondrial failure have been recognized as a prominent, early event in Alzheimer's disease (AD). Recently, we demonstrated that chronic exposure to amyloid-beta (Aβ) in human neuroblastoma cells over-expressing human wild-type amyloid precursor protein (APP) resulted in (i) activity changes of complexes III and IV of the oxidative phosphorylation system (OXPHOS) and in (ii) a drop of ATP levels which may finally instigate loss of synapses and neuronal cell death in AD. Therefore, the aim of the present study was to investigate whether standardized Ginkgo biloba extract LI 1370 (GBE) is able to rescue Aβ-induced defects in energy metabolism. Methodology/Principal Findings We used a high-resolution respiratory protocol to evaluate OXPHOS respiratory capacity under physiological condition in control (stably transfected with the empty vector) and APP cells after treatment with GBE. In addition, oxygen consumption of isolated mitochondria, activities of mitochondrial respiratory enzymes, ATP and reactive oxygen species (ROS) levels as well as mitochondrial membrane mass and mitochondrial DNA content were determined. We observed a general antioxidant effect of GBE leading to an increase of the coupling state of mitochondria as well as energy homeostasis and a reduction of ROS levels in control cells and in APP cells. GBE effect on OXPHOS was even preserved in mitochondria after isolation from treated cells. Moreover, these functional data were paralleled by an up-regulation of mitochondrial DNA. Improvement of the OXPHOS efficiency was stronger in APP cells than in control cells. In APP cells, the GBE-induced amelioration of oxygen consumption most likely arose from the modulation and respective normalization of the Aβ-induced disturbance in the activity of mitochondrial complexes III and IV restoring impaired ATP levels possibly through decreasing Aβ and oxidative stress level. Conclusions/Significance Although the underlying

  10. Ginkgo biloba extract ameliorates oxidative phosphorylation performance and rescues abeta-induced failure.

    Directory of Open Access Journals (Sweden)

    Virginie Rhein

    Full Text Available BACKGROUND: Energy deficiency and mitochondrial failure have been recognized as a prominent, early event in Alzheimer's disease (AD. Recently, we demonstrated that chronic exposure to amyloid-beta (Abeta in human neuroblastoma cells over-expressing human wild-type amyloid precursor protein (APP resulted in (i activity changes of complexes III and IV of the oxidative phosphorylation system (OXPHOS and in (ii a drop of ATP levels which may finally instigate loss of synapses and neuronal cell death in AD. Therefore, the aim of the present study was to investigate whether standardized Ginkgo biloba extract LI 1370 (GBE is able to rescue Abeta-induced defects in energy metabolism. METHODOLOGY/PRINCIPAL FINDINGS: We used a high-resolution respiratory protocol to evaluate OXPHOS respiratory capacity under physiological condition in control (stably transfected with the empty vector and APP cells after treatment with GBE. In addition, oxygen consumption of isolated mitochondria, activities of mitochondrial respiratory enzymes, ATP and reactive oxygen species (ROS levels as well as mitochondrial membrane mass and mitochondrial DNA content were determined. We observed a general antioxidant effect of GBE leading to an increase of the coupling state of mitochondria as well as energy homeostasis and a reduction of ROS levels in control cells and in APP cells. GBE effect on OXPHOS was even preserved in mitochondria after isolation from treated cells. Moreover, these functional data were paralleled by an up-regulation of mitochondrial DNA. Improvement of the OXPHOS efficiency was stronger in APP cells than in control cells. In APP cells, the GBE-induced amelioration of oxygen consumption most likely arose from the modulation and respective normalization of the Abeta-induced disturbance in the activity of mitochondrial complexes III and IV restoring impaired ATP levels possibly through decreasing Abeta and oxidative stress level. CONCLUSIONS

  11. TCR-Induced Akt Serine 473 Phosphorylation Is Regulated by Protein Kinase C-Alpha

    OpenAIRE

    Yang, Lifen; Qiao, Guilin; Ying, Haiyan; Zhang, Jian; Yin, Fei

    2010-01-01

    Akt signaling plays a central role in T cell functions, such as proliferation, apoptosis, and regulatory T cell development. Phosphorylation at Ser473 in the hydrophobic motif, along with Thr308 in its activation loop, is considered necessary for Akt function. It is widely accepted that Phosphoinositide-dependent kinase 1 (PDK-1) phosphorylates Akt at Thr308, but the kinase(s) responsible for phosphorylating Akt at Ser473 (PDK-2) remains elusive. The existence of PDK-2 is considered to be spe...

  12. Protein Kinase D1-Dependent Phosphorylation of Dopamine D1 Receptor Regulates Cocaine-Induced Behavioral Responses

    Science.gov (United States)

    Wang, Ning; Su, Ping; Zhang, Ying; Lu, Jie; Xing, Baoming; Kang, Kai; Li, Wenqi; Wang, Yun

    2014-01-01

    The dopamine (DA) D1 receptor (D1R) is critically involved in reward and drug addiction. Phosphorylation-mediated desensitization or internalization of D1R has been extensively investigated. However, the potential for upregulation of D1R function through phosphorylation remains to be determined. Here we report that acute cocaine exposure induces protein kinase D1 (PKD1) activation in the rat striatum, and knockdown of PKD1 in the rat dorsal striatum attenuates cocaine-induced locomotor hyperactivity. Moreover, PKD1-mediated phosphorylation of serine 421 (S421) of D1R promotes surface localization of D1R and enhances downstream extracellular signal-regulated kinase signaling in D1R-transfected HEK 293 cells. Importantly, injection of the peptide Tat-S421, an engineered Tat fusion-peptide targeting S421 (Tat-S421), into the rat dorsal striatum inhibits cocaine-induced locomotor hyperactivity and injection of Tat-S421 into the rat hippocampus or the shell of the nucleus accumbens (NAc) also inhibits cocaine-induced conditioned place preference (CPP). However, injection of Tat-S421 into the rat NAc shell does not establish CPP by itself and injection of Tat-S421 into the hippocampus does not influence spatial learning and memory. Thus, targeting S421 of D1R represents a promising strategy for the development of pharmacotherapeutic treatments for drug addiction and other disorders that result from DA imbalances. PMID:24362306

  13. Constitutive phosphorylation of aurora-a on ser51 induces its stabilization and consequent overexpression in cancer.

    Directory of Open Access Journals (Sweden)

    Shojiro Kitajima

    Full Text Available BACKGROUND: The serine/threonine kinase Aurora-A (Aur-A is a proto-oncoprotein overexpressed in a wide range of human cancers. Overexpression of Aur-A is thought to be caused by gene amplification or mRNA overexpression. However, recent evidence revealed that the discrepancies between amplification of Aur-A and overexpression rates of Aur-A mRNA were observed in breast cancer, gastric cancer, hepatocellular carcinoma, and ovarian cancer. We found that aggressive head and neck cancers exhibited overexpression and stabilization of Aur-A protein without gene amplification or mRNA overexpression. Here we tested the hypothesis that aberration of the protein destruction system induces accumulation and consequently overexpression of Aur-A in cancer. PRINCIPAL FINDINGS: Aur-A protein was ubiquitinylated by APC(Cdh1 and consequently degraded when cells exited mitosis, and phosphorylation of Aur-A on Ser51 was observed during mitosis. Phosphorylation of Aur-A on Ser51 inhibited its APC(Cdh1-mediated ubiquitylation and consequent degradation. Interestingly, constitutive phosphorylation on Ser51 was observed in head and neck cancer cells with protein overexpression and stabilization. Indeed, phosphorylation on Ser51 was observed in head and neck cancer tissues with Aur-A protein overexpression. Moreover, an Aur-A Ser51 phospho-mimetic mutant displayed stabilization of protein during cell cycle progression and enhanced ability to cell transformation. CONCLUSIONS/SIGNIFICANCE: Broadly, this study identifies a new mode of Aur-A overexpression in cancer through phosphorylation-dependent inhibition of its proteolysis in addition to gene amplification and mRNA overexpression. We suggest that the inhibition of Aur-A phosphorylation can represent a novel way to decrease Aur-A levels in cancer therapy.

  14. Observation of fine particle aggregating behavior induced by high intensity conditioning using high speed CCD

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    The aggregating behavior between bubbles and particles induced by high intensity conditioning (HIC) was studied using high speed CCD technique. Bubble size measurement was conducted, and the attachment behavior between bubbles and particles in HIC cell and flotation cell were observed. The results show that in HIC cell, high intensity conditioning creates an advantage environment for the formation of small size bubble due to hydrodynamic cavitations, and these fine bubbles have high probability of bubble-particle collision,which will enhance fine particle flotation. The bubble-particle attachment experiments indicate that in high intensity conditioning cell, a lot of fine bubbles are produced in situ on the surface of fine particles, and most of fine particles are aggregated under the bridging action of fine bubbles. The observation of bubble-particle interaction in flotation cell illustrates that aggregates created by HIC can be loaded more easily by big air bubble in flotation cell than those created by normal conditioning.

  15. Rapid Formation of Cell Aggregates and Spheroids Induced by a "Smart" Boronic Acid Copolymer.

    Science.gov (United States)

    Amaral, Adérito J R; Pasparakis, George

    2016-09-01

    Cell surface engineering has emerged as a powerful approach to forming cell aggregates/spheroids and cell-biomaterial ensembles with significant uses in tissue engineering and cell therapeutics. Herein, we demonstrate that cell membrane remodeling with a thermoresponsive boronic acid copolymer induces the rapid formation of spheroids using either cancer or cardiac cell lines under conventional cell culture conditions at minute concentrations. It is shown that the formation of well-defined spheroids is accelerated by at least 24 h compared to non-polymer-treated controls, and, more importantly, the polymer allows for fine control of the aggregation kinetics owing to its stimulus response to temperature and glucose content. On the basis of its simplicity and effectiveness to promote cellular aggregation, this platform holds promise in three-dimensional tissue/tumor modeling and tissue engineering applications. PMID:27571512

  16. Degradation and aggregation of delta sleep-inducing peptide (DSIP) and two analogs in plasma and serum

    Energy Technology Data Exchange (ETDEWEB)

    Graf, M.V.; Saegesser, B.; Schoenenberger, G.A.

    1987-07-01

    The biostability of DSIP (delta sleep-inducing peptide) and two analogs in blood was investigated in order to determine if rates of inactivation contribute to variable effects in vivo. Incubation of DSIP in human or rat blood led to release of products having retention times on a gel filtration column equivalent to Trp. Formation of products was dependent on temperature, time, and species. Incubation of /sup 125/I-N-Tyr-DSIP and /sup 125/I-N-Tyr-P-DSIP, a phosphorylated analog, revealed slower degradation and, in contrast to DSIP, produced complex formation. An excess of unlabeled material did not displace the radioactivity supporting the assumption of non-specific binding/aggregation. It was concluded that the rapid disappearance of injected DSIP in blood was due to degradation, whereas complex formation together with slower degradation resulted in longer persistence of apparently intact analogs. Whether this could explain the sometimes stronger and more consistent effects of DSIP-analogs remains to be examined.

  17. Kinetics for Cu(2+) induced Sepia pharaonis arginine kinase inactivation and aggregation.

    Science.gov (United States)

    Shi, Xiao-Yu; Zhang, Li-Li; Wu, Feng; Fu, Yang-Yong; Yin, Shang-Jun; Si, Yue-Xiu; Park, Yong-Doo

    2016-10-01

    Arginine kinase plays an important role in cellular energy metabolism and is closely related to the environmental stress response in marine invertebrates. We studied the Cu(2+)-mediated inhibition and aggregation of Sepia pharaonis arginine kinase (SPAK) and found that Cu(2+) markedly inhibited the SPAK activity along with mixed-type inhibition against the arginine substrate and noncompetitive inhibition against the ATP cofactor. Spectrofluorimetry results showed that Cu(2+) induced a tertiary structure change in SPAK, resulting in exposure of the hydrophobic surface and increased aggregation. Cu(2+)-mediated SPAK aggregation followed first-order kinetics consistent with monophasic and a biphasic processes. Addition of osmolytes, including glycine and proline, effectively blocked SPAK aggregation and restored SPAK activity. Our results demonstrated the effects of Cu(2+) on SPAK catalytic function, conformation, and aggregation, as well as the protective effects of osmolytes on SPAK folding. This study provided important insights into the role of Cu(2+) as a negative effector of the S. pharaonis metabolic enzyme AK and the possible responses of cephalopods to unfavorable environmental conditions. PMID:27318110

  18. Sorbitol crystallization-induced aggregation in frozen mAb formulations.

    Science.gov (United States)

    Piedmonte, Deirdre Murphy; Hair, Alison; Baker, Priti; Brych, Lejla; Nagapudi, Karthik; Lin, Hong; Cao, Wenjin; Hershenson, Susan; Ratnaswamy, Gayathri

    2015-02-01

    Sorbitol crystallization-induced aggregation of mAbs in the frozen state was evaluated. The effect of protein aggregation resulting from sorbitol crystallization was measured as a function of formulation variables such as protein concentration and pH. Long-term studies were performed on both IgG1 and IgG2 mAbs over the protein concentration range of 0.1-120 mg/mL. Protein aggregation was measured by size-exclusion HPLC (SE-HPLC) and further characterized by capillary-electrophoresis SDS. Sorbitol crystallization was monitored and characterized by subambient differential scanning calorimetry and X-ray diffraction. Aggregation due to sorbitol crystallization is inversely proportional to both protein concentration and formulation pH. At high protein concentrations, sorbitol crystallization was suppressed, and minimal aggregation by SE-HPLC resulted, presumably because of self-stabilization of the mAbs. The glass transition temperature (Tg ') and fragility index measurements were made to assess the influence of molecular mobility on the crystallization of sorbitol. Tg ' increased with increasing protein concentration for both mAbs. The fragility index decreased with increasing protein concentration, suggesting that it is increasingly difficult for sorbitol to crystallize at high protein concentrations.

  19. Expression and radiation-induced phosphorylation of histone H2AX in mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Yoshida, Kayo; Morita, Takashi [Osaka City Univ. (Japan). Graduate School of Medicine; Yoshida, Shu-hei; Shimoda, Chikashi [Osaka City Univ. (Japan). Graduate School of Science

    2003-03-01

    The mouse histone H2AX (H2AX) has unique C-terminal Ser residues, which are phosphorylated in response to DNA double-strand breaks (DSBs) by ionizing radiation, suggesting that it plays a role in the maintenance of genomic stability. Here, we show that the H2AX protein was detected in most cells in various tissues, and was abundant in the S phase of the cell cycle. Following X-ray irradiation, H2AX was phosphorylated ({gamma}-H2AX) in the thymus, small intestine and testis. However, H2AX in epithelial cells in the villi of the small intestine were not strongly phosphorylated, even after X-irradiation. Thus, H2AX was expressed in almost all cells. However, the cells that expressed H2AX were not always phosphorylated by X-irradiation, suggesting a different mechanism of kination in those cells. (author)

  20. A Requirement for SOCS-1 and SOCS-3 Phosphorylation in Bcr-Abl-Induced Tumorigenesis

    Directory of Open Access Journals (Sweden)

    Xiaoxue Qiu

    2012-06-01

    Full Text Available Suppressors of cytokine signaling 1 and 3 (SOCS-1 and SOCS-3 are inhibitors of the Janus tyrosine kinase (JAK/signal transducers and activators of transcription (STAT pathway and function in a negative feedback loop during cytokine signaling. Abl transformation is associated with constitutive activation of JAK/STAT-dependent signaling. However, the mechanism by which Abl oncoproteins bypass SOCS inhibitory regulation remains poorly defined. Here, we demonstrate that coexpression of Bcr-Abl with SOCS-1 or SOCS-3 results in tyrosine phosphorylation of these SOCS proteins. Interestingly, SOCS-1 is highly tyrosine phosphorylated in one of five primary chronic myelogenous leukemia samples. Bcr-Abl-dependent tyrosine phosphorylation of SOCS-1 and SOCS-3 occurs mainly on Tyr 155 and Tyr 204 residues of SOCS-1 and on Tyr 221 residue of SOCS-3. We observed that phosphorylation of these SOCS proteins was associated with their binding to Bcr-Abl. Bcr-Abl-dependent phosphorylation of SOCS-1 and SOCS-3 diminished their inhibitory effects on the activation of JAK and STAT5 and thereby enhanced JAK/STAT5 signaling. Strikingly, disrupting the tyrosine phosphorylation of SOCS-1 or SOCS-3 impaired the expression of Bcl-XL protein and sensitized K562 leukemic cells to undergo apoptosis. Moreover, selective mutation of tyrosine phosphorylation sites of SOCS-1 or SOCS-3 significantly blocked Bcr-Abl-mediated tumorigenesis in nude mice and inhibited Bcr-Abl-mediated murine bone marrow transformation. Together, these results reveal a mechanism of how Bcr-Abl may overcome SOCS-1 and SOCS-3 inhibition to constitutively activate the JAK/STAT-dependent signaling, and suggest that Bcr-Abl may critically requires tyrosine phosphorylation of SOCS-1 and SOCS-3 to mediate tumorigenesis when these SOCS proteins are present in cells.

  1. Salt-induced aggregation of gold nanoparticles for photoacoustic imaging and photothermal therapy of cancer

    Science.gov (United States)

    Sun, Mengmeng; Liu, Fei; Zhu, Yukun; Wang, Wansheng; Hu, Jin; Liu, Jing; Dai, Zhifei; Wang, Kun; Wei, Yen; Bai, Jing; Gao, Weiping

    2016-02-01

    The challenge in photothermal therapy (PTT) is to develop biocompatible photothermal transducers that can absorb and convert near-infrared (NIR) light into heat with high efficiency. Herein, we report salt-induced aggregation of gold nanoparticles (GNPs) in biological media to form highly efficient and biocompatible NIR photothermal transducers for PTT and photothermal/photoacoustic (PT/PA) imaging of cancer. The GNP depots in situ formed by salt-induced aggregation of GNPs show strong NIR absorption induced by plasmonic coupling between adjacent GNPs and very high photothermal conversion efficiency (52%), enabling photothermal destruction of tumor cells. More interestingly, GNPs in situ aggregate in tumors to form GNP depots, enabling simultaneous PT/PA imaging and PTT of the tumors. These findings may provide a simple and effective way to develop a new class of intelligent and biocompatible NIR photothermal transducers with high efficiency for PT/PA imaging and PTT.The challenge in photothermal therapy (PTT) is to develop biocompatible photothermal transducers that can absorb and convert near-infrared (NIR) light into heat with high efficiency. Herein, we report salt-induced aggregation of gold nanoparticles (GNPs) in biological media to form highly efficient and biocompatible NIR photothermal transducers for PTT and photothermal/photoacoustic (PT/PA) imaging of cancer. The GNP depots in situ formed by salt-induced aggregation of GNPs show strong NIR absorption induced by plasmonic coupling between adjacent GNPs and very high photothermal conversion efficiency (52%), enabling photothermal destruction of tumor cells. More interestingly, GNPs in situ aggregate in tumors to form GNP depots, enabling simultaneous PT/PA imaging and PTT of the tumors. These findings may provide a simple and effective way to develop a new class of intelligent and biocompatible NIR photothermal transducers with high efficiency for PT/PA imaging and PTT. Electronic supplementary

  2. Ischemia/reperfusion-induced myosin light chain 1 phosphorylation increases its degradation by matrix metalloproteinase-2

    Science.gov (United States)

    Cadete, Virgilio J. J.; Sawicka, Jolanta; Jaswal, Jagdip; Lopaschuk, Gary D.; Schulz, Richard; Szczesna-Cordary, Danuta; Sawicki, Grzegorz

    2012-01-01

    Summary Degradation of myosin light chain 1 (MLC1) by matrix metalloproteinase-2 (MMP-2) during myocardial ischemia/reperfusion (I/R) injury has been established. However, the exact mechanisms controlling this process remain unknown. I/R increases the phosphorylation of MLC1, but the consequences of this modification are not known. We hypothesized that phosphorylation of MLC1 plays an important role in its degradation by MMP-2. To examine this, isolated perfused rat hearts were subjected to 20 min global ischemia followed by 30 min of aerobic reperfusion. I/R increased phosphorylation of MLC1 (as measured by mass spectrometry). If hearts were subjected to I/R in the presence of ML-7 (a myosin light chain kinase (MLCK) inhibitor) or doxycycline (a MMP inhibitor) an improved recovery of contractile function was seen compared to aerobic hearts and MLC1 was protected from degradation. Enzyme kinetic studies revealed an increased affinity of MMP-2 for the phosphorylated form of MLC1 compared to non-phosphorylated MLC1. We conclude that MLC1 phosphorylation is important mechanism controlling the intracellular action of MMP-2 and promoting the degradation of MLC1. These results further support previous findings implicating posttranslational modifications of contractile proteins as a key factor in the pathology of cardiac dysfunction during and following ischemia. PMID:22564771

  3. Kit- and Fc epsilonRI-induced differential phosphorylation of the transmembrane adaptor molecule NTAL/LAB/LAT2 allows flexibility in its scaffolding function in mast cells

    DEFF Research Database (Denmark)

    Iwaki, Shoko; Spicka, Jiri; Tkaczyk, Christine;

    2008-01-01

    and Kit phosphorylated other tyrosines, both inside and outside of these motifs. Pull down studies revealed that PLCgamma1 associated with the two terminal Syk-phosphorylated Grb2-binding sites, which would help to explain the observed decrease in antigen-induced calcium signal and degranulation in NTAL...

  4. Downregulation of IL-6-induced STAT3 tyrosine phosphorylation by TGF-β1 is mediated by caspase-dependent and -independent processes

    NARCIS (Netherlands)

    Wierenga, A.T.J.; Schuringa, J.J.; Eggen, B.J.L.; Kruijer, W.; Vellenga, E.

    2002-01-01

    To explore the possible cross-talk between the IL-6 and TGF-β1 pathways in AML blast cells, the effect of TGF-β1 pretreatment on IL-6-induced STAT3 tyrosine phosphorylation was studied. A reduction of STAT3 tyrosine phosphorylation after TGF-β1 pretreatment was observed in four out of 40 AML cases (

  5. Downregulation of IL-6-induced STAT3 tyrosine phosphorylation by TGF-beta 1 is mediated by caspase-dependent and -independent processes

    NARCIS (Netherlands)

    Wierenga, ATJ; Schuringa, JJ; Eggen, BJL; Kruijer, W; Vellenga, E

    2002-01-01

    To explore the possible cross-talk between the IL-6 and TGF-beta1 pathways in AML blast cells, the effect of TGF-beta1 pretreatment on IL-6-induced STAT3 tyrosine phosphorylation was studied. A reduction of STAT3 tyrosine phosphorylation after TGF-beta1 pretreatment was observed in four out of 40 AM

  6. Relationship between G-proteins associated transmembrane cascades and MAPK phosphorylation induced by recombinant interleukin-α in fibroblast-like synoviocytes from rats with adjuvant arthritis

    Institute of Scientific and Technical Information of China (English)

    Yong-qiuZHENG; WeiWEI; LeiZHU; Xiao-yiJIA

    2004-01-01

    AIM: To elucidate the relationship between G protein-associ-ated transmembrane cascade and mitogen-activated protein kinases (MAPKs) phosphorylation in recombinant rat IL-1α(rIL-1α)-induced fibroblast-like synoviocytes (FLS) from rats with adjuvant arthritis (AA). METHODS: The expressions of MAPKs phosphorylation, the stimulatory subunit of G alpha

  7. mTORC2 phosphorylation of Akt1: a possible mechanism for hydrogen sulfide-induced cardioprotection.

    Directory of Open Access Journals (Sweden)

    Yue Zhou

    Full Text Available Hydrogen sulfide (H2S is known to have cardiac protective effects through Akt activation. Akt acts as a 'central sensor' for myocyte survival or death; its activity is regulated by multiple kinases including PI3K, mTORC2, PDK1 and phosphatases including PTEN, PP2A and PHLPPL. Based on the previous finding that PI3K inhibitor LY294002 abolishes H2S-induced Akt phosphorylation and cardioprotection, it is accepted that PI3K is the mediator of H2S-induced Akt phosphorylation. However, LY294002 inhibits both PI3K and mTOR, and PI3K only recruits Akt to the membrane where Akt is phosphorylated by Akt kinases. We undertook a series of experiments to further evaluate the role of mTORC2, PDK1, PTEN, PP2A and PHLPPL in H2S-induced Akt phosphorylation and cardioprotection, which, we believe, has not been investigated before. Hearts from adult Sprague-Dawley rats were isolated and subjected to (i normoxia, (ii global ischemia and (iii ischemia/reperfusion in the presence or absence of 50 µM of H2S donor NaHS. Cardiac mechanical function and lactate dehydrogenase (LDH release were assessed. All hearts also were Western analyzed at the end of perfusion for Akt and a panel of appropriate Akt regulators and targets. Hearts pretreated with 50 µM NaHS had improved function at the end of reperfusion (Rate pressure product; 19±4×10(3 vs. 10±3×10(3 mmHg/min, p<0.05 and reduced cell injury (LDH release 19±10 vs. 170±87 mU/ml p<0.05 compared to untreated hearts. NaHS significantly increased phospho-Akt, phospho-mTOR, phospho-Bim and Bcl-2 in reperfused hearts (P<0.05. Furthermore using H9c2 cells we demonstrate that NaHS pretreatment reduces apoptosis following hypoxia/re-oxygenation. Importantly, PP242, a specific mTOR inhibitor, abolished both cardioprotection and protein phosphorylation in isolated heart and reduced apoptotic effects in H9c2 cells. Treating hearts with NaHS only during reperfusion produced less cardioprotection through a similar mechanism

  8. Induced nanoparticle aggregation for short nucleic acid quantification by depletion isotachophoresis.

    Science.gov (United States)

    Marczak, Steven; Senapati, Satyajyoti; Slouka, Zdenek; Chang, Hsueh-Chia

    2016-12-15

    A rapid (assay with probe-functionalized gold nanoparticles and their separation into concentrated bands by depletion-generated gel isotachophoresis. The platform sequentially exploits the enrichment and depletion phenomena of an ion-selective cation-exchange membrane created under an applied electric field. Enrichment is used to concentrate the nanoparticles and targets at a localized position at the gel-membrane interface for rapid hybridization. The depletion generates an isotachophoretic zone without the need for different conductivity buffers, and is used to separate linked nanoparticles from isolated ones in the gel medium and then by field-enhanced aggregation of only the linked particles at the depletion front. The selective field-induced aggregation of the linked nanoparticles during the subsequent depletion step produces two lateral-flow like bands within 1cm for easy visualization and quantification as the aggregates have negligible electrophoretic mobility in the gel and the isolated nanoparticles are isotachophoretically packed against the migrating depletion front. The detection limit for 69-base single-stranded DNA targets is 10 pM (about 10 million copies for our sample volume) with high selectivity against nontargets and a three decade linear range for quantification. The selectivity and signal intensity are maintained in heterogeneous mixtures where the nontargets outnumber the targets 10,000 to 1. The selective field-induced aggregation of DNA-linked nanoparticles at the ion depletion front is attributed to their trailing position at the isotachophoretic front with a large field gradient. PMID:27494807

  9. Oxaliplatin-induced loss of phosphorylated heavy neurofilament subunit neuronal immunoreactivity in rat DRG tissue

    Directory of Open Access Journals (Sweden)

    Connor Bronwen

    2009-11-01

    Full Text Available Abstract Background Oxaliplatin and related chemotherapeutic drugs cause painful chronic peripheral neuropathies in cancer patients. We investigated changes in neuronal size profiles and neurofilament immunoreactivity in L5 dorsal root ganglion (DRG tissue of adult female Wistar rats after multiple-dose treatment with oxaliplatin, cisplatin, carboplatin or paclitaxel. Results After treatment with oxaliplatin, phosphorylated neurofilament heavy subunit (pNF-H immunoreactivity was reduced in neuronal cell bodies, but unchanged in nerve fibres, of the L5 DRG. Morphometric analysis confirmed significant changes in the number (-75%; P P P = 0.82, NF-M (-1%, P = 0.96 or NF-H (0%; P = 0.93 after oxaliplatin treatment, although the sizes of parvalbumin (-29%, P = 0.047, NF-M (-11%, P = 0.038 and NF-H (-28%; P = 0.0033 immunoreactive neurons were reduced. In an independent comparison of different chemotherapeutic agents, the number of pNF-H-immunoreactive neurons was significantly altered by oxaliplatin (-77.2%; P P = 0.03 but not by carboplatin or paclitaxel, and their mean cell body area was significantly changed by oxaliplatin (-31.1%; P = 0.008 but not by cisplatin, carboplatin or paclitaxel. Conclusion This study has demonstrated a specific pattern of loss of pNF-H immunoreactivity in rat DRG tissue that corresponds with the relative neurotoxicity of oxaliplatin, cisplatin and carboplatin. Loss of pNF-H may be mechanistically linked to oxaliplatin-induced neuronal atrophy, and serves as a readily measureable endpoint of its neurotoxicity in the rat model.

  10. Kinome analysis of receptor-induced phosphorylation in human natural killer cells.

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    Sebastian König

    Full Text Available BACKGROUND: Natural killer (NK cells contribute to the defense against infected and transformed cells through the engagement of multiple germline-encoded activation receptors. Stimulation of the Fc receptor CD16 alone is sufficient for NK cell activation, whereas other receptors, such as 2B4 (CD244 and DNAM-1 (CD226, act synergistically. After receptor engagement, protein kinases play a major role in signaling networks controlling NK cell effector functions. However, it has not been characterized systematically which of all kinases encoded by the human genome (kinome are involved in NK cell activation. RESULTS: A kinase-selective phosphoproteome approach enabled the determination of 188 kinases expressed in human NK cells. Crosslinking of CD16 as well as 2B4 and DNAM-1 revealed a total of 313 distinct kinase phosphorylation sites on 109 different kinases. Phosphorylation sites on 21 kinases were similarly regulated after engagement of either CD16 or co-engagement of 2B4 and DNAM-1. Among those, increased phosphorylation of FYN, KCC2G (CAMK2, FES, and AAK1, as well as the reduced phosphorylation of MARK2, were reproducibly observed both after engagement of CD16 and co-engagement of 2B4 and DNAM-1. Notably, only one phosphorylation on PAK4 was differentally regulated. CONCLUSIONS: The present study has identified a significant portion of the NK cell kinome and defined novel phosphorylation sites in primary lymphocytes. Regulated phosphorylations observed in the early phase of NK cell activation imply these kinases are involved in NK cell signaling. Taken together, this study suggests a largely shared signaling pathway downstream of distinct activation receptors and constitutes a valuable resource for further elucidating the regulation of NK cell effector responses.

  11. Double-stranded RNA induces biphasic STAT1 phosphorylation by both type I interferon (IFN)-dependent and type I IFN-independent pathways.

    Science.gov (United States)

    Dempoya, Junichi; Matsumiya, Tomoh; Imaizumi, Tadaatsu; Hayakari, Ryo; Xing, Fei; Yoshida, Hidemi; Okumura, Ken; Satoh, Kei

    2012-12-01

    Upon viral infection, pattern recognition receptors sense viral nucleic acids, leading to the production of type I interferons (IFNs), which initiate antiviral activities. Type I IFNs bind to their cognate receptor, IFNAR, resulting in the activation of signal-transducing activators of transcription 1 (STAT1). Thus, it has long been thought that double-stranded RNA (dsRNA)-induced STAT1 phosphorylation is mediated by the transactivation of type I IFN signaling. Foreign RNA, such as viral RNA, in cells is sensed by the cytoplasmic sensors retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA-5). In this study, we explored the molecular mechanism responsible for STAT1 phosphorylation in response to the sensing of dsRNA by cytosolic RNA sensors. Polyinosinic-poly(C) [poly(I:C)], a synthetic dsRNA that is sensed by both RIG-I and MDA-5, induces STAT1 phosphorylation. We found that the poly(I:C)-induced initial phosphorylation of STAT1 is dependent on the RIG-I pathway and that MDA-5 is not involved in STAT1 phosphorylation. Furthermore, pretreatment of the cells with neutralizing antibody targeting the IFN receptor suppressed the initial STAT1 phosphorylation in response to poly(I:C), suggesting that this initial phosphorylation event is predominantly type I IFN dependent. In contrast, neither the known RIG-I pathway nor type I IFN is involved in the late phosphorylation of STAT1. In addition, poly(I:C) stimulated STAT1 phosphorylation in type I IFN receptor-deficient U5A cells with delayed kinetics. Collectively, our study provides evidence of a comprehensive regulatory mechanism in which dsRNA induces STAT1 phosphorylation, indicating the importance of STAT1 in maintaining very tight regulation of the innate immune system.

  12. Multistimuli-Responsive Luminescence of Naphthalazine Based on Aggregation-Induced Emission

    OpenAIRE

    Yao, Xiang; Ru, Jia-Xi; Xu, Cong; Liu, Ya-Ming; Dou, Wei; Tang, Xiao-Liang; Zhang, Guo-lin; Liu, Wei-Sheng

    2015-01-01

    Stimuli-responsive luminescent materials, which are dependent on changes in physical molecular packing modes, have attracted more and more interest over the past ten years. In this study, 2,2-dihydroxy-1,1-naphthalazine was synthesized and shown to exhibit different fluorescence emission in solution and solid states with characteristic aggregation-induced emission (AIE) properties. A remarkable change in the fluorescence of 2,2-dihydroxy-1,1-naphthalazine occurred upon mechanical grinding, he...

  13. An Aggregation-Induced-Emission Platform for Direct Visualization of Interfacial Dynamic Self-Assembly**

    OpenAIRE

    Li, Junwei; Li, Yuan; Chan, Carrie Y.K.; Ryan T. K. Kwok; Li, Hongkun; Zrazhevskiy, Pavel; Gao, Xiaohu; Sun, Jing Zhi; Qin, Anjun; Tang, Ben Zhong

    2014-01-01

    An in-depth understanding of dynamic interfacial self-assembly processes is essential for a wide range of topics in theoretical physics, materials design, and biomedical research. However, direct monitoring of such processes is hampered by the poor imaging contrast of a thin interfacial layer. We report in situ imaging technology capable of selectively highlighting self-assembly at the phase boundary in real time by employing the unique photophysical properties of aggregation-induced emission...

  14. Hypertension alters phosphorylation of VASP in brain endothelial cells.

    Science.gov (United States)

    Arlier, Zulfikar; Basar, Murat; Kocamaz, Erdogan; Kiraz, Kemal; Tanriover, Gamze; Kocer, Gunnur; Arlier, Sefa; Giray, Semih; Nasırcılar, Seher; Gunduz, Filiz; Senturk, Umit K; Demir, Necdet

    2015-04-01

    Hypertension impairs cerebral vascular function. Vasodilator-stimulated phosphoprotein (VASP) mediates active reorganization of the cytoskeleton via membrane ruffling, aggregation and tethering of actin filaments. VASP regulation of endothelial barrier function has been demonstrated by studies using VASP(-/-) animals under conditions associated with tissue hypoxia. We hypothesize that hypertension regulates VASP expression and/or phosphorylation in endothelial cells, thereby contributing to dysfunction in the cerebral vasculature. Because exercise has direct and indirect salutary effects on vascular systems that have been damaged by hypertension, we also investigated the effect of exercise on maintenance of VASP expression and/or phosphorylation. We used immunohistochemistry, Western blotting and immunocytochemistry to examine the effect of hypertension on VASP expression and phosphorylation in brain endothelial cells in normotensive [Wistar-Kyoto (WKY)] and spontaneously hypertensive (SH) rats under normal and exercise conditions. In addition, we analyzed VASP regulation in normoxia- and hypoxia-induced endothelial cells. Brain endothelial cells exhibited significantly lower VASP immunoreactivity and phosphorylation at the Ser157 residue in SHR versus WKY rats. Exercise reversed hypertension-induced alterations in VASP phosphorylation. Western blotting and immunocytochemistry indicated reduction in VASP phosphorylation in hypoxic versus normoxic endothelial cells. These results suggest that diminished VASP expression and/or Ser157 phosphorylation mediates endothelial changes associated with hypertension and exercise may normalize these changes, at least in part, by restoring VASP phosphorylation. PMID:24894047

  15. Vitamin C Prevents Cigarette Smoke-Induced Leukocyte Aggregation and Adhesion to Endothelium in vivo

    Science.gov (United States)

    Lehr, Hans-Anton; Frei, Balz; Arfors, Karl-E.

    1994-08-01

    A common feature of cigarette-smoke (CS)-associated diseases such as atherosclerosis and pulmonary emphysema is the activation, aggregation, and adhesion of leukocytes to micro- and macrovascular endothelium. A previous study, using a skinfold chamber model for intravital fluorescence microscopy in awake hamsters, has shown that exposure of hamsters to the smoke generated by one research cigarette elicits the adhesion of fluorescently labeled leukocytes to the endothelium of arterioles and small venules. By the combined use of intravital microscopy and scanning electron microscopy, we now demonstrate in the same animal model that (i) CS-induced leukocyte adhesion is not confined to the microcirculation, but that leukocytes also adhere singly and in clusters to the aortic endothelium; (ii) CS induces the formation in the bloodstream of aggregates between leukocytes and platelets; and (iii) CS-induced leukocyte adhesion to micro- and macrovascular endothelium and leukocyte-platelet aggregate formation are almost entirely prevented by dietary or intravenous pretreatment with the water-soluble antioxidant vitamin C (venules, 21.4 ± 11.0 vs. 149.6 ± 38.7 leukocytes per mm^2, P dietary means or supplementation, suggesting that vitamin C effectively contributes to protection from CS-associated cardiovascular and pulmonary diseases in humans.

  16. 20-Hydroxyeicosatetraenoic acid impairs endothelial insulin signaling by inducing phosphorylation of the insulin receptor substrate-1 at Ser616.

    Directory of Open Access Journals (Sweden)

    Xuguang Li

    Full Text Available 20-Hydroxyeicosatetraenoic acid (20-HETE induces endothelial dysfunction and is correlated with diabetes. This study was designed to investigate the effects of 20-HETE on endothelial insulin signaling.Human umbilical vein endothelial cells (HUVECs or C57BL/6J mice were treated with 20-HETE in the presence or absence of insulin, and p-ERK1/2, p-JNK, IRS-1/PI3K/AKT/eNOS pathway, were examined in endothelial cells and aortas by immunoblotting. eNOS activity and nitric oxide production were measured. 20-HETE increased ERK1/2 phosphorylation and IRS-1 phosphorylation at Ser616; these effects were reversed by ERK1/2 inhibition. We further observed that 20-HETE treatment resulted in impaired insulin-stimulated IRS-1 phosphorylation at Tyr632 and subsequent PI3-kinase/Akt activation. Furthermore, 20-HETE treatment blocked insulin-stimulated phosphorylation of eNOS at the stimulatory Ser1177 site, eNOS activation and NO production; these effects were reversed by inhibiting ERK1/2. Treatment of C57BL/6J mice with 20-HETE resulted in ERK1/2 activation and impaired insulin-dependent activation of the IRS-1/PI3K/Akt/eNOS pathway in the aorta. Our data suggest that the 20-HETE activation of IRS-1 phosphorylation at Ser616 is dependent on ERK1/2 and leads to impaired insulin-stimulated vasodilator effects that are mediated by the IRS-1/PI3K/AKT/eNOS pathway.

  17. Contractions induce phosphorylation of the AMPK site Ser565 in hormone-sensitive lipase in muscle

    DEFF Research Database (Denmark)

    Donsmark, Morten; Langfort, Jozef; Holm, Cecilia;

    2004-01-01

    Intramyocellular triglyceride is an important energy store which is related to insulin resistance. Mobilization of fatty acids from this pool is probably regulated by hormone-sensitive lipase (HSL), which has recently been shown to exist in muscle and to be activated by epinephrine via PKA...... and by contractions via PKC and ERK. 5' AMP-activated protein kinase (AMPK) is an intracellular fuel gauge which regulates metabolism. In this study we incubated rat soleus muscle to investigate if AMPK influences HSL during 5min of repeated tetanic contractions. An eightfold increase in AMPK activity was accompanied...... by a 2.5-fold increase in phosphorylation of the AMPK-site Ser(565) in HSL (pHSL activation while HSL-Ser(565) phosphorylation was not reduced. The study indicates that during contractions AMPK phosphorylates HSL in Ser(565...

  18. [Research on expression and function of phosphorylated DARPP-32 on pentylenetetrazol-induced epilepsy model of rat].

    Science.gov (United States)

    Wang, Weiwen; Liao, Xiaoyang; Yang, Zhenghui; Lin, Hang; Wang, Qingsong; Wu, Yuxian; Liu, Yu

    2014-06-01

    The present study is to explore the change process and distribution of phosphorylated DARPP-32 (p-DARPP-32) in rat brain including cortex, hippocampus and striatum and to further deduce whether p-DARPP-32 was possibly involved in epilepsy induced by repetitive low doses of pentylenetetrazol (PTZ). PTZ-induced epilepsy model in rat was established with 30 male SD rats randomly divided into 6 groups, control group and five trial groups [PTZ 1 h, PTZ 6 h, PTZ 24 h, PTZ 48 h and PTZ 72 h respectively, after onset of status epilepticus (SE)]. Immunohistochemistry and immunofluorescence double-labeling were used to detect the temporal time change and distribution of p-DARPP-32 expression and to analyze the coexpression of DARPP-32 and p-DARPP-32 in rat brain after the onset of PTZ-induced generalized SE. The results showed that there was a temporal time change of p-DARPP-32 expression in rat brain after the onset of SE. The number of p-DARPP-32-positive cells increased significantly and reached the peaks at the ends of 1 hour and 6 hours after the onset of SE, but decreased at the end of 24 hours. The moderate to strong p-DARPP-32-immunopositive neurons were observed in cortex, hippocampus and striatum, and located in cell cytoplasm and cell nucleus. Further immunofluorescence double-labeling revealed that denser colocalization of p-DARPP-32 and DARPP-32 in the neurons existed in the area mentioned above. Therefore, PTZ-induced SE may cause phosphorylation of DARPP-32 in rat brain. The temporal time change and distribution of p-DARPP-32 suggest that phosphorylation of DARPP-32 may be involved in PTZ-induced epilepsy in rat brain including cortex, hippocampus and striatum, and p-DARPP-32 may play a central role in the onset of SE.

  19. Strategy of ring-shaped aggregates in excitation energy transfer for removing disorder-induced shielding

    International Nuclear Information System (INIS)

    Peripheral light harvesting complex (LH2), which is found in photosynthetic antenna systems of purple photosynthetic bacteria, has important functions in the photosynthetic process, such as harvesting sunlight and transferring its energy to the photosynthetic reaction center. The key component in excitation energy transfer (EET) between LH2s is B850, which is a characteristic ring-shaped aggregate of pigments usually formed by 18 or 16 bacteriochlorophylls in LH2. We theoretically study the strategy of the ring-shaped aggregate structure, which maximizes EET efficiency, by using the standard Frenkel exciton model and the self-consistent calculation method for the Markovian quantum master equation and Maxwell equation. As a result, we have revealed a simple but ingenious strategy of the ring-shaped aggregate structure. The combination of three key properties of the ring unit system maximizes the EET efficiency, namely the large dipole moment of aggregates causes the basic improvement of EET efficiency, and the isotropic nature and the large occupying area are critically effective to remove the disorder-induced shielding that inhibits EET in the presence of the randomness of orientation and alignment of carriers of excitation energy. (paper)

  20. Profiling DNA damage-induced phosphorylation in budding yeast reveals diverse signaling networks.

    Science.gov (United States)

    Zhou, Chunshui; Elia, Andrew E H; Naylor, Maria L; Dephoure, Noah; Ballif, Bryan A; Goel, Gautam; Xu, Qikai; Ng, Aylwin; Chou, Danny M; Xavier, Ramnik J; Gygi, Steven P; Elledge, Stephen J

    2016-06-28

    The DNA damage response (DDR) is regulated by a protein kinase signaling cascade that orchestrates DNA repair and other processes. Identifying the substrate effectors of these kinases is critical for understanding the underlying physiology and mechanism of the response. We have used quantitative mass spectrometry to profile DDR-dependent phosphorylation in budding yeast and genetically explored the dependency of these phosphorylation events on the DDR kinases MEC1, RAD53, CHK1, and DUN1. Based on these screens, a database containing many novel DDR-regulated phosphorylation events has been established. Phosphorylation of many of these proteins has been validated by quantitative peptide phospho-immunoprecipitation and examined for functional relevance to the DDR through large-scale analysis of sensitivity to DNA damage in yeast deletion strains. We reveal a link between DDR signaling and the metabolic pathways of inositol phosphate and phosphatidyl inositol synthesis, which are required for resistance to DNA damage. We also uncover links between the DDR and TOR signaling as well as translation regulation. Taken together, these data shed new light on the organization of DDR signaling in budding yeast. PMID:27298372

  1. Reconstruction and analysis of nutrient-induced phosphorylation networks in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Guangyou eDuan

    2013-12-01

    Full Text Available Elucidating the dynamics of molecular processes in living organisms in response to external perturbations is a central goal in modern systems biology. We investigated the dynamics of protein phosphorylation events in Arabidopsis thaliana exposed to changing nutrient conditions. Phosphopeptide expression levels were detected at five consecutive time points over a time interval of 30 minutes after nutrient resupply following prior starvation. The three tested inorganic, ionic nutrients NH4+, NO3-, PO43- elicited similar phosphosignaling responses that were distinguishable from those invoked by the sugars mannitol, sucrose. When embedded in the protein-protein interaction network of Arabidopsis thaliana, phosphoproteins were found to exhibit a higher degree compared to average proteins. Based on the time-series data, we reconstructed a network of regulatory interactions mediated by phosphorylation. The performance of different network inference methods was evaluated by the observed likelihood of physical interactions within and across different subcellular compartments and based on gene ontology semantic similarity. The dynamic phosphorylation network was then reconstructed using a Pearson correlation method with added directionality based on partial variance differences. The topology of the inferred integrated network corresponds to an information dissemination architecture, in which the phosphorylation signal is passed on to an increasing number of phosphoproteins stratified into an initiation, processing, and effector layer. Specific phosphorylation peptide motifs associated with the distinct layers were identified indicating the action of layer-specific kinases. Despite the limited temporal resolution, combined with information on subcellular location, the available time-series data proved useful for reconstructing the dynamics of the molecular signaling cascade in response to nutrient stress conditions in the plant Arabidopsis thaliana.

  2. Detection of Gold Nanoparticles Aggregation Growth Induced by Nucleic Acid through Laser Scanning Confocal Microscopy

    Science.gov (United States)

    Gary, Ramla; Carbone, Giovani; Petriashvili, Gia; De Santo, Maria Penelope; Barberi, Riccardo

    2016-01-01

    The gold nanoparticle (GNP) aggregation growth induced by deoxyribonucleic acid (DNA) is studied by laser scanning confocal and environmental scanning electron microscopies. As in the investigated case the direct light scattering analysis is not suitable, we observe the behavior of the fluorescence produced by a dye and we detect the aggregation by the shift and the broadening of the fluorescence peak. Results of laser scanning confocal microscopy images and the fluorescence emission spectra from lambda scan mode suggest, in fact, that the intruding of the hydrophobic moiety of the probe within the cationic surfactants bilayer film coating GNPs results in a Förster resonance energy transfer. The environmental scanning electron microscopy images show that DNA molecules act as template to assemble GNPs into three-dimensional structures which are reminiscent of the DNA helix. This study is useful to design better nanobiotechnological devices using GNPs and DNA. PMID:26907286

  3. Detection of Gold Nanoparticles Aggregation Growth Induced by Nucleic Acid through Laser Scanning Confocal Microscopy

    Directory of Open Access Journals (Sweden)

    Ramla Gary

    2016-02-01

    Full Text Available The gold nanoparticle (GNP aggregation growth induced by deoxyribonucleic acid (DNA is studied by laser scanning confocal and environmental scanning electron microscopies. As in the investigated case the direct light scattering analysis is not suitable, we observe the behavior of the fluorescence produced by a dye and we detect the aggregation by the shift and the broadening of the fluorescence peak. Results of laser scanning confocal microscopy images and the fluorescence emission spectra from lambda scan mode suggest, in fact, that the intruding of the hydrophobic moiety of the probe within the cationic surfactants bilayer film coating GNPs results in a Förster resonance energy transfer. The environmental scanning electron microscopy images show that DNA molecules act as template to assemble GNPs into three-dimensional structures which are reminiscent of the DNA helix. This study is useful to design better nanobiotechnological devices using GNPs and DNA.

  4. Study of molecular mechanisms of UV-induced aggregation of crystallins and possibility of maintaining eye lens transparency

    Science.gov (United States)

    Soustov, L. V.; Chelnokov, E. V.; Bityurin, N. M.; Kiselev, A. L.; Nemov, V. V.; Sergeev, Yu. V.; Ostrovsky, M. A.

    2006-03-01

    The effect of D-pantethine and L-carnosine on the rate of UV-induced (XeC1 laser λ = 308 nm) aggregation of a mixture of βL-crystallin and α-crystallin is studied. We also demonstrate that the suggested by us combination of short-chain peptides shows better protective properties with respect to UV-induced aggregation than known anti-cataract agents.

  5. Active protein aggregates induced by terminally attached self-assembling peptide ELK16 in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Zhou Bihong

    2011-02-01

    Full Text Available Abstract Background In recent years, it has been gradually realized that bacterial inclusion bodies (IBs could be biologically active. In particular, several proteins including green fluorescent protein, β-galactosidase, β-lactamase, alkaline phosphatase, D-amino acid oxidase, polyphosphate kinase 3, maltodextrin phosphorylase, and sialic acid aldolase have been successfully produced as active IBs when fused to an appropriate partner such as the foot-and-mouth disease virus capsid protein VP1, or the human β-amyloid peptide Aβ42(F19D. As active IBs may have many attractive advantages in enzyme production and industrial applications, it is of considerable interest to explore them further. Results In this paper, we report that an ionic self-assembling peptide ELK16 (LELELKLK2 was able to effectively induce the formation of cytoplasmic inclusion bodies in Escherichia coli (E. coli when attached to the carboxyl termini of four model proteins including lipase A, amadoriase II, β-xylosidase, and green fluorescent protein. These aggregates had a general appearance similar to the usually reported cytoplasmic inclusion bodies (IBs under transmission electron microscopy or fluorescence confocal microscopy. Except for lipase A-ELK16 fusion, the three other fusion protein aggregates retained comparable specific activities with the native counterparts. Conformational analyses by Fourier transform infrared spectroscopy revealed the existence of newly formed antiparallel beta-sheet structures in these ELK16 peptide-induced inclusion bodies, which is consistent with the reported assembly of the ELK16 peptide. Conclusions This has been the first report where a terminally attached self-assembling β peptide ELK16 can promote the formation of active inclusion bodies or active protein aggregates in E. coli. It has the potential to render E. coli and other recombinant hosts more efficient as microbial cell factories for protein production. Our observation might

  6. Frizzled-Induced Van Gogh Phosphorylation by CK1ε Promotes Asymmetric Localization of Core PCP Factors in Drosophila

    Directory of Open Access Journals (Sweden)

    Lindsay K. Kelly

    2016-07-01

    Full Text Available Epithelial tissues are polarized along two axes. In addition to apical-basal polarity, they are often polarized within the plane of the epithelium, so-called Planar Cell Polarity (PCP. PCP depends upon Wnt/Frizzled (Fz signaling factors, including Fz itself and Van Gogh (Vang/Vangl. We sought to understand how Vang interaction with other core PCP factors affects Vang function. We find that Fz induces Vang phosphorylation in a cell-autonomous manner. Vang phosphorylation occurs on conserved N-terminal serine/threonine residues, is mediated by CK1ε/Dco, and is critical for polarized membrane localization of Vang and other PCP proteins. This regulatory mechanism does not require Fz signaling through Dishevelled and thus represents a cell-autonomous upstream interaction between Fz and Vang. Furthermore, this signaling event appears to be related to Wnt5a-mediated Vangl2 phosphorylation during mouse limb patterning and may thus be a general mechanism underlying Wnt-regulated PCP establishment.

  7. Cholecystokinin (CCK) stimulates S6 phosphorylation and induced activation of S6 protein kinase in rat pancreatic acini

    Energy Technology Data Exchange (ETDEWEB)

    Sung, C.; Okabayashi, Y.; Williams, J.

    1987-05-01

    CCK and insulin stimulate pancreatic protein synthesis at a post transcriptional step. To better understand this regulation the authors evaluated the phosphorylation state of ribosomal protein S6 and the presence of a specific S6 protein kinase in pancreatic acini from diabetic rats. Both CCK and insulin increased S6 phosphorylation by up to 400% in intact TSP-labelled acini. The phorbol ester 12-0-tetradecanoylphorbol 13-acetate also stimulated both protein synthesis and S6 phosphorlyation suggesting a role for protein kinase C in mediating the effect of CCK. By contrast, the CaS ionophore ionomycin had no effect on either parameter. Recently, insulin has been shown to activate a unique S6 kinase in various cells. To test for its presence, cytosolic extracts were prepared from acini stimulated with CCK and insulin by homogenization in US -glycerophosphate buffer and assayed for the kinase using el-TSP ATP and rat pancreatic ribosomes followed by SDS-polyacrylamide gel electrophoresis. CCK and insulin both increased S6 kinase activity which required neither CaS or phospholipid. The dose response for CCk was similar to S6 phosphorlyation in the intact acini. TPA did not stimulate the S6 kinase. Thus, CCK may induce S6 phosphorylation both via C kinase and by activation of a unique S6 kinase.

  8. Crk adaptor protein-induced phosphorylation of Gab1 on tyrosine 307 via Src is important for organization of focal adhesions and enhanced cell migration

    Institute of Scientific and Technical Information of China (English)

    Takuya Watanabe; Masumi Tsuda; Yoshinori Makino; Tassos Konstantinou; Hiroshi Nishihara; Tokifumi Majima; Akio Minami; Stephan M Feller; Shinya Tanaka

    2009-01-01

    Upon growth factor stimulation, the scaffold protein, Gabl, is tyrosine phosphorylated and subsequently the adaptor protein, Crk, transmits signals from Gabl. We have previously shown that Crk overexpression, which is detectable in various human cancers, induces tyrosine phosphorylation of Gab1 without extraceilular stimuli. In the present study, the underlying mechanisms were further investigated. Mutational analyses of Crkll demonstrated that the SH2 domain, but not the SH3(N) or the regulatory Y221 residue of Crkll, is critical for the induction of Gabl-Y307 phosphorylation. SH2 mutation of Crkll also decreased the interaction with Gab1. In GST pull-down assay, Crk-SH2 bound to wild-type Gabl, whereas Crk-SH3(N) interacted with the Gabl mutant, which lacks the clus-tered tyrosine region (residues 242-410). Tyrosine phosphorylation of Gabl was induced by all Crk family proteins, but not other SH2-containing signalling adaptors. Src-family kinase inhibitor, PP2, abrogates Crk-induced tyrosine phosphorylations of Gabl. Y307 phosphorylation was undetectable in fibroblasts lacking Src, Yes, and Fyn, even upon overexpression of Crk, whereas cells lacking only Yes and Fyn still contained Gabl with phosphorylated Y307. Furthermore, Crk induced the phosphorylation of Src-Y416; accordingly the interaction between Crk and Csk was increased. The GabI-Y307F mutant failed to localize near the plasma membrane even upon HGF stimulation and decreased cell migration. Moreover, Gabl-Y307F disturbed the localization of Crk, FAK, and paxiilin, which are the typical components of focal adhesions. Taken together, these results indicate that Crk facilitates tyrosine phosphory-lation of Gabl-Y307 through Src, contributing to the organization of focal adhesions and enhanced cell migration, thereby possibly promoting human cancer development.

  9. Compressive stress induces dephosphorylation of the myosin regulatory light chain via RhoA phosphorylation by the adenylyl cyclase/protein kinase A signaling pathway.

    Directory of Open Access Journals (Sweden)

    Kenji Takemoto

    Full Text Available Mechanical stress that arises due to deformation of the extracellular matrix (ECM either stretches or compresses cells. The cellular response to stretching has been actively studied. For example, stretching induces phosphorylation of the myosin regulatory light chain (MRLC via the RhoA/RhoA-associated protein kinase (ROCK pathway, resulting in increased cellular tension. In contrast, the effects of compressive stress on cellular functions are not fully resolved. The mechanisms for sensing and differentially responding to stretching and compressive stress are not known. To address these questions, we investigated whether phosphorylation levels of MRLC were affected by compressive stress. Contrary to the response in stretching cells, MRLC was dephosphorylated 5 min after cells were subjected to compressive stress. Compressive loading induced activation of myosin phosphatase mediated via the dephosphorylation of myosin phosphatase targeting subunit 1 (Thr853. Because myosin phosphatase targeting subunit 1 (Thr853 is phosphorylated only by ROCK, compressive loading may have induced inactivation of ROCK. However, GTP-bound RhoA (active form increased in response to compressive stress. The compression-induced activation of RhoA and inactivation of its effector ROCK are contradictory. This inconsistency was due to phosphorylation of RhoA (Ser188 that reduced affinity of RhoA to ROCK. Treatment with the inhibitor of protein kinase A that phosphorylates RhoA (Ser188 induced suppression of compression-stimulated MRLC dephosphorylation. Incidentally, stretching induced phosphorylation of MRLC, but did not affect phosphorylation levels of RhoA (Ser188. Together, our results suggest that RhoA phosphorylation is an important process for MRLC dephosphorylation by compressive loading, and for distinguishing between stretching and compressing cells.

  10. A high-fructose diet induces changes in pp185 phosphorylation in muscle and liver of rats

    Directory of Open Access Journals (Sweden)

    M. Ueno

    2000-12-01

    Full Text Available Insulin stimulates the tyrosine kinase activity of its receptor resulting in the tyrosine phosphorylation of pp185, which contains insulin receptor substrates IRS-1 and IRS-2. These early steps in insulin action are essential for the metabolic effects of insulin. Feeding animals a high-fructose diet results in insulin resistance. However, the exact molecular mechanism underlying this effect is unknown. In the present study, we determined the levels and phosphorylation status of the insulin receptor and pp185 (IRS-1/2 in liver and muscle of rats submitted to a high-fructose diet evaluated by immunoblotting with specific antibodies. Feeding fructose (28 days induced a discrete insulin resistance, as demonstrated by the insulin tolerance test. Plasma glucose and serum insulin and cholesterol levels of the two groups of rats, fructose-fed and control, were similar, whereas plasma triacylglycerol concentration was significantly increased in the rats submitted to the fructose diet (P<0.05. There were no changes in insulin receptor concentration in the liver or muscle of either group. However, insulin-stimulated receptor autophosphorylation was reduced to 72 ± 4% (P<0.05 in the liver of high-fructose rats. The IRS-1 protein levels were similar in both liver and muscle of the two groups of rats. In contrast, there was a significant decrease in insulin-induced pp185 (IRS-1/2 phosphorylation, to 83 ± 5% (P<0.05 in liver and to 77 ± 4% (P<0.05 in muscle of the high-fructose rats. These data suggest that changes in the early steps of insulin signal transduction may have an important role in the insulin resistance induced by high-fructose feeding.

  11. Cryopreservation-induced alterations in protein tyrosine phosphorylation of spermatozoa from different portions of the boar ejaculate.

    Science.gov (United States)

    Kumaresan, A; Siqueira, A P; Hossain, M S; Bergqvist, A S

    2011-12-01

    Previous studies have shown that boar sperm quality after cryopreservation differs depending on the ejaculate fraction used and that spermatozoa contained in the first 10mL (P1) of the sperm-rich fraction (SRF) show better cryosurvival than those in the SRF-P1. Since protein tyrosine phosphorylation (PTP) in spermatozoa is related with the tolerance of spermatozoa to frozen storage and cryocapacitation, we assessed the dynamics of cryopreservation-induced PTP and intracellular calcium ([Ca(2+)]i) in spermatozoa, using flow cytometry, from P1 and SRF-P1 of the boar ejaculate at different stages of cryopreservation. Sperm kinetics, assessed using a computer-assisted semen analyzer, did not differ between P1 and SRF-P1 during cryopreservation but the decrease in sperm velocity during cryopreservation was significant (Psemen. A higher (Pboar ejaculate. However at any given step during cryopreservation the percentage of spermatozoa with PTP was comparatively higher in SRF-P1 than P1. A 32kDa tyrosine phosphorylated protein, associated with capacitation, appeared after cooling suggesting that cooling induces capacitation-like changes in boar spermatozoa. In conclusion, the study has shown that the cryopreservation process induced PTP in spermatozoa and their proportions were similar between portions of SRF. PMID:21893053

  12. Phosphorylation of PTEN increase in pathological right ventricular hypertrophy in rats with chronic hypoxia induced pulmonary hypertension

    Institute of Scientific and Technical Information of China (English)

    Nie Xin; Shi Yiwei; Yu Wenyan; Xu Jianying; Hu Xiaoyun; Du Yongcheng

    2014-01-01

    Background Phosphatase and tensin homologue on chromosome ten (PTEN) acts as a convergent nodal signalling point for cardiomyocyte hypertrophy,growth and survival.However,the role of PTEN in cardiac conditions such as right ventricular hypertrophy caused by chronic hypoxic pulmonary,hypertension remains unclear.This study preliminarily discussed the role of PTEN in the cardiac response to increased pulmonary vascular resistance using the hypoxia-induced PH rats.Methods Male Sprague Dawley rats were exposed to 10% oxygen for 1,3,7,14 or 21 days to induce hypertension and right ventricular hypertrophy.Right ventricular systolic pressure was measured via catheterization.Hypertrophy index was calculated as the ratio of right ventricular mass to left ventricle plus septum mass.Tissue morphology and fibrosis were measured using hematoxylin,eosin and picrosirius red staining.The expression and phosphorylation levels of PTEN in ventricles were determined by real time PCR and Western blotting.Results Hypoxic exposure of rats resulted in pathological hypertrophy,interstitial fibrosis and remodelling of the right ventricle.The phosphorylation of PTEN increased significantly in the hypertrophic right ventricle compared to the normoxic control group.There were no changes in protein expression in either ventricle.Conclusion Hypoxia induced pulmonary hypertension developed pathological right ventricular hypertrophy and remodelling probablv related to an increased phosohorvlation of PTEN.

  13. Structural Characterizations of Glycerol Kinase: Unraveling Phosphorylation-Induced Long-Range Activation

    Energy Technology Data Exchange (ETDEWEB)

    Yeh, Joanne I.; Kettering, Regina; Saxl, Ruth; Bourand, Alexa; Darbon, Emmanuelle; Joly, Nathalie; Briozzo, Pierre; Deutscher, Josef; (Pitt); (CNRS-CRMD)

    2009-09-11

    Glycerol metabolism provides a central link between sugar and fatty acid catabolism. In most bacteria, glycerol kinase plays a crucial role in regulating channel/facilitator-dependent uptake of glycerol into the cell. In the firmicute Enterococcus casseliflavus, this enzyme's activity is enhanced by phosphorylation of the histidine residue (His232) located in its activation loop, approximately 25 A from its catalytic cleft. We reported earlier that some mutations of His232 altered enzyme activities; we present here the crystal structures of these mutant GlpK enzymes. The structure of a mutant enzyme with enhanced enzymatic activity, His232Arg, reveals that residues at the catalytic cleft are more optimally aligned to bind ATP and mediate phosphoryl transfer. Specifically, the position of Arg18 in His232Arg shifts by approximately 1 A when compared to its position in wild-type (WT), His232Ala, and His232Glu enzymes. This new conformation of Arg18 is more optimally positioned at the presumed gamma-phosphate location of ATP, close to the glycerol substrate. In addition to structural changes exhibited at the active site, the conformational stability of the activation loop is decreased, as reflected by an approximately 35% increase in B factors ('thermal factors') in a mutant enzyme displaying diminished activity, His232Glu. Correlating conformational changes to alteration of enzymatic activities in the mutant enzymes identifies distinct localized regions that can have profound effects on intramolecular signal transduction. Alterations in pairwise interactions across the dimer interface can communicate phosphorylation states over 25 A from the activation loop to the catalytic cleft, positioning Arg18 to form favorable interactions at the beta,gamma-bridging position with ATP. This would offset loss of the hydrogen bonds at the gamma-phosphate of ATP during phosphoryl transfer to glycerol, suggesting that appropriate alignment of the second substrate of

  14. Mitochondrial Oxidative Phosphorylation Reserve Is Required for Hormone- and PPARγ Agonist-Induced Adipogenesis

    OpenAIRE

    Ryu, Min Jeong; Kim, Soung Jung; Choi, Min Jeong; Kim, Yong Kyung; Lee, Min Hee; Lee, Seong Eun; Chung, Hyo Kyun; Jung, Saet Byel; Kim, Hyun-Jin; Kim, Koon Soon; Jo, Young Suk; Kweon, Gi Ryang; Lee, Chul-Ho; Shong, Minho

    2013-01-01

    Adipocyte differentiation requires the coordinated activities of several nuclear transcription factors. Recently, mitochondria biogenesis was reported to occur during adipocyte differentiation and following treatment with thiazolidinediones in vitro and in vivo. Crif1 is a translational factor for mitochondrial DNA (mtDNA) and is important for transcription of the mitochondrial oxidative phosphorylation (OXPHOS) complex. To investigate the role of OXPHOS in adipogenesis, we analyzed adipocyte...

  15. Phosphorylation of DGCR8 Increases Its Intracellular Stability and Induces a Progrowth miRNA Profile

    Directory of Open Access Journals (Sweden)

    Kristina M. Herbert

    2013-11-01

    Full Text Available During miRNA biogenesis, the microprocessor complex (MC, which is composed minimally of Drosha, an RNase III enzyme, and DGCR8, a double-stranded RNA-binding protein, cleaves the primary miRNA (pri-miRNA in order to release the pre-miRNA stem-loop structure. Using phosphoproteomics, we mapped 23 phosphorylation sites on full-length human DGCR8 expressed in insect or mammalian cells. DGCR8 can be phosphorylated by mitogenic ERK/MAPK, indicating that DGCR8 phosphorylation may respond to and integrate extracellular cues. The expression of phosphomimetic DGCR8 or inhibition of phosphatases increased the cellular levels of DGCR8 and Drosha proteins. Increased levels of phosphomimetic DGCR8 were not due to higher mRNA levels, altered DGCR8 localization, or DGCR8’s ability to self-associate, but rather to an increase in protein stability. MCs incorporating phosphomutant or phosphomimetic DGCR8 were not altered in specific processing activity. However, HeLa cells expressing phosphomimetic DGCR8 exhibited a progrowth miRNA expression profile and increased proliferation and scratch closure rates relative to cells expressing phosphomutant DGCR8.

  16. Human herpesvirus 6B induces phosphorylation of p53 in its regulatory domain by a CK2- and p38-independent pathway

    DEFF Research Database (Denmark)

    Øster, Bodil; Bundgaard, Bettina; Hupp, TR;

    2008-01-01

    Here, we demonstrate that human herpesvirus 6B (HHV-6B) infection upregulates the tumour suppressor p53 and induces phosphorylation of p53 at Ser392. Interestingly, phosphorylation at the equivalent site has previously been shown to correlate with p53 tumour suppression in murine models. Although...... or Cdk9, eluted in column fractions that phosphorylated p53 at Ser392. However, treatment of cells with neither the CK2 and Cdk9 inhibitor 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole (DRB) nor p38 kinase inhibitors reduced HHV-6B-induced Ser392 phosphorylation significantly. Knockdown of the CK2......beta subunit or p38alpha by small interfering RNA had no effect on HHV-6B-induced phosphorylation of p53 at Ser392. Thus, HHV-6B induces p53 Ser392 phosphorylation by an atypical pathway independent of CK2 and p38 kinases, whereas mitogen-activated protein (MAP) kinase signalling pathways are involved...

  17. White-Light Electroluminescence with Tetraphenylethylene as Emitting Layer of Aggregation-Induced Emissions Enhancement

    Institute of Scientific and Technical Information of China (English)

    罗建芳; 王晓宏; 王筱梅; 苏文明; 陶绪堂; 陈志刚

    2012-01-01

    Tetraphenylethylene (TPE) based molecules with easy synthesis, good thermal stability, and especially their aggregation-induced emissions enhancement (AIEE) effect recently become attractive organic emitting materials due to their potentially practical application in OLEDs. Herein, the AIEE behaviors of tetraphenylethylene dyes (TMTPE and TBTPE) were investigated. Fabricated luminesent device using TMTPE dye as emitting layer displays two strong emitting bands: the blue emission coming from the first-step aggregation and the yellow emission attrib- uted to the second-step aggregation. Thus, it can be utilized to fabricate the white-light OLEDs (WOLEDs) of the single-emitting-component. A three-layer device with the brightness of 1200 cd·m^-2 and current efficiency of 0.78 cd·A^-1 emits the close to white light with the CIE coordinates of x=0.333 and y=0.358, when applied voltage from 8-13 V, verifying that the TPE-based dyes of AIEE effect can be effectively applied in single-emitting- component WOLEDs fabrication.

  18. Aggregation-Induced Emission Luminogen-Embedded Silica Nanoparticles Containing DNA Aptamers for Targeted Cell Imaging.

    Science.gov (United States)

    Wang, Xiaoyan; Song, Panshu; Peng, Lu; Tong, Aijun; Xiang, Yu

    2016-01-13

    Conventional fluorophores usually undergo aggregation-caused quenching (ACQ), which limits the loading amount of these fluorophores in nanoparticles for bright fluorescence imaging. On the contrary, fluorophores with aggregation-induced emission (AIE) characteristics are strongly fluorescent in their aggregate states and have been an ideal platform for developing highly fluorescent nanomaterials, such as fluorescent silica nanoparticles (FSNPs). In this work, AIE luminogens based on salicylaldehyde hydrazones were embedded in silica nanoparticles through a facile noncovalent approach, which afforded AIE-FSNPs emitting much brighter fluorescence than that of some commercial fluorescein-doped silica and polystyrene nanoparticles. These AIE-FSNPs displaying multiple fluorescence colors were fabricated by a general method, and they underwent much less fluorescence variation due to environmental pH changes compared with fluorescein-hybridized FSNPs. In addition, a DNA aptamer specific to nucleolin was functionalized on the surface of AIE-FSNPs for targeted cell imaging. Fluorescent microscopy and flow cytometry studies both revealed highly selective fluorescence staining of MCF-7 (a cancer cell line with nucleolin overexpression) over MCF-10A (normal) cells by the aptamer-functionalized AIE-FSNPs. The fluorescence imaging in different color channels was achieved using AIE-FSNPs containing each of the AIE luminogens, as well as photoactivatable fluorescent imaging of target cells by the caged AIE fluorophore. PMID:26653325

  19. Aggregation-induced emission active tetraphenylethene-based sensor for uranyl ion detection.

    Science.gov (United States)

    Wen, Jun; Huang, Zeng; Hu, Sheng; Li, Shuo; Li, Weiyi; Wang, Xiaolin

    2016-11-15

    A novel tetraphenylethene-based fluorescent sensor, TPE-T, was developed for the detection of uranyl ions. The selective binding of TPE-T to uranyl ions resulted in a detectable signal owing to the quenching of its aggregation-induced emission. The developed sensor could be used to visually distinguish UO2(2+) from lanthanides, transition metals, and alkali metals under UV light; the presence of other metal ions did not interfere with the detection of uranyl ions. In addition, TPE-T was successfully used for the detection of uranyl ions in river water, illustrating its potential applications in environmental systems. PMID:27439180

  20. Chlamydia trachomatis serovar L2 induces protein tyrosine phosphorylation during uptake by HeLa cells

    DEFF Research Database (Denmark)

    Birkelund, Svend; Johnsen, H; Christiansen, Gunna

    1994-01-01

    inactive. Attachment of EBs to host cells is medicated by a heparan sulfate-like glycosaminoglycan. Following attachment, the EB is internalized within a membrane-bound vesicle, and during the first 8 h of infection the vesicles are transported to a perinuclear location where they aggregate and fuse...

  1. Effect of nitric oxide-induced tyrosine phosphorylation of calcium-activated potassium channel α subunit on vascular hyporesponsiveness in rats

    Institute of Scientific and Technical Information of China (English)

    ZHOU Rong; LIU Liang-ming; HU De-yao

    2005-01-01

    Objective: To study the effect of nitric oxide-induced tyrosine phosphorylation of large-conductance calcium-activated potassium (BKCa) channel α subunit on vascular hyporesponsiveness in rats. Methods: A total of 46 Wistar rats of either sex, weighing 250 g±20 g, were used in this study. Models of vascular hyporesponsiveness induced by hemorrhagic shock (30 mm Hg for 2 hours) in vivo and by L-arginine in vitro were established respectively. The vascular responsiveness of isolated superior mesenteric arteries to norepinephrine was observed. Tyrosine phosphorylation of BKCa α subunit was evaluated with methods of immunoprecipitation and Western blotting. Results: In the smooth muscle cells of the superior mesenteric arteries, the expression of BKCa α subunit tyrosine phosphorylation increased following hemorrhagic shock, and L-arginine could induce BKCa channel α subunit tyrosine phosphorylation in a time- and dose-dependent manner. L-NAME (Nω-nitro-L-arginine-methyl-ester), a nitric oxide synthetase inhibitor, could partly restore the decreased vasoresponsiveness of the superior mesenteric arteries after hemorrhagic shock in rats. Down-regulating the protein tyrosine phosphorylation with genistein, a widely-used special protein tyrosine kinase inhibitor, could partly improve the decreased vasoresponsiveness of the superior mesenteric arteries induced by L-arginine in vitro, while up-regulating the protein tyrosine phosphorylation with Na3VO4, a protein tyrosine phosphatase inhibitor, could further decrease the nitric oxide-induced vascular hyporesponsiveness, which could be partly ameliorated by 0.1 mmol/L tetrabutylammonium chloride (TEA), a selective BKCa inhibitor at this concentration. Conclusions: Nitric oxide can induce the tyrosine phosphorylation of BKCa α subunit, which influences the vascular hyporesponsiveness in hemorrhagic shock rats or induced by L-arginine in vitro.

  2. Maintained activity of glycogen synthase kinase-3{beta} despite of its phosphorylation at serine-9 in okadaic acid-induced neurodegenerative model

    Energy Technology Data Exchange (ETDEWEB)

    Lim, Yong-Whan [Department of Anatomy and Cell Biology, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Yoon, Seung-Yong, E-mail: ysy@amc.seoul.kr [Department of Anatomy and Cell Biology, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Institute for Biomacromolecules, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Choi, Jung-Eun [Department of Anatomy and Cell Biology, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Institute for Biomacromolecules, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Kim, Sang-Min [Department of Anatomy and Cell Biology, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Lee, Hui-Sun; Choe, Han [Department of Physiology, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Institute for Biomacromolecules, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Lee, Seung-Chul [CrystalGenomics, Seoul (Korea, Republic of); Kim, Dong-Hou, E-mail: dhkim@amc.seoul.kr [Department of Anatomy and Cell Biology, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Institute for Biomacromolecules, University of Ulsan College of Medicine, Seoul (Korea, Republic of)

    2010-04-30

    Glycogen synthase kinase-3{beta} (GSK3{beta}) is recognized as one of major kinases to phosphorylate tau in Alzheimer's disease (AD), thus lots of AD drug discoveries target GSK3{beta}. However, the inactive form of GSK3{beta} which is phosphorylated at serine-9 is increased in AD brains. This is also inconsistent with phosphorylation status of other GSK3{beta} substrates, such as {beta}-catenin and collapsin response mediator protein-2 (CRMP2) since their phosphorylation is all increased in AD brains. Thus, we addressed this paradoxical condition of AD in rat neurons treated with okadaic acid (OA) which inhibits protein phosphatase-2A (PP2A) and induces tau hyperphosphorylation and cell death. Interestingly, OA also induces phosphorylation of GSK3{beta} at serine-9 and other substrates including tau, {beta}-catenin and CRMP2 like in AD brains. In this context, we observed that GSK3{beta} inhibitors such as lithium chloride and 6-bromoindirubin-3'-monoxime (6-BIO) reversed those phosphorylation events and protected neurons. These data suggest that GSK3{beta} may still have its kinase activity despite increase of its phosphorylation at serine-9 in AD brains at least in PP2A-compromised conditions and that GSK3{beta} inhibitors could be a valuable drug candidate in AD.

  3. Hyperosmotic stress induces Rho/Rho kinase/LIM kinase-mediated cofilin phosphorylation in tubular cells: key role in the osmotically triggered F-actin response

    DEFF Research Database (Denmark)

    Thirone, Ana C P; Speight, Pam; Zulys, Matthew;

    2009-01-01

    treatment. Inhibition of cofilin phosphorylation by Y-27632 prevented the hyperosmolarity-provoked F-actin increase. Taken together, cofilin is necessary for maintaining the osmotic responsiveness of the cytoskeleton in tubular cells, and the Rho/ROCK/LIMK-mediated cofilin phosphorylation is a key mechanism......Hyperosmotic stress induces cytoskeleton reorganization and a net increase in cellular F-actin, but the underlying mechanisms are incompletely understood. While de novo F-actin polymerization likely contributes to the actin response, the role of F-actin severing is unknown. To address this problem...... cofilin. Here we show that hyperosmolarity induced rapid, sustained and reversible phosphorylation of cofilin in kidney tubular (LLC-PK1 and MDCK) cells. Hyperosmolarity-provoked cofilin phosphorylation was mediated by the Rho/Rho kinase (ROCK)/LIM Kinase (LIMK) but not the Rac/PAK/LIMK pathway, because a...

  4. Cigarette sidestream smoke induces histone H3 phosphorylation via JNK and PI3K/Akt pathways, leading to the expression of proto-oncogenes.

    Science.gov (United States)

    Ibuki, Yuko; Toyooka, Tatsushi; Zhao, Xiaoxu; Yoshida, Ikuma

    2014-06-01

    Post-translational modifications in histones have been associated with cancer. Although cigarette sidestream smoke (CSS) as well as mainstream smoke are carcinogens, the relationship between carcinogenicity and histone modifications has not yet been clarified. Here, we demonstrated that CSS induced phosphorylation of histones, involving a carcinogenic process. Treatment with CSS markedly induced the phosphorylation of histone H3 at serine 10 and 28 residues (H3S10 and H3S28), which was independent from the cell cycle, in the human pulmonary epithelial cell model, A549 and normal human lung fibroblasts, MRC-5 and WI-38. Using specific inhibitors and small interfering RNA, the phosphorylation of H3S10 was found to be mediated by c-jun N-terminal kinase (JNK) and phosphoinositide 3-kinase (PI3K)/Akt pathways. These pathways were different from that of the CSS-induced phosphorylation of histone H2AX (γ-H2AX) mediated by Ataxia telangiectasia-mutated (ATM) and ATM-Rad3-related (ATR) protein kinases. A chromatin immunoprecipitation assay revealed that the phosphorylation of H3S10 was increased in the promoter sites of the proto-oncogenes, c-fos and c-jun, which indicated that CSS plays a role in tumor promotion. Because the phosphorylation of H3S10 was decreased in the aldehyde-removed CSS and was significantly induced by treatment with formaldehyde, aldehydes are suspected to partially contribute to this phosphorylation. These findings suggested that any chemicals in CSS, including aldehydes, phosphorylate H3S10 via JNK and PI3K/Akt pathways, which is different from the DNA damage response, resulting in tumor promotion.

  5. Inhibition of Emodin on LPS-induced Nitric Oxide Generation by Suppressing PLC-γ Phosphorylation in Rat Peritoneal Macrophages

    Institute of Scientific and Technical Information of China (English)

    WANG Xin-yu; CAI Shou-guang; WU Yi-fen; LI Jun-ying; YANG Wen-xiu; HU Fen

    2010-01-01

    Objective To investigate the inhibitory mechanism of emodin on lipopolysaccharide(LPS)-induced nitric oxide(NO)generation in rat peritoneal macrophages.Methods NO production and iNOS expression were measured through nitrite assay and Western blotting assay,respectively.NF-kB activity and nuclei P65 expression were estimated by dual-luciferase and Western blotting assay,respectively.Intracellular free Ca2+([Ca2+]i)was detected using the ratiometric fluorescent calcium indicator dye,Fura-2,and a microspectrofluorometer.PLC-γphosporylation was analyzed by Western blotting assay.Results First,emodin was found playing active roles in suppressing LPS-induced NF-kB activation in rat peritoneal macrophages.Second,emodin down-regulated transient[Ca2*]i and could increase in NF-kB upstream signal.Finally,emodin suppressed phosphorylation of PLC-γ by LPS stimulation in the upstream of[Ca2+]i.Conclusion Suppression of PLC-γ phosphorylation is involved in emodin inhibiting NO generation by LPS stimulation in rat peritoneal macrophages.

  6. Tyrosine-phosphorylated caveolin-1 blocks bacterial uptake by inducing Vav2-RhoA-mediated cytoskeletal rearrangements.

    Directory of Open Access Journals (Sweden)

    Jan Peter Boettcher

    Full Text Available Certain bacterial adhesins appear to promote a pathogen's extracellular lifestyle rather than its entry into host cells. However, little is known about the stimuli elicited upon such pathogen host-cell interactions. Here, we report that type IV pili (Tfp-producing Neisseria gonorrhoeae (P(+GC induces an immediate recruitment of caveolin-1 (Cav1 in the host cell, which subsequently prevents bacterial internalization by triggering cytoskeletal rearrangements via downstream phosphotyrosine signaling. A broad and unbiased analysis of potential interaction partners for tyrosine-phosphorylated Cav1 revealed a direct interaction with the Rho-family guanine nucleotide exchange factor Vav2. Both Vav2 and its substrate, the small GTPase RhoA, were found to play a direct role in the Cav1-mediated prevention of bacterial uptake. Our findings, which have been extended to enteropathogenic Escherichia coli, highlight how Tfp-producing bacteria avoid host cell uptake. Further, our data establish a mechanistic link between Cav1 phosphorylation and pathogen-induced cytoskeleton reorganization and advance our understanding of caveolin function.

  7. Protein phosphorylation is involved in the water stress induced ABA accumulation in the roots of Malus hupehensis Rehd

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Water stress-induced ABA accumulation plays a key role in the root to shoot communication and/or the cell to cell signaling under the soil stresses. The signaling of the water stress itself that leads to the accumulation, however, is less known. In this study, we subjected the roots of Malus hupehensis seedlings to water stress treatment and investigated the ABA accumulation in relation to protein phosphorylation. Our results showed that ABA accumulation could be substantially triggered in 40 min and reached 4 folds in 100 min after treatment with 30% PEG 6000 (polyethylene glycol). The water stress treatment also led to a substantial enhancement of total kinase activity, assessed with histone-Ⅲ as substrate, in 15 min and a maximum enhancement in 30 min before it declined to initial level. The Ca2+-dependent kinase activity showed a similar, if not more sensitive, trend. When the roots were fed with labeled 32pATP, water stress enhanced the labeling of proteins, which showed a maximum labeling at 40 min. Two inhibitors of protein kinases, Quercetin and H7, effectively diminished or completely blocked the ABA accumulation under the stress treatment. It is therefore suggest that protein phosphorylation is involved in the signaling of the water stress-induced ABA accumulation.

  8. Dasatinib promotes paclitaxel-induced necroptosis in lung adenocarcinoma with phosphorylated caspase-8 by c-Src.

    Science.gov (United States)

    Diao, Yan; Ma, Xiaobin; Min, WeiLi; Lin, Shuai; Kang, HuaFeng; Dai, ZhiJun; Wang, Xijing; Zhao, Yang

    2016-08-28

    Cisplatin and paclitaxel are considered to be the backbone of chemotherapy in lung adenocarcinoma. These agents show pleiotropic effects on cell death. However, the precise mechanisms remain unclear. The present study reported that phosphorylated caspase-8 at tyrosine 380 (p-Casp8) was characterized as a biomarker of chemoresistance to TP regimen (cisplatin and paclitaxel) in patients with resectable lung adenocarcinoma with significantly poorer 5-year disease-free survival (DFS) and overall survival (OS). Cisplatin killed lung adenocarcinoma cells regardless of c-Src-induced caspase-8 phosphorylation at tyrosine 380. Subsequently, we identified a novel mechanism by which paclitaxel induced necroptosis in lung adenocarcinoma cells that was dependent upon p-Casp8, receptor-interacting protein kinase 1 (RIPK1), and RIPK3. Moreover, dasatinib, a c-Src inhibitor, dephosphorylated caspase-8 to facilitate necroptosis, rather than apoptosis, in paclitaxel-treated p-Casp8-expressing lung adenocarcinoma cells. The data from our study revealed previously unrecognized roles of p-Casp8 as a positive effector in the initiation of necroptosis and as a negative effector in the repression of the interaction between RIPK1 and RIPK3. Moreover, these outcomes supported the need for further clinical studies with the goal of evaluating the efficacy of dasatinib plus paclitaxel in the treatment of lung adenocarcinoma. PMID:27195913

  9. Spinal D1-like dopamine receptors modulate NMDA receptor-induced hyperexcitability and NR1 subunit phosphorylation at serine 889.

    Science.gov (United States)

    Aira, Zigor; Barrenetxea, Teresa; Buesa, Itsaso; Martínez, Endika; Azkue, Jon Jatsu

    2016-04-01

    Activation of the N-methyl-d-aspartate receptor (NMDAR) in dorsal horn neurons is recognized as a fundamental mechanism of central sensitization and pathologic pain. This study assessed the influence of dopaminergic, D1-like receptor-mediated input to the spinal dorsal horn on NMDAR function. Spinal superfusion with selective NMDAR agonist cis-ACPD significantly increased C-fiber-evoked field potentials in rats subjected to spinal nerve ligation (SNL), but not in sham-operated rats. Simultaneous application of D1LR antagonist SCH 23390 dramatically reduced hyperexcitability induced by cis-ACPD. Furthermore, cis-ACPD-induced hyperexcitability seen in nerve-ligated rats could be mimicked in unin-jured rats during stimulation of D1LRs by agonist SKF 38393 at subthreshold concentration. Phosphorylation of NMDAR subunit NR1 at serine 889 at postsynaptic sites was found to be increased in dorsal horn neurons 90 min after SNL, as assessed by increased co-localization with postsynaptic marker PSD-95. Increased NR1 phosphorylation was attenuated in the presence of SCH 23390 in the spinal superfusate. The present results support that D1LRs regulate most basic determinants of NMDAR function in dorsal horn neurons, suggesting a potential mechanism whereby dopaminergic input to the dorsal horn can modulate central sensitization and pathologic pain.

  10. Effect of molecular aggregation on the photo-induced anisotropy in amorphous polymethacrylate bearing an aminonitroazobenzene moiety

    CERN Document Server

    Kim, B J; Choi, D H

    2001-01-01

    We investigated H-type molecular aggregation in a simply spin-coated amorphous homopolymer film of polymethacrylate containing push-pull azobenzene moieties. It was found that the aggregate formation was strongly influenced by thermal treatment and that the aggregate created in the polymer film could be easily disrupted by irradiation of a linearly polarized light. In the first writing cycle of aggregated polymer film, photo-induced birefringence showed a steep increase to the highest value followed by a gradual decrease to the certain asymptotic value under longer irradiation of linearly polarized light. This unique behavior could be attributed to the cooperative motion and the disruption of the aggregated molecules under continuous irradiation of light.

  11. Activation of ERK induces phosphorylation of MAPK phosphatase-7, a JNK specific phosphatase, at Ser-446.

    Science.gov (United States)

    Masuda, Kouhei; Shima, Hiroshi; Katagiri, Chiaki; Kikuchi, Kunimi

    2003-08-22

    We previously showed that MKP-7 suppresses MAPK activation in COS-7 cells in the order of selectivity, JNK > p38 > ERK, but interacts with ERK as well as JNK and p38. In this study we found that, when expressed in COS-7 cells with HA-ERK2, the mobility of FLAG-MKP-7 was decreased on SDS-PAGE gels depending on several stimuli, including phorbol 12-myristate 13-acetate, fetal bovine serum, epidermal growth factor, H2O2, and ionomycin. By using U0126, a MEK inhibitor, and introducing several point mutations, we demonstrated that this upward mobility shift is because of phosphorylation and identified Ser-446 of MKP-7 as the phosphorylation site targeted by ERK activation. To determine how MKP-7 interacts with MAPKs, we identified three domains in MKP-7 required for interaction with MAPKs, namely, putative MAP kinase docking domains (D-domain) I and II and a long COOH-terminal stretch unique to MKP-7. The D-domain I is required for interaction with ERK and p38, whereas the D-domain II is required for interaction with JNK and p38, which is likely to be important for MKP-7 to suppress JNK and p38 activations. The COOH-terminal stretch of MKP-7 was shown to determine JNK preference for MKP-7 by masking MKP-7 activity toward p38 and is a domain bound by ERK. These data strongly suggested that Ser-446 of MKP-7 is phosphorylated by ERK. PMID:12794087

  12. The mitogen-activated protein (MAP) kinase ERK induces tRNA synthesis by phosphorylating TFIIIB

    OpenAIRE

    Felton-Edkins, Zoe A.; Fairley, Jennifer A.; Graham, Emma L.; Johnston, Imogen M.; White, Robert J.; Scott, Pamela H.

    2003-01-01

    RNA polymerase (pol) III transcription increases within minutes of serum addition to growth-arrested fibroblasts. We show that ERK mitogen-activated protein kinases regulate pol III output by directly binding and phosphorylating the BRF1 subunit of transcription factor TFIIIB. Blocking the ERK signalling cascade inhibits TFIIIB binding to pol III and to transcription factor TFIIIC2. Chromatin immunoprecipitation shows that the association of BRF1 and pol III with tRNALeu genes in cells decrea...

  13. Cell nonhomologous end joining capacity controls SAF-A phosphorylation by DNA-PK in response to DNA double-strand breaks inducers.

    Science.gov (United States)

    Britton, Sébastien; Froment, Carine; Frit, Philippe; Monsarrat, Bernard; Salles, Bernard; Calsou, Patrick

    2009-11-15

    Aiming to identify novel phosphorylation sites in response to DNA double-strand breaks (DSB) inducers, we have isolated a phosphorylation site on KU70. Unexpectedly, a rabbit antiserum raised against this site cross-reacted with a 120 kDa protein in cells treated by DNA DSB inducers. We identified this protein as SAF-A/hnRNP U, an abundant and essential nuclear protein containing regions binding DNA or RNA. The phosphorylation site was mapped at S59 position in a sequence context favoring a "S-hydrophobic" consensus model for DNA-PK phosphorylation site in vivo. This site was exclusively phosphorylated by DNA-PK in response to DNA DSB inducers. In addition, the extent and duration of this phosphorylation was in inverse correlation with the capacity of the cells to repair DSB by Nonhomologous End Joining. These results bring a new link between the hnRNP family and the DNA damage response. Addtionaly, the mapped phospho-site on SAF-A might serve as a potential bio-marker for DNA-PK activity in academic studies and clinical analyses of DNA-PK activators or inhibitors. PMID:19844162

  14. Phosphorylation-triggered CUEDC2 degradation promotes UV-induced G1 arrest through APC/C(Cdh1) regulation.

    Science.gov (United States)

    Zhang, Wei-Na; Zhou, Jie; Zhou, Tao; Li, Ai-Ling; Wang, Na; Xu, Jin-Jing; Chang, Yan; Man, Jiang-Hong; Pan, Xin; Li, Tao; Li, Wei-Hua; Mu, Rui; Liang, Bing; Chen, Liang; Jin, Bao-Feng; Xia, Qing; Gong, Wei-Li; Zhang, Xue-Min; Wang, Li; Li, Hui-Yan

    2013-07-01

    DNA damage triggers cell cycle arrest to provide a time window for DNA repair. Failure of arrest could lead to genomic instability and tumorigenesis. DNA damage-induced G1 arrest is generally achieved by the accumulation of Cyclin-dependent kinase inhibitor 1 (p21). However, p21 is degraded and does not play a role in UV-induced G1 arrest. The mechanism of UV-induced G1 arrest thus remains elusive. Here, we have identified a critical role for CUE domain-containing protein 2 (CUEDC2) in this process. CUEDC2 binds to and inhibits anaphase-promoting complex/cyclosome-Cdh1 (APC/C(Cdh1)), a critical ubiquitin ligase in G1 phase, thereby stabilizing Cyclin A and promoting G1-S transition. In response to UV irradiation, CUEDC2 undergoes ERK1/2-dependent phosphorylation and ubiquitin-dependent degradation, leading to APC/C(Cdh1)-mediated Cyclin A destruction, Cyclin-dependent kinase 2 inactivation, and G1 arrest. A nonphosphorylatable CUEDC2 mutant is resistant to UV-induced degradation. Expression of this stable mutant effectively overrides UV-induced G1-S block. These results establish CUEDC2 as an APC/C(Cdh1) inhibitor and indicate that regulated CUEDC2 degradation is critical for UV-induced G1 arrest.

  15. Changes of conformation and aggregation state induced by binding of lanthanide ions to insulin

    Institute of Scientific and Technical Information of China (English)

    程驿; 李荣昌; 王夔

    2002-01-01

    To clarify the mechanism of lanthanide ions (Ln3+) on the across-membrane transport of insulin and subsequent reducing blood glucose, the interactions of Ln3+ with Zn-insulin and Zn-free insulin are investigated by spectroscopic methods. The results reveal that the binding of Ln3+ to insulin can induce its structure changes from secondary to quaternary structure, depending on the Ln3+ concentration. In the lower concentration, it triggers the conformational changes of insulin monomer in the binding region with insulin receptor (B(24-30)). It would affect insulin-insulin receptor interaction. Moreover, Ln3+ binding promotes the assembly of insulin monomer from dimer to polymer. The potency of Ln3+ in inducing insulin's aggregation is stronger than that of Zn2+. Furthermore, the aggregation can be reversed partly by EDTA-treatment, indicating that it is not due to denaturation. Similar to Zn2+ effect, Ln3+ can stabilize insulin hexamer in a certain range of concentration, but is stronger than the former.

  16. Changes of conformation and aggregation state induced by binding of lanthanide ions to insulin

    Institute of Scientific and Technical Information of China (English)

    程驿; 李荣昌; 王夔

    2002-01-01

    To clarify the mechanism of lanthanide ions (Ln3+) on the across-membrane transport of insulin and subsequent reducing blood glucose, the interactions of Ln3+with Zn-insulin and Zn-free insulin are investigated by spectroscopic methods. The results reveal that the binding of Ln3+ to insulin can induce its structure changes from secondary to quaternary structure, depending on the Ln3+ concentration. In the lower concentration, it triggers the conformational changes of insulin monomer in the binding region with insulin receptor (B(24-30)). It would affect insulin-insulin receptor interaction. Moreover, Ln3+ binding promotes the assembly of insulin monomer from dimer to polymer. The potency of Ln3+ in inducing insulin’s aggregation is stronger than that of Zn2+. Furthermore, the aggregation can be reversed partly by EDTA-treatment, indicating that it is not due to denaturation. Similar to Zn2+ effect, Ln3+ can stabilize insulin hexamer in a certain range of concentration, but is stronger than the former.

  17. Composite alginate hydrogel microparticulate delivery system of zidovudine hydrochloride based on counter ion induced aggregation

    Science.gov (United States)

    Roy, Harekrishna; Rao, P. Venkateswar; Panda, Sanjay Kumar; Biswal, Asim Kumar; Parida, Kirti Ranjan; Dash, Jharana

    2014-01-01

    Aim: The present study deals with preparation of zidovudine loaded microparticle by counter ion induced aggregation method. During this study effect of polyacrylates and hypromellose polymers on release study were investigated. Materials and Methods: The ion induced aggregated alginate based microparticles were characterized for surface morphology, particle size analysis, drug entrapment study, in-vitro study, Fourier-transform infrared (FTIR) spectroscopy, and differential scanning calorimetry (DSC) study. Results and Discussion: The result showed Eudragit RL-100 (ERL) based formulations had smoother surface as well as their mean particle sizes were found greater compared with Eudragit RS-100 (ERS) microparticles. Furthermore, drug entrapments were found to be more in ERL formulae as compared with ERS. RL3 released 101.05% drug over a period of 8th h and followed Higuchi profile and Fickian diffusion. Moreover, data obtained illustrated that, higher amount of quaternary ammonium group, alkali value, and glass transition temperature may be possible reason for improving permeability of ERL based formulations. It was also noticed, hyroxypropyl methylcellulose (HPMC) K4M premium grade polymer sustained drug release more than HPMC K15M. In addition, drug-excipient interaction study was carried out by FTIR and DSC study. PMID:25298940

  18. Imatinib induces H2AX phosphorylation and apoptosis in chronic myelogenous leukemia cells in vitro via caspase-3/Mst1 pathway

    Institute of Scientific and Technical Information of China (English)

    Yan-jun ZHANG; Lian-ning DUAN; Cheng-rong LU; Yan CAO; Yuan LUO; Rong-feng BAO; Shu YAN; Mei XUE; Feng ZHU; Zhe WANG

    2012-01-01

    Aim:Histone H2AX is a novel tumor suppressor and its phosphorylation at the C terminus (Ser139 and Tyr142)is required for tumor cell apoptosis.The aim of the present study was to elucidate the mechanisms underlying imatinib-induced C-terminal phosphorylation of H2AX in chronic myelogenous leukemia cells in vitro.Methods:BCR-ABL-positive K562 cells were used.Microscopy,Western blotting and flow cytometry were used to study the signaling pathways that regulate imatinib-induced H2AX phosphorylation and the apoptotic mechanisms.Results:Treatment of K562 cells with imatinib (1-8 μmol/L)induced phosphorylation of H2AX at Ser139 and Tyr142 in time-and dose-dependent manners.In contrast,imatinib at the same concentrations did not affect H2AX acetylation at Lys 5,and the acetylated H2AX maintained a higher level in the cells.Meanwhile,imatinib (1-8 μmol/L)activated caspase-3 and its downstream mammalian STE20-like kinase 1 (Mst1),and induced apoptosis of K562 cells.The caspase-3 inhibitor Z-VAD (40 μmol/L)reduced imatinibinduced H2AX phosphorylation at Ser139 and Tyr142 and blocked imatinib-induced apoptosis of K562 cells.Imatinib (4 μmol/L)induced expression of Williams-Beuren syndrome transcription factor (WSTF),but not wild-type p53-induced phosphatase 1 (Wip1)in K562 cells.Conclusion:The caspase-3/Mst1 pathway is required for H2AX C-terminal phosphorylation at Ser139 and Tyr142 and subsequent apoptosis in Bcr-Abl-positive K562 cells induced by imatinib.

  19. The secondary and aggregation structural changes of BSA induced by trivalent chromium: A biophysical study

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Mingmao [Institute of Biomedical and Pharmaceutical Technology, Fuzhou University, Fuzhou 350002, Fujian (China); Liu, Yan, E-mail: liuyan@fjirsm.ac.cn [The State Key Lab of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou 350002, Fujian (China); Cao, Huan [Institute of Biomedical and Pharmaceutical Technology, Fuzhou University, Fuzhou 350002, Fujian (China); Song, Ling [The State Key Lab of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou 350002, Fujian (China); Zhang, Qiqing, E-mail: zhangqiq@126.com [Institute of Biomedical and Pharmaceutical Technology, Fuzhou University, Fuzhou 350002, Fujian (China)

    2015-02-15

    Trivalent chromium Cr(III), which was originally considered to be innocuous as a nutriment, has been suspected to induce some abnormalities in human body recently. In the present work, the effects of Cr(III) on the structural state of BSA were comprehensively investigated through a series of appropriate methods in combination, including X-ray photoelectron spectroscopy (XPS), fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), UV–vis absorption, synchronous fluorescence, fluorescence lifetime analysis, resonance light scattering (RLS), dynamic light scattering (DLS) and excitation–emission matrix spectroscopy (EEMS) methods. XPS accurately described the binding activity of Cr(III) with protein C, N and O atoms. The structural analysis according to FTIR and CD methods showed that the Cr(III) binding altered BSA conformation with a major reduction of α-helix. RLS and DLS analyses demonstrated that the presence of Cr(III) with low concentration could induce the aggregation structural changes of BSA. UV–vis absorption, EEMS and synchronous fluorescence suggested that the interaction between Cr(III) and BSA induced a slight unfolding of the polypeptide backbone and altered the microenvironments of Trp and Tyr residues in BSA. This research is helpful for understanding the structure-function relationship involved in metal ion-protein bioconjugate process. - Highlights: • The effect of Cr(III) on the conformational state of BSA was comprehensively studied. • XPS described the binding activity of Cr(III) with protein C, N and O atoms. • FTIR and CD data revealed secondary structural alteration in BSA. • Cr(III) complexation induced microenvironmental changes of Trp and Tyr. • RLS, DLS and EEMS presented the aggregational states of Cr(III)–BSA complex.

  20. The secondary and aggregation structural changes of BSA induced by trivalent chromium: A biophysical study

    International Nuclear Information System (INIS)

    Trivalent chromium Cr(III), which was originally considered to be innocuous as a nutriment, has been suspected to induce some abnormalities in human body recently. In the present work, the effects of Cr(III) on the structural state of BSA were comprehensively investigated through a series of appropriate methods in combination, including X-ray photoelectron spectroscopy (XPS), fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), UV–vis absorption, synchronous fluorescence, fluorescence lifetime analysis, resonance light scattering (RLS), dynamic light scattering (DLS) and excitation–emission matrix spectroscopy (EEMS) methods. XPS accurately described the binding activity of Cr(III) with protein C, N and O atoms. The structural analysis according to FTIR and CD methods showed that the Cr(III) binding altered BSA conformation with a major reduction of α-helix. RLS and DLS analyses demonstrated that the presence of Cr(III) with low concentration could induce the aggregation structural changes of BSA. UV–vis absorption, EEMS and synchronous fluorescence suggested that the interaction between Cr(III) and BSA induced a slight unfolding of the polypeptide backbone and altered the microenvironments of Trp and Tyr residues in BSA. This research is helpful for understanding the structure-function relationship involved in metal ion-protein bioconjugate process. - Highlights: • The effect of Cr(III) on the conformational state of BSA was comprehensively studied. • XPS described the binding activity of Cr(III) with protein C, N and O atoms. • FTIR and CD data revealed secondary structural alteration in BSA. • Cr(III) complexation induced microenvironmental changes of Trp and Tyr. • RLS, DLS and EEMS presented the aggregational states of Cr(III)–BSA complex

  1. Dermcidin isoform-2 induced nullification of the effect of acetyl salicylic acid in platelet aggregation in acute myocardial infarction

    OpenAIRE

    Sarbashri Bank; Pradipta Jana; Smarajit Maiti; Santanu Guha; Sinha, A.K.

    2014-01-01

    The aggregation of platelets on the plaque rupture site on the coronary artery is reported to cause both acute coronary syndromes (ACS) and acute myocardial infarction (AMI). While the inhibition of platelet aggregation by acetyl salicylic acid was reported to produce beneficial effects in ACS, it failed to do in AMI. The concentration of a stress induced protein (dermcidin isoform-2) was much higher in AMI than that in ACS. Incubation of normal platelet rich plasma (PRP) with dermcidin showe...

  2. RNA-dependent protein kinase (PKR) depletes nutrients, inducing phosphorylation of AMP-activated kinase in lung cancer.

    Science.gov (United States)

    Guo, Chengcheng; Hao, Chuncheng; Shao, RuPing; Fang, Bingliang; Correa, Arlene M; Hofstetter, Wayne L; Roth, Jack A; Behrens, Carmen; Kalhor, Neda; Wistuba, Ignacio I; Swisher, Stephen G; Pataer, Apar

    2015-05-10

    We have demonstrated that RNA-dependent protein kinase (PKR) and its downstream protein p-eIF2α are independent prognostic markers for overall survival in lung cancer. In the current study, we further investigate the interaction between PKR and AMPK in lung tumor tissue and cancer cell lines. We examined PKR protein expression in 55 frozen primary lung tumor tissues by Western blotting and analyzed the association between PKR expression and expression of 139 proteins on tissue samples examined previously by Reverse Phase Protein Array (RPPA) from the same 55 patients. We observed that biomarkers were either positively (phosphorylated AMP-activated kinase(T172) [p-AMPK]) or negatively (insulin receptor substrate 1, meiotic recombination 11, ATR interacting protein, telomerase, checkpoint kinase 1, and cyclin E1) correlated with PKR. We further confirmed that induction of PKR with expression vectors in lung cancer cells causes activation of the AMPK protein independent of the LKB1, TAK1, and CaMKKβ pathway. We found that PKR causes nutrient depletion, which increases AMP levels and decreases ATP levels, causing AMPK phosphorylation. We further demonstrated that inhibiting AMPK expression with compound C or siRNA enhanced PKR-mediated cell death. We next explored the combination of PKR and p-AMPK expression in NSCLC patients and observed that expression of p-AMPK predicted a poor outcome for adenocarcinoma patients with high PKR expression and a better prognosis for those with low PKR expression. These findings were consistent with our in vitro results. AMPK might rescue cells facing metabolic stresses, such as ATP depletion caused by PKR. Our data indicate that PKR causes nutrient depletion, which induces the phosphorylation of AMPK. AMPK might act as a protective response to metabolic stresses, such as nutrient deprivation. PMID:25798539

  3. Nuclear translocation of phosphorylated STAT3 regulates VEGF-A-induced lymphatic endothelial cell migration and tube formation

    Energy Technology Data Exchange (ETDEWEB)

    Okazaki, Hideki; Tokumaru, Sho; Hanakawa, Yasushi; Shiraishi, Ken; Shirakata, Yuji; Dai, Xiuju; Yang, Lijun; Tohyama, Mikiko; Hashimoto, Koji [Department of Dermatology, Ehime University Graduate School of Medicine, Toon, Ehime 791-0295 (Japan); Sayama, Koji, E-mail: sayama@m.ehime-u.ac.jp [Department of Dermatology, Ehime University Graduate School of Medicine, Toon, Ehime 791-0295 (Japan)

    2011-09-02

    Highlights: {yields} VEGF-A enhanced lymphatic endothelial cell migration and increased tube formation. {yields} VEGF-A treated lymphatic endothelial cell showed activation of STAT3. {yields} Dominant-negative STAT3 inhibited VEGF-A-induced lymphatic endothelial cell migration and tube formation. -- Abstract: Vascular endothelial growth factor (VEGF) is an endothelial cell-specific growth factor that regulates endothelial functions, and signal transducers and activators of transcription (STATs) are known to be important during VEGF receptor signaling. The aim of this study was to determine whether STAT3 regulates VEGF-induced lymphatic endothelial cell (LEC) migration and tube formation. VEGF-A (33 ng/ml) enhanced LEC migration by 2-fold and increased tube length by 25% compared with the control, as analyzed using a Boyden chamber and Matrigel assay, respectively. Western blot analysis and immunostaining revealed that VEGF-A induced the nuclear translocation of phosphorylated STAT3 in LECs, and this translocation was blocked by the transfection of LECs with an adenovirus vector expressing a dominant-negative mutant of STAT3 (Ax-STAT3F). Transfection with Ax-STAT3F also almost completely inhibited VEGF-A-induced LEC migration and tube formation. These results indicate that STAT3 is essential for VEGF-A-induced LEC migration and tube formation and that STAT3 regulates LEC functions.

  4. Role of pH-induced structural change in protein aggregation in foam fractionation of bovine serum albumin

    Directory of Open Access Journals (Sweden)

    Rui Li

    2016-03-01

    Full Text Available For reducing protein aggregation in foam fractionation, the role of pH-induced structural change in the interface-induced protein aggregation was analyzed using bovine serum albumin (BSA as a model protein. The results show that the decrease in pH from 7.0 to 3.0 gradually unfolded the BSA structure to increase the molecular size and the relative content of β-sheet and thus reduced the stability of BSA in the aqueous solution. At the isoelectric point (pH 4.7, BSA suffered the lowest level in protein aggregation induced by the gas–liquid interface. In the pH range from 7.0 to 4.7, most BSA aggregates were formed in the defoaming process while in the pH range from 4.7 to 3.0, the BSA aggregates were formed at the gas–liquid interface due to the unfolded BSA structure and they further aggregated to form insoluble ones in the desorption process.

  5. Decreased hepatic phosphorylated p38 mitogen-activated protein kinase contributes to attenuation of thioacetamide-induced hepatic necrosis in diet-induced obese mice.

    Science.gov (United States)

    Shirai, Makoto; Arakawa, Shingo; Teranishi, Munehiro; Kai, Kiyonori

    2016-04-01

    We previously reported that thioacetamide (TA)-induced hepatocellular necrosis was attenuated in mice fed a high-fat diet (HFD mice) compared with mice fed a normal rodent diet (ND mice). In this study, we investigated whether p38 mitogen-activated protein kinase (p38 MAPK) was involved in this attenuation. Western blot analysis revealed that hepatic phosphorylated p38 MAPK protein decreased at 8 and 24 hours (hr) after TA dosing in the HFD mice, while it decreased only at 24 hr in the ND mice in comparison to the time- and diet-matched, vehicle-treated mice. p38 MAPK regulates various biological functions including inflammation, therefore, hepatic metabolomics analysis focusing on pro-inflammatory lipid mediators was performed. At 24 hr after TA dosing, only one pro-inflammatory mediator, 12-hydroxyeicosatetraenoic acid (HETE), was higher in the HFD mice. On the other hand, in addition to 12-HETE, 15-HETE and 12-hydroxyeicosapentaenoic acid (HEPE) were higher and omega-3/omega-6 polyunsaturated fatty acids ratios were lower in the ND mice at 24 hr. These results of metabolomics indicated that less pro-inflammatory state was seen in HFD mice than in ND mice at 24 hr. Finally, to confirm whether the observed decrease in phosphorylated p38 MAPK could attenuate TA-induced hepatocellular necrosis, we showed that SB203580 hydrochloride, an inhibitor of p38 MAPK, partially attenuated TA-induced hepatic necrosis in ND mice. Collectively, these results suggest that a prompt decrease in phosphorylation of p38 MAPK after TA administration is one of the factors that attenuate TA-induced hepatic necrosis in HFD mice. PMID:26961609

  6. Trichothecin induces cell death in NF-κB constitutively activated human cancer cells via inhibition of IKKβ phosphorylation.

    Directory of Open Access Journals (Sweden)

    Jia Su

    Full Text Available Constitutive activation of the transcription factor nuclear factor-κB (NF-κB is involved in tumorigenesis and chemo-resistance. As the key regulator of NF-κB, IKKβ is a major therapeutic target for various cancers. Trichothecin (TCN is a metabolite isolated from an endophytic fungus of the herbal plant Maytenus hookeri Loes. In this study, we evaluated the anti-tumor activity of TCN and found that TCN markedly inhibits the growth of cancer cells with constitutively activated NF-κB. TCN induces G0/G1 cell cycle arrest and apoptosis in cancer cells, activating pro-apoptotic proteins, including caspase-3, -8 and PARP-1, and decreasing the expression of anti-apoptotic proteins Bcl-2, Bcl-xL, and survivin. Reporter activity assay and target genes expression analysis illustrated that TCN works as a potent inhibitor of the NF-κB signaling pathway. TCN inhibits the phosphorylation and degradation of IκBα and blocks the nuclear translocation of p65, and thus inhibits the expression of NF-κB target genes XIAP, cyclin D1, and Bcl-xL. Though TCN does not directly interfere with IKKβ kinase, it suppresses the phosphorylation of IKKβ. Overexpression of constitutively activated IKKβ aborted TCN induced cancer cell apoptosis, whereas knockdown of endogenous IKKβ with siRNA sensitized cancer cells toward apoptosis induced by TCN. Moreover, TCN showed a markedly weaker effect on normal cells. These findings suggest that TCN may be a potential therapeutic candidate for cancer treatment, targeting NF-κB signaling.

  7. Acute exercise and physiological insulin induce distinct phosphorylation signatures on TBC1D1 and TBC1D4 in human skeletal muscle

    DEFF Research Database (Denmark)

    Treebak, Jonas Thue; Pehmøller, Christian; Kristensen, Jonas Møller;

    2014-01-01

    We investigated the phosphorylation signatures of two Rab GTPase activating proteins TBC1D1 and TBC1D4 in human skeletal muscle in response to physical exercise and physiological insulin levels induced by a carbohydrate rich meal using a paired experimental design. Eight healthy male volunteers...... in response to physiological stimuli in human skeletal muscle and support the idea that Akt and AMPK are upstream kinases. TBC1D1 phosphorylation signatures were comparable between in vitro contracted mouse skeletal muscle and exercised human muscle, and we show that AMPK was regulating phosphorylation...... of these sites in mouse muscle. Contraction and exercise elicited a different phosphorylation pattern of TBC1D4 in mouse compared with human muscle, and although different circumstances in our experimental setup may contribute to this difference, the observation exemplifies that transferring findings between...

  8. R-Ras Inhibits VEGF-Induced p38MAPK Activation and HSP27 Phosphorylation in Endothelial Cells.

    Science.gov (United States)

    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

  9. Gab2 is phosphorylated on tyrosine upon interleukin-2/interleukin-15 stimulation in mycosis-fungoides-derived tumor T cells and associates inducibly with SHP-2 and Stat5a

    DEFF Research Database (Denmark)

    Brockdorff, J L; Gu, H; Mustelin, T;

    2001-01-01

    mycosis fungoides (MF) tumor T cells upon IL-2 stimulation and that SHP2 as well as Stat5a associated inducibly with Gab2. IL-15, but not IL-4, also induced tyrosine phosphorylation of Gab2, suggesting that the IL-2 receptor beta-chain is important for IL-2-induced Gab2 phosphorylation. Preincubation...

  10. Interplay between desolvation and secondary structure in mediating cosolvent and temperature induced alpha-synuclein aggregation

    Science.gov (United States)

    Anderson, V. L.; Webb, W. W.; Eliezer, D.

    2012-10-01

    Both increased temperature and moderate concentrations of fluorinated alcohols enhance aggregation of the Parkinson's disease-associated protein α-synuclein (αS). Here, we investigate the secondary structural rearrangements induced by heating and trifluoroethanol [TFE]. At low TFE concentrations, CD spectra feature a negative peak characteristic of disordered polypeptides near 200 nm and a slight shoulder around 220 nm suggesting some polyproline-II content. Upon heating, these peaks weaken, while a weak negative signal develops at 222 nm. At high TFE concentrations, the spectra show distinct minima at 208 and 222 nm, indicative of considerable α-helical structure, which diminish upon heating. We observe a crossover between the low-TFE and high-TFE behavior near 15% TFE, where we previously showed that a partially helical intermediate is populated. We postulate that the protein is well solvated by water at low TFE concentrations and by TFE at high TFE concentrations, but may become desolvated at the crossover point. We discuss the potential roles and interplay of desolvation and helical secondary structure in driving αS aggregation.

  11. Aggregation-induced emissive nanoparticles for fluorescence signaling in a low cost paper-based immunoassay.

    Science.gov (United States)

    Engels, Jan F; Roose, Jesse; Zhai, Demi Shuang; Yip, Ka Man; Lee, Mei Suet; Tang, Ben Zhong; Renneberg, Reinhard

    2016-07-01

    Low cost paper based immunoassays are receiving interest due to their fast performance and small amounts of biomolecules needed for developing an immunoassay complex. In this work aggregation-induced emissive (AIE) nanoparticles, obtained from a diastereoisomeric mixture of 1,2-di-(4-hydroxyphenyl)-1,2-diphenylethene (TPEDH) in a one-step top-down method, are characterized through Dynamic Light Scattering (DLS), Scanning Electron Microscopy (SEM), and Zeta potential. By measuring the Zeta potential before and after labeling the nanoparticles with antibodies we demonstrate that the colloidal system is stable in a wide pH-range. The AIE-active nanoparticles are deposited on chitosan and glutaraldehyde modified paper pads overcoming the common aggregation-caused quenching (ACQ) effect. Analyte concentrations from 1000ng and below are applied in a model immunocomplex using Goat anti-Rabbit IgG and Rabbit IgG. In the range of 7.81ng-250ng, linear trends with a high R(2) are observed, which leads to a strong increase of the blue fluorescence from the TPEDH nanoparticles. PMID:27037781

  12. The differential DRP1 phosphorylation and mitochondrial dynamics in the regional specific astroglial death induced by status epilepticus

    Directory of Open Access Journals (Sweden)

    Ah-Reum eKo

    2016-05-01

    Full Text Available The response and susceptibility to astroglial degenerations are relevant to the distinctive properties of astrocytes in a hemodynamic-independent manner following status epilepticus (SE.Since impaired mitochondrial fission plays an important role in mitosis, apoptosis and programmed necrosis, we investigated whether the unique pattern of mitochondrial dynamics is involved in the characteristics of astroglial death induced by SE. In the present study, SE induced astroglial apoptosis in the molecular layer of the dentate gyrus, accompanied by decreased mitochondrial length. In contrast, clasmatodendritic (autophagic astrocytes in the CA1 region showed mitochondrial elongation induced by SE. Mdivi-1 (an inhibitor of mitochondrial fission effectively attenuated astroglial apoptosis, but WY14643 (an enhancer of mitochondrial fissionaggravated it. In addition, Mdivi-1accelerated clasmatodendritic changes in astrocytes. These regional specific mitochondrial dynamics in astrocytes were closely correlated with dynamin-related protein (DRP1, a mitochondrial fission protein phosphorylation, not optic atrophy 1 (a mitochondrial fusion protein expression. To the best of our knowledge, the present data demonstrate for the first time the novel role of DRP1-mediated mitochondrial fission in astroglial loss. Thus, the present findings suggest that the differential astroglial mitochondrial dynamics may participate in the distinct characteristics of astroglial death induced by SE.

  13. Intense resistance exercise induces early and transient increases in ryanodine receptor 1 phosphorylation in human skeletal muscle.

    Directory of Open Access Journals (Sweden)

    Sebastian Gehlert

    Full Text Available BACKGROUND: While ryanodine receptor 1 (RyR1 critically contributes to skeletal muscle contraction abilities by mediating Ca²⁺ion oscillation between sarcoplasmatic and myofibrillar compartments, AMP-activated protein kinase (AMPK senses contraction-induced energetic stress by phosphorylation at Thr¹⁷². Phosphorylation of RyR1 at serine²⁸⁴³ (pRyR1Ser²⁸⁴³ results in leaky RyR1 channels and impaired Ca²⁺homeostasis. Because acute resistance exercise exerts decreased contraction performance in skeletal muscle, preceded by high rates of Ca²⁺-oscillation and energetic stress, intense myofiber contractions may induce increased RyR1 and AMPK phosphorylation. However, no data are available regarding the time-course and magnitude of early RyR1 and AMPK phosphorylation in human myofibers in response to acute resistance exercise. PURPOSE: Determine the effects and early time-course of resistance exercise on pRyR1Ser²⁸⁴³ and pAMPKThr¹⁷² in type I and II myofibers. METHODS: 7 male subjects (age 23±2 years, height: 185±7 cm, weight: 82±5 kg performed 3 sets of 8 repetitions of maximum eccentric knee extensions. Muscle biopsies were taken at rest, 15, 30 and 60 min post exercise. pRyR1Ser²⁸⁴³ and pAMPKThr¹⁷² levels were determined by western blot and semi-quantitative immunohistochemistry techniques. RESULTS: While total RyR1 and total AMPK levels remained unchanged, RyR1 was significantly more abundant in type II than type I myofibers. pRyR1Ser²⁸⁴³ increased 15 min and peaked 30 min (p<0.01 post exercise in both myofiber types. Type I fibers showed relatively higher increases in pRyR1Ser²⁸⁴³ levels than type II myofibers and remained elevated up to 60 min post resistance exercise (p<0.05. pAMPKThr¹⁷² also increased 15 to 30 min post exercise (p<0.01 in type I and II myofibers and in whole skeletal muscle. CONCLUSION: Resistance exercise induces acutely increased pRyR1Ser²⁸⁴³ and

  14. Highly selective fluorogenic multianalyte biosensors constructed via enzyme-catalyzed coupling and aggregation-induced emission.

    Science.gov (United States)

    Wang, Xiaorui; Hu, Jinming; Zhang, Guoying; Liu, Shiyong

    2014-07-16

    The development of a highly selective and fast responsive fluorogenic biosensor for diverse analytes ranging from bioactive small molecules to specific antigens is highly desirable but remains a considerable challenge. We herein propose a new approach by integrating substrate-selective enzymatic reactions with fluorogens exhibiting aggregation-induced emission feature. Tyrosine-functionalized tetraphenylethene, TPE-Tyr, molecularly dissolves in aqueous media with negligible fluorescence emission; upon addition of horseradish peroxidase (HRP) and H2O2, effective cross-linking occurs due to HRP-catalyzed oxidative coupling of tyrosine moieties in TPE-Tyr. This leads to fluorescence emission turn-on and fast detection of H2O2 with high sensitivity and selectivity. As a validation of the new strategy's generality, we further configure it into the biosensor design for glucose through cascade enzymatic reactions and for pathologically relevant antigens (e.g., human carcinoembryonic antigen) by combining with the ELISA kit. PMID:24983204

  15. An aggregation-induced-emission platform for direct visualization of interfacial dynamic self-assembly.

    Science.gov (United States)

    Li, Junwei; Li, Yuan; Chan, Carrie Y K; Kwok, Ryan T K; Li, Hongkun; Zrazhevskiy, Pavel; Gao, Xiaohu; Sun, Jing Zhi; Qin, Anjun; Tang, Ben Zhong

    2014-12-01

    An in-depth understanding of dynamic interfacial self-assembly processes is essential for a wide range of topics in theoretical physics, materials design, and biomedical research. However, direct monitoring of such processes is hampered by the poor imaging contrast of a thin interfacial layer. We report in situ imaging technology capable of selectively highlighting self-assembly at the phase boundary in real time by employing the unique photophysical properties of aggregation-induced emission. Its application to the study of breath-figure formation, an immensely useful yet poorly understood phenomenon, provided a mechanistic model supported by direct visualization of all main steps and fully corroborated by simulation and theoretical analysis. This platform is expected to advance the understanding of the dynamic phase-transition phenomena, offer insights into interfacial biological processes, and guide development of novel self-assembly technologies. PMID:25363745

  16. An Aggregation-Induced-Emission Platform for Direct Visualization of Interfacial Dynamic Self-Assembly**

    Science.gov (United States)

    Chan, Carrie Y.K.; Kwok, Ryan T.K.; Li, Hongkun; Zrazhevskiy, Pavel; Gao, Xiaohu; Sun, Jing Zhi; Qin, Anjun; Tang, Ben Zhong

    2015-01-01

    An in-depth understanding of dynamic interfacial self-assembly processes is essential for a wide range of topics in theoretical physics, materials design, and biomedical research. However, direct monitoring of such processes is hampered by the poor imaging contrast of a thin interfacial layer. We report in situ imaging technology capable of selectively highlighting self-assembly at the phase boundary in real time by employing the unique photophysical properties of aggregation-induced emission. Its application to the study of breath-figure formation, an immensely useful yet poorly understood phenomenon, provided a mechanistic model supported by direct visualization of all main steps and fully corroborated by simulation and theoretical analysis. This platform is expected to advance the understanding of the dynamic phase-transition phenomena, offer insights into interfacial biological processes, and guide development of novel self-assembly technologies. PMID:25363745

  17. AGGREGATION-INDUCED EMISSION ENHANCEMENT IN POLY(PHENYLENE-ETHYNYLENE)S BEARING ANILINE GROUPS

    Institute of Scientific and Technical Information of China (English)

    Yang Liu; Xiao Feng; Jian-bing Shi; Jun-ge Zhi; Bin Tong; Yu-ping Dong

    2012-01-01

    Poly(phenylene ethynylene)s (P1) with 4-vinylaniline pendant groups were successfully prepared by the Sonogashira coupling polymerization between 1,4-diethynyl-2,5-bis(pentyloxy)bcnzene and 4-[2-(2,5-dibromophenyl)vinyl]-aniline.In comparison with its analogue P2 without amino group,the emission of P1 is only enhanccd by aggregation when adding n-hexane into its THF solution,exhibiting an aggrcgation-induced emission enhancement (AIEE) effect.When methanol or water instead ofhexane was added into THF solution,P1,however,didn't show AIEE.The results indicated that amino groups strengthen the inter-chain and intra-chain interactions in P1 and restrict the non-radiative energy transition.This strategy can provide a platform for developing highly sensitive and efficient bio- and chemosensors.

  18. Time-dependent inhibitory effects of cGMP-analogues on thrombin-induced platelet-derived microparticles formation, platelet aggregation, and P-selectin expression.

    Science.gov (United States)

    Nygaard, Gyrid; Herfindal, Lars; Kopperud, Reidun; Aragay, Anna M; Holmsen, Holm; Døskeland, Stein Ove; Kleppe, Rune; Selheim, Frode

    2014-07-01

    In platelets, nitric oxide (NO) activates cGMP/PKG signalling, whereas prostaglandins and adenosine signal through cAMP/PKA. Cyclic nucleotide signalling has been considered to play an inhibitory role in platelets. However, an early stimulatory effect of NO and cGMP-PKG signalling in low dose agonist-induced platelet activation have recently been suggested. Here, we investigated whether different experimental conditions could explain some of the discrepancy reported for platelet cGMP-PKG-signalling. We treated gel-filtered human platelets with cGMP and cAMP analogues, and used flow cytometric assays to detect low dose thrombin-induced formation of small platelet aggregates, single platelet disappearance (SPD), platelet-derived microparticles (PMP) and thrombin receptor agonist peptide (TRAP)-induced P-selectin expression. All four agonist-induced platelet activation phases were blocked when platelets were costimulated with the PKG activators 8-Br-PET-cGMP or 8-pCPT-cGMP and low-doses of thrombin or TRAP. However, extended incubation with 8-Br-PET-cGMP decreased its inhibition of TRAP-induced P-selectin expression in a time-dependent manner. This effect did not involve desensitisation of PKG or PKA activity, measured as site-specific VASP phosphorylation. Moreover, PKG activators in combination with the PKA activator Sp-5,6-DCL-cBIMPS revealed additive inhibitory effect on TRAP-induced P-selectin expression. Taken together, we found no evidence for a stimulatory role of cGMP/PKG in platelets activation and conclude rather that cGMP/PKG signalling has an important inhibitory function in human platelet activation.

  19. PKC phosphorylates residues in the N-terminal of the DA transporter to regulate amphetamine-induced DA efflux.

    Science.gov (United States)

    Wang, Qiang; Bubula, Nancy; Brown, Jason; Wang, Yunliang; Kondev, Veronika; Vezina, Paul

    2016-05-27

    The DA transporter (DAT), a phosphoprotein, controls extracellular dopamine (DA) levels in the central nervous system through transport or reverse transport (efflux). Multiple lines of evidence support the claim that PKC significantly contributes to amphetamine-induced DA efflux. Other signaling pathways, involving CaMKII and ERK, have also been shown to regulate DAT mediated efflux. Here we assessed the contribution of putative PKC residues (S4, S7, S13) in the N-terminal of the DAT to amphetamine-induced DA efflux by transfecting DATs containing different serine to alanine (S-A) point mutations into DA pre-loaded HEK-293 cells and incubating these cells in amphetamine (2μM). The effects of a S-A mutation at the non-PKC residue S12 and a threonine to alanine (T-A) mutation at the ERK T53 residue were also assessed for comparison. WT-DATs were used as controls. In an initial experiment, we confirmed that inhibiting PKC with Go6976 (130nM) significantly reduced amphetamine-induced DA efflux. In subsequent experiments, cells transfected with the S4A, S12A, S13A, T53A and S4,7,13A mutants showed a reduction in amphetamine-induced DA efflux similar to that observed with Go6976. Interestingly, cells transfected with the S7A mutant, identified by some as a PKC-PKA residue, showed unperturbed WT-DAT levels of amphetamine-induced DA efflux. These results indicate that phosphorylation by PKC of select residues in the DAT N-terminal can regulate amphetamine-induced efflux. PKC can act either independently or in concert with other kinases such as ERK to produce this effect.

  20. Exercise-induced TBC1D1 Ser237 phosphorylation and 14-3-3 protein binding capacity in human skeletal muscle

    DEFF Research Database (Denmark)

    Frøsig, Christian; Pehmøller, Christian; Birk, Jesper Bratz;

    2010-01-01

    TBC1D1 is a Rab-GTPase activating protein involved in regulation of GLUT4 translocation in skeletal muscle. We here evaluated exercise-induced regulation of TBC1D1 Ser237 phosphorylation and 14-3-3 protein binding capacity in human skeletal muscle. In separate experiments healthy men performed all......-out cycle exercise lasting either 30 sec, 2 min or 20 min. After all exercise protocols, TBC1D1 Ser237 phosphorylation increased (~70 - 230%, Pprotein showed a similar pattern of regulation...... increasing 60 - 250% (Pprotein kinase (AMPK) induced both Ser237 phosphorylation and 14-3-3 binding properties on human TBC1D1 when evaluated in vitro. To further characterize the role of AMPK as an upstream kinase regulating TBC1D1, extensor digitorum longus...

  1. Loss of p53-regulatory protein IFI16 induces NBS1 leading to activation of p53-mediated checkpoint by phosphorylation of p53 SER37.

    Science.gov (United States)

    Tawara, Hideyuki; Fujiuchi, Nobuko; Sironi, Juan; Martin, Sarah; Aglipay, Jason; Ouchi, Mutsuko; Taga, Makoto; Chen, Phang-Lang; Ouchi, Toru

    2008-01-01

    Our previous results that IFI16 is involved in p53 transcription activity under conditions of ionizing radiation (IR), and that the protein is frequently lost in human breast cancer cell lines and breast adenocarcinoma tissues suggesting that IFI16 plays a crucial role in controlling cell growth. Here, we show that loss of IFI16 by RNA interference in cell culture causes elevated phosphorylation of p53 Ser37 and accumulated NBS1 (nibrin) and p21WAF1, leading to growth retardation. Consistent with these observations, doxycyclin-induced NBS1 caused accumulation of p21WAF1 and increased phosphorylation of p53 Ser37, leading to cell cycle arrest in G1 phase. Wortmannin treatment was found to decrease p53 Ser37 phosphorylation in NBS-induced cells. These results suggest that loss of IFI16 activates p53 checkpoint through NBS1-DNA-PKcs pathway. PMID:17981542

  2. Serine 105 phosphorylation of transcription factor GATA4 is necessary for stress-induced cardiac hypertrophy in vivo.

    Science.gov (United States)

    van Berlo, Jop H; Elrod, John W; Aronow, Bruce J; Pu, William T; Molkentin, Jeffery D

    2011-07-26

    Cardiac hypertrophy is an adaptive growth process that occurs in response to stress stimulation or injury wherein multiple signal transduction pathways are induced, culminating in transcription factor activation and the reprogramming of gene expression. GATA4 is a critical transcription factor in the heart that is known to induce/regulate the hypertrophic program, in part, by receiving signals from MAPKs. Here we generated knock-in mice in which a known MAPK phosphorylation site at serine 105 (S105) in Gata4 that augments activity was mutated to alanine. Homozygous Gata4-S105A mutant mice were viable as adults, although they showed a compromised stress response of the myocardium. For example, cardiac hypertrophy in response to phenylephrine agonist infusion for 2 wk was largely blunted in Gata4-S105A mice, as was the hypertrophic response to pressure overload at 1 and 2 wk of applied stimulation. Gata4-S105A mice were also more susceptible to heart failure and cardiac dilation after 2 wk of pressure overload. With respect to the upstream pathway, hearts from Gata4-S105A mice did not efficiently hypertrophy following direct ERK1/2 activation using an activated MEK1 transgene in vivo. Mechanistically, GATA4 mutant protein from these hearts failed to show enhanced DNA binding in response to hypertrophic stimulation. Moreover, hearts from Gata4-S105A mice had significant changes in the expression of hypertrophy-inducible, fetal, and remodeling-related genes.

  3. Focal adhesion kinase and Src phosphorylations in HGF-induced proliferation and invasion of human cholangiocarcinoma cell line, HuCCA-1

    Institute of Scientific and Technical Information of China (English)

    Urai Pongchairerk; Jun-Lin Guan; Vijittra Leardkamolkarn

    2005-01-01

    AIM: To study the role of focal adhesion kinase (FAK) and its association with Src in hepatocyte growth factor (HGF)-induced cell signaling in cholangiocarcinoma progression.METHODS: Previously isolated HuCCA-1 cells were re-characterized by immunofluorescent staining and reverse transcriptase-polymerase chain reaction assay for the expression of cytokeratin 19, HGF and c-Met mRNA. Cultured HuCCA-1 cells were treated with HGF and determined for cell proliferation and invasion effects by MTT and invasion assays. Western blotting, immunoprecipitation, and co-immunoprecipitation were also performed to study the phosphorylation and interaction of FAK and Src. A novel Src inhibitor (AZM555130) was applied in cultures to investigate the effects on FAK phosphorylation inhibition and on cell proliferation and invasion.RESULTS: HGF enhanced HuCCA-1 cell proliferation and invasion by mediating FAK and Src phosphorylations.FAK-Src interaction occurred in a time-dependent manner that Src was proved to be an upstream signaling molecule to FAK. The inhibitor to Src decreased FAK phosphorylation level in correlation with the reduction of cell proliferation and invasion.CONCLUSION: FAK plays a significant role in signaling pathway of HGF-responsive cell line derived from cholangiocarcinoma. Autophosphorylated Src, induced by HGF, mediates Src kinase activation, which subsequently phosphorylates its substrate, FAK, and signals to cell proliferation and invasion.

  4. Expression of phosphorylated cAMP response element binding protein (p-CREB) in bladder afferent pathways in VIP-/- mice with cyclophosphamide (CYP)-induced cystitis

    DEFF Research Database (Denmark)

    Jensen, Dorthe G; Studeny, Simon; May, Victor;

    2008-01-01

    The expression of phosphorylated cAMP response element binding protein (p-CREB) in dorsal root ganglia (DRG) with and without cyclophosphamide (CYP)-induced cystitis (150 mg/kg, i.p; 48 h) was determined in VIP(-/-) and wild-type (WT) mice. p-CREB immunoreactivity (IR) was determined in bladder...

  5. Pressure effects on the structure, kinetic, and thermodynamic properties of heat-induced aggregation of protein studied by FT-IR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Taniguchi, Y [Applied Chemistry Department, Ritsumeikan University, Kusatsu, Shiga 525-8577 (Japan); Okuno, A [Research Department 3, Central Research, Bridgestone Co. Kodaira, Tokyo 187-8531 (Japan); Kato, M, E-mail: taniguti@sk.ritsumei.ac.j [Pharmaceutical Sciences Department, Ritsumeikan University, Kusatsu, Shiga 525-8577 (Japan)

    2010-03-01

    Pressure can retrain the heat-induced aggregation and dissociate the heat-induced aggregates. We observed the aggregation-preventing pressure effect and the aggregates-dissociating pressure effect to characterize the heat-induced aggregation of equine serum albumin (ESA) by FT-IR spectroscopy. The results suggest the {alpha}-helical structure collapses at the beginning of heat-induced aggregation through the swollen structure, and then the rearrangement of structure to the intermolecular {beta}-sheet takes place through partially unfolded structure. We determined the activation volume for the heat-induced aggregation ({Delta}V'' = +93 ml/mol) and the partial molar volume difference between native state and heat-induced aggregates ({Delta}V=+32 ml/mol). This positive partial molar volume difference suggests that the heat-induced aggregates have larger internal voids than the native structure. Moreover, the positive volume change implies that the formation of the intermolecular {beta}-sheet is unfavorable under high pressure.

  6. Temperature induced structural transitions from native to unfolded aggregated states of tobacco etch virus protease

    Science.gov (United States)

    Zhu, Guo-Fei; Ren, Si-Yan; Xi, Lei; Du, Lin-Fang; Zhu, Xiao-Feng

    2015-02-01

    Tobacco etch virus protease (TEVp) is widely used to remove fusion tags from recombinant proteins because of its high and unique specificity. This work describes the conformational and the thermodynamic properties in the unfolding/refolding process of TEVp3M (three-point mutant: L56V/S135G/S219V) induced by temperature. With temperature increasing from 20 to 100 °C, the CD spectra showed a transition trend from α-helix to β-sheet, and the fluorescence emission, synchronous fluorescence, ANS and RLS spectroscopy consistently revealed that the temperature-induced unfolding process behaved in a three-state manner, for there was a relatively stable intermediate state observed around 50 °C. The reversibility of thermal unfolding of TEVp3M further showed that the transition from the native to the intermediate state was reversible (below 50 °C), however the transition from the intermediate to the unfolded state was irreversible (above 60 °C). Moreover, aggregates were observed above 60 °C as revealed by SDS-PAGE, Thioflavin-T fluorescence and Congo red absorbance.

  7. Protein carbonylation and aggregation precede neuronal apoptosis induced by partial glutathione depletion

    Directory of Open Access Journals (Sweden)

    Jianzheng Zheng

    2012-04-01

    Full Text Available While the build-up of oxidized proteins within cells is believed to be toxic, there is currently no evidence linking protein carbonylation and cell death. In the present study, we show that incubation of nPC12 (neuron-like PC12 cells with 50 μM DEM (diethyl maleate leads to a partial and transient depletion of glutathione (GSH. Concomitant with GSH disappearance there is increased accumulation of PCOs (protein carbonyls and cell death (both by necrosis and apoptosis. Immunocytochemical studies also revealed a temporal/spatial relationship between carbonylation and cellular apoptosis. In addition, the extent of all three, PCO accumulation, protein aggregation and cell death, augments if oxidized proteins are not removed by proteasomal degradation. Furthermore, the effectiveness of the carbonyl scavengers hydralazine, histidine hydrazide and methoxylamine at preventing cell death identifies PCOs as the toxic species. Experiments using well-characterized apoptosis inhibitors place protein carbonylation downstream of the mitochondrial transition pore opening and upstream of caspase activation. While the study focused mostly on nPC12 cells, experiments in primary neuronal cultures yielded the same results. The findings are also not restricted to DEM-induced cell death, since a similar relationship between carbonylation and apoptosis was found in staurosporine- and buthionine sulfoximine-treated nPC12 cells. In sum, the above results show for the first time a causal relationship between carbonylation, protein aggregation and apoptosis of neurons undergoing oxidative damage. To the best of our knowledge, this is the first study to place direct (oxidative protein carbonylation within the apoptotic pathway.

  8. Liquid-Crystalline Star-Shaped Supergelator Exhibiting Aggregation-Induced Blue Light Emission.

    Science.gov (United States)

    Pathak, Suraj Kumar; Pradhan, Balaram; Gupta, Monika; Pal, Santanu Kumar; Sudhakar, Achalkumar Ammathnadu

    2016-09-13

    A family of closely related star-shaped stilbene-based molecules containing an amide linkage are synthesized, and their self-assembly in liquid-crystalline and gel states was investigated. The number and position of the peripheral alkyl tails were systematically varied to understand the structure-property relation. Interestingly, one of the molecules with seven peripheral chains was bimesomorphic, exhibiting columnar hexagonal and columnar rectangular phases, whereas the rest of them stabilized the room-temperature columnar hexagonal phase. The self-assembly of these molecules in liquid-crystalline and organogel states is extremely sensitive to the position and number of alkoxy tails in the periphery. Two of the compounds with six and seven peripheral tails exhibited supergelation behavior in long-chain hydrocarbon solvents. One of these compounds with seven alkyl chains was investigated further, and it has shown higher stability and moldability in the gel state. The xerogel of the same compound was characterized with the help of extensive microscopic and X-ray diffraction studies. The nanofibers in the xerogel are found to consist of molecules arranged in a lamellar fashion. Furthermore, this compound shows very weak emission in solution but an aggregation-induced emission property in the gel state. Considering the dearth of solid-state blue-light-emitting organic materials, this molecular design is promising where the self-assembly and emission in the aggregated state can be preserved. The nonsymmetric design lowers the phase-transition temperatures.The presence of an amide bond helps to stabilize columnar packing over a long range because of its polarity and intermolecular hydrogen bonding in addition to promoting organogelation.

  9. Temperature-Induced Aggregate Transitions in Mixtures of Cationic Ammonium Gemini Surfactant with Anionic Glutamic Acid Surfactant in Aqueous Solution.

    Science.gov (United States)

    Ji, Xiuling; Tian, Maozhang; Wang, Yilin

    2016-02-01

    The aggregation behaviors of the mixtures of cationic gemini surfactant 1,4-bis(dodecyl-N,N-dimethylammonium bromide)-2,3-butanediol (C12C4(OH)2C12Br2) and anionic amino acid surfactant N-dodecanoylglutamic acid (C12Glu) in aqueous solution of pH = 10.0 have been studied. The mixture forms spherical micelles, vesicles, and wormlike micelles at 25 °C by changing mixing ratios and/or total surfactant concentration. Then these aggregates undergo a series of transitions upon increasing the temperature. Smaller spherical micelles transfer into larger vesicles, vesicles transfer into solid spherical aggregates and then into larger irregular aggregates, and entangled wormlike micelles transfer into branched wormlike micelles. Moreover, the larger irregular aggregates and branched micelles finally lead to precipitation and clouding phenomenon, respectively. All these transitions are thermally reversible, and the transition temperatures can be tuned by varying the mixing ratios and/or total concentration. These temperature-dependent aggregate transitions can be elucidated on the basis of the temperature-induced variations in the dehydration, electrostatic interaction, and hydrogen bonds of the headgroup area and in the hydrophobic interaction between the hydrocarbon chains. The results suggest that the surfactants carrying multiple binding sites will greatly improve the regulation ability and temperature sensitivity. PMID:26750978

  10. Self-assembly of diphenylalanine peptides into microtubes with "turn on" fluorescence using an aggregation-induced emission molecule.

    Science.gov (United States)

    Na, Na; Mu, Xiaoyan; Liu, Qiuling; Wen, Jiying; Wang, Fangfang; Ouyang, Jin

    2013-10-01

    The self-assembly of diphenylalanine peptides (l-Phe-l-Phe) into microtubes with "turn on" bright yellow green fluorescence was described, which was achieved using an aggregation-induced emission (AIE) molecule of 9,10-bis[4-(3-sulfonatopropoxyl)-styryl] anthracene (BSPSA) sodium. PMID:24045462

  11. The unfolded protein response mediates reversible tau phosphorylation induced by metabolic stress

    NARCIS (Netherlands)

    van der Harg, J. M.; Nolle, A.; Zwart, R.; Boerema, A. S.; van Haastert, E. S.; Strijkstra, A. M.; Hoozemans, J. J. M.; Scheper, W.

    2014-01-01

    The unfolded protein response (UPR) is activated in neurodegenerative tauopathies such as Alzheimer's disease (AD) in close connection with early stages of tau pathology. Metabolic disturbances are strongly associated with increased risk for AD and are a potent inducer of the UPR. Here, we demonstra

  12. Blockade of the formation of insoluble ubiquitinated protein aggregates by EGCG3"Me in the alloxan-induced diabetic kidney.

    Directory of Open Access Journals (Sweden)

    Shuxian Cai

    Full Text Available BACKGROUND: Renal accumulation of reactive carbonyl compounds (RCCs has been linked to the progression of diabetic nephropathy. We previously demonstrated that carbonyl stress induces the formation of amino-carbonyl cross-links and sharply increases the content of β-sheet-rich structures, which is the seed of insoluble aggregates formation, and tea catechin (--epigallocatechin 3-gallate (EGCG can reverse this process in vitro and in vivo. In this study, methylated derivative (--epigallocatechin-3-O-(3-O-methyl-gallate (EGCG3"Me was hypothesized to neutralize carbonyl stress mediating the formation of insoluble ubiquitinated protein (IUP aggregates, and reduce the early development of diabetic nephropathy. METHODS AND RESULTS: Diabetes was induced in mice by intraperitoneally injecting alloxan monohydrate (200 mg/kg/d twice and administering EGCG3"Me by gavage for 15 d. Reagent case and western blot results showed that, in diabetic kidneys, the carbonyl proteins in the serum increased; and in insoluble protein fraction, 4-hydroxynonenal-modified proteins, IUP aggregates and p62 accumulated; FT-IR study demonstrated that the lipid content, anti-parallel β-sheet structure and aggregates increased. EGCG3"Me treatment could effectively reverse this process, even better than the negative control treatment. CONCLUSIONS: EGCG3"Me exhibiting anti-β-sheet-rich IUP aggregate properties, maybe represents a new strategy to impede the progression of diabetic nephropathy and other diabetic complications.

  13. Quantitative Label-Free Phosphoproteomics Reveals Differentially Regulated Protein Phosphorylation Involved in West Nile Virus-Induced Host Inflammatory Response.

    Science.gov (United States)

    Zhang, Hao; Sun, Jun; Ye, Jing; Ashraf, Usama; Chen, Zheng; Zhu, Bibo; He, Wen; Xu, Qiuping; Wei, Yanming; Chen, Huanchun; Fu, Zhen F; Liu, Rong; Cao, Shengbo

    2015-12-01

    West Nile virus (WNV) can cause neuro-invasive and febrile illness that may be fatal to humans. The production of inflammatory cytokines is key to mediating WNV-induced immunopathology in the central nervous system. Elucidating the host factors utilized by WNV for productive infection would provide valuable insights into the evasion strategies used by this virus. Although attempts have been made to determine these host factors, proteomic data depicting WNV-host protein interactions are limited. We applied liquid chromatography-tandem mass spectrometry for label-free, quantitative phosphoproteomics to systematically investigate the global phosphorylation events induced by WNV infection. Quantifiable changes to 1,657 phosphoproteins were found; of these, 626 were significantly upregulated and 227 were downregulated at 12 h postinfection. The phosphoproteomic data were subjected to gene ontology enrichment analysis, which returned the inflammation-related spliceosome, ErbB, mitogen-activated protein kinase, nuclear factor kappa B, and mechanistic target of rapamycin signaling pathways. We used short interfering RNAs to decrease the levels of glycogen synthase kinase-3 beta, bifunctional polynucleotide phosphatase/kinase, and retinoblastoma 1 and found that the activity of nuclear factor kappa B (p65) is significantly decreased in WNV-infected U251 cells, which in turn led to markedly reduced inflammatory cytokine production. Our results provide a better understanding of the host response to WNV infection and highlight multiple targets for the development of antiviral and anti-inflammatory therapies.

  14. Ilex latifolia Prevents Amyloid β Protein (25-35)-Induced Memory Impairment by Inhibiting Apoptosis and Tau Phosphorylation in Mice.

    Science.gov (United States)

    Kim, Joo Youn; Lee, Hong Kyu; Jang, Ji Yeon; Yoo, Jae Kuk; Seong, Yeon Hee

    2015-12-01

    Ilex latifolia Thunb. (Aquifoliaceae), a Chinese bitter tea called "kudingcha," has been widely consumed as a health beverage and found to possess antioxidant, antidiabetic, antihypertensive, anti-inflammatory, and anti-ischemic activities. The aim of the present study was to investigate the neuroprotective effects of an ethanol extract of I. latifolia against amyloid β protein (Aβ)-induced memory impairment in mice and neurotoxicity in cultured rat cortical neurons. Memory impairment in mice was induced by intracerebroventricular injection of 15 nmol Aβ (25-35) and measured by the passive avoidance test and Morris water maze test. Chronic administration of I. latifolia (25-100 mg/kg, p.o.) significantly prevented Aβ (25-35)-induced memory loss. I. latifolia also prevented the decrease of glutathione concentrations, increased lipid peroxidation, expression of phosphorylated tau (p-tau), and changes in apoptosis-associated proteins in the memory-impaired mouse brain. Exposure of cultured cortical neurons to 10 μM Aβ (25-35) for 36 h induced neuronal apoptotic death. The neuronal cell death, elevation of intracellular Ca(2+) concentration, generation of reactive oxygen species, and expression of proapoptotic proteins caused by Aβ (25-35) in the cultured neurons were inhibited by treatment with I. latifolia (1-50 μg/mL). These results suggest that I. latifolia may have a possible therapeutic role in managing cognitive impairment associated with Alzheimer's disease. The underlying mechanism might involve the antiapoptotic effects mediated by antioxidant activity and inhibition of p-tau formation.

  15. Proteasome inhibition-induced p38 MAPK/ERK signaling regulates autophagy and apoptosis through the dual phosphorylation of glycogen synthase kinase 3{beta}

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Cheol-Hee [Research Center for Resistant Cells, Chosun University, Seosuk-dong, Dong-gu, Gwangju 501-759 (Korea, Republic of); Department of Pharmacology, College of Medicine, Chosun University, Seosuk-dong, Dong-gu, Gwangju 501-759 (Korea, Republic of); Lee, Byung-Hoon [College of Pharmacy and Multiscreening Center for Drug Development, Seoul National University, Seoul 151-742 (Korea, Republic of); Ahn, Sang-Gun [Department of Pathology, College of Dentistry, Chosun University, Gwangju 501-759 (Korea, Republic of); Oh, Seon-Hee, E-mail: oshccw@hanmail.net [Research Center for Resistant Cells, Chosun University, Seosuk-dong, Dong-gu, Gwangju 501-759 (Korea, Republic of)

    2012-02-24

    Highlights: Black-Right-Pointing-Pointer MG132 induces the phosphorylation of GSK3{beta}{sup Ser9} and, to a lesser extent, of GSK3{beta}{sup Thr390}. Black-Right-Pointing-Pointer MG132 induces dephosphorylation of p70S6K{sup Thr389} and phosphorylation of p70S6K{sup Thr421/Ser424}. Black-Right-Pointing-Pointer Inactivation of p38 dephosphorylates GSK3{beta}{sup Ser9} and phosphorylates GSK3{beta}{sup Thr390}. Black-Right-Pointing-Pointer Inactivation of p38 phosphorylates p70S6K{sup Thr389} and increases the phosphorylation of p70S6K{sup Thr421/Ser424}. Black-Right-Pointing-Pointer Inactivation of p38 decreases autophagy and increases apoptosis induced by MG132. -- Abstract: Proteasome inhibition is a promising approach for cancer treatment; however, the underlying mechanisms involved have not been fully elucidated. Here, we show that proteasome inhibition-induced p38 mitogen-activated protein kinase regulates autophagy and apoptosis by modulating the phosphorylation status of glycogen synthase kinase 3{beta} (GSK3{beta}) and 70 kDa ribosomal S6 kinase (p70S6K). The treatment of MDA-MB-231 cells with MG132 induced endoplasmic reticulum stress through the induction of ATF6a, PERK phosphorylation, and CHOP, and apoptosis through the cleavage of Bax and procaspase-3. MG132 caused the phosphorylation of GSK3{beta} at Ser{sup 9} and, to a lesser extent, Thr{sup 390}, the dephosphorylation of p70S6K at Thr{sup 389}, and the phosphorylation of p70S6K at Thr{sup 421} and Ser{sup 424}. The specific p38 inhibitor SB203080 reduced the p-GSK3{beta}{sup Ser9} and autophagy through the phosphorylation of p70S6K{sup Thr389}; however, it augmented the levels of p-ERK, p-GSK3{beta}{sup Thr390}, and p-70S6K{sup Thr421/Ser424} induced by MG132, and increased apoptotic cell death. The GSK inhibitor SB216763, but not lithium, inhibited the MG132-induced phosphorylation of p38, and the downstream signaling pathway was consistent with that in SB203580-treated cells. Taken together, our

  16. Acute phencyclidine treatment induces extensive and distinct protein phosphorylation in rat frontal cortex

    DEFF Research Database (Denmark)

    Palmowski, Pawel; Rogowska-Wrzesinska, Adelina; Williamson, James;

    2014-01-01

    data sets to date of a preclinical model of schizophrenia. Our findings contribute to the understanding of alterations in glutamatergic neurotransmission in schizophrenia and provide a foundation for discovery of novel targets for pharmacological intervention. © 2014 American Chemical Society.......Phencyclidine (PCP), a noncompetitive N-methyl-d-aspartate receptor antagonist, induces psychotomimetic effects in humans and animals. Administration of PCP to rodents is used as a preclinical model for schizophrenia; however, the molecular mechanisms underlying the symptoms remain largely unknown....... Acute PCP treatment rapidly induces behavioral and cognitive deficits; therefore, post-translational regulation of protein activity is expected to play a role at early time points. We performed mass-spectrometry- driven quantitative analysis of rat frontal cortex 15, 30, or 240 min after...

  17. Mitochondrial role of Apoptosis-Inducing Factor (AIF): Oxidative Phosphorylation and Reactive Oxygen Species.

    OpenAIRE

    Apostolova, Nadezda

    2008-01-01

    The apoptotic function of Apoptosis-inducing factor (AIF) is well documented in the literature, but its physiological role in the mitochondrion is less certain. Using a small interfering RNA (siRNA) strategy, we studied whether modulation of AIF expression in cultured cells influenced the production of reactive oxygen species (ROS). We found that siAIF-transfected cells had reduced AIF protein levels and this was paralleled by a significant increase in ROS. We tested the genera...

  18. Enhanced Ca2+ Entry and Tyrosine Phosphorylation Mediate Nanostructure-Induced Endothelial Proliferation

    Directory of Open Access Journals (Sweden)

    Michaela Schernthaner

    2013-01-01

    Full Text Available Nanostructured substrates have been recognized to initiate transcriptional programs promoting cell proliferation. Specifically β-catenin has been identified as transcriptional regulator, activated by adhesion to nanostructures. We set out to identify processes responsible for nanostructure-induced endothelial β-catenin signaling. Transmission electron microscopy (TEM of cell contacts to differently sized polyethylene terephthalate (PET surface structures (ripples with 250 to 300 nm and walls with 1.5 µm periodicity revealed different patterns of cell-substrate interactions. Cell adhesion to ripples occurred exclusively on ripple peaks, while cells were attached to walls continuously. The Src kinase inhibitor PP2 was active only in cells grown on ripples, while the Abl inhibitors dasatinib and imatinib suppressed β-catenin translocation on both structures. Moreover, Gd3+ sensitive Ca2+ entry was observed in response to mechanical stimulation or Ca2+ store depletion exclusively in cells grown on ripples. Both PP2 and Gd3+ suppressed β-catenin nuclear translocation along with proliferation in cells grown on ripples but not on walls. Our results suggest that adhesion of endothelial cells to ripple structured PET induces highly specific, interface topology-dependent changes in cellular signalling, characterized by promotion of Gd3+-sensitive Ca2+ entry and Src/Abl activation. We propose that these signaling events are crucially involved in nanostructure-induced promotion of cell proliferation.

  19. Structural phase transition of merocyanine J-aggregate induced by ion-recombination in the aqueous sub-phase

    Science.gov (United States)

    Kato, Noritaka; Saito, Kentaro; Uesu, Yoshiaki

    2000-08-01

    By using the sub-phase, which contains two different kinds of counter-ions, we found a reversible thermochromic transition between different J-aggregate states of amphiphilic merocyanine dye (MD) molecules in the monolayer at the air-water interface. This chromatic change is attributed to the structural phase transition of MD J-aggregate crystallites induced by the mutual recombination of different counter-ions to MD molecules. The drastic morphological change of the MD monolayer during the transition is revealed by the in-situ observation using a multipurpose non-linear optical microscope.

  20. Selective PKCalpha inhibition uncouples platelet angiogenesis promotion from collagen-induced aggregation

    OpenAIRE

    Radomski, Marek

    2013-01-01

    Platelets promote angiogenesis by releasing angiogenesis-regulating factors from their α-granules upon aggregation. This effect has both physiologic and pathologic significance as it may contribute to carcinogenesis. Platelet α-granule release and aggregation are regulated, in part, via protein kinase C (PKC) α and β signaling. Our study investigated the effects of PKC inhibition on aggregation, angiogenesis-regulator secretion from α-granules, and platelet-stimulated angiogenesis. We hypothe...

  1. Inhibition of ALK enzymatic activity in T-cell lymphoma cells induces apoptosis and suppresses proliferation and STAT3 phosphorylation independently of Jak3

    DEFF Research Database (Denmark)

    Marzec, Michal; Kasprzycka, Monika; Ptasznik, Andrzej;

    2005-01-01

    Aberrant expression of the ALK tyrosine kinase as a chimeric protein with nucleophosmin (NPM) and other partners plays a key role in malignant cell transformation of T-lymphocytes and other cells. Here we report that two small-molecule, structurally related, quinazoline-type compounds, WHI-131...... and WHI-154, directly inhibit enzymatic activity of NPM/ALK as demonstrated by in vitro kinase assays using a synthetic tyrosine-rich oligopeptide and the kinase itself as the substrates. The inhibition of NPM/ALK activity resulted in malignant T cells in suppression of their growth, induction...... of apoptosis and inhibition of tyrosine phosphorylation of STAT3, the key effector of the NPM/ALK-induced oncogenesis. We also show that the STAT3 tyrosine phosphorylation is mediated in the malignant T cells by NPM/ALK independently of Jak3 kinase as evidenced by the presence of STAT3 phosphorylation...

  2. Inhibitory effects and mechanisms of high molecular-weight phlorotannins from Sargassum thunbergii on ADP-induced platelet aggregation

    Institute of Scientific and Technical Information of China (English)

    WEI Yuxi; WANG Changyun; LI Jing; GUO Qi; QI Hongtao

    2009-01-01

    We evaluated the effects of high molecular-weight phlorotannins from Sargassum thunbergii (STP) on ADP-induced platelet aggregation and arachidonic acid (AA) metabolism in New Zealand white rabbits and Wistar rats. The inhibition of STP on platelet aggregation was investigated using a turbidimetric method, and the levels of the terminal products of AA metabolism were measured using the corresponding kits for maleic dialdehyde (MDA), thromboxane B2 (TXB2) and 6-keto-prostaglandin F1α (6-keto-PGF1α) by colorimetry and radioimmunoassay, as appropriate. We found that STP could inhibit ADP-induced platelet aggregation, and the inhibitory ratio was 91.50% at the STP concentration of 4.0 mg/mL. Furthermore, STP markedly affected AA metabolism by decreasing the synthesis of MDA (P<0.01) and increasing the synthesis of 6-keto-PGF1α, thus changing the plasma TXB2/6-keto-PGF1α balance when the platelets were activated (P<0.01). Therefore, STP altered AA metabolism and these findings partly revealed the molecular mechanism by which STP inhibits ADP-induced platelet aggregation.

  3. Combination of PKCε Activation and PTP1B Inhibition Effectively Suppresses Aβ-Induced GSK-3β Activation and Tau Phosphorylation.

    Science.gov (United States)

    Kanno, Takeshi; Tsuchiya, Ayako; Tanaka, Akito; Nishizaki, Tomoyuki

    2016-09-01

    Glycogen synthase kinase-3β (GSK-3β) is a key element to phosphorylate tau and form neurofibrillary tangles (NFTs) found in tauopathies including Alzheimer's disease (AD). A current topic for AD therapy is focused upon how to prevent tau phosphorylation. In the present study, PKCε activated Akt and inactivated GSK-3β by directly interacting with each protein. Inhibition of protein tyrosine phosphatase 1B (PTP1B), alternatively, caused an enhancement in the tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1), allowing activation of Akt through a pathway along an IRS-1/phosphatidylinositol 3 kinase (PI3K)/3-phosphoinositide-dependent protein kinase-1 (PDK1)/Akt axis, to phosphorylate and inactivate GSK-3β. Combination of PKCε activation and PTP1B inhibition more sufficiently activated Akt and inactivated GSK-3β than each independent treatment, to suppress amyloid β (Aβ)-induced tau phosphorylation and ameliorate spatial learning and memory impairment in 5xFAD transgenic mice, an animal model of AD. This may represent an innovative strategy for AD therapy. PMID:26328540

  4. Bevacizumab reduced auto-phosphorylation of VEGFR2 to protect HDM-induced asthma mice.

    Science.gov (United States)

    Huang, Chaowen; Dong, Hangming; Zou, Mengchen; Luo, Lishan; Hu, Yahui; Xie, Zhefan; Le, Yanqing; Liu, Laiyu; Zou, Fei; Cai, Shaoxi

    2016-09-01

    Vascular endothelial growth factor (VEFG) is a major angiogenic factor involved in both normal physiological processes, such as embryonic development and wound healing, and in diseases, like cancer. Recent studies have revealed the functions of VEGF in inflammation and immunoregulation. Asthma is a chronic inflammation of the airways characterized by airway epithelial barrier dysfunction and imbalance in T-helper (Th) 1/Th2 during immunoregulation. We hypothesized that VEGF plays an important role in asthma. Utilizing a house dust mite extract (HDM)-induced murine model of asthma, we investigated whether bevacizumab, a humanized anti-VEGF monoclonal antibody, could protect the epithelial barrier in murine airways. We found that bevacizumab reduced airway hyper-responsiveness (AHR) and airway inflammation induced by HDM. In addition, HDM exposure promoted expression of VEGF, and caused AHR, disruptions of the epithelial barrier, and airway inflammation. Bevacizumab ameliorated AHR and the release of Th2 cytokines, thereby protecting the epithelial barrier. Our data suggest that bevacizumab may be a new therapeutic strategy for asthma. PMID:27453339

  5. The phosphorylation status of extracellular-regulated kinase 1/2 in astrocytes and neurons from rat hippocampus determines the thrombin-induced calcium release and ROS generation.

    Science.gov (United States)

    Zündorf, Gregor; Reiser, Georg

    2011-12-01

    Challenge of protease-activated receptors induces cytosolic Ca(2+) concentration ([Ca(2+) ](c)) increase, mitogen-activated protein kinase activation and reactive oxygen species (ROS) formation with a bandwidth of responses in individual cells. We detected in this study in situ the thrombin-induced [Ca(2+) ](c) rise and ROS formation in dissociated hippocampal astrocytes and neurons in a mixed culture. In identified cells, single cell responses were correlated with extracellular-regulated kinase (ERK)1/2 phosphorylation level. On average, in astrocytes, thrombin induced a transient [Ca(2+) ](c) rise with concentration-dependent increase in amplitude and extrusion rate and high ERK1/2 phosphorylation level. Correlation analysis of [Ca(2+) ](c) response characteristics of single astrocytes reveals that astrocytes with nuclear phosphoERK1/2 localization have a smaller Ca(2+) amplitude and extrusion rate compared with cells with a cytosolic phosphoERK1/2 localization. In naive neurons, without thrombin challenge, variable ERK1/2 phosphorylation patterns are observed. ROS were detected by hydroethidine. Only in neurons with increased ERK1/2 phosphorylation level, we see sustained intracellular rise in fluorescence of the dye lasting over several minutes. ROS formation was abolished by pre-incubation with the NADPH oxidase inhibitor apocynin. Additionally, thrombin induced an immediate, transient hydroethidine fluorescence increase. This was interpreted as NADPH oxidase-mediated O(2) (•-) -release into the extracellular milieu, because it was decreased by pre-incubation with apocynin, and could be eluted by superfusion. In conclusion, the phosphorylation status of ERK1/2 determines the thrombin-dependent [Ca(2+) ](c) increase and ROS formation and, thus, influences the capacity of thrombin to regulate neuroprotection or neurodegeneration. PMID:21988180

  6. Glutamine supplementation prevents exercise-induced neutrophil apoptosis and reduces p38 MAPK and JNK phosphorylation and p53 and caspase 3 expression.

    Science.gov (United States)

    Lagranha, Claudia J; Hirabara, Sandro M; Curi, Rui; Pithon-Curi, Tania C

    2007-01-01

    We have previously shown that a single session of exercise induces DNA fragmentation, mitochondrial membrane depolarization, increases expression of pro-apoptotic genes (bax and bcl-xS) and decreases expression of anti-apoptotic genes (bcl-xL) in rat neutrophils. Glutamine supplementation had a protective effect in the apoptosis induced by a single session of exercise. The mechanism involved in the effect of single session of exercise to induce apoptosis was investigated by measuring expression of p53 and caspase 3 and phosphorylation of p38 mitogen-activated protein kinases (MAPK) and cJun NH(2)-terminal kinase (JNK) in neutrophils from rats supplemented or not with glutamine. Exercise was carried out on a treadmill for 1 h and the rats were killed by decapitation. Neutrophils were obtained by intraperitoneal (i.p.) lavage with PBS, 4 h after injection of oyster glycogen solution. Glutamine supplementation (1g per Kg b.w.) was given by gavage 1 h before the exercise session. Gene expression and protein phosphorylation were then analyzed by reverse transcriptase chain reaction (RT-PCR) and Western blotting, respectively. A single session of exercise increased p38 MAPK and JNK phosphorylation and p53 and caspase 3 expression. Glutamine supplementation partially prevented the increase in p38 MAPK and JNK phosphorylation and p53 expression, and fully abolished the increase in caspase 3 expression. Thus, neutrophil apoptosis induced by a single session of exercise is accompanied by increased p53 and caspase 3 expression and p38 MAPK and JNK phosphorylation. Glutamine supplementation prevents these effects of exercise and reduces apoptosis. PMID:17542038

  7. Lipopolysaccharide-induced activation of NF-{kappa}B non-canonical pathway requires BCL10 serine 138 and NIK phosphorylations

    Energy Technology Data Exchange (ETDEWEB)

    Bhattacharyya, Sumit; Borthakur, Alip; Dudeja, Pradeep K. [Department of Medicine, University of Illinois at Chicago and Jesse Brown VA Medical Center, Chicago, IL 60612-7227 (United States); Tobacman, Joanne K., E-mail: jkt@uic.edu [Department of Medicine, University of Illinois at Chicago and Jesse Brown VA Medical Center, Chicago, IL 60612-7227 (United States)

    2010-11-15

    Background and aims: B-cell lymphoma/leukemia (BCL)-10 and reactive oxygen species mediate two pathways of NF-{kappa}B (RelA) activation by lipopolysaccharide (LPS) in human colonic epithelial cells. The pathway for LPS activation of RelB by the non-canonical pathway (RelB) in non-myeloid cells was not yet reported, but important for understanding the range of potential microbial LPS-induced effects in inflammatory bowel disease. Methods: Experiments were performed in human colonic epithelial cells and in mouse embryonic fibroblasts deficient in components of the IkappaB kinase (IKK) signalosome, in order to detect mediators of the non-canonical pathway of NF-{kappa}B activation, including nuclear RelB and p52 and phospho- and total NF-{kappa}B inducing kinase (NIK). BCL10 was silenced by siRNA and effects of mutations of specific phosphorylation sites of BCL10 (Ser138Gly and Ser218Gly) were determined. Results: By the non-canonical pathway, LPS exposure increased nuclear RelB and p52, and phospho-NIK, with no change in total NIK. Phosphorylation of BCL10 serine 138 was required for NIK phosphorylation, since mutation of this residue eliminated the increases in phospho-NIK and nuclear RelB and p52. Mutations of either serine 138 or serine 218 reduced RelA, p50, and phospho-I{kappa}B{alpha} of the canonical pathway. Effects of LPS stimulation and BCL10 silencing on NIK phosphorylation were demonstrated in confocal images. Conclusions: LPS induces activation of both canonical and non-canonical pathways of NF-{kappa}B in human colonic epithelial cells, and the non-canonical pathway requires phosphorylations of BCL10 (serine 138) and NIK. These findings demonstrate the important role of BCL10 in mediating LPS-induced inflammation in human colonic epithelial cells and may open new avenues for therapeutic interventions.

  8. Glutamine supplementation prevents exercise-induced neutrophil apoptosis and reduces p38 MAPK and JNK phosphorylation and p53 and caspase 3 expression.

    Science.gov (United States)

    Lagranha, Claudia J; Hirabara, Sandro M; Curi, Rui; Pithon-Curi, Tania C

    2007-01-01

    We have previously shown that a single session of exercise induces DNA fragmentation, mitochondrial membrane depolarization, increases expression of pro-apoptotic genes (bax and bcl-xS) and decreases expression of anti-apoptotic genes (bcl-xL) in rat neutrophils. Glutamine supplementation had a protective effect in the apoptosis induced by a single session of exercise. The mechanism involved in the effect of single session of exercise to induce apoptosis was investigated by measuring expression of p53 and caspase 3 and phosphorylation of p38 mitogen-activated protein kinases (MAPK) and cJun NH(2)-terminal kinase (JNK) in neutrophils from rats supplemented or not with glutamine. Exercise was carried out on a treadmill for 1 h and the rats were killed by decapitation. Neutrophils were obtained by intraperitoneal (i.p.) lavage with PBS, 4 h after injection of oyster glycogen solution. Glutamine supplementation (1g per Kg b.w.) was given by gavage 1 h before the exercise session. Gene expression and protein phosphorylation were then analyzed by reverse transcriptase chain reaction (RT-PCR) and Western blotting, respectively. A single session of exercise increased p38 MAPK and JNK phosphorylation and p53 and caspase 3 expression. Glutamine supplementation partially prevented the increase in p38 MAPK and JNK phosphorylation and p53 expression, and fully abolished the increase in caspase 3 expression. Thus, neutrophil apoptosis induced by a single session of exercise is accompanied by increased p53 and caspase 3 expression and p38 MAPK and JNK phosphorylation. Glutamine supplementation prevents these effects of exercise and reduces apoptosis.

  9. Induced growth of dendrite gold nanostructure by controlling self-assembly aggregation dynamics.

    Science.gov (United States)

    Abdellatif, M H; Abdelrasoul, G N; Scarpellini, A; Marras, S; Diaspro, A

    2015-11-15

    Self-assembly of gold nanoparticles (AuNPs) is an important growth mode for fabricating functional materials. In this work we report a dendrite structure formed by slowing down the aggregation dynamics of AuNPs self-assembly. The obtained results show that the aggregation dynamics is dominated by the Reaction Limited Aggregation Model (RLA) more than the Diffusion Limited Aggregation Model (DLA). In which the repulsion due to electrostatic forces is dominant by the Van Der Walls attraction forces, and low sticking probability of nanoparticles. The aggregation dynamics of AuNPs can be slowed down if the water evaporation of the drop casted colloidal AuNPs on a quartz substrate is slowed. Slowing down the evaporation allows electrostatic repulsion forces to decrease gradually. At certain point, the attraction forces become higher than the electrostatic repulsion and hence cluster aggregation take place slowly. The slow aggregation dynamics allows the nanoparticles to sample all possible orientation in the sticking site, searching for the lowest energy configuration. The size distribution of the nanoparticles in liquid is confirmed using dynamic light scattering based on Stokes-Einstein equation for diffusion coefficient in water. X-ray and photoluminescence (PL) spectra of the sample after aggregation showed a shift which is related to the aggregation compared with non-aggregated colloidal nanoparticles in the solution. The study shows that dendrite self similar structure can be formed by slowing down the aggregation dynamics of nanoparticles as a result of minimizing the Helmholtz free surface energy of the system. PMID:26233557

  10. Unusual Aggregation-Induced Emission of a Coumarin Derivative as a Result of the Restriction of an Intramolecular Twisting Motion.

    Science.gov (United States)

    Bu, Fan; Duan, Ruihong; Xie, Yujun; Yi, Yuanping; Peng, Qian; Hu, Rongrong; Qin, Anjun; Zhao, Zujin; Tang, Ben Zhong

    2015-11-23

    Aggregation-induced emission (AIE) is commonly observed for propeller-like luminogens with aromatic rotors and stators. Herein, we report that a coumarin derivative containing a seven-membered aliphatic ring (CD-7) but no rotors showed typical AIE characteristics, whereas its analogue with a five-membered aliphatic ring (CD-5) exhibited an opposite aggregation-caused quenching (ACQ) effect. Experimental and theoretical results revealed that a large aliphatic ring in CD-7 weakens structural rigidity and promotes out-of-plane twisting of the molecular backbone to drastically accelerate nonradiative excited-state decay, thus resulting in poor emission in solution. The restriction of twisting motion in aggregates blocks the nonradiative decay channels and enables CD-7 to fluoresce strongly. The results also show that AIE is a general phenomenon and not peculiar to propeller-like molecules. The AIE and ACQ effects can be switched readily by the modulation of molecular rigidity. PMID:26439884

  11. Small heat shock proteins protect against α-synuclein-induced toxicity and aggregation

    International Nuclear Information System (INIS)

    Protein misfolding and inclusion formation are common events in neurodegenerative diseases, such as Parkinson's disease (PD), Alzheimer's disease (AD) or Huntington's disease (HD). α-Synuclein (aSyn) is the main protein component of inclusions called Lewy bodies (LB) which are pathognomic of PD, Dementia with Lewy bodies (DLB), and other diseases collectively known as LB diseases. Heat shock proteins (HSPs) are one class of the cellular quality control system that mediate protein folding, remodeling, and even disaggregation. Here, we investigated the role of the small heat shock proteins Hsp27 and αB-crystallin, in LB diseases. We demonstrate, via quantitative PCR, that Hsp27 messenger RNA levels are ∼2-3-fold higher in DLB cases compared to control. We also show a corresponding increase in Hsp27 protein levels. Furthermore, we found that Hsp27 reduces aSyn-induced toxicity by ∼80% in a culture model while αB-crystallin reduces toxicity by ∼20%. In addition, intracellular inclusions were immunopositive for endogenous Hsp27, and overexpression of this protein reduced aSyn aggregation in a cell culture model

  12. Inhalation of nitric oxide inhibits ADP-induced platelet aggregation and alpha-granule release.

    Science.gov (United States)

    Hagberg, I A; Sølvik, U Ø; Opdahl, H; Roald, H E; Lyberg, T

    1999-01-01

    To gather further information about the effects on blood platelet activation of in vivo exposure to nitric oxide (NO), platelet reactivity was studied in blood from healthy, non-smoking male volunteers before and after 30 min inhalation of 40 ppm NO. Whole blood was stimulated in vitro with adenosine diphosphate or thrombin receptor activation peptide (TRAP-6). In an ex vivo perfusion model, non-anticoagulated blood was exposed to immobilised collagen at arterial blood flow conditions (2600 s(-1)). Blood samples from both the in vitro and ex vivo experiments were stained with fluorochrome-labelled Annexin-V and antibodies against CD42a, CD45, CD49b, CD61, CD62P and fibrinogen, and analysed with a three-colour flow cytometry technique. NO inhalation reduced the platelet activation response to adenosine diphosphate (ADP) stimulation by decreasing platelet-platelet aggregation, alpha-granule release and platelet-leukocyte conjugate formation. TRAP-stimulated platelet activation, collagen-induced platelet activation and thrombus growth was unaffected by NO inhalation. We therefore suggest an ADP receptor inhibitor mode of action of inhaled NO, selective on the newly suggested G protein- and phospholipase C-coupled P2Y1 receptor. Our results demonstrate that blood platelet activation in healthy subjects is modulated by inhalation of NO in therapeutically relevant doses, although the clinical impact of our findings remains unclear. PMID:16801117

  13. Fabrication of aggregation induced emission active luminescent chitosan nanoparticles via a "one-pot" multicomponent reaction.

    Science.gov (United States)

    Wan, Qing; Liu, Meiying; Xu, Dazhuang; Mao, Liucheng; Tian, Jianwen; Huang, Hongye; Gao, Peng; Deng, Fengjie; Zhang, Xiaoyong; Wei, Yen

    2016-11-01

    Chitosan based nanomaterials have been extensively examined for biomedical applications for their biodegradability, low toxicity, biological activity and low cost. In this work, a novel strategy for fabrication of luminescent polymeric nanoparticles (LPNs) based on aggregation induced emission (AIE) dye and water soluble chitosan (WS-Chitosan) were firstly developed via a highly efficient mercaptoacetic acid (MA) locking imine reaction. In this multicomponent reaction (MCR), MA serves as "lock" to connect 9,10-Bis(aldehydephenl)anthracene dye (An-CHO) and amino-containing WS-Chitosan under mild reaction conditions. The obtained WS-Chitosan@An-CHO LPNs show strong yellow emission and great water dispersibility. Biological evaluation results demonstrated that synthetic luminescent polymeric nanoparticles possess desirable cytocompatibility and distinct imaging properties. Therefore, we have developed a facile and useful method to fabricate AIE active nanoprobes with desirable properties for various biomedical applications. This strategy should be a general and easy handling tool to fabricate many other AIE dye based materials. PMID:27516264

  14. Membrane aggregation and perturbation induced by antimicrobial peptide of S-thanatin

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Guoqiu, E-mail: guoqiuwu@163.com [Center of Clinical Laboratory Medicine of Zhongda Hospital, Southeast University, Nanjing (China); Wu, Hongbin; Li, Linxian; Fan, Xiaobo; Ding, Jiaxuan; Li, Xiaofang; Xi, Tao [Biotechnology Center, Department of Life Science and Biotechnology, China Pharmaceutical University, Nanjing 210009 (China); Shen, Zilong, E-mail: Zilongshen@sina.com [Biotechnology Center, Department of Life Science and Biotechnology, China Pharmaceutical University, Nanjing 210009 (China)

    2010-04-23

    Thanatin, a 21-residue peptide, is an inducible insect peptide. In our previous study, we have identified a novel thanatin analog of S-thanatin, which exhibited a broad antimicrobial activity against bacteria and fungi with low hemolytic activity. This study was aimed to delineate the antimicrobial mechanism of S-thanatin and identify its interaction with bacterial membranes. In this study, membrane phospholipid was found to be the target for S-thanatin. In the presence of vesicles, S-thanatin interestingly led to the aggregation of anionic vesicles and sonicated bacteria. Adding S-thanatin to Escherichia coli suspension would result in the collapse of membrane and kill bacteria. The sensitivity assay of protoplast elucidated the importance of outer membrane (OM) for S-thanatin's antimicrobial activity. Compared with other antimicrobial peptide, S-thanatin produced chaotic membrane morphology and cell debris in electron microscopic appearance. These results supported our hypothesis that S-thanatin bound to negatively charged LPS and anionic lipid, impeded membrane respiration, exhausted the intracellular potential, and released periplasmic material, which led to cell death.

  15. Thermally-induced aggregation and fusion of protein-free lipid vesicles.

    Science.gov (United States)

    Ibarguren, Maitane; Bomans, Paul H H; Ruiz-Mirazo, Kepa; Frederik, Peter M; Alonso, Alicia; Goñi, Félix M

    2015-12-01

    Membrane fusion is an important phenomenon in cell biology and pathology. This phenomenon can be modeled using vesicles of defined size and lipid composition. Up to now fusion models typically required the use of chemical (polyethyleneglycol, cations) or enzymatic catalysts (phospholipases). We present here a model of lipid vesicle fusion induced by heat. Large unilamellar vesicles consisting of a phospholipid (dioleoylphosphatidylcholine), cholesterol and diacylglycerol in a 43:57:3 mol ratio were employed. In this simple system, fusion was the result of thermal fluctuations, above 60 °C. A similar system containing phospholipid and cholesterol but no diacylglycerol was observed to aggregate at and above 60 °C, in the absence of fusion. Vesicle fusion occurred under our experimental conditions only when (31)P NMR and cryo-transmission electron microscopy of the lipid mixtures used in vesicle preparation showed non-lamellar lipid phase formation (hexagonal and cubic). Non-lamellar structures are probably the result of lipid reassembly of the products of individual fusion events, or of fusion intermediates. A temperature-triggered mechanism of lipid reassembly might have occurred at various stages of protocellular evolution.

  16. Enhanced hydrophobicity of polyurethane via non-solvent induced surface aggregation of silica nanoparticles.

    Science.gov (United States)

    Seyfi, Javad; Hejazi, Iman; Jafari, Seyed Hassan; Khonakdar, Hossein Ali; Simon, Frank

    2016-09-15

    Fabrication of superhydrophobic surfaces from hydrophilic polymers has always been regarded as a challenge. In this study, to achieve superhydrophobic polyurethane (PU) surfaces, silica nanoparticles and ethanol as non-solvent were simultaneously utilized during a solution casting-based process. Such modified version of phase separation process was found to be highly efficient, and also it required much lower concentration of nanoparticles to achieve superhydrophobicity as compared to the previously reported methods in the literature. According to the proposed mechanism, non-solvent induces a more profound aggregation of silica nanoparticles at the surface's top layer causing the surface energy to be highly diminished, and thus, the water repellency is improved. Morphology and topography results showed that a unique "triple-sized" structure was formed on the surface of superhydrophobic samples. X-ray photoelectron spectroscopy results proved that both PU macromolecules and silica nanoparticles were concurrently present at the surface layer of the superhydrophobic sample. It was concluded that surface composition and roughness could be regarded as competing factors in achieving superhydrophobicity. Based on the obtained results, the proposed method exhibits a promising potential in large-scale fabrication of surface layers with superhydrophobic property. Moreover, a mechanism was also presented to further explicate the physics behind the suggested method. PMID:27288577

  17. Synthetic Quorum Sensing and Induced Aggregation in Model Microcapsule Colonies with Repressilator Feedback

    Science.gov (United States)

    Shum, Henry; Yashin, Victor; Balazs, Anna

    We model a system of synthetic microcapsules that communicate chemically by releasing nanoparticles or signaling molecules. These signaling species bind to receptors on the shells of capsules and modulate the target shell's permeability, thereby controlling nanoparticle release from the target capsule. Using the repressilator regulatory network motif, whereby three species suppress the production of the next in a cyclic fashion, we show that large amplitude oscillations in nanoparticle release can emerge when many capsules are close together. This exemplifies quorum sensing, which is the ability of cells to gauge their population density and collectively initiate a new behavior once a critical density is reached. We present a physically realizable model in which the oscillations exhibited in crowded populations induce aggregation of the microcapsules, mimicking complex biological behavior of the slime mold Dictyostelium discoideum with only simple, synthetic components. We also show that the clusters can be dispersed and reformed repeatedly and controllably by addition of chemical stimuli, demonstrating possible applications in creating reconfigurable or programmable materials.

  18. Comparison of Tooth Discoloration Induced by Calcium-Enriched Mixture and Mineral Trioxide Aggregate

    Science.gov (United States)

    Rouhani, Armita; Akbari, Majid; Farhadi-faz, Aida

    2016-01-01

    Introduction: The aim of this in vitro study was to evaluate the tooth discoloration induced by calcium-enriched mixture (CEM) cement and mineral trioxide aggregate (MTA). Methods and Materials: Forty five endodontically treated human maxillary central incisors were selected and divided into three groups (n=15) after removing the coronal 3 mm of the obturating materials. In the MTA group, white MTA plug was placed in pulp chamber and coronal zone of the root canal. In CEM cement group, CEM plug was placed in the tooth in the same manner. In both groups, a wet cotton pellet was placed in the access cavity and the teeth were temporarily sealed. After 24 h the teeth were restored with resin composite. In the negative control group the teeth were also restored with resin composite. The color change in the cervical third of teeth was measured with a colorimeter and was repeated 3 times for each specimen. The teeth were kept in artificial saliva for 6 months. After this period, the color change was measured again. Data were collected by Commission International de I'Eclairage's L*a*b color values, and corresponding ΔE values were calculated. The results were analyzed using the one-way ANOVA and post-hoc Tukey’s test with the significance level defined as 0.05. Results: There was no significant differences between CEM group and control group in mean discoloration. The mean tooth discoloration in MTA group was significantly greater than CEM and control groups (P<0.05). Conclusion: According to the result of the present study CEM cement did not induce tooth discoloration after six months. Therefore it can be used in vital pulp therapy of esthetically sensitive teeth. PMID:27471526

  19. Tyrosine phosphorylation of the N-Methyl-D-Aspartate receptor 2B subunit in spinal cord contributes to remifentanil-induced postoperative hyperalgesia: the preventive effect of ketamine

    Directory of Open Access Journals (Sweden)

    Cui Songqin

    2009-12-01

    Full Text Available Abstract Background Experimental and clinical studies showed that intraoperative infusionof remifentanil has been associated with postoperative hyperalgesia. Previous reports suggested that spinal N-methyl-D-aspartate (NMDA receptors may contribute to the development and maintenance of opioid-induced hyperalgesia. In the present study, we used a rat model of postoperative pain to investigate the role of tyrosine phosphorylation of NMDA receptor 2B (NR2B subunit in spinal cord in the postoperative hyperalgesia induced by remifentanil and the intervention of pretreatment with ketamine. Results Intraoperative infusion of remifentanil (0.04 mg/kg, subcutaneous significantly enhanced mechanical allodynia and thermal hyperalgesia induced by the plantar incision during the postoperative period (each lasting between 2 h and 48 h, which was attenuated by pretreatment with ketamine (10 mg/kg, subcutaneous. Correlated with the pain behavior changes, immunocytochemical and western blotting experiments in our study revealed that there was a marked increase in NR2B phosphorylation at Tyr1472 in the superficial dorsal horn after intraoperative infusion of remifentanil, which was attenuated by pretreatment with ketamine. Conclusions This study provides direct evidence that tyrosine phosphorylation of the NR2B at Tyr1472 in spinal dosal horn contributes to postoperative hyperalgesia induced by remifentanil and supports the potential therapeutic value of ketamine for improving postoperative hyperalgesia induced by remifentanil.

  20. Milk protein suspensions enriched with three essential minerals: Physicochemical characterization and aggregation induced by a novel enzymatic pool.

    Science.gov (United States)

    Lombardi, Julia; Spelzini, Darío; Corrêa, Ana Paula Folmer; Brandelli, Adriano; Risso, Patricia; Boeris, Valeria

    2016-04-01

    Structural changes of casein micelles and their aggregation induced by a novel enzymatic pool isolated from Bacillus spp. in the presence of calcium, magnesium or zinc were investigated. The effect of cations on milk protein structure was studied using fluorescence and dynamic light scattering. In the presence of cations, milk protein structure rearrangements and larger casein micelle size were observed. The interaction of milk proteins with zinc appears to be of a different nature than that with calcium or magnesium. Under the experimental conditions assayed, the affinity of each cation for some groups present in milk proteins seems to play an important role, besides electrostatic interaction. On the other hand, the lowest aggregation times were achieved at the highest calcium and zinc concentrations (15 mM and 0.25 mM, respectively). The study found that the faster the aggregation of casein micelles, the less compact the gel matrix obtained. Cation concentrations affected milk protein aggregation kinetics and the structure of the aggregates formed. PMID:26803666

  1. Milk protein suspensions enriched with three essential minerals: Physicochemical characterization and aggregation induced by a novel enzymatic pool.

    Science.gov (United States)

    Lombardi, Julia; Spelzini, Darío; Corrêa, Ana Paula Folmer; Brandelli, Adriano; Risso, Patricia; Boeris, Valeria

    2016-04-01

    Structural changes of casein micelles and their aggregation induced by a novel enzymatic pool isolated from Bacillus spp. in the presence of calcium, magnesium or zinc were investigated. The effect of cations on milk protein structure was studied using fluorescence and dynamic light scattering. In the presence of cations, milk protein structure rearrangements and larger casein micelle size were observed. The interaction of milk proteins with zinc appears to be of a different nature than that with calcium or magnesium. Under the experimental conditions assayed, the affinity of each cation for some groups present in milk proteins seems to play an important role, besides electrostatic interaction. On the other hand, the lowest aggregation times were achieved at the highest calcium and zinc concentrations (15 mM and 0.25 mM, respectively). The study found that the faster the aggregation of casein micelles, the less compact the gel matrix obtained. Cation concentrations affected milk protein aggregation kinetics and the structure of the aggregates formed.

  2. Thiosemicarbazone modification of 3-acetyl coumarin inhibits Aβ peptide aggregation and protect against Aβ-induced cytotoxicity.

    Science.gov (United States)

    Ranade, Dnyanesh S; Bapat, Archika M; Ramteke, Shefali N; Joshi, Bimba N; Roussel, Pascal; Tomas, Alain; Deschamps, Patrick; Kulkarni, Prasad P

    2016-10-01

    Aggregation of amyloid β peptide (Aβ) is an important event in the progression of Alzheimer's disease. Therefore, among the available therapeutic approaches to fight with disease, inhibition of Aβ aggregation is widely studied and one of the promising approach for the development of treatments for Alzheimer's disease. Thiosemicarbazone compounds are known for their variety of biological activities. However, the potential of thiosemicarbazone compounds towards inhibition of Aβ peptide aggregation and the subsequent toxicity is little explored. Herein, we report synthesis and x-ray crystal structure of novel compound 3-acetyl coumarin thiosemicarbazone and its efficacy toward inhibition of Aβ(1-42) peptide aggregation. Our results indicate that 3-acetyl coumarin thiosemicarbazone inhibits Aβ(1-42) peptide aggregation up to 80% compared to the parent 3-acetyl coumarin which inhibits 52%. Further, 3-acetyl coumarin thiosemicarbazone provides neuroprotection against Aβ-induced cytotoxicity in SH-SY5Y cell line. These findings indicate that thiosemicarbazone modification renders 3-acetyl coumarin neuroprotective properties.

  3. Rosin Surfactant QRMAE Can Be Utilized as an Amorphous Aggregate Inducer: A Case Study of Mammalian Serum Albumin.

    Directory of Open Access Journals (Sweden)

    Mohd Ishtikhar

    Full Text Available Quaternary amine of diethylaminoethyl rosin ester (QRMAE, chemically synthesized biocompatible rosin based cationic surfactant, has various biological applications including its use as a food product additive. In this study, we examined the amorphous aggregation behavior of mammalian serum albumins at pH 7.5, i.e., two units above their isoelectric points (pI ~5.5, and the roles played by positive charge and hydrophobicity of exogenously added rosin surfactant QRMAE. The study was carried out on five mammalian serum albumins, using various spectroscopic methods, dye binding assay, circular dichroism and electron microscopy. The thermodynamics of the binding of mammalian serum albumins to cationic rosin modified surfactant were established using isothermal titration calorimetry (ITC. It was observed that a suitable molar ratio of protein to QRMAE surfactant enthusiastically induces amorphous aggregate formation at a pH above two units of pI. Rosin surfactant QRMAE-albumins interactions revealed a unique interplay between the initial electrostatic and the subsequent hydrophobic interactions that play an important role towards the formation of hydrophobic interactions-driven amorphous aggregate. Amorphous aggregation of proteins is associated with varying diseases, from the formation of protein wine haze to the expansion of the eye lenses in cataract, during the expression and purification of recombinant proteins. This study can be used for the design of novel biomolecules or drugs with the ability to neutralize factor(s responsible for the aggregate formation, in addition to various other industrial applications.

  4. Rosin Surfactant QRMAE Can Be Utilized as an Amorphous Aggregate Inducer: A Case Study of Mammalian Serum Albumin.

    Science.gov (United States)

    Ishtikhar, Mohd; Chandel, Tajjali Ilm; Ahmad, Aamir; Ali, Mohd Sajid; Al-Lohadan, Hamad A; Atta, Ayman M; Khan, Rizwan Hasan

    2015-01-01

    Quaternary amine of diethylaminoethyl rosin ester (QRMAE), chemically synthesized biocompatible rosin based cationic surfactant, has various biological applications including its use as a food product additive. In this study, we examined the amorphous aggregation behavior of mammalian serum albumins at pH 7.5, i.e., two units above their isoelectric points (pI ~5.5), and the roles played by positive charge and hydrophobicity of exogenously added rosin surfactant QRMAE. The study was carried out on five mammalian serum albumins, using various spectroscopic methods, dye binding assay, circular dichroism and electron microscopy. The thermodynamics of the binding of mammalian serum albumins to cationic rosin modified surfactant were established using isothermal titration calorimetry (ITC). It was observed that a suitable molar ratio of protein to QRMAE surfactant enthusiastically induces amorphous aggregate formation at a pH above two units of pI. Rosin surfactant QRMAE-albumins interactions revealed a unique interplay between the initial electrostatic and the subsequent hydrophobic interactions that play an important role towards the formation of hydrophobic interactions-driven amorphous aggregate. Amorphous aggregation of proteins is associated with varying diseases, from the formation of protein wine haze to the expansion of the eye lenses in cataract, during the expression and purification of recombinant proteins. This study can be used for the design of novel biomolecules or drugs with the ability to neutralize factor(s) responsible for the aggregate formation, in addition to various other industrial applications.

  5. Human recombinant H2 relaxin induces AKT and GSK3β phosphorylation and HTR-8/SVneo cell proliferation.

    Science.gov (United States)

    Astuti, Yoni; Nakabayashi, Koji; Deguchi, Masashi; Ebina, Yasuhiko; Yamada, Hideto

    2015-01-01

    Relaxin is essential for trophoblast development during pregnancy. Evidence shows that relaxin increases trophoblast cell migration capacity. Here, we show the effect of relaxin on protein kinase B (AKT) activation and glycogen synthase kinase 3-beta (GSK3β) inactivation as well as on the proliferation of HTR-8/SVneo cells, a model of human extravillous trophoblast (EVT). HTR-8/SVneo cells were treated with different doses of human recombinant (rH2) relaxin in serum-deprived conditions and treated for increasing time with 1 ng/mL of rH2 relaxin. Western blot analysis was performed to detect pAKT, AKT, pGSK3β, GSK3β, and actin expression. Proliferation of HTR-8/SVneo cells was analyzed by MTS assay. rH2 relaxin treatment increased the ratio of pAKT/AKT, pGSK3β/GSK3β, and proliferation in HTR-8/SVneo cells. Furthermore, AKT and GSK3β activation by rH2 relaxin was inhibited by a phosphoinositide 3-kinase (PI3K) inhibitor. This study suggests that rH2 relaxin induces AKT and GSK3β phosphorylation as well as proliferation in HTR-8/SVneo cells. PMID:25868609

  6. Tunneling electron induced molecular electroluminescence from individual porphyrin J-aggregates

    Energy Technology Data Exchange (ETDEWEB)

    Meng, Qiushi; Zhang, Chao; Zhang, Yang, E-mail: zhyangnano@ustc.edu.cn, E-mail: zcdong@ustc.edu.cn; Zhang, Yao; Liao, Yuan; Dong, Zhenchao, E-mail: zhyangnano@ustc.edu.cn, E-mail: zcdong@ustc.edu.cn [Hefei National Laboratory for Physical Sciences at the Microscale and Synergetic Innovation Center of Quantum Information and Quantum Physics, University of Science and Technology of China, Hefei, Anhui 230026 (China)

    2015-07-27

    We investigate molecular electroluminescence from individual tubular porphyrin J-aggregates on Au(111) by tunneling electron excitations in an ultrahigh-vacuum scanning tunneling microscope (STM). High-resolution STM images suggest a spiral tubular structure for the porphyrin J-aggregate with highly ordered “brickwork”-like arrangements. Such aggregated nanotube is found to behave like a self-decoupled molecular architecture and shows red-shifted electroluminescence characteristics of J-aggregates originated from the delocalized excitons. The positions of the emission peaks are found to shift slightly depending on the excitation sites, which, together with the changes in the observed spectral profiles with vibronic progressions, suggest a limited exciton coherence number within several molecules. The J-aggregate electroluminescence is also found unipolar, occurring only at negative sample voltages, which is presumably related to the junction asymmetry in the context of molecular excitations via the carrier injection mechanism.

  7. Modulation of opiate-related signaling molecules in morphine-dependent conditioned behavior: conditioned place preference to morphine induces CREB phosphorylation.

    Science.gov (United States)

    Morón, José A; Gullapalli, Srinivas; Taylor, Chirisse; Gupta, Achla; Gomes, Ivone; Devi, Lakshmi A

    2010-03-01

    Opiate addiction is a chronic, relapsing behavioral disorder where learned associations that develop between the abused opiate and the environment in which it is consumed are brought about through Pavlovian (classical) conditioning processes. However, the signaling mechanisms/pathways regulating the mechanisms that underlie the responses to opiate-associated cues or the development of sensitization as a consequence of repeated context-independent administration of opiates are unknown. In this study we examined the phosphorylation levels of various classic signaling molecules in brain regions implicated in addictive behaviors after acute and repeated morphine administration. An unbiased place conditioning protocol was used to examine changes in phosphorylation that are associated with (1) the expression of the rewarding effects of morphine and (2) the sensitization that develops to this effect. We also examined the effects of a delta-receptor antagonist on morphine-induced conditioned behavior and on the phosphorylation of classic signaling molecules in view of data showing that blockade of delta-opioid receptor (deltaOR) prevents the development of sensitization to the rewarding effects of morphine. We find that CREB phosphorylation is specifically induced upon the expression of a sensitized response to morphine-induced conditioned behavior in brain areas related to memory consolidation, such as the hippocampus and cortex. A similar effect is also observed, albeit to a lesser extent, in the case of the GluR1 subunit of AMPA glutamate receptor. These increases in the phosphorylation levels of CREB and pGluR1 are significantly blocked by pretreatment with a deltaOR antagonist. These results indicate a critical role for phospho-CREB, AMPA, and deltaOR activities in mediating the expression of a sensitized response to morphine-dependent conditioned behavior. PMID:19956087

  8. Modulation of opiate-related signaling molecules in morphine-dependent conditioned behavior: conditioned place preference to morphine induces CREB phosphorylation.

    Science.gov (United States)

    Morón, José A; Gullapalli, Srinivas; Taylor, Chirisse; Gupta, Achla; Gomes, Ivone; Devi, Lakshmi A

    2010-03-01

    Opiate addiction is a chronic, relapsing behavioral disorder where learned associations that develop between the abused opiate and the environment in which it is consumed are brought about through Pavlovian (classical) conditioning processes. However, the signaling mechanisms/pathways regulating the mechanisms that underlie the responses to opiate-associated cues or the development of sensitization as a consequence of repeated context-independent administration of opiates are unknown. In this study we examined the phosphorylation levels of various classic signaling molecules in brain regions implicated in addictive behaviors after acute and repeated morphine administration. An unbiased place conditioning protocol was used to examine changes in phosphorylation that are associated with (1) the expression of the rewarding effects of morphine and (2) the sensitization that develops to this effect. We also examined the effects of a delta-receptor antagonist on morphine-induced conditioned behavior and on the phosphorylation of classic signaling molecules in view of data showing that blockade of delta-opioid receptor (deltaOR) prevents the development of sensitization to the rewarding effects of morphine. We find that CREB phosphorylation is specifically induced upon the expression of a sensitized response to morphine-induced conditioned behavior in brain areas related to memory consolidation, such as the hippocampus and cortex. A similar effect is also observed, albeit to a lesser extent, in the case of the GluR1 subunit of AMPA glutamate receptor. These increases in the phosphorylation levels of CREB and pGluR1 are significantly blocked by pretreatment with a deltaOR antagonist. These results indicate a critical role for phospho-CREB, AMPA, and deltaOR activities in mediating the expression of a sensitized response to morphine-dependent conditioned behavior.

  9. Differential inhibition of tumour cell-induced platelet aggregation by the nicotinate aspirin prodrug (ST0702) and aspirin

    Science.gov (United States)

    Medina, Carlos; Harmon, Shona; Inkielewicz, Iwona; Santos-Martinez, Maria Jose; Jones, Michael; Cantwell, Paula; Bazou, Despina; Ledwidge, Mark; Radomski, Marek W; Gilmer, John F

    2012-01-01

    BACKGROUND AND PURPOSE Tumour cell-induced platelet aggregation (TCIPA) facilitates cancer cell invasion, angiogenesis and the formation of metastatic foci. TCIPA can be modulated by pharmacological inhibitors of MMP-2 and ADP; however, the COX inhibitor aspirin did not prevent TCIPA. In this study, we have tested the pharmacological effects of a new group of isosorbide-based aspirin prodrugs on TCIPA. EXPERIMENTAL APPROACH TCIPA was induced in human platelets by mixing with human adenocarcinoma or fibrosarcoma cells under no flow and flow conditions. The release of gelatinases and P-selectin expression during TCIPA were studied by zymography and flow cytometry respectively. KEY RESULTS Tumour cells caused platelet aggregation. This aggregation resulted in the release of MMP-2 and a significant up-regulation of P-selectin on platelets, indicative of platelet activation. Pharmacological modulation of TCIPA revealed that ST0702, one of the aspirin prodrugs, down-regulated TCIPA while aspirin was ineffective. The deacetylated metabolite of ST0702, 5-nicotinate salicylate (ST0702 salicylate), down-regulated both ADP-stimulated platelet aggregation and TCIPA. CONCLUSIONS AND IMPLICATIONS Our results show that ST0702 was an effective inhibitor of TCIPA in vitro. Its deacetylated metabolite may contribute to the effects of ST0702 by inhibiting ADP-mediated TCIPA. PMID:22122360

  10. Aggregation induced emission enhancement from Bathophenanthroline microstructures and its potential use as sensor of mercury ions in water.

    Science.gov (United States)

    Mazumdar, Prativa; Das, Debasish; Sahoo, Gobinda Prasad; Salgado-Morán, Guillermo; Misra, Ajay

    2014-04-01

    Bathophenanthroline (BA) microstructures of various morphologies have been synthesized using a reprecipitation method. The morphologies of the particles are characterized using optical and scanning electron microscopy (SEM) methods. An aqueous dispersion of BA microstructures shows aggregation induced emission enhancement (AIEE) compared to BA in a good solvent, THF. This luminescent property of aggregated BA hydrosol is used for the selective detection of trace amounts of mercury ion (Hg(2+)) in water. It is observed that Hg(2+) ions can quench the photoluminescence (PL) intensity of BA aggregates even at very low concentrations, compared to other heavy metal ions e.g. nickel (Ni(2+)), manganese (Mn(2+)), cadmium (Cd(2+)), cobalt (Co(2+)), copper (Cu(2+)), ferrous (Fe(2+)) and zinc (Zn(2+)). This strong fluorescence quenching of aggregated BA in the presence of Hg(2+) ions has been explained as a complex interplay between the ground state complexation between BA and Hg(2+) ions and external heavy atom induced perturbation by Hg(2+) ions on the excited states of the fluorophore BA. PMID:24569390

  11. Acute ethanol intake induces superoxide anion generation and mitogen-activated protein kinase phosphorylation in rat aorta: A role for angiotensin type 1 receptor

    Energy Technology Data Exchange (ETDEWEB)

    Yogi, Alvaro; Callera, Glaucia E. [Kidney Research Centre, Ottawa Hospital Research Institute, University of Ottawa, Ontario (Canada); Mecawi, André S. [Department of Physiology, Faculty of Medicine of Ribeirão Preto, University of São Paulo (USP), Ribeirão Preto, SP (Brazil); Batalhão, Marcelo E.; Carnio, Evelin C. [Department of General and Specialized Nursing, College of Nursing of Ribeirão Preto, USP, São Paulo (Brazil); Antunes-Rodrigues, José [Department of Physiology, Faculty of Medicine of Ribeirão Preto, University of São Paulo (USP), Ribeirão Preto, SP (Brazil); Queiroz, Regina H. [Department of Clinical, Toxicological and Food Science Analysis, Faculty of Pharmaceutical Sciences, USP, São Paulo (Brazil); Touyz, Rhian M. [Kidney Research Centre, Ottawa Hospital Research Institute, University of Ottawa, Ontario (Canada); Tirapelli, Carlos R., E-mail: crtirapelli@eerp.usp.br [Department of Psychiatric Nursing and Human Sciences, Laboratory of Pharmacology, College of Nursing of Ribeirão Preto, USP, Ribeirão Preto, SP (Brazil)

    2012-11-01

    Ethanol intake is associated with increase in blood pressure, through unknown mechanisms. We hypothesized that acute ethanol intake enhances vascular oxidative stress and induces vascular dysfunction through renin–angiotensin system (RAS) activation. Ethanol (1 g/kg; p.o. gavage) effects were assessed within 30 min in male Wistar rats. The transient decrease in blood pressure induced by ethanol was not affected by the previous administration of losartan (10 mg/kg; p.o. gavage), a selective AT{sub 1} receptor antagonist. Acute ethanol intake increased plasma renin activity (PRA), angiotensin converting enzyme (ACE) activity, plasma angiotensin I (ANG I) and angiotensin II (ANG II) levels. Ethanol induced systemic and vascular oxidative stress, evidenced by increased plasma thiobarbituric acid-reacting substances (TBARS) levels, NAD(P)H oxidase‐mediated vascular generation of superoxide anion and p47phox translocation (cytosol to membrane). These effects were prevented by losartan. Isolated aortas from ethanol-treated rats displayed increased p38MAPK and SAPK/JNK phosphorylation. Losartan inhibited ethanol-induced increase in the phosphorylation of these kinases. Ethanol intake decreased acetylcholine-induced relaxation and increased phenylephrine-induced contraction in endothelium-intact aortas. Ethanol significantly decreased plasma and aortic nitrate levels. These changes in vascular reactivity and in the end product of endogenous nitric oxide metabolism were not affected by losartan. Our study provides novel evidence that acute ethanol intake stimulates RAS activity and induces vascular oxidative stress and redox-signaling activation through AT{sub 1}-dependent mechanisms. These findings highlight the importance of RAS in acute ethanol-induced oxidative damage. -- Highlights: ► Acute ethanol intake stimulates RAS activity and vascular oxidative stress. ► RAS plays a role in acute ethanol-induced oxidative damage via AT{sub 1} receptor activation.

  12. Acute ethanol intake induces superoxide anion generation and mitogen-activated protein kinase phosphorylation in rat aorta: A role for angiotensin type 1 receptor

    International Nuclear Information System (INIS)

    Ethanol intake is associated with increase in blood pressure, through unknown mechanisms. We hypothesized that acute ethanol intake enhances vascular oxidative stress and induces vascular dysfunction through renin–angiotensin system (RAS) activation. Ethanol (1 g/kg; p.o. gavage) effects were assessed within 30 min in male Wistar rats. The transient decrease in blood pressure induced by ethanol was not affected by the previous administration of losartan (10 mg/kg; p.o. gavage), a selective AT1 receptor antagonist. Acute ethanol intake increased plasma renin activity (PRA), angiotensin converting enzyme (ACE) activity, plasma angiotensin I (ANG I) and angiotensin II (ANG II) levels. Ethanol induced systemic and vascular oxidative stress, evidenced by increased plasma thiobarbituric acid-reacting substances (TBARS) levels, NAD(P)H oxidase‐mediated vascular generation of superoxide anion and p47phox translocation (cytosol to membrane). These effects were prevented by losartan. Isolated aortas from ethanol-treated rats displayed increased p38MAPK and SAPK/JNK phosphorylation. Losartan inhibited ethanol-induced increase in the phosphorylation of these kinases. Ethanol intake decreased acetylcholine-induced relaxation and increased phenylephrine-induced contraction in endothelium-intact aortas. Ethanol significantly decreased plasma and aortic nitrate levels. These changes in vascular reactivity and in the end product of endogenous nitric oxide metabolism were not affected by losartan. Our study provides novel evidence that acute ethanol intake stimulates RAS activity and induces vascular oxidative stress and redox-signaling activation through AT1-dependent mechanisms. These findings highlight the importance of RAS in acute ethanol-induced oxidative damage. -- Highlights: ► Acute ethanol intake stimulates RAS activity and vascular oxidative stress. ► RAS plays a role in acute ethanol-induced oxidative damage via AT1 receptor activation. ► Translocation of p47

  13. Cathepsin G Induces Cell Aggregation of Human Breast Cancer MCF-7 Cells via a 2-Step Mechanism: Catalytic Site-Independent Binding to the Cell Surface and Enzymatic Activity-Dependent Induction of the Cell Aggregation

    Directory of Open Access Journals (Sweden)

    Riyo Morimoto-Kamata

    2012-01-01

    Full Text Available Neutrophils often invade various tumor tissues and affect tumor progression and metastasis. Cathepsin G (CG is a serine protease secreted from activated neutrophils. Previously, we have shown that CG induces the formation of E-cadherin-mediated multicellular spheroids of human breast cancer MCF-7 cells; however, the molecular mechanisms involved in this process are unknown. In this study, we investigated whether CG required its enzymatic activity to induce MCF-7 cell aggregation. The cell aggregation-inducing activity of CG was inhibited by pretreatment of CG with the serine protease inhibitors chymostatin and phenylmethylsulfonyl fluoride. In addition, an enzymatically inactive S195G (chymotrypsinogen numbering CG did not induce cell aggregation. Furthermore, CG specifically bound to the cell surface of MCF-7 cells via a catalytic site-independent mechanism because the binding was not affected by pretreatment of CG with serine protease inhibitors, and cell surface binding was also detected with S195G CG. Therefore, we propose that the CG-induced aggregation of MCF-7 cells occurs via a 2-step process, in which CG binds to the cell surface, independently of its catalytic site, and then induces cell aggregation, which is dependent on its enzymatic activity.

  14. Resistance exercise, but not endurance exercise, induces IKKβ phosphorylation in human skeletal muscle of training-accustomed individuals

    DEFF Research Database (Denmark)

    Møller, Andreas Buch; Vendelbo, Mikkel Holm; Rahbek, Stine Klejs;

    2013-01-01

    kinases (MAPKs) Erk1/2 and p38 has been suggested to link inflammation and cellular stress to activation of mTORC1 through the tuberous sclerosis 1 (TSC1)/tuberous sclerosis 2 (TSC2) complex. Consequently, activation of these proteins constitutes potential alternative mechanisms of mTORC1 activation...... finding was that IKKβ phosphorylation increased exclusively after resistance exercise. No changes in TSC1, Erk1/2, insulin receptor, or insulin receptor substrate 1 phosphorylation were observed in any of the groups, while p38 phosphorylation was higher in the resistance exercise group compared to both...

  15. p38 mitogen-activated protein kinase up-regulates NF-{kappa}B transcriptional activation through RelA phosphorylation during stretch-induced myogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Ji, Guoping [Department of Orthodontics, College of Stomatology, Ninth People' s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Research Institute of Stomatology, Shanghai 200011 (China); Liu, Dongxu [Department of Orthodontics, College of Stomatology, Shandong University, Jinan, Shandong Province 250012 (China); Liu, Jing [Department of Orthodontics, The Affiliated Qingdao Municipal Hospital, Qingdao University, Qingdao, Shandong Province 266075 (China); Gao, Hui [Department of Orthodontics, Tianjin Stomatological Hospital, Tianjin 300041 (China); Yuan, Xiao, E-mail: yuanxiaoqd@163.com [Department of Orthodontics, The Affiliated Qingdao Municipal Hospital, Qingdao University, Qingdao, Shandong Province 266075 (China); Shen, Gang, E-mail: ganshen2007@163.com [Department of Orthodontics, College of Stomatology, Ninth People' s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Research Institute of Stomatology, Shanghai 200011 (China)

    2010-01-01

    p38 MAPK and nuclear factor-B (NF-B) signaling pathways play an indispensable role in the control of skeletal myogenesis. The specific contribution of these signaling pathways to the response of myoblast to the mechanical stimulation and the molecular mechanisms underlying this response remain unresolved. Using an established in vitro model, we now show that p38 MAP kinase activity regulates the transcriptional activation of NF-{kappa}B in response to mechanical stimulation of myoblasts. Furthermore, SB203580 blocked stretch-induced NF-{kappa}B activation during myogenesis, not through down-regulation of degradation of I{kappa}B-{alpha}, and consequent translocation of the p65 subunit of NF-{kappa}B to the nucleus. It is likely that stretch-induced NF-{kappa}B activation by phosphorylation of p65 NF-{kappa}B. Moreover, depletion of p38{alpha} using siRNA significantly reduces stretch-induced phosphorylation of RelA and NF-{kappa}B activity. These results provides the first evidence of a cross-talk between p38 MAPK and NF-{kappa}B signaling pathways during stretch-induced myogenesis, with phosphorylation of RelA being one of the effectors of this promyogenic mechanism. The {alpha} isoform of p38MAP kinase regulates the transcriptional activation of NF-{kappa}B following stimulation with cyclic stretch.

  16. p38 mitogen-activated protein kinase up-regulates NF-κB transcriptional activation through RelA phosphorylation during stretch-induced myogenesis

    International Nuclear Information System (INIS)

    p38 MAPK and nuclear factor-B (NF-B) signaling pathways play an indispensable role in the control of skeletal myogenesis. The specific contribution of these signaling pathways to the response of myoblast to the mechanical stimulation and the molecular mechanisms underlying this response remain unresolved. Using an established in vitro model, we now show that p38 MAP kinase activity regulates the transcriptional activation of NF-κB in response to mechanical stimulation of myoblasts. Furthermore, SB203580 blocked stretch-induced NF-κB activation during myogenesis, not through down-regulation of degradation of IκB-α, and consequent translocation of the p65 subunit of NF-κB to the nucleus. It is likely that stretch-induced NF-κB activation by phosphorylation of p65 NF-κB. Moreover, depletion of p38α using siRNA significantly reduces stretch-induced phosphorylation of RelA and NF-κB activity. These results provides the first evidence of a cross-talk between p38 MAPK and NF-κB signaling pathways during stretch-induced myogenesis, with phosphorylation of RelA being one of the effectors of this promyogenic mechanism. The α isoform of p38MAP kinase regulates the transcriptional activation of NF-κB following stimulation with cyclic stretch.

  17. Dabigatran and rivaroxaban do not affect AA- and ADP-induced platelet aggregation in patients receiving concomitant platelet inhibitors.

    Science.gov (United States)

    Olivier, Christoph B; Weik, Patrick; Meyer, Melanie; Weber, Susanne; Diehl, Philipp; Bode, Christoph; Moser, Martin; Zhou, Qian

    2016-08-01

    Dabigatran and rivaroxaban are novel, vitamin K-independent oral anticoagulants (NOACs) and act via antagonism of the coagulation factor (F) IIa (dabigatran) or FXa (rivaroxaban), respectively. Compared to vitamin-K-antagonists, NOACs have shown non-inferiority of risk and benefit in patients with non valvular atrial fibrillation (AF). In clinical practice there is increasing use of NOACs combined with platelet inhibitors in patients with AF and coronary artery disease. However, whether NOACs affect the function of platelet inhibitors remains incompletely known. This observational study aimed to assess the platelet function in patients receiving dabigatran or rivaroxaban and concomitant platelet inhibitors. A single centre observational study was performed analysing the platelet aggregation of patients treated with dabigatran or rivaroxaban with or without concomitant platelet inhibitors. Measurements before the initiation of NOAC therapy served as the respective control group. Platelet aggregation was measured by multiple electrode aggregometry and was induced with adenosine diphosphate (ADP, 6.5 µM) and arachidonic acid (AA, 0.5 mM), respectively. In order to evaluate whether NOACs interact with platelet inhibition by ASA or the P2Y12-antagonist clopidogrel, 87 patients were grouped according to their concomitant antiplatelet medication. Comparing the ADP- and AA-induced platelet aggregation in patients without concomitant platelet inhibitors (n = 45) no significant differences under therapy with dabigatran (d) or rivaroxaban (r) compared to the control group (c) were observed. In patients taking clopidogrel as a concomitant platelet inhibitor (n = 21), neither dabigatran nor rivaroxaban affected the ADP-induced platelet aggregation (c 20 ± 11, d 21 ± 14, r 18 ± 8 AU*min, p = 0.200). Patients receiving dabigatran or rivaroxaban in combination with ASA (n = 42; 21 ASA only, 21 ASA + clopidogrel) showed no significant differences of the AA-induced

  18. A Diffusion Model of Field-Induced Aggregation in Ferrofluid Film

    Institute of Scientific and Technical Information of China (English)

    FANG Wen-Xiao; HE Zhen-Hui; CHEN Di-Hu; ZHAO Yan-E

    2008-01-01

    By introducing Arrhenius behaviour to the ferroparticles on the surface of the aggregated columnar structure in a diffusion model, equilibrium equations are set up. The solution of the equations shows that to keep the aggregated structures stable, a characteristic fleld is needed. The aggregation is enhanced by magnetic fields, yet suppressed as the temperature increases. Analysing the influence of the magnetic field on the interaction energy between the dipolar particles, we estimate the portion of the diffusing particles, and provide the agreeable ratio of the column radius over the centre-to-centre spacing between columns in a hexagonal columnar structure formed under a perpendicular magnetic field.

  19. Intensive aggregate formation with low vertical flux during an upwelling-induced diatom bloom

    DEFF Research Database (Denmark)

    Kiørboe, Thomas; Tiselius, P.; Mitchell-Innes, B.;

    1998-01-01

    The surfaces of most pelagic diatoms are sticky at times and may therefore form rapidly settling aggregates by physical coagulation. Stickiness and aggregate formation may be particularly adaptive in upwelling systems by allowing the retention of diatom populations in the vicinity of the upwelling...... center. We therefore hypothesized that upwelling diatom blooms are terminated by aggregate formation and rapid sedimentation. We monitored the development of a maturing diatom (mainly Chaetoceros spp.) bloom in the Benguela upwelling current during 7 d in February. Chlorophyll concentrations remained...

  20. Break-Induced Replication Requires DNA Damage-Induced Phosphorylation of Pif1 and Leads to Telomere Lengthening

    OpenAIRE

    Yulia Vasianovich; Harrington, Lea A.; Svetlana Makovets

    2014-01-01

    Broken replication forks result in DNA breaks that are normally repaired via homologous recombination or break induced replication (BIR). Mild insufficiency in the replicative ligase Cdc9 in budding yeast Saccharomyces cerevisiae resulted in a population of cells with persistent DNA damage, most likely due to broken replication forks, constitutive activation of the DNA damage checkpoint and longer telomeres. This telomere lengthening required functional telomerase, the core DNA damage signali...

  1. Kinetics of self-induced aggregation of Brownian particles: non-Markovian and non-Gaussian features

    CERN Document Server

    Ghosh, Pulak Kumar; Bag, Bidhan Chandra

    2012-01-01

    In this paper we have studied a model for self-induced aggregation in Brownian particle incorporating the non-Markovian and non-Gaussian character of the associated random noise process. In this model the time evolution of each individual is guided by an over-damped Langevin equation of motion with a non-local drift resulting from the local unbalance distributions of the other individuals. Our simulation result shows that colored nose can induce the cluster formation even at large noise strength. Another observation is that critical noise strength grows very rapidly with increase of noise correlation time for Gaussian noise than non Gaussian one. However, at long time limit the cluster number in aggregation process decreases with time following a power law. The exponent in the power law increases remarkable for switching from Markovian to non Markovian noise process.

  2. A neuroligin-1-derived peptide stimulates phosphorylation of the NMDA receptor NR1 subunit and rescues MK-801-induced decrease in long-term potentiation and memory impairment

    DEFF Research Database (Denmark)

    Korshunova, Irina; Gjørlund, Michelle D; Jacobsen, Sylwia Owczarek;

    2015-01-01

    neurolide-1 effects on short- and long-term social and spatial memory in social recognition, Morris water-maze, and Y-maze tests. We found that subcutaneous neurolide-1 administration, restored hippocampal LTP compromised by NMDA receptor inhibitor MK-801. It counteracted MK-801-induced memory deficit...... in the water-maze and Y-maze tests after long-term treatment (24 h and 1-2 h before the test), but not after short-term exposure (1-2 h). Long-term exposure to neurolide-1 also facilitated social recognition memory. In addition, neurolide-1-induced phosphorylation of the NMDA receptor NR1 subunit on a site...... receptor phosphorylation after treatment with NL1 or a mimetic peptide, neurolide-1, was quantified by immunoblotting. Subsequently, we investigated effects of neurolide-1 on long-term potentiation (LTP) induction in hippocampal slices compromised by NMDA receptor inhibitor MK-801. Finally, we investigated...

  3. Cdk5 phosphorylates non-genotoxically overexpressed p53 following inhibition of PP2A to induce cell cycle arrest/apoptosis and inhibits tumor progression

    Directory of Open Access Journals (Sweden)

    Kumari Ratna

    2010-07-01

    Full Text Available Abstract Background p53 is the most studied tumor suppressor and its overexpression may or may not cause cell death depending upon the genetic background of the cells. p53 is degraded by human papillomavirus (HPV E6 protein in cervical carcinoma. Several stress activated kinases are known to phosphorylate p53 and, among them cyclin dependent kinase 5 (Cdk5 is one of the kinase studied in neuronal cell system. Recently, the involvement of Cdk5 in phosphorylating p53 has been shown in certain cancer types. Phosphorylation at specific serine residues in p53 is essential for it to cause cell growth inhibition. Activation of p53 under non stress conditions is poorly understood. Therefore, the activation of p53 and detection of upstream kinases that phosphorylate non-genotoxically overexpressed p53 will be of therapeutic importance for cancer treatment. Results To determine the non-genotoxic effect of p53; Tet-On system was utilized and p53 inducible HPV-positive HeLa cells were developed. p53 overexpression in HPV-positive cells did not induce cell cycle arrest or apoptosis. However, we demonstrate that overexpressed p53 can be activated to upregulate p21 and Bax which causes G2 arrest and apoptosis, by inhibiting protein phosphatase 2A. Additionally, we report that the upstream kinase cyclin dependent kinase 5 interacts with p53 to phosphorylate it at Serine20 and Serine46 residues thereby promoting its recruitment on p21 and bax promoters. Upregulation and translocation of Bax causes apoptosis through intrinsic mitochondrial pathway. Interestingly, overexpressed activated p53 specifically inhibits cell-growth and causes regression in vivo tumor growth as well. Conclusion Present study details the mechanism of activation of p53 and puts forth the possibility of p53 gene therapy to work in HPV positive cervical carcinoma.

  4. Solution structure of copper ion-induced molecular aggregates of tyrosine melanin.

    OpenAIRE

    Gallas, J M; Littrell, K. C.; Seifert, S; Zajac, G W; Thiyagarajan, P.

    1999-01-01

    Melanin, the ubiquitous biological pigment, provides photoprotection by efficient filtration of light and also by its antioxidant behavior. In solutions of synthetic melanin, both optical and antioxidant behavior are affected by the aggregation states of melanin. We have utilized small-angle x-ray and neutron scattering to determine the molecular dimensions of synthetic tyrosine melanin in its unaggregated state in D(2)O and H(2)O to study the structure of melanin aggregates formed in the pre...

  5. Mucin 3 is involved in intestinal epithelial cell apoptosis via N-(3-oxododecanoyl)-L-homoserine lactone-induced suppression of Akt phosphorylation.

    Science.gov (United States)

    Taguchi, Ryoko; Tanaka, Shinya; Joe, Ga-Hyun; Maseda, Hideaki; Nomura, Nobuhiko; Ohnishi, Junji; Ishizuka, Satoshi; Shimizu, Hidehisa; Miyazaki, Hitoshi

    2014-07-15

    N-acyl-homoserine lactones (AHL) are quorum-sensing molecules in bacteria that play important roles in regulating virulence gene expression in pathogens such as Pseudomonas aeruginosa. The present study compared responses between undifferentiated and differentiated Caco-2 cells to N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL). A low concentration of 3-oxo-C12-HSL (30 μM) is sufficient to reduce viability accompanied by apoptosis via the suppression of phosphorylation by Akt in undifferentiated Caco-2 cells. The suppression of Akt phosphorylation appears specific in 3-oxo-C12-HSL, because other AHLs did not influence the phosphorylation status of Akt. The reduced viability induced by 3-oxo-C12-HSL was partially recovered by constitutively active Akt overexpression in undifferentiated Caco-2 cells. Since mucin is considered a vital component of the gut barrier, we investigated whether mucin protects cellular functions induced by 3-oxo-C12-HSL in undifferentiated Caco-2 cells. The results showed that mucin protected undifferentiated Caco-2 cells from apoptosis induced by 3-oxo-C12-HSL. 3-Oxo-C12-HSL did not induce cell death in differentiated Caco-2 cells that expressed higher levels of mucin 3 (MUC3) than undifferentiated Caco-2 cells. In addition, 3-oxo-C12-HSL promoted cell death in undifferentiated Caco-2 cells transfected with MUC3 siRNA and reduced MUC3 expression in undifferentiated Caco-2 cells. Therefore, MUC3 might be responsible for the survival of undifferentiated intestinal epithelial cells in the presence of 3-oxo-C12-HSL through regulating Akt phosphorylation. In conclusion, 3-oxo-C12-HSL might influence the survival of undifferentiated intestinal epithelial cells as well as interactions between these cells and pathogens.

  6. Inhibitory effect of puerarin on vascular smooth muscle cells proliferation induced by oxidised low-density lipoprotein via suppressing ERK 1/2 phosphorylation and PCNA expression.

    Science.gov (United States)

    Hu, Yanwu; Liu, Kai; Bo, Sun; Yan, Mengtong; Zhang, Yang; Miao, Chunsheng; Ren, Liqun

    2016-02-01

    Puerarin, an isoflavonoid isolated from the traditional Chinese herbal medicine Pueraria lobata (Wild.) Ohwi, has been shown to process antioxidant, anti-inflammatory, anti-cancer, anti-hypercholesterolemic, and anti-hyperglycemic activities in vivo and in vitro. The aim of the present study was to investigate the antiproliferative effects and the possible mechanisms of puerarin in vascular smooth muscle cells (VSMCs) stimulated with oxidised low-density lipoprotein (ox-LDL). VSMCs were cultured and pretreated with different concentrations of puerarin (0, 1, 10, 50 µM) before stimulated by ox-LDL (50 µg/mL). Cell proliferation was evaluated by MTT assay. Flow cytometry was used to study the influence of puerarin on cell cycle. Proliferating cell nuclear antigen (PCNA) expression and phosphorylation levels of extracellular signal-regulated kinase (ERK) 1/2 were detected by western blotting analysis. The results indicated that puerarin significantly inhibited VSMCs proliferation induced by ox-LDL and phosphorylation of ERK 1/2. Furthermore, puerarin also blocked the ox-LDL-induced cell-cycle progression at G1/S-interphase and down-regulated the expression of PCNA of VSMCs. The results suggest puerarin inhibits ox-LDL-induced proliferation of VSMCs by suppressing ERK 1/2 phosphorylation and PCNA expression.

  7. Arginine-aromatic interactions and their effects on arginine-induced solubilization of aromatic solutes and suppression of protein aggregation

    KAUST Repository

    Shah, Dhawal

    2011-09-21

    We examine the interaction of aromatic residues of proteins with arginine, an additive commonly used to suppress protein aggregation, using experiments and molecular dynamics simulations. An aromatic-rich peptide, FFYTP (a segment of insulin), and lysozyme and insulin are used as model systems. Mass spectrometry shows that arginine increases the solubility of FFYTP by binding to the peptide, with the simulations revealing the predominant association of arginine to be with the aromatic residues. The calculations further show a positive preferential interaction coefficient, Γ XP, contrary to conventional thinking that positive Γ XP\\'s indicate aggregation rather than suppression of aggregation. Simulations with lysozyme and insulin also show arginine\\'s preference for aromatic residues, in addition to acidic residues. We use these observations and earlier results reported by us and others to discuss the possible implications of arginine\\'s interactions with aromatic residues on the solubilization of aromatic moieties and proteins. Our results also highlight the fact that explanations based purely on Γ XP, which measures average affinity of an additive to a protein, could obscure or misinterpret the underlying molecular mechanisms behind additive-induced suppression of protein aggregation. © 2011 American Institute of Chemical Engineers (AIChE).

  8. Time-dependent inhibitory effects of cGMP-analogues on thrombin-induced platelet-derived microparticles formation, platelet aggregation, and P-selectin expression

    Energy Technology Data Exchange (ETDEWEB)

    Nygaard, Gyrid [Proteomic Unit at University of Bergen (PROBE), University of Bergen, Bergen (Norway); Department of Biomedicine, University of Bergen, Bergen (Norway); Herfindal, Lars; Kopperud, Reidun [Department of Biomedicine, University of Bergen, Bergen (Norway); Aragay, Anna M. [Department of Biomedicine, University of Bergen, Bergen (Norway); Molecular Biology Institute of Barcelona (IBMB, CSIC), Barcelona (Spain); Holmsen, Holm; Døskeland, Stein Ove; Kleppe, Rune [Department of Biomedicine, University of Bergen, Bergen (Norway); Selheim, Frode, E-mail: Frode.Selheim@biomed.uib.no [Proteomic Unit at University of Bergen (PROBE), University of Bergen, Bergen (Norway); Department of Biomedicine, University of Bergen, Bergen (Norway)

    2014-07-04

    Highlights: • We investigated the impact of cyclic nucleotide analogues on platelet activation. • Different time dependence were found for inhibition of platelet activation. • Additive effect was found using PKA- and PKG-activating analogues. • Our results may explain some of the discrepancies reported for cNMP signalling. - Abstract: In platelets, nitric oxide (NO) activates cGMP/PKG signalling, whereas prostaglandins and adenosine signal through cAMP/PKA. Cyclic nucleotide signalling has been considered to play an inhibitory role in platelets. However, an early stimulatory effect of NO and cGMP-PKG signalling in low dose agonist-induced platelet activation have recently been suggested. Here, we investigated whether different experimental conditions could explain some of the discrepancy reported for platelet cGMP-PKG-signalling. We treated gel-filtered human platelets with cGMP and cAMP analogues, and used flow cytometric assays to detect low dose thrombin-induced formation of small platelet aggregates, single platelet disappearance (SPD), platelet-derived microparticles (PMP) and thrombin receptor agonist peptide (TRAP)-induced P-selectin expression. All four agonist-induced platelet activation phases were blocked when platelets were costimulated with the PKG activators 8-Br-PET-cGMP or 8-pCPT-cGMP and low-doses of thrombin or TRAP. However, extended incubation with 8-Br-PET-cGMP decreased its inhibition of TRAP-induced P-selectin expression in a time-dependent manner. This effect did not involve desensitisation of PKG or PKA activity, measured as site-specific VASP phosphorylation. Moreover, PKG activators in combination with the PKA activator Sp-5,6-DCL-cBIMPS revealed additive inhibitory effect on TRAP-induced P-selectin expression. Taken together, we found no evidence for a stimulatory role of cGMP/PKG in platelets activation and conclude rather that cGMP/PKG signalling has an important inhibitory function in human platelet activation.

  9. Aggregation-induced reversal of transport distances of soil organic matter: are our balances correct?

    Science.gov (United States)

    Hu, Yaxian; Kuhn, Nikolaus

    2014-05-01

    The effect of soil erosion on global carbon cycling, especially as a source or sink of green-house gases (GHGs), is the subject of intense debate. The controversy arises mostly from the lack of information on the fate of eroded soil organic carbon (SOC) as it moves from the site of erosion to the site of longer-term deposition. This requires improved understanding the transport distances of eroded SOC, which is principally related to the settling velocities of sediment fractions that carry the eroded SOC. For aggregated soils, settling velocities are affected by their actual aggregate size rather than the mineral grain size distribution. Aggregate stability is, in turn, strongly influenced by soil organic matter. This study aims at identifying the effect of aggregation on the transport distances of eroded SOC and its susceptibility to mineralization after transport and deposition. A rainfall simulation was carried out on a silty loam soil. The eroded sediments were fractionated by a settling tube apparatus into six different size classes according to their settling velocities and likely transport distances. Weight, SOC concentration and instantaneous respiration rates of the fractions of the six classes were measured. Our results show that: 1) 41% of the eroded SOC was transported with coarse aggregates that would be likely re-distributed across landscapes; 2) erosion was prone to accelerate the mineralization of eroded organic carbon immediately after erosion, compared to undisturbed aggregates; 3) erosion might make a higher contribution to atmospheric CO2 than the estimation made without considering the effects of aggregation and extra SOC mineralization during transport.

  10. Monitoring of Intracellular Tau Aggregation Regulated by OGA/OGT Inhibitors

    Directory of Open Access Journals (Sweden)

    Sungsu Lim

    2015-08-01

    Full Text Available Abnormal phosphorylation of tau has been considered as a key pathogenic mechanism inducing tau aggregation in multiple neurodegenerative disorders, collectively called tauopathies. Recent evidence showed that tau phosphorylation sites are protected with O-linked β-N-acetylglucosamine (O-GlcNAc in normal brain. In pathological condition, tau is de-glycosylated and becomes a substrate for kinases. Despite the importance of O-GlcNAcylation in tau pathology, O-GlcNAc transferase (OGT, and an enzyme catalyzing O-GlcNAc to tau, has not been carefully investigated in the context of tau aggregation. Here, we investigated intracellular tau aggregation regulated by BZX2, an inhibitor of OGT. Upon the inhibition of OGT, tau phosphorylation increased 2.0-fold at Ser199 and 1.5-fold at Ser396, resulting in increased tau aggregation. Moreover, the BZX2 induced tau aggregation was efficiently reduced by the treatment of Thiamet G, an inhibitor of O-GlcNAcase (OGA. Our results demonstrated the protective role of OGT in tau aggregation and also suggest the counter-regulatory mechanism of OGA and OGT in tau pathology.

  11. Adenovirus-induced extracellular signal-regulated kinase phosphorylation during the late phase of infection enhances viral protein levels and virus progeny

    DEFF Research Database (Denmark)

    Schümann, Michael; Dobbelstein, Matthias

    2006-01-01

    . Hence, adenovirus induces the oncogenic Raf/MEK/ERK signaling pathway to enhance viral progeny by sustaining the levels of viral proteins. Concerning therapy, our results suggest that the use of Raf/MEK/ERK inhibitors will interfere with the propagation of oncolytic adenoviruses.......The Raf/mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK signaling cascade enhances tumor cell proliferation in many cases. Here, we show that adenovirus type 5, a small DNA tumor virus used in experimental cancer therapy, strongly induces ERK phosphorylation...

  12. HDAC2 phosphorylation-dependent KIf5 deacetylation and RARα acetylation induced by RAR agonist switch the transcription regulatory programs of p21 in VSMCs

    Institute of Scientific and Technical Information of China (English)

    Bin Zheng; Mei Han; Ya-nan Shu; Yimg-jie Li; Sui-bing Miao; Xin-hua Zhang; Hui-jing Shi; Tian zhang; Jin-kun Wen

    2011-01-01

    Abnormal proliferation of vascular smooth muscle cells (VSMCs) occurs in hypertension,atherosclerosis and restenosis after angioplasty,leading to pathophysiological vascular remodeling.As an important growth arrest gene,p21 plays critical roles in vascular remodeling.Regulation of p21 expression by retinoic acid receptor (RAR) and its ligand has important implications for control of pathological vascular remodeling.Nevertheless,the mechanism of RAR-mediated p21 expression in VSMCs remains poorly understood.Here,we show that,under basal conditions,RARa forms a complex with histone deacetylase 2 (HDAC2) and Krüppel-like factor 5 (KIf5) at the p21 promoter to inhibit its expression.Upon RARα agonist stimulation,HDAC2 is phosphorylated by CK2α.Phosphorylation of HDAC2,on the one hand,promotes its dissociation from RARα,thus allowing the liganded-RARα to interact with co-activators; on the other hand,it increases its interaction with KIf5,thus leading to deacetylation of Klf5.Deacetylation of KIf5 facilitates its dissociation from thep21 promoter,relieving its repressive effect on thep21 promoter.Interference with HDAC2 phosphorylation by either CK2α knockdown or the use of phosphorylation-deficient mutant of HDAC2 prevents the dissociation of KIf5 from the p21 promoter and impairs RAR agonist-induced p21 activation.Our results reveal a novel mechanism involving a phosphorylation-deacetylation cascade that functions to remove the basal repression complex from the p21 promoter upon RAR agonlst treatment,allowing for optimum agonistinduced p21 expression.

  13. Nitric oxide inhibited the melanophore aggregation induced by extracellular calcium concentration in snakehead fish, Channa punctatus.

    Science.gov (United States)

    Biswas, Saikat P; Palande, Nikhil V; Jadhao, Arun G

    2011-12-01

    We studied the role of nitric oxide (NO) and extra-cellular Ca(2+) on the melanophores in Indian snakehead teleost, Channa punctatus. Increase of Ca(2+) level in the external medium causes pigment aggregation in melanophores. This pigment-aggregating effect was found to be inhibited when the external medium contained spontaneous NO donor, sodium nitro prusside (SNP) at all the levels of concentration tested. Furthermore, it has been observed that SNP keeps the pigment in dispersed state even after increasing the amount of Ca(2+). In order to test whether NO donor SNP causes dispersion of pigments or not is checked by adding the inhibitor of nitric oxide synthase, N-omega-Nitro-L-arginine (L-NNA) in the medium. It has been noted that the inhibitor L-NNA blocked the effect of NO donor SNP causing aggregation of pigments. In that way NO is inhibiting the effect of extracellular Ca(2+), keeping the pigment dispersed.

  14. Role of amorphous and aggregate phases on field-induced exciton dissociation in a conjugated polymer

    Science.gov (United States)

    Mróz, Marta M.; Lüer, Larry; Houarner-Rassin, Coralie; Anderson, Harry L.; Cabanillas-Gonzalez, Juan

    2013-01-01

    We have applied electric field assisted pump-probe spectroscopy in order to unravel the interplay of amorphous and aggregate phases on the polaron-pair photogeneration process in a conjugated porphyrin polymer. We find that excitons photogenerated in both phases are precursors for polaron pairs with different yields. Kinetic modeling indicates a substantially larger barrier for exciton dissociation in aggregates compared to amorphous areas. The majority of polaron pairs are however formed in aggregate phases due to efficient energy transfer from the amorphous phase. Based on the change in the Stark shift associated with the photogenerated polaron density, we provide a picture of the motion of polaron pairs under the external electric field.

  15. CFD simulation of shear-induced aggregation and breakage in turbulent Taylor-Couette flow.

    Science.gov (United States)

    Wang, Liguang; Vigil, R Dennis; Fox, Rodney O

    2005-05-01

    An experimental and computational investigation of the effects of local fluid shear rate on the aggregation and breakage of approximately 10 microm latex spheres suspended in an aqueous solution undergoing turbulent Taylor-Couette flow was carried out. First, computational fluid dynamics (CFD) simulations were performed and the flow field predictions were validated with data from particle image velocimetry experiments. Subsequently, the quadrature method of moments (QMOM) was implemented into the CFD code to obtain predictions for mean particle size that account for the effects of local shear rate on the aggregation and breakage. These predictions were then compared with experimental data for latex sphere aggregates (using an in situ optical imaging method). Excellent agreement between the CFD-QMOM and experimental results was observed for two Reynolds numbers in the turbulent-flow regime. PMID:15797411

  16. Direct observation of electric field induced pattern formation and particle aggregation in ferrofluids

    Energy Technology Data Exchange (ETDEWEB)

    Rajnak, Michal; Kopcansky, Peter; Timko, Milan [Institute of Experimental Physics SAS, Watsonova 47, 04001 Košice (Slovakia); Petrenko, Viktor I. [Joint Institute for Nuclear Research, Joliot-Curie 6, 141980 Dubna, Moscow Region (Russian Federation); Kyiv Taras Shevchenko National University, Volodymyrska Street 64, Kyiv 01033 (Ukraine); Avdeev, Mikhail V. [Joint Institute for Nuclear Research, Joliot-Curie 6, 141980 Dubna, Moscow Region (Russian Federation); Ivankov, Olexandr I. [Joint Institute for Nuclear Research, Joliot-Curie 6, 141980 Dubna, Moscow Region (Russian Federation); Kyiv Taras Shevchenko National University, Volodymyrska Street 64, Kyiv 01033 (Ukraine); Moscow Institute of Physics and Technology, Institutskiy per. 9, Dolgoprudniy 141700 (Russian Federation); Feoktystov, Artem [Jülich Centre for Neutron Science (JCNS) at Heinz Maier-Leibnitz Zentrum (MLZ), Forschungszentrum Jülich GmbH, Lichtenbergstr. 1, 85747 Garching (Germany); Dolnik, Bystrik; Kurimsky, Juraj [Faculty of Electrical Engineering and Informatics, Technical University of Košice, Letná 9, 04200 Košice (Slovakia)

    2015-08-17

    Ferrofluids typically respond to magnetic fields and can be manipulated by external magnetic fields. Here, we report on formation of visually observable patterns in a diluted low-polarity ferrofluid exposed to external electric fields. This presents a specific type of ferrofluid structure driven by a combined effect of electrohydrodynamics and electrical body forces. The free charge and permittivity variation are considered to play a key role in the observed phenomenon. The corresponding changes in the ferrofluid structure have been found at nanoscale as well. By small-angle neutron scattering (SANS), we show that the magnetic nanoparticles aggregate in direct current (dc) electric field with a strong dependence on the field intensity. The anisotropic aggregates preferably orient in the direction of the applied electric field. Conducting SANS experiments with alternating current (ac) electric fields of various frequencies, we found a critical frequency triggering the aggregation process. Our experimental study could open future applications of ferrofluids based on insulating liquids.

  17. Direct observation of electric field induced pattern formation and particle aggregation in ferrofluids

    Science.gov (United States)

    Rajnak, Michal; Petrenko, Viktor I.; Avdeev, Mikhail V.; Ivankov, Olexandr I.; Feoktystov, Artem; Dolnik, Bystrik; Kurimsky, Juraj; Kopcansky, Peter; Timko, Milan

    2015-08-01

    Ferrofluids typically respond to magnetic fields and can be manipulated by external magnetic fields. Here, we report on formation of visually observable patterns in a diluted low-polarity ferrofluid exposed to external electric fields. This presents a specific type of ferrofluid structure driven by a combined effect of electrohydrodynamics and electrical body forces. The free charge and permittivity variation are considered to play a key role in the observed phenomenon. The corresponding changes in the ferrofluid structure have been found at nanoscale as well. By small-angle neutron scattering (SANS), we show that the magnetic nanoparticles aggregate in direct current (dc) electric field with a strong dependence on the field intensity. The anisotropic aggregates preferably orient in the direction of the applied electric field. Conducting SANS experiments with alternating current (ac) electric fields of various frequencies, we found a critical frequency triggering the aggregation process. Our experimental study could open future applications of ferrofluids based on insulating liquids.

  18. Sodium butyrate modulates pRb phosphorylation and induces cell death in human vestibular schwannomas in vitro.

    Science.gov (United States)

    Mitra, Rohan; Devi, B Indira; Gope, Mohan L; Subbakrishna, D K; Gope, Rajalakshmi

    2012-01-01

    In the present study, effect of Na-Bu on the pRb phosphorylation was analysed in the primary cultures of 12 VS tumors. Primary cultures of VS tumors were established from the fresh tumor tissues removed surgically and were treated with Na-Bu. Na-Bu treatment for 48 h led to morphological changes and apoptotic cell death in VS tumor cells. Na-Bu treatment decreased level of total pRb and phosphorylated form of pRb and caused specific dephosphorylation at Ser 249/Thr 252 and Ser 567. In the untreated and Na-Bu treated cells (when present), pRb was localised in the nucleus. Moreover, in Na-Bu treated cells the nucleus appeared highly condensed as compared to untreated cells. Results of the present study indicated that Na-Bu treatment modulated pRb phosphorylation status and caused apoptotic cell death in VS tumors.

  19. Ultrasound-induced emission enhancement based on structure-dependent homo- and heterochiral aggregations of chiral binuclear platinum complexes.

    Science.gov (United States)

    Komiya, Naruyoshi; Muraoka, Takako; Iida, Masayuki; Miyanaga, Maiko; Takahashi, Koichi; Naota, Takeshi

    2011-10-12

    Instant and precise control of phosphorescent emission can be performed by ultrasound-induced gelation of organic liquids with chiral, clothespin-shaped trans-bis(salicylaldiminato)Pt(II) complexes, anti-1. Nonemissive solutions of racemic, short-linked anti-1a (n = 5) and optically pure, long-linked anti-1c (n = 7) in organic liquids are transformed immediately into stable phosphorescent gels upon brief irradiation of low-power ultrasound. Emission from the gels can be controlled by sonication time, linker length, and optical activity of the complexes. Several experimental results indicated that structure-dependent homo- and heterochiral aggregations and ultrasound-control of the aggregate morphology are key factors for emission enhancement. PMID:21894951

  20. Aggregation-Induced Delayed Fluorescence Based on Donor/Acceptor-Tethered Janus Carborane Triads: Unique Photophysical Properties of Nondoped OLEDs.

    Science.gov (United States)

    Furue, Ryuhei; Nishimoto, Takuro; Park, In Seob; Lee, Jiyoung; Yasuda, Takuma

    2016-06-13

    Luminescent materials consisting of boron clusters, such as carboranes, have attracted immense interest in recent years. In this study, luminescent organic-inorganic conjugated systems based on o-carboranes directly bonded to electron-donating and electron-accepting π-conjugated units were elaborated as novel optoelectronic materials. These o-carborane derivatives simultaneously possessed aggregation-induced emission (AIE) and thermally activated delayed fluorescence (TADF) capabilities, and showed strong yellow-to-red emissions with high photoluminescence quantum efficiencies of up to 97 % in their aggregated states or in solid neat films. Organic light-emitting diodes utilizing these o-carborane derivatives as a nondoped emission layer exhibited maximum external electroluminescence quantum efficiencies as high as 11 %, originating from TADF. PMID:27145481

  1. Damage Simulation of a Random Aggregate Model Induced by Microwave under Different Discontinuous Ratios and Exposure Times

    Directory of Open Access Journals (Sweden)

    Yang Tang

    2016-01-01

    Full Text Available A random aggregate algorithmic method and a numerical model for two-phase materials (composed of quartz and plagioclase with different discontinuous ratios and irradiation times were studied based on the discrete element method using two-dimensional particle flow code (PFC2D. The results showed that this algorithm can simulate random irregular aggregate shapes. Furthermore, crack initiation and development and the coalescence process of microwave-induced material damage could be predicted using the discrete element method. After analysis of this study, the micro crack originated from the boundary of the high-absorption-phase plagioclase crystal and expanded around the plagioclase, extending into the quartz material. The crack morphology presented a radial network.

  2. ADHESION-INDUCE PROTEIN TYROSINE PHOSPHORY-LATION IS ASSOCIATED WITH INVASIVE AND METASTATIC POTENTIALS IN B16-BL6 MELANOMA CELLS

    Institute of Scientific and Technical Information of China (English)

    Yan Chunhong; Han Rui

    1998-01-01

    Objective: The interaction of cancer cell with extracellular matrix (ECM) happens as an earlier and specific event in the invasive and metastatic cascade. To explore the key element(s) in cancer metastasis and observe the cell-ECM interaction and its role. Methods:To interrupt the cell-ECM interaction by suppression of adhesion-induced protein tyrosine phosphorylation with protein tyrosine kinase inhibitor genistein in B16-B16mouse melanoma cells. Results: When B16-BL6 cells attached to Matrigel, a solubilized basement membrane preparation from EHS sarcoma, a 125 kDa protein increased its phosphotyrosine content dramatically. In contrast, when the cells were pretreated with 20μM or 30μM genistein for 3 days, it was revealed a less increase in the phosphotyrosine content of this 125 kDa protein inresponse to cell attachment to ECM was revealed with immunoblot analysis. Accompanied by the lower level of adhesion-induced protein tyrosine phosphorylation the genistein-treated cells exhibited a decrease in their capabilities of adhesion to Matrigel and invasion through reconstituted basement membrane. The potentials of and forming lung metastatic nodules were also shown to be decreased dramatically in these genistein-treated cells.Conclusion: It was suggested that protein tyrosine phosphorylation in cell-ECM interaction might be associated with invasive and metastatic potentials in cancer cells.

  3. Prometaphase arrest-dependent phosphorylation of Bcl-2 family proteins and activation of mitochondrial apoptotic pathway are associated with 17α-estradiol-induced apoptosis in human Jurkat T cells.

    Science.gov (United States)

    Han, Cho Rong; Jun, Do Youn; Kim, Yoon Hee; Lee, Ji Young; Kim, Young Ho

    2013-10-01

    In Jurkat T cell clone (JT/Neo), G2/M arrest, apoptotic sub-G1 peak, mitochondrial membrane potential (Δψm) loss, and TUNEL-positive DNA fragmentation were induced following exposure to 17α-estradiol (17α-E2), whereas none of these events (except for G2/M arrest) were induced in Jurkat cells overexpressing Bcl-2 (JT/Bcl-2). Under these conditions, phosphorylation at Thr161 and dephosphorylation at Tyr15 of Cdk1, upregulation of cyclin B1 level, histone H1 phosphorylation, Cdc25C phosphorylation at Thr-48, Bcl-2 phosphorylation at Thr-56 and Ser-70, Mcl-1 phosphorylation, and Bim phosphorylation were detected in the presence of Bcl-2 overexpression. However, the 17α-E2-induced upregulation of Bak levels, activation of Bak, activation of caspase-3, and PARP degradation were abrogated by Bcl-2 overexpression. In the presence of the G1/S blocking agent hydroxyurea, 17α-E2 failed to induce G2/M arrest and all apoptotic events including Cdk1 activation and phosphorylation of Bcl-2, Mcl-1 and Bim. The 17α-E2-induced phosphorylation of Bcl-2 family proteins and mitochondrial apoptotic events were suppressed by a Cdk1 inhibitor but not by aurora A and aurora B kinase inhibitors. Immunofluorescence microscopic analysis showed that an aberrant bipolar microtubule array, incomplete chromosome congression at the metaphase plate, and prometaphase arrest, which was reversible, were the underlying factors for 17α-E2-induced mitotic arrest. The in vitro microtubule polymerization assay showed that 17α-E2 could directly inhibit microtubule formation. These results show that the apoptogenic activity of 17α-E2 was due to the impaired mitotic spindle assembly causing prometaphase arrest and prolonged Cdk1 activation, the phosphorylation of Bcl-2, Mcl-1 and Bim, and the activation of Bak and mitochondria-dependent caspase cascade. PMID:23707954

  4. SB203580 Modulates p38 MAPK Signaling and Dengue Virus-Induced Liver Injury by Reducing MAPKAPK2, HSP27, and ATF2 Phosphorylation.

    Directory of Open Access Journals (Sweden)

    Gopinathan Pillai Sreekanth

    Full Text Available Dengue virus (DENV infection causes organ injuries, and the liver is one of the most important sites of DENV infection, where viral replication generates a high viral load. The molecular mechanism of DENV-induced liver injury is still under investigation. The mitogen activated protein kinases (MAPKs, including p38 MAPK, have roles in the hepatic cell apoptosis induced by DENV. However, the in vivo role of p38 MAPK in DENV-induced liver injury is not fully understood. In this study, we investigated the role of SB203580, a p38 MAPK inhibitor, in a mouse model of DENV infection. Both the hematological parameters, leucopenia and thrombocytopenia, were improved by SB203580 treatment and liver transaminases and histopathology were also improved. We used a real-time PCR microarray to profile the expression of apoptosis-related genes. Tumor necrosis factor α, caspase 9, caspase 8, and caspase 3 proteins were significantly lower in the SB203580-treated DENV-infected mice than that in the infected control mice. Increased expressions of cytokines including TNF-α, IL-6 and IL-10, and chemokines including RANTES and IP-10 in DENV infection were reduced by SB203580 treatment. DENV infection induced the phosphorylation of p38MAPK, and its downstream signals including MAPKAPK2, HSP27 and ATF-2. SB203580 treatment did not decrease the phosphorylation of p38 MAPK, but it significantly reduced the phosphorylation of MAPKAPK2, HSP27, and ATF2. Therefore, SB203580 modulates the downstream signals to p38 MAPK and reduces DENV-induced liver injury.

  5. Hippocampal expression of synaptic structural proteins and phosphorylated cAMP response element-binding protein in a rat model of vascular dementia induced by chronic cerebral hypoperfusion

    Institute of Scientific and Technical Information of China (English)

    Hui Zhao; Zhiyong Li; Yali Wang; Qiuxia Zhang

    2012-01-01

    The present study established a rat model of vascular dementia induced by chronic cerebral hy-poperfusion through permanent ligation of bilateral common carotid arteries. At 60 days after mod-eling, escape latency and swimming path length during hidden-platform acquisition training in Morris water maze significantly increased in the model group. In addition, the number of accurate crossings over the original platform significantly decreased, hippocampal CA1 synaptophysin and growth-associated protein 43 expression significantly decreased, cAMP response element-binding protein expression remained unchanged, and phosphorylated cAMP response element-binding protein expression significantly decreased. Results suggested that abnormal expression of hippo-campal synaptic structural protein and cAMP response element-binding protein phosphorylation played a role in cognitive impairment following chronic cerebral hypoperfusion.

  6. Earthworm-induced distribution of organic matter in macro-aggregates from differently managed arable fields.

    NARCIS (Netherlands)

    Marinissen, J.C.Y.; Hillenaar, S.I.

    1997-01-01

    To study the influence of soil structure on organic matter decomposition, and the possible role of earthworms therein, aggregates of the size of earthworm casts (3-4.8 mm) were sieved from air-dry soil of three arable fields. Due to different management histories (in terms of manuring and pesticide

  7. Direct Observation of Aggregation-Induced Backbone Conformational Changes in Tau Peptides.

    Science.gov (United States)

    Jiji, A C; Shine, A; Vijayan, Vinesh

    2016-09-12

    In tau proteins, the hexapeptides in the R2 and R3 repeats are known to initiate tau fibril formation, which causes a class of neurodegenerative diseases called the taupathies. We show that in R3, in addition to the presence of the hexapeptides, the correct turn conformation upstream to it is also essential for producing prion-like fibrils that are capable of propagation. A time-dependent NMR aggregation assay of a slow fibril forming R3-S316P peptide revealed a trans to cis equilibrium shift in the peptide-bond conformation preceding P316 during the growth phase of the aggregation process. S316 was identified as the key residue in the turn that confers templating capacity on R3 fibrils to accelerate the aggregation of the R3-S316P peptide. These results on the specific interactions and conformational changes responsible for tau aggregation could prove useful for developing an efficient therapeutic intervention in Alzheimer's disease. PMID:27513615

  8. Lactic Acid Reduces LPS-Induced TNF- and IL-6 mRNA Levels Through Decreasing IB Phosphorylation

    Institute of Scientific and Technical Information of China (English)

    XU Guang-yong; JIANG Jin-qi; WANG Ming; LI Jie; SU Jing-liang; REN Xiao-ming

    2013-01-01

    This study explored the effects over time of lactic acid (LA) on IBαphosphorylation and nuclear factor-kappa B(NF-B) p65 protein expression, and on tumor necrosis factor (TNF-α) and interleukin-6 (IL-6) mRNA levels in rat intestinal mucosa microvascular endothelial cells (RIMMVECs) stimulated by lipopolysaccharide (LPS). IB , phosphorylated IB (p-IB ) and p65 protein levels were monitored by Western blot analysis, and TNF- and IL-6 mRNA levels were analyzed using real-time PCR. LA treatment reduced TNF- and IL-6 mRNA levels in LPS-stimulated RIMMVECs, with the greatest effect being after 3 h. The highest inhibitory effect of LA on IB phosphorylation to prevent activation of NF-B was after 6 h. These results suggest that LA reduces TNF- and IL-6 mRNA levels through decreasing IB phosphorylation and blocking the dissociation of IKK complex, which prevents activation of NF-B.

  9. Inhibition of protein phosphatase 2A induces serine/threonine phosphorylation, subcellular redistribution, and functional inhibition of STAT3

    DEFF Research Database (Denmark)

    Woetmann, A; Nielsen, M; Christensen, S T;

    1999-01-01

    Signal transducers and activators of transcription (STATs) are rapidly phosphorylated on tyrosine residues in response to cytokine and growth factor stimulation of cell surface receptors. STATs hereafter are translocated to the nucleus where they act as transcription factors. Recent reports sugge...

  10. INTERLEUKIN-6-INDUCED SERINE PHOSPHORYLATION OF TRANSCRIPTION FACTOR APRF - EVIDENCE FOR A ROLE IN INTERLEUKIN-6 TARGET GENE INDUCTION

    NARCIS (Netherlands)

    LUTTICKEN, C; COFFER, P; YUAN, JP; SCHWARTZ, C; CALDENHOVEN, E; SCHINDLER, C; KRUIJER, W; HEINRICH, PC; HORN, F

    1995-01-01

    The cytokine interleukin-6 (IL-6) rapidly activates a latent cytoplasmic transcription factor, acute-phase response factor (APRF), by tyrosine phosphorylation. Activation and DNA binding of APRF are inhibited by inhibitors of protein tyrosine kinases but not serine/threonine kinases. However, immedi

  11. The Prolyl Isomerase Pin1 Promotes the Herpesvirus-Induced Phosphorylation-Dependent Disassembly of the Nuclear Lamina Required for Nucleocytoplasmic Egress.

    Science.gov (United States)

    Milbradt, Jens; Hutterer, Corina; Bahsi, Hanife; Wagner, Sabrina; Sonntag, Eric; Horn, Anselm H C; Kaufer, Benedikt B; Mori, Yasuko; Sticht, Heinrich; Fossen, Torgils; Marschall, Manfred

    2016-08-01

    The nuclear lamina lines the inner nuclear membrane providing a structural framework for the nucleus. Cellular processes, such as nuclear envelope breakdown during mitosis or nuclear export of large ribonucleoprotein complexes, are functionally linked to the disassembly of the nuclear lamina. In general, lamina disassembly is mediated by phosphorylation, but the precise molecular mechanism is still not completely understood. Recently, we suggested a novel mechanism for lamina disassembly during the nuclear egress of herpesviral capsids which involves the cellular isomerase Pin1. In this study, we focused on mechanistic details of herpesviral nuclear replication to demonstrate the general importance of Pin1 for lamina disassembly. In particular, Ser22-specific lamin phosphorylation consistently generates a Pin1-binding motif in cells infected with human and animal alpha-, beta-, and gammaherpesviruses. Using nuclear magnetic resonance spectroscopy, we showed that binding of Pin1 to a synthetic lamin peptide induces its cis/trans isomerization in vitro. A detailed bioinformatic evaluation strongly suggests that this structural conversion induces large-scale secondary structural changes in the lamin N-terminus. Thus, we concluded that a Pin1-induced conformational change of lamins may represent the molecular trigger responsible for lamina disassembly. Consistent with this concept, pharmacological inhibition of Pin1 activity blocked lamina disassembly in herpesvirus-infected fibroblasts and consequently impaired virus replication. In addition, a phospho-mimetic Ser22Glu lamin mutant was still able to form a regular lamina structure and overexpression of a Ser22-phosphorylating kinase did not induce lamina disassembly in Pin1 knockout cells. Intriguingly, this was observed in absence of herpesvirus infection proposing a broader importance of Pin1 for lamina constitution. Thus, our results suggest a functional model of similar events leading to disassembly of the nuclear

  12. Hydrogen sulfide prevents OGD/R-induced apoptosis by suppressing the phosphorylation of p38 and secretion of IL-6 in PC12 cells.

    Science.gov (United States)

    Li, Chong; Liu, Yue; Tang, Peng; Liu, Peng; Hou, Chen; Zhang, Xin; Chen, Li; Zhang, Lina; Gu, Chaochao

    2016-03-01

    Hydrogen sulfide (H2S), a well-known endogenous mediator, has been shown to exert protective effects against neuronal damage caused by brain ischemia, but the mechanism of its action remains unclear. We have reported the neuroprotective properties of H2S against oxygen-glucose deprivation/reoxygenation (OGD/R)-induced injury by inhibiting the phosphorylation of p38. The present study evaluates the effect of H2S on OGD/R-induced cell injury or apoptosis and the mechanisms for its action in PC12 cells. Pretreatment of PC12 cells with exogenous sodium hydrosulfide (NaHS) (a H2S donor, 100 or 300 µM) for 12 h before exposure to OGD/R markedly attenuated p38 phosphorylation. Activation of p38 MAPK by transfection of activated p38α, but not p38β, reversed the protective effect of NaHS, as measured by enzyme-linked immunosorbent assay analysis. Importantly, SB203580 (a p38 MAPK inhibitor) also reversed the protective effects of p38α-activated p38 MAPK. Interleukin-6 secretion after OGD/R decreased significantly with NaHS compared with without NaHS. Taken together, we show that the p38 pathway contributes toward OGD/R-induced cell death and p38α plays a key role in OGD/R-induced interleukin-6 secretion.

  13. Aristolochia manshuriensis Kom ethyl acetate extract protects against high-fat diet-induced non-alcoholic steatohepatitis by regulating kinase phosphorylation in mouse

    Science.gov (United States)

    Kwak, Dong Hoon; Kim, Ji-Su; Chang, Kyu-Tae

    2016-01-01

    Aristolochia manshuriensis Kom (AMK) is an herb used as a traditional medicine; however, it causes side effects such as nephrotoxicity and carcinogenicity. Nevertheless, AMK can be applied in specific ways medicinally, including via ingestion of low doses for short periods of time. Non-alcoholic steatohepatitis (NASH) induced the hepatocyte injury and inflammation. The protective effects of AMK against NASH are unclear; therefore, in this study, the protective effects of AMK ethyl acetate extract were investigated in a high-fat diet (HFD)-induced NASH model. We found decreased hepatic steatosis and inflammation, as well as increased levels of lipoproteins during AMK extract treatment. We also observed decreased hepatic lipid peroxidation and triglycerides, as well as suppressed hepatic expression of lipogenic genes in extract-treated livers. Treatment with extract decreased the activation of c-jun N-terminal kinase 1/2 (JNK1/2) and increased the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). These results demonstrate that the protective effect of the extract against HFD-induced NASH occurred via reductions in reactive oxygen species production, inflammation suppression, and apoptosis related to the suppression of JNK1/2 activation and increased ERK1/2 phosphorylation. Taken together, these results indicate that that ethyl acetate extract of AMK has potential therapeutic effects in the HFD-induced NASH mouse model. PMID:26726030

  14. Dermcidin isoform-2 induced nullification of the effect of acetyl salicylic acid in platelet aggregation in acute myocardial infarction

    Science.gov (United States)

    Bank, Sarbashri; Jana, Pradipta; Maiti, Smarajit; Guha, Santanu; Sinha, A. K.

    2014-01-01

    The aggregation of platelets on the plaque rupture site on the coronary artery is reported to cause both acute coronary syndromes (ACS) and acute myocardial infarction (AMI). While the inhibition of platelet aggregation by acetyl salicylic acid was reported to produce beneficial effects in ACS, it failed to do in AMI. The concentration of a stress induced protein (dermcidin isoform-2) was much higher in AMI than that in ACS. Incubation of normal platelet rich plasma (PRP) with dermcidin showed one high affinity (Kd = 40 nM) and one low affinity binding sites (Kd = 333 nM). When normal PRP was incubated with 0.4 μM dermcidin, the platelets became resistant to the inhibitory effect of aspirin similar to that in the case of AMI. Incubation of PRP from AMI with dermcidin antibody restored the sensitivity of the platelets to the aspirin effect. Incubation of AMI PRP pretreated with 15 μM aspirin, a stimulator of the NO synthesis, resulted in the increased production of NO in the platelets that removed the bound dermcidin by 40% from the high affinity binding sites of AMI platelets. When the same AMI PRP was retreated with 10 μM aspirin, the aggregation of platelets was completely inhibited by NO synthesis. PMID:25055737

  15. A pyrene-benzthiazolium conjugate portraying aggregation induced emission, a ratiometric detection and live cell visualization of HSO3(.).

    Science.gov (United States)

    Diwan, Uzra; Kumar, Virendra; Mishra, Rakesh K; Rana, Nishant Kumar; Koch, Biplob; Singh, Manish Kumar; Upadhyay, K K

    2016-07-27

    The present study deals with the photophysical property of a pyrene-benzthiazolium conjugate R1, as a strong intramolecular charge transfer (ICT) probe exhibiting long wavelength emission in the red region. Unlike traditional planar polyaromatic hydrocarbons whose aggregation generally quenches the light emission, the pyrene based R1 was found to display aggregation-induced emission (AIE) property along with simultaneous increase in its quantum yield upon increasing the water content of the medium. The R1 exhibits high specificity towards HSO3(-)/SO3(2-) by interrupting its own ICT producing there upon a large ratiometric blue shift of ∼220 nm in its emission spectrum. The lowest detection limit for the above measurement was found to be 8.90 × 10(-8) M. The fluorescent detection of HSO3(-) was also demonstrated excellently by test paper strip and silica coated TLC plate incorporating R1. The live cell imaging of HSO3(─) through R1 in HeLa cells was studied using fluorescence microscopic studies. The particle size and morphological features of R1 and R1-HSO3(-) aggregates in aqueous solution were characterized by DLS along with SEM analysis. PMID:27251947

  16. Effect of Vitamin C Supplementation on Platelet Aggregation and Serum Electrolytes Levels in Streptozotocin-Induced Diabetes Mellitus in Rats.

    Science.gov (United States)

    Owu, Daniel U; Nwokocha, Chukwuemeka R; Ikpi, Daniel E; Ogar, Emmanuel I

    2016-01-01

    Diabetes mellitus (DM) is a disease condition characterised by hyperglycemia; free radical and abnormalhaematological indices. Vitamin C can reduce free radical generation and ameliorate adverse conditions of diabetes mellitus.The aim of the present study is to investigate the effect of vitamin C on platelet aggregation and electrolyte levels in Type 1DM. Male Wistar rats were divided into four groups namely control, DM, DM +Vitamin C and Vitamin C groups. Rats weremade diabetic with a single dose of streptozotocin (65 mg/kg) intraperitoneally. Vitamin C was administered orally todiabetic and normal rats at 200 mg/kg body weight for 28 days. Blood samples were analyzed for hematological parameters,platelet aggregation, and serum electrolyte levels. Blood glucose in DM+ Vitamin C group (9.9 ± 1.8 mmol/L) wassignificantly reduced (pcontrol(4.4 ± 0.8 mmol/L). Haemoglobin (Hb) concentration in DM group (12 ± 0.1 g/dL) was significantly reduced (pcontrol groups (14 ± 0.24 g/dL) and significantly increased (pcontrol (59.49 ± 0.5fL). Platelet count in DM group (523± 8.5 x109/L) was significantly raised (pcontrol (356 ± 6.2 x109/L) and significantly reduced(pglucose level, platelet count, serumsodium/potassium ion ratio and inhibits platelet aggregation in streptozotocin-induced DM in rats. PMID:27574765

  17. The Kinetics of Dislocation Loop Formation in Ferritic Alloys Through the Aggregation of Irradiation Induced Defects

    Science.gov (United States)

    Kohnert, Aaron Anthony

    The mechanical properties of materials are often degraded over time by exposure to irradiation environments, a phenomenon that has hindered the development of multiple nuclear reactor design concepts. Such property changes are the result of microstructural changes induced by the collision of high energy particles with the atoms in a material. The lattice defects generated in these recoil events migrate and interact to form extended damage structures. This study has used theoretical models based on the mean field chemical reaction rate theory to analyze the aggregation of isolated lattice defects into larger microstructural features that are responsible for long term property changes, focusing on the development of black dot damage in ferritic iron based alloys. The purpose of such endeavors is two-fold. Primarily, such models explain and quantify the processes through which these microstructures form. Additionally, models provide insight into the behavior and properties of the point defects and defect clusters which drive general microstructural evolution processes. The modeling effort presented in this work has focused on physical fidelity, drawing from a variety of sources of information to characterize the unobservable defect generation and agglomeration processes that give rise to the observable features reported in experimental data. As such, the models are based not solely on isolated point defect creation, as is the case with many older rate theory approaches, but instead on realistic estimates of the defect cluster population produced in high energy cascade damage events. Experimental assessments of the microstructural changes evident in transmission electron microscopy studies provide a means to measure the efficacy of the kinetic models. Using common assumptions of the mobility of defect clusters generated in cascade damage conditions, an unphysically high density of damage features develops at the temperatures of interest with a temperature dependence

  18. Bivalent Copper Ions Promote Fibrillar Aggregation of KCTD1 and Induce Cytotoxicity.

    Science.gov (United States)

    Liu, Zhepeng; Song, Feifei; Ma, Zhi-Li; Xiong, Qiushuang; Wang, Jingwen; Guo, Deyin; Sun, Guihong

    2016-01-01

    Potassium channel tetramerization domain containing 1 (KCTD1) family members have a BTB/POZ domain, which can facilitate protein-protein interactions involved in the regulation of different signaling pathways. KCTD proteins have potential Zn(2+)/Cu(2+) binding sites with currently unknown structural and functional roles. We investigated potential Cu(2+)-specific effects on KCTD1 using circular dichroism, turbidity measurement, fluorescent dye binding, proteinase K (PK) digestion, cell proliferation and apoptosis assays. These experiments indicate that the KCTD1 secondary structure assumes greater β-sheet content and the proteins aggregate into a PK-resistant form under 20 μM Cu(2+), and this β-sheet-rich aggregation with Cu(2+) promotes fibril formation, which results in increased cell toxicity by apoptosis. Our results reveal a novel role for Cu(2+) in determining the structure and function of KCTD1.

  19. Zinc induces unfolding and aggregation of dimeric arginine kinase by trapping reversible unfolding intermediate.

    Science.gov (United States)

    Liu, Taotao; Wang, Xicheng

    2010-11-01

    Arginine kinase plays an important role in the cellular energy metabolism of invertebrates. Dimeric arginine kinase (dAK) is unique in some marine invertebrates. The effects of Zn²(+) on the unfolding and aggregation of dAK from the sea cucumber Stichopus japonicus were investigated. Our results indicated that Zn²(+) caused dAK inactivation accompanied by conformational unfolding, the exposure of hydrophobic surface, and aggregation. Kinetic studies showed the inactivation and unfolding of dAK followed biphasic kinetic courses. Zn²(+) can affect unfolding and refolding of dAK by trapping the reversible intermediate. Our study provides important information regarding the effect of Zn²(+) on metabolic enzymes in marine invertebrates.

  20. Bivalent Copper Ions Promote Fibrillar Aggregation of KCTD1 and Induce Cytotoxicity.

    Science.gov (United States)

    Liu, Zhepeng; Song, Feifei; Ma, Zhi-Li; Xiong, Qiushuang; Wang, Jingwen; Guo, Deyin; Sun, Guihong

    2016-01-01

    Potassium channel tetramerization domain containing 1 (KCTD1) family members have a BTB/POZ domain, which can facilitate protein-protein interactions involved in the regulation of different signaling pathways. KCTD proteins have potential Zn(2+)/Cu(2+) binding sites with currently unknown structural and functional roles. We investigated potential Cu(2+)-specific effects on KCTD1 using circular dichroism, turbidity measurement, fluorescent dye binding, proteinase K (PK) digestion, cell proliferation and apoptosis assays. These experiments indicate that the KCTD1 secondary structure assumes greater β-sheet content and the proteins aggregate into a PK-resistant form under 20 μM Cu(2+), and this β-sheet-rich aggregation with Cu(2+) promotes fibril formation, which results in increased cell toxicity by apoptosis. Our results reveal a novel role for Cu(2+) in determining the structure and function of KCTD1. PMID:27596723

  1. Pistacia chinensis Methanolic Extract Attenuated MAPK and Akt Phosphorylations in ADP Stimulated Rat Platelets In Vitro

    Directory of Open Access Journals (Sweden)

    Ji Young Park

    2012-01-01

    (2.5–20 μg/mL inhibited ADP-induced platelet aggregation. While PCME diminished [Ca2+]i, ATP, and TXA2 release in ADP-activated platelets, it enhanced cAMP production in resting platelets. Likewise, PCME inhibited fibrinogen binding to αIIbβ3 and downregulated JNK, ERK, and Akt phosphorylations. Thus, PCME contains potential antiplatelet compounds that could be deployed for their therapeutic values in cardiovascular pathology.

  2. Casein Kinase 2 (CK2)-mediated Phosphorylation of Hsp90β as a Novel Mechanism of Rifampin-induced MDR1 Expression.

    Science.gov (United States)

    Kim, So Won; Hasanuzzaman, Md; Cho, Munju; Heo, Ye Rang; Ryu, Min-Jung; Ha, Na-Young; Park, Hyun June; Park, Hyung-Yeon; Shin, Jae-Gook

    2015-07-01

    The P-glycoprotein (P-gp) encoded by the MDR1 gene is a drug-exporting transporter located in the cellular membrane. P-gp induction is regarded as one of the main mechanisms underlying drug-induced resistance. Although there is great interest in the regulation of P-gp expression, little is known about its underlying regulatory mechanisms. In this study, we demonstrate that casein kinase 2 (CK2)-mediated phosphorylation of heat shock protein 90β (Hsp90β) and subsequent stabilization of PXR is a key mechanism in the regulation of MDR1 expression. Furthermore, we show that CK2 is directly activated by rifampin. Upon exposure to rifampin, CK2 catalyzes the phosphorylation of Hsp90β at the Ser-225/254 residues. Phosphorylated Hsp90β then interacts with PXR, causing a subsequent increase in its stability, leading to the induction of P-gp expression. In addition, inhibition of CK2 and Hsp90β enhances the down-regulation of PXR and P-gp expression. The results of this study may facilitate the development of new strategies to prevent multidrug resistance and provide a plausible mechanism for acquired drug resistance by CK2-mediated regulation of P-gp expression. PMID:25995454

  3. Amino acid induced fractal aggregation of gold nanoparticles: Why and how.

    Science.gov (United States)

    Doyen, Matthieu; Goole, Jonathan; Bartik, Kristin; Bruylants, Gilles

    2016-02-15

    Gold colloids are the object of many studies as they are reported to have potential biological sensing, imaging and drug delivery applications. In the presence of certain amino acids the aggregation of the gold nanoparticles into linear structures is observed, as highlighted by the appearance of a second plasmon band in the UV-Vis spectra of the colloid. The mechanism behind this phenomenon is still under debate. In order to help elucidate this issue, the interaction between gold colloids and different amino acids, modified amino acids and molecules mimicking their side-chain was monitored by UV-Vis absorption, DLS and TEM. The results show that phenomenon can be rationalized in terms of the Diffusion Limited Colloid Aggregation (DLCA) model which gives rise to the fractal aggregation colloids. The global charge of the compound, which influences the ionic strength of the solution, and the ease with which the compound can interact with the GNPs and affect their surface potential, are, the two parameters which control the DLCA regime. Calculations based on the Derjaguin, Landau, Verwey and Overbeek (DLVO) theory confirm all the experimental observations.

  4. Role of Au nanoparticle aggregation in laser induced anisotropy of ITO transparent substrates

    Energy Technology Data Exchange (ETDEWEB)

    Kityk, I.V., E-mail: iwank74@gmail.com [Electrical Engineering Department, Czetochowa University Technology, Armii Krajowej 17, Czestochowa (Poland); Ebothe, J.; Bercu, N-B. [Laboratoire de Recherche en Nanosciences, E.A. 4682, Université de Reims, 21 rue Clément Ader, 51685 Reims cedex 02 (France); Aziz, Md. Abdul [Center of Research Excellence in Nanotechnology, King Fahd University of Petroleum and Minerals, Dhahran 31261 (Saudi Arabia); Oyama, Munetaka [Department of Material Chemistry, Graduate School of Engineering, Kyoto University, Nishikyo-ku, Kyoto 615-8520 (Japan)

    2014-02-05

    Highlights: • Principal role of Au NP surface aggregation on the output photoinduced birefringence is shown. • The higher changes are obtained for the samples with sizes 30 nm. • The process is slowly relaxed. -- Abstract: A principal possibility to achieve the photoinduced anisotropy in the Au NP deposited onto the ITO substrate is experimentally shown. The sizes of the Au NP forming the corresponding nanocomposites were 20 nm, 30 nm and 40 nm. As a photoinducing light source we have used two coherent beam originating from the Er:glass laser generating at 1540 nm with frequency repetition 15 Hz as well as its second harmonic doubled frequency signal at 770 nm. The effect is sensitive to the angle between the two laser beams as well as to the Au NP sizes, inter-particle distances and topology connected with their aggregation. The effect shows slow relaxation to the initial state. The optimal conditions are achieved for nanocomposites formed by 30 nm despite the expected 20 nm. This one may be caused by crucial role of the partial aggregation which even changes the effective grain sizes. The contribution of the dipole–dipole as well as quadrupole–dipole interactions to the changes of the anisotropy is discussed. The excitation is far from the resonance which allow to predict that effective role play overlap with nanotrapping levels. So principal role may belongs to surface topology and which is studied using the birefringence directly connected with the anisotropy.

  5. Varicella-zoster virus induces the formation of dynamic nuclear capsid aggregates

    Energy Technology Data Exchange (ETDEWEB)

    Lebrun, Marielle [University of Liege (ULg), GIGA-Infection Immunity and Inflammation, Laboratory of Virology and Immunology, Liege (Belgium); Thelen, Nicolas; Thiry, Marc [University of Liege (ULg), GIGA-Neurosciences, Laboratory of Cellular and Tissular Biology, Liege (Belgium); Riva, Laura; Ote, Isabelle; Condé, Claude; Vandevenne, Patricia [University of Liege (ULg), GIGA-Infection Immunity and Inflammation, Laboratory of Virology and Immunology, Liege (Belgium); Di Valentin, Emmanuel [University of Liege (ULg), GIGA-Viral Vectors Platform, Liege (Belgium); Bontems, Sébastien [University of Liege (ULg), GIGA-Infection Immunity and Inflammation, Laboratory of Virology and Immunology, Liege (Belgium); Sadzot-Delvaux, Catherine, E-mail: csadzot@ulg.ac.be [University of Liege (ULg), GIGA-Infection Immunity and Inflammation, Laboratory of Virology and Immunology, Liege (Belgium)

    2014-04-15

    The first step of herpesviruses virion assembly occurs in the nucleus. However, the exact site where nucleocapsids are assembled, where the genome and the inner tegument are acquired, remains controversial. We created a recombinant VZV expressing ORF23 (homologous to HSV-1 VP26) fused to the eGFP and dually fluorescent viruses with a tegument protein additionally fused to a red tag (ORF9, ORF21 and ORF22 corresponding to HSV-1 UL49, UL37 and UL36). We identified nuclear dense structures containing the major capsid protein, the scaffold protein and maturing protease, as well as ORF21 and ORF22. Correlative microscopy demonstrated that the structures correspond to capsid aggregates and time-lapse video imaging showed that they appear prior to the accumulation of cytoplasmic capsids, presumably undergoing the secondary egress, and are highly dynamic. Our observations suggest that these structures might represent a nuclear area important for capsid assembly and/or maturation before the budding at the inner nuclear membrane. - Highlights: • We created a recombinant VZV expressing the small capsid protein fused to the eGFP. • We identified nuclear dense structures containing capsid and procapsid proteins. • Correlative microscopy showed that the structures correspond to capsid aggregates. • Procapsids and partial capsids are found within the aggregates of WT and eGFP-23 VZV. • FRAP and FLIP experiments demonstrated that they are dynamic structures.

  6. Varicella-zoster virus induces the formation of dynamic nuclear capsid aggregates

    International Nuclear Information System (INIS)

    The first step of herpesviruses virion assembly occurs in the nucleus. However, the exact site where nucleocapsids are assembled, where the genome and the inner tegument are acquired, remains controversial. We created a recombinant VZV expressing ORF23 (homologous to HSV-1 VP26) fused to the eGFP and dually fluorescent viruses with a tegument protein additionally fused to a red tag (ORF9, ORF21 and ORF22 corresponding to HSV-1 UL49, UL37 and UL36). We identified nuclear dense structures containing the major capsid protein, the scaffold protein and maturing protease, as well as ORF21 and ORF22. Correlative microscopy demonstrated that the structures correspond to capsid aggregates and time-lapse video imaging showed that they appear prior to the accumulation of cytoplasmic capsids, presumably undergoing the secondary egress, and are highly dynamic. Our observations suggest that these structures might represent a nuclear area important for capsid assembly and/or maturation before the budding at the inner nuclear membrane. - Highlights: • We created a recombinant VZV expressing the small capsid protein fused to the eGFP. • We identified nuclear dense structures containing capsid and procapsid proteins. • Correlative microscopy showed that the structures correspond to capsid aggregates. • Procapsids and partial capsids are found within the aggregates of WT and eGFP-23 VZV. • FRAP and FLIP experiments demonstrated that they are dynamic structures

  7. A Phosphorylation-Induced Turn Defines the Alzheimer's Disease AT8 Antibody Epitope on the Tau Protein.

    Science.gov (United States)

    Gandhi, Neha S; Landrieu, Isabelle; Byrne, Cillian; Kukic, Predrag; Amniai, Laziza; Cantrelle, François-Xavier; Wieruszeski, Jean-Michel; Mancera, Ricardo L; Jacquot, Yves; Lippens, Guy

    2015-06-01

    Post mortem biochemical staging of Alzheimer's disease is currently based on immunochemical analysis of brain slices with the AT8 antibody. The epitope of AT8 is described around the pSer202/pThr205 region of the hyperphosphorylated form of the neuronal protein tau. In this study, NMR spectroscopy was used to precisely map the AT8 epitope on phosphorylated tau, and derive its defining structural features by a combination of NMR analyses and molecular dynamics. A particular turn conformation is stabilized by a hydrogen bond of the phosphorylated Thr205 residue to the amide proton of Gly207, and is further stabilized by the two Arg residues opposing the pSer202/pThr205. PMID:25881502

  8. PDGF-induced phosphorylation of Tyr28 in the N-terminus of Fyn affects Fyn activation

    DEFF Research Database (Denmark)

    Hansen, Klaus; Alonso, G; Courtneidge, S A;

    1997-01-01

    Binding of platelet-derived growth factor (PDGF) to its receptors leads to the activation of members of the Src family of protein tyrosine kinases. We show here that Fyn, a member of the Src family, is phosphorylated on Tyr28 in the unique N-terminal part of the molecule after interaction...... with the intracellular domain of the PDGF beta-receptor. Activated Fyn furthermore undergoes autophosphorylation on Tyr30, Tyr39 and Tyr420. When Fyn mutants with Tyr28, Tyr30 or Tyr39 replaced with phenylalanine residues were transfected into NIH3T3 cells a decreased activation after PDGF stimulation was seen......, suggesting a functional importance of the N-terminal tyrosine phosphorylation of Fyn....

  9. FV-429 induces apoptosis and inhibits glycolysis by inhibiting Akt-mediated phosphorylation of hexokinase II in MDA-MB-231 cells.

    Science.gov (United States)

    Zhou, Yuxin; Lu, Na; Qiao, Chen; Ni, Ting; Li, Zhiyu; Yu, Boyang; Guo, Qinglong; Wei, Libin

    2016-09-01

    In this study, the anticancer effect of a newly synthesized flavonoid FV-429, against human breast cancer MDA-MB-231 cells, and the underlying mechanisms were investigated. FV-429 triggered the apoptosis and simultaneously inhibited the glycolysis of MDA-MB-231 cells. Both the HK II activity and its level in mitochondria were significantly down regulated by FV-429. Moreover, FV-429 weakened the interaction between HKII and VDAC, stimulated the detachment of HK II from the mitochondria, and resulted in the opening of the mitochondrial permeability transition pores. Thus FV-429 induced the mitochondrial-mediated apoptosis, showing increased Bax/Bcl-2 ratio, loss of mitochondrial membrane potential (MMP) and activation of caspase-3 and -9, cytochrome c (Cyt c) release, and apoptosis inducing factor (AIF) transposition. Further research revealed that the phosphorylation of mitochondrial HKII via Akt was responsible for the dissociation of HKII and the decreased HKII activity induced by FV-429. Taken together, FV-429 inhibited the phosphorylation of HKII, down-regulated its activity, and stimulated the release of HKII from the mitochondria, resulting the inhibited glycolysis and mitochondrial-mediated apoptosis. The studies provide a molecular basis for the development of flavonoid compounds as novel anticancer agents for breast cancer. © 2015 Wiley Periodicals, Inc. PMID:26258875

  10. Squamocin modulates histone H3 phosphorylation levels and induces G1 phase arrest and apoptosis in cancer cells

    Directory of Open Access Journals (Sweden)

    Wu Yang-Chang

    2011-02-01

    Full Text Available Abstract Background Histone modifications in tumorigenesis are increasingly recognized as important epigenetic factors leading to cancer. Increased phosphorylation levels of histone H3 as a result of aurora B and pMSK1 overexpression were observed in various tumors. We selected aurora B and MSK1 as representatives for testing various compounds and drugs, and found that squamocin, a bis-tetrahydrofuran annonaceous acetogenin, exerted a potent effect on histone H3 phosphorylation. Methods GBM8401, Huh-7, and SW620 cells were incubated with 15, 30, and 60 μM squamocin for 24 h. The expressions of mRNA and proteins were analyzed by qRT-PCR and Western blotting, respectively. The cell viability was determined by an MTT assay. Cell cycle distribution and apoptotic cells were analyzed by flow cytometry. Results Our results showed that squamocin inhibited the proliferation of GBM8401, Huh-7, and SW620 cells, arrested the cell cycle at the G1 phase, and activated both intrinsic and extrinsic pathways to apoptosis. In addition, we demonstrated that squamocin had the ability to modulate the phosphorylation levels of H3S10 (H3S10p and H3S28 (H3S28p in association with the downregulation of aurora B and pMSK1 expressions. Conclusions This study is the first to show that squamocin affects epigenetic alterations by modulating histone H3 phosphorylation at S10 and S28, providing a novel view of the antitumor mechanism of squamocin.

  11. Studies on the photophysical properties of 1,8-naphthalimide derivative and aggregation induced emission recognition for casein

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Yang, E-mail: 66160692@qq.com [Department of Chemistry and Chemical Engineering, Xi' an University of Arts and Science, No. 168, Taibai South Road, Xi' an, Shaanxi 710065 (China); Liang, Xuhua; Fan, Jun [School of Chemical Engineering, Northwest University, No. 229, Taibai North Road, Xi' an, Shaanxi 710069 (China); Han, Quan, E-mail: xahanq@hotmail.com [Department of Chemistry and Chemical Engineering, Xi' an University of Arts and Science, No. 168, Taibai South Road, Xi' an, Shaanxi 710065 (China)

    2013-09-15

    A novel water-soluble 1,8-naphthalimide derivative 1, bearing two acetic carboxylic groups, exhibited fluorescent turn-on recognition for casein micelle based on the aggregation induced emission (AIE) character. The photophysical properties of 1 consisting of donor and acceptor units were investigated by the solvation effect. Changing from polar to non-polar solvent increased the solvent interaction; both the excitation and emission spectra were shifted to shorter wavelength and intensity decreased through taking advantage of twisted intramolecular charge transfer (TICT) and self-association fluorescence emission. Moreover, the red-shift and quenching in protic solvent were caused by the excited-state hydrogen bond strengthening effect. The density functional theory (DFT) and time dependent density functional theory (TDDFT) were used to obtain the most stable structure, electronic excitation energy, dipole moments and charge distribution. The AIE mechanism of 1 with casein micelle was due to 1 docked in the hydrophobic cavity between sub-micelles and bound with amino acid residues, resulting in the aggregation of 1 on the casein micelle surface and emission enhancement, based on which, a novel casein assay method was developed. The proposed method exhibited a good linear range from 0.1 to 10.5 μg mL{sup −1}, with the detection limit of 3.0 ng mL{sup −1}. Satisfactory reproducibility, reversibility and a short response time were realized. This method was applied for the determination of casein in milk powder samples, avoiding the interferences from other components and illegal additives in milk. -- Highlights: • A water-soluble 1,8-naphthalimide-based fluorescent probe 1 was synthesized. • Photophysical characterization of 1 was studied. • Aggregation induced emission enhancement of 1 with casein was investigated. • A novel casein quantification method was developed.

  12. A fluorescent assay for γ-glutamyltranspeptidase via aggregation induced emission and its applications in real samples.

    Science.gov (United States)

    Hou, Xianfeng; Zeng, Fang; Wu, Shuizhu

    2016-11-15

    γ-Glutamyl transpeptidase (GGT) plays crucial roles in some physiological processes. Herein a turn-on fluorescent probe for γ-glutamyl transpeptidase (GGT) assay based on aggregation-induced-emission (AIE) effect and the enzyme-induced transformation of hydrophilicity to hydrophobicity has been developed by functionalizing tetraphenylethylene (TPE) derivative with two γ-glutamyl amide groups, which simultaneously work as recognition units and hydrophilic groups. When the γ-glutamyl amide groups are cleaved through GGT enzymatic reaction, the hydrophobic reaction product readily aggregate and correspondingly strong blue fluorescence can be observed, as a result of activated AIE process. By virtue of the probe's good solubility in totally aqueous solution, high sensitivity and excellent photostability, the probe can be employed to detect GGT level in human serum samples. Furthermore, the probe can be used for imaging endogenous GGT in living A2780 cells. Hence, the probe holds great promise for acting as a convenient one-step straightforward assay for GGT detection in diagnostic-related applications, and also it could provide a useful approach for conducting pathological analysis for diseases involving GGT. PMID:27183282

  13. Probe Intracellular Trafficking of a Polymeric DNA Delivery Vehicle by Functionalization with an Aggregation-Induced Emissive Tetraphenylethene Derivative.

    Science.gov (United States)

    Han, Xiongqi; Chen, Qixian; Lu, Hongguang; Ma, Jianbiao; Gao, Hui

    2015-12-30

    Characteristic aggregation-induced quenching of π-fluorophores imposed substantial hindrance to their utilization in nanomedicine for insight into microscopic intracellular trafficking of therapeutic payload. To address this obstacle, we attempted to introduce a novel aggregation-induced emission (AIE) fluorophore into the cationic polymer, which was further used for formulation of a gene delivery carrier. Note that the selective restriction of the intramolecular rotation of the AIE fluorophore through its covalent bond to the polymer conduced to immense AIE. Furthermore, DNA payload labeled with the appropriate fluorophore as the Förster resonance energy transfer (FRET) acceptor verified a facile strategy to trace intracellular DNA releasing activity relying on the distance limitation requested by FRET (AIE fluorophore as FRET donor). Moreover, the hydrophobic nature of the AIE fluorophore appeared to promote colloidal stability of the constructed formulation. Together with other chemistry functionalization strategies (including endosome escape), the ultimate formulation exerted dramatic gene transfection efficiency. Hence, this report manifested a first nanomedicine platform combining AIE and FRET for microscopic insight into DNA intracellular trafficking activity. PMID:26634294

  14. Creation of reduced fat foods: influence of calcium-induced droplet aggregation on microstructure and rheology of mixed food dispersions.

    Science.gov (United States)

    Wu, Bi-cheng; Degner, Brian; McClements, David Julian

    2013-12-15

    The impact of calcium-induced fat droplet aggregation on the microstructure and physicochemical properties of model mixed colloidal dispersions was investigated. These systems consisted of 2 wt% whey protein-coated fat droplets and 4 wt% modified starch granules heated to induce starch swelling (pH 7). Optical and confocal microscopy showed that the fat droplets were dispersed within the interstitial region between the swollen starch granules. The structural organisation of the fat droplets within these interstitial regions could be modulated by controlling the calcium concentration: (i) at a low calcium concentration the droplets were evenly distributed; (ii) at an intermediate calcium concentration they formed a layer around the starch granules; (iii) at a high calcium concentration they formed a network of aggregated droplets. Paste-like materials were produced when the fat droplets formed a three-dimensional network in the interstitial region. The properties of fat droplet-starch granule suspensions can be modulated by altering the electrostatic interactions to alter microstructure.

  15. Modelling Ser129 phosphorylation inhibits membrane binding of pore-forming alpha-synuclein oligomers.

    Directory of Open Access Journals (Sweden)

    Georg Sebastian Nübling

    Full Text Available BACKGROUND: In several neurodegenerative diseases, hyperphosphorylation at position Ser129 is found in fibrillar deposits of alpha-synuclein (asyn, implying a pathophysiological role of asyn phosphorylation in neurodegeneration. However, recent animal models applying asyn phosphorylation mimics demonstrated a protective effect of phosphorylation. Since metal-ion induced asyn oligomers were identified as a potential neurotoxic aggregate species with membrane pore-forming abilities, the current study was undertaken to determine effects of asyn phosphorylation on oligomer membrane binding. METHODS: We investigated the influence of S129 phosphorylation on interactions of metal-ion induced asyn oligomers with small unilamellar lipid vesicles (SUV composed of POPC and DPPC applying the phosphorylation mimic asyn129E. Confocal single-particle fluorescence techniques were used to monitor membrane binding at the single-particle level. RESULTS: Binding of asyn129E monomers to gel-state membranes (DPPC-SUV is slightly reduced compared to wild-type asyn, while no interactions with membranes in the liquid-crystalline state (POPC-SUV are seen for both asyn and asyn129E. Conversely, metal-ion induced oligomer formation is markedly increased in asyn129E. Surprisingly, membrane binding to POPC-SUV is nearly absent in Fe(3+ induced asyn129E oligomers and markedly reduced in Al(3+ induced oligomers. CONCLUSION: The protective effect of pseudophosphorylation seen in animal models may be due to impeded oligomer membrane binding. Phosphorylation at Ser129 may thus have a protective effect against neurotoxic asyn oligomers by preventing oligomer membrane binding and disruption of the cellular electrophysiological equilibrium. Importantly, these findings put a new complexion on experimental pharmaceutical interventions against POLO-2 kinase.

  16. Overexpression of KAI1 induces autophagy and increases MiaPaCa-2 cell survival through the phosphorylation of extracellular signal-regulated kinases

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Chun-Yan [State Key Laboratory of Cancer Biology and Institute of Digestive Diseases, Xijing Hospital of Digestive Disease, Fourth Military Medical University, Xi' an 710032 (China); Department of Gastroenterology, Shenyang General Hospital of PLA, 83 Wenhua Road, Shenyang 110016 (China); Yan, Jun; Yang, Yue-Feng; Xiao, Feng-Jun; Li, Qing-Fang; Zhang, Qun-Wei; Wang, Li-Sheng [Department of Experimental Hematology, Beijing Institute of Radiation Medicine, Beijing 100850 (China); Guo, Xiao-Zhong, E-mail: guoxiaozhong1962@163.com [Department of Gastroenterology, Shenyang General Hospital of PLA, 83 Wenhua Road, Shenyang 110016 (China); Wang, Hua, E-mail: wanghua@bmi.ac.cn [Department of Experimental Hematology, Beijing Institute of Radiation Medicine, Beijing 100850 (China)

    2011-01-21

    Research highlights: {yields} We first investigate the effects of KAI1 on autophagy in MiaPaCa-2 cells. {yields} Our findings demonstrate that KAI1 induces autophagy, which in turn inhibits KAI1-induced apoptosis. {yields} This study also supplies a possible novel therapeutic method for the treatment of pancreatic cancer using autophagy inhibitors. -- Abstract: KAI1, a metastasis-suppressor gene belonging to the tetraspanin family, is known to inhibit cancer metastasis without affecting the primary tumorigenicity by inhibiting the epidermal growth factor (EGF) signaling pathway. Recent studies have shown that hypoxic conditions of solid tumors induce high-level autophagy and KAI1 expression. However, the relationship between autophagy and KAI1 remains unclear. By using transmission electron microscopy, confocal microscopy, and Western blotting, we found that KAI1 can induce autophagy in a dose- and time-dependent manner in the human pancreatic cell line MiaPaCa-2. KAI1-induced autophagy was confirmed by the expression of autophagy-related proteins LC3 and Beclin 1. KAI1 induces autophagy through phosphorylation of extracellular signal-related kinases rather than that of AKT. KAI1-induced autophagy protects MiaPaCa-2 cells from apoptosis and proliferation inhibition partially through the downregulation of poly [adenosine diphosphate (ADP)-ribose] polymerase (PARP) cleavage and caspase-3 activation.

  17. Neurotensin-induced Erk1/2 phosphorylation and growth of human colonic cancer cells are independent from growth factors receptors activation

    Energy Technology Data Exchange (ETDEWEB)

    Massa, Fabienne; Tormo, Aurelie; Beraud-Dufour, Sophie; Coppola, Thierry [Institut de Pharmacologie Moleculaire et Cellulaire, Universite de Nice-Sophia Antipolis, CNRS UMR 6097, 660 route des Lucioles, 06560 Valbonne (France); Mazella, Jean, E-mail: mazella@ipmc.cnrs.fr [Institut de Pharmacologie Moleculaire et Cellulaire, Universite de Nice-Sophia Antipolis, CNRS UMR 6097, 660 route des Lucioles, 06560 Valbonne (France)

    2011-10-14

    Highlights: {yields} We compare intracellular pathways of NT and EGF in HT29 cells. {yields} NT does not transactivate EGFR. {yields} Transactivation of EGFR is not a general rule in cancer cell growth. -- Abstract: Neurotensin (NT) promotes the proliferation of human colonic cancer cells by undefined mechanisms. We already demonstrated that, in the human colon adenocarcinoma cell line HT29, the effects of NT were mediated by a complex formed between the NT receptor-1 (NTSR1) and-3 (NTSR3). Here we examined cellular mechanisms that led to NT-induced MAP kinase phosphorylation and growth factors receptors transactivation in colonic cancer cells and proliferation in HT29 cells. With the aim to identify upstream signaling involved in NT-elicited MAP kinase activation, we found that the stimulatory effects of the peptide were totally independent from the activation of the epidermal growth factor receptor (EGFR) both in the HT29 and the HCT116 cells. NT was unable to promote phosphorylation of EGFR and to compete with EGF for its binding to the receptor. Pharmacological approaches allowed us to differentiate EGF and NT signaling in HT29 cells since only NT activation of Erk1/2 was shown to be sensitive to PKC inhibitors and since only NT increased the intracellular level of calcium. We also observed that NT was not able to transactivate Insulin-like growth factor receptor. Our findings indicate that, in the HT29 and HCT116 cell lines, NT stimulates MAP kinase phosphorylation and cell growth by a pathway which does not involve EGF system but rather NT receptors which transduce their own intracellular effectors. These results indicate that depending on the cell line used, blocking EGFR is not the general rule to inhibit NT-induced cancer cell proliferation.

  18. Binding of Folic Acid Induces Specific Self-Aggregation of Lactoferrin: Thermodynamic Characterization.

    Science.gov (United States)

    Tavares, Guilherme M; Croguennec, Thomas; Lê, Sébastien; Lerideau, Olivia; Hamon, Pascaline; Carvalho, Antônio F; Bouhallab, Saïd

    2015-11-17

    In the study presented here, we investigated the interaction at pH 5.5 between folic acid (FA) and lactoferrin (LF), a positively charged protein. We found a binding constant Ka of 10(5) M(-1) and a high stoichiometry of 10 mol of FA/mol of LF. The size and charge of the complexes formed evolved during titration experiments. Increasing the ionic strength to 50 mM completely abolished the isothermal titration calorimetry (ITC) signal, suggesting the predominance of electrostatic interactions in the exothermic binding obtained. We developed a theoretical model that explains the complex triphasic ITC profile. Our results revealed a two-step mechanism: FA/LF interaction followed by self-association of the complexes thus formed. We suggest that 10 FA molecules bind to LF to form saturated reactive complexes (FA10/LF) that further self-associate into aggregates with a finite size of around 15 nm. There is thus a critical saturation degree of the protein, above which the self-association can take place. We present here the first results that provide comprehensive details of the thermodynamics of FA/LF complexation-association. Given the high stoichiometry, allowing a load of 55 mg of FA/g of LF, we suggest that FA/LF aggregates would be an effective vehicle for FA in fortified drinks. PMID:26488446

  19. Serotonin2C receptor stimulation inhibits cocaine-induced Fos expression and DARPP-32 phosphorylation in the rat striatum independently of dopamine outflow.

    Science.gov (United States)

    Devroye, Céline; Cathala, Adeline; Maitre, Marlène; Piazza, Pier Vincenzo; Abrous, Djoher Nora; Revest, Jean-Michel; Spampinato, Umberto

    2015-02-01

    The serotonin(2C) receptor (5-HT(2C)R) is known to control dopamine (DA) neuron function by modulating DA neuronal firing and DA exocytosis at terminals. Recent studies assessing the influence of 5-HT(2C)Rs on cocaine-induced neurochemical and behavioral responses have shown that 5-HT2CRs can also modulate mesoaccumbens DA pathway activity at post-synaptic level, by controlling DA transmission in the nucleus accumbens (NAc), independently of DA release itself. A similar mechanism has been proposed to occur at the level of the nigrostriatal DA system. Here, using in vivo microdialysis in freely moving rats and molecular approaches, we assessed this hypothesis by studying the influence of the 5-HT(2C)R agonist Ro 60-0175 on cocaine-induced responses in the striatum. The intraperitoneal (i.p.) administration of 1 mg/kg Ro 60-0175 had no effect on the increase in striatal DA outflow induced by cocaine (15 mg/kg, i.p.). Conversely, Ro 60-0175 inhibited cocaine-induced Fos immunoreactivity and phosphorylation of the DA and c-AMP regulated phosphoprotein of Mr 32 kDa (DARPP-32) at threonine 75 residue in the striatum. Finally, the suppressant effect of Ro 60-0175 on cocaine-induced DARPP-32 phosphorylation was reversed by the selective 5-HT(2C)R antagonist SB 242084 (0.5 mg/kg, i.p.). In keeping with the key role of DARPP-32 in DA neurotransmission, our results demonstrate that 5-HT(2C)Rs are capable of modulating nigrostriatal DA pathway activity at post-synaptic level, by specifically controlling DA signaling in the striatum. PMID:25446572

  20. A novel BH3 mimetic efficiently induces apoptosis in melanoma cells through direct binding to anti-apoptotic Bcl-2 family proteins, including phosphorylated Mcl-1.

    Science.gov (United States)

    Liu, Yubo; Xie, Mingzhou; Song, Ting; Sheng, Hongkun; Yu, Xiaoyan; Zhang, Zhichao

    2015-03-01

    The Bcl-2 family modulates sensitivity to chemotherapy in many cancers, including melanoma, in which the RAS/BRAF/MEK/ERK pathway is constitutively activated. Mcl-1, a major anti-apoptotic protein in the Bcl-2 family, is extensively expressed in melanoma and contributes to melanoma's well-documented chemoresistance. Here, we provide the first evidence that Mcl-1 phosphorylation at T163 by ERK1/2 and JNK is associated with the resistance of melanoma cell lines to the existing BH3 mimetics gossypol, S1 and ABT-737, and a novel anti-apoptotic mechanism of phosphorylated Mcl-1 (pMcl-1) is revealed. pMcl-1 antagonized the known BH3 mimetics by sequestering pro-apoptotic proteins that were released from Bcl-2/Mcl-1. Furthermore, an anthraquinone BH3 mimetic, compound 6, was identified to be the first small molecule to that induces endogenous apoptosis in melanoma cells by directly binding Bcl-2, Mcl-1, and pMcl-1 and disrupting the heterodimers of these proteins. Although compound 6 induced upregulation of the pro-apoptotic protein Noxa, its apoptotic induction was independent of Noxa. These data reveal the promising therapeutic potential of targeting pMcl-1 to treat melanoma. Compound 6 is therefore a potent drug that targets pMcl-1 in melanoma.

  1. BAFF induces spleen CD4+ T cell proliferation by down-regulating phosphorylation of FOXO3A and activates cyclin D2 and D3 expression

    International Nuclear Information System (INIS)

    Highlights: ► Firstly analyze the mechanism of BAFF and anti-CD3 co-stimulation on purified mouse splenic CD4+ T cells. ► Carrying out siRNA technology to study FOXO3A protein function. ► Helpful to understand the T cell especially CD4+ T cell‘s role in immunological reaction. -- Abstract: The TNF ligand family member “B cell-activating factor belonging to the TNF family” (BAFF, also called BLyS, TALL-1, zTNF-4, and THANK) is an important survival factor for B and T cells. In this study, we show that BAFF is able to induce CD4+ spleen T cell proliferation when co-stimulated with anti-CD3. Expression of phosphorylated FOXO3A was notably down-regulated and cyclins D2 and D3 were up-regulated and higher in the CD4+ T cells when treated with BAFF and anti-CD3, as assessed by Western blotting. Furthermore, after FOXO3A was knocked down, expression of cyclin D1 was unchanged, compared with control group levels, but the expression of cyclins D2 and D3 increased, compared with the control group. In conclusion, our results suggest that BAFF induced CD4+ spleen T cell proliferation by down-regulating the phosphorylation of FOXO3A and then activating cyclin D2 and D3 expression, leading to CD4+ T cell proliferation.

  2. Suppression of Akt1 phosphorylation by adenoviral transfer of the PTEN gene inhibits hypoxia-induced proliferation of rat pulmonary arterial smooth muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Luo, Chunxia [Department of Neurosurgery, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China); Yi, Bin, E-mail: yibin1974@163.com [Department of Anesthesia, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China); Institute of Respiratory Disease, Xinqiao Hospital, Third Military Medical University, Chongqing 400037 (China); Bai, Li [Institute of Respiratory Disease, Xinqiao Hospital, Third Military Medical University, Chongqing 400037 (China); Xia, Yongzhi [Department of Neurosurgery, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China); Wang, Guansong; Qian, Guisheng [Institute of Respiratory Disease, Xinqiao Hospital, Third Military Medical University, Chongqing 400037 (China); Feng, Hua [Department of Neurosurgery, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China)

    2010-07-02

    Recent findings identify the role of proliferation of pulmonary artery smooth muscle cells (PASMCs) in pulmonary vascular remodeling. Phosphoinositide 3 kinase (PI3K) and serine/threonine kinase (Akt) proteins are expressed in vascular smooth muscle cells. In addition, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) has been identified as a negative regulator of cytokine signaling that inhibits the PI3K-Akt pathway. However, little is known about the role of PTEN/Akt signaling in hypoxia-associated vascular remodeling. In this study, we found that hypoxia-induced the expression of Akt1 mRNA and phosphorylated protein by at least twofold in rat PASMCs. Phospho-PTEN significantly decreased in the nuclei of PASMCs after hypoxic stimulation. After forcing over-expression of PTEN by adenovirus-mediated PTEN (Ad-PTEN) transfection, the expression of phospho-Akt1 was significantly suppressed in PASMCs at all time-points measured. Additionally, we showed here that hypoxia increased proliferation of PASMCs by nearly twofold and over-expression of PTEN significantly inhibited hypoxia-induced PASMCs proliferation. These findings suggest that phospho-PTEN loss in the nuclei of PASMCs under hypoxic conditions may be the major cause of aberrant activation of Akt1 and may, therefore, play an important role in hypoxia-associated pulmonary arterial remodeling. Finally, the fact that transfection with Ad-PTEN inhibits the phosphorylation of Akt1 in PASMCs suggests a potential therapeutic effect on hypoxia-associated pulmonary arterial remodeling.

  3. Differential Regulation of MAPK Phosphorylation in the Dorsal Hippocampus in Response to Prolonged Morphine Withdrawal-Induced Depressive-Like Symptoms in Mice.

    Directory of Open Access Journals (Sweden)

    Wei Jia

    Full Text Available Depression is one of the most frequent neuropsychiatric comorbidities associated with opiate addiction. Mitogen activated protein kinase (MAPK and MAPK phosphatase (MKP are involved in drug addiction and depression. However, the potential role of MAPK and MKP in depression caused by morphine withdrawal remains unclear. We utilized a mouse model of repeated morphine administration to examine the molecular mechanisms that contribute to prolonged withdrawal induced depressive-like behaviors. Depressive-like behaviors were significant at 1 week after withdrawal and worsened over time. Phospho-ERK (extracellular signal-regulated protein kinase was decreased and MKP-1 was elevated in the hippocampus, and JNK (c-Jun N-terminal protein kinase, p38 (p38 protein kinase and MKP-3 were unaffected. A pharmacological blockade of MKP-1 by intra-hippocampal sanguinarine (SA infusion prevented the development of depressive-like behaviors and resulted in relatively normal levels of MKP-1 and phospho-ERK after withdrawal. Our findings support the association between hippocampal MAPK phosphorylation and prolonged morphine withdrawal-induced depression, and emphasize the MKP-1 as an negative regulator of the ERK phosphorylation that contributes to depression.

  4. The phosphorylated C-terminus of cAR1 plays a role in cell-type-specific gene expression and STATa tyrosine phosphorylation.

    Science.gov (United States)

    Briscoe, C; Moniakis, J; Kim, J Y; Brown, J M; Hereld, D; Devreotes, P N; Firtel, R A

    2001-05-01

    cAMP receptors mediate some signaling pathways via coupled heterotrimeric G proteins, while others are G-protein-independent. This latter class includes the activation of the transcription factors GBF and STATa. Within the cellular mounds formed by aggregation of Dictyostelium, micromolar levels of cAMP activate GBF function, thereby inducing the transcription of postaggregative genes and initiating multicellular differentiation. Activation of STATa, a regulator of culmination and ecmB expression, results from cAMP receptor-dependent tyrosine phosphorylation and nuclear localization, also in mound-stage cells. During mound development, the cAMP receptor cAR1 is in a low-affinity state and is phosphorylated on multiple serine residues in its C-terminus. This paper addresses possible roles of cAMP receptor phosphorylation in the cAMP-mediated stimulation of GBF activity, STATa tyrosine phosphorylation, and cell-type-specific gene expression. To accomplish this, we have expressed cAR1 mutants in a strain in which the endogenous cAMP receptors that mediate postaggregative gene expression in vivo are deleted. We then examined the ability of these cells to undergo morphogenesis and induce postaggregative and cell-type-specific gene expression and STATa tyrosine phosphorylation. Analysis of cAR1 mutants in which the C-terminal tail is deleted or the ligand-mediated phosphorylation sites are mutated suggests that the cAR1 C-terminus is not essential for GBF-mediated postaggregative gene expression or STATa tyrosine phosphorylation, but may play a role in regulating cell-type-specific gene expression and morphogenesis. A mutant receptor, in which the C-terminal tail is constitutively phosphorylated, exhibits constitutive activation of STATa tyrosine phosphorylation in pulsed cells in suspension and a significantly impaired ability to induce cell-type-specific gene expression. The constitutively phosphorylated receptor also exerts a partial dominant negative effect on

  5. Involvement of the Octadecanoid Pathway and Protein Phosphorylation in Fungal Elicitor-Induced Expression of Terpenoid Indole Alkaloid Biosynthetic Genes in Catharanthus roseus

    Science.gov (United States)

    Menke, Frank L.H.; Parchmann, Stefanie; Mueller, Martin J.; Kijne, Jan W.; Memelink, Johan

    1999-01-01

    Two key genes in terpenoid indole alkaloid biosynthesis, Tdc and Str, encoding tryptophan decarboxylase and strictosidine synthase, respectively, are coordinately induced by fungal elicitors in suspension-cultured Catharanthus roseus cells. We have studied the roles of the jasmonate biosynthetic pathway and of protein phosphorylation in signal transduction initiated by a partially purified elicitor from yeast extract. In addition to activating Tdc and Str gene expression, the elicitor also induced the biosynthesis of jasmonic acid. The jasmonate precursor α-linolenic acid or methyl jasmonate (MeJA) itself induced Tdc and Str gene expression when added exogenously . Diethyldithiocarbamic acid, an inhibitor of jasmonate biosynthesis, blocked both the elicitor-induced formation of jasmonic acid and the activation of terpenoid indole alkaloid biosynthetic genes. The protein kinase inhibitor K-252a abolished both elicitor-induced jasmonate biosynthesis and MeJA-induced Tdc and Str gene expression. Analysis of the expression of Str promoter/gusA fusions in transgenic C. roseus cells showed that the elicitor and MeJA act at the transcriptional level. These results demonstrate that the jasmonate biosynthetic pathway is an integral part of the elicitor-triggered signal transduction pathway that results in the coordinate expression of the Tdc and Str genes and that protein kinases act both upstream and downstream of jasmonates. PMID:10198087

  6. pH-Induced aggregated melanin nanoparticles for photoacoustic signal amplification

    Science.gov (United States)

    Ju, Kuk-Youn; Kang, Jeeun; Pyo, Jung; Lim, Joohyun; Chang, Jin Ho; Lee, Jin-Kyu

    2016-07-01

    We present a new melanin-like nanoparticle (MelNP) and its performance evaluation results. This particle is proposed as an exogenous contrast agent for photoacoustic (PA) imaging. Conventional PA contrast agents are based on non-biological materials. In contrast, the MelNPs are organic nanoparticles inspired by natural melanin. Melanin is an endogenous chromophore that has the ability to produce a PA signal in vivo. The developed MelNPs are capable of aggregating with one another under mildly acidic conditions after introducing hydrolysis-susceptible citraconic amide on the surface of bare MelNPs. We ascertained that the physical aggregation of the MelNPs resulted in an increased PA signal strength in the near-infrared window of biological tissue (i.e., 700 nm) without absorption tuning. This phenomenon is likely because of the overlapping thermal fields of the developed MelNPs. The PA signal produced from the developed MelNPs, after exposure to mildly acidic conditions (i.e., pH 6), is 8.1 times stronger than under neutral conditions. This unique characteristic found in this study can be utilized in a practical strategy for highly sensitive in vivo cancer target imaging in response to its acidic microenvironment. This approach to amplify the PA response of MelNPs in clusters could accelerate the use of MelNPs as an alternative to non-biological nanoprobes, so that MelNPs may be applicable in PA imaging and functional PA imaging such as stimuli sensitive, multimodal, and theranostic imaging.We present a new melanin-like nanoparticle (MelNP) and its performance evaluation results. This particle is proposed as an exogenous contrast agent for photoacoustic (PA) imaging. Conventional PA contrast agents are based on non-biological materials. In contrast, the MelNPs are organic nanoparticles inspired by natural melanin. Melanin is an endogenous chromophore that has the ability to produce a PA signal in vivo. The developed MelNPs are capable of aggregating with one

  7. Latent heat induced rotation limited aggregation in 2D ice nanocrystals

    Science.gov (United States)

    Bampoulis, Pantelis; Siekman, Martin H.; Kooij, E. Stefan; Lohse, Detlef; Zandvliet, Harold J. W.; Poelsema, Bene

    2015-07-01

    The basic science responsible for the fascinating shapes of ice crystals and snowflakes is still not understood. Insufficient knowledge of the interaction potentials and the lack of relevant experimental access to the growth process are to blame for this failure. Here, we study the growth of fractal nanostructures in a two-dimensional (2D) system, intercalated between mica and graphene. Based on our scanning tunneling spectroscopy data, we provide compelling evidence that these fractals are 2D ice. They grow while they are in material contact with the atmosphere at 20 °C and without significant thermal contact to the ambient. The growth is studied in situ, in real time and space at the nanoscale. We find that the growing 2D ice nanocrystals assume a fractal shape, which is conventionally attributed to Diffusion Limited Aggregation (DLA). However, DLA requires a low mass density mother phase, in contrast to the actual currently present high mass density mother phase. Latent heat effects and consequent transport of heat and molecules are found to be key ingredients for understanding the evolution of the snow (ice) flakes. We conclude that not the local availability of water molecules (DLA), but rather them having the locally required orientation is the key factor for incorporation into the 2D ice nanocrystal. In combination with the transport of latent heat, we attribute the evolution of fractal 2D ice nanocrystals to local temperature dependent rotation limited aggregation. The ice growth occurs under extreme supersaturation, i.e., the conditions closely resemble the natural ones for the growth of complex 2D snow (ice) flakes and we consider our findings crucial for solving the "perennial" snow (ice) flake enigma.

  8. Neuregulin1–β decreases interleukin–1β–induced RhoA activation, myosin light chain phosphorylation, and endothelial hyperpermeability

    Science.gov (United States)

    Wu, Limin; Ramirez, Servio H.; Andrews, Allison M.; Leung, Wendy; Itoh, Kanako; Wu, Jiang; Arai, Ken; Lo, Eng H.; Lok, Josephine

    2016-01-01

    Neuregulin-1 (NRG1) is an endogenous growth factor with multiple functions in the embryonic and postnatal brain. The NRG1 gene is large and complex, transcribing more than twenty transmembrane proteins and generating a large number of isoforms in tissue and cell type-specific patterns. Within the brain, NRG1 functions have been studied most extensively in neurons and glia, as well as in the peripheral vasculature. Recently, NRG1 signaling has been found to be important in the function of brain microvascular endothelial cells, decreasing IL-1β-induced increases in endothelial permeability. In the current experiments, we have investigated the pathways through which the NRG1-β isoform acts on IL-1β-induced endothelial permeability. Our data show that NRG1-β increases barrier function, measured by transendothelial electrical resistance, and decreases IL-1β-induced hyperpermeability, measured by dextran-40 extravasation through a monolayer of brain microvascular endothelial cells plated on transwells. An investigation of key signaling proteins suggests that the effect of NRG1-β on endothelial permeability is mediated through RhoA activation and myosin light chain phosphorylation, events which affect filamentous actin morphology. In addition, AG825, an inhibitor of the erbB2-associated tyrosine kinase, reduces the effect of NRG1-β on IL-1β-induced RhoA activation and myosin light chain phosphorylation. These data add to the evidence that NRG1-β signaling affects changes in the brain microvasculature in the setting of neuroinflammation. PMID:26438054

  9. (-)-Epigallocatechin-3-gallate decreases thrombin/paclitaxel-induced endothelial tissue factor expression via the inhibition of c-Jun terminal NH2 kinase phosphorylation

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Huang-Joe [Institute of Biotechnology, National Tsing Hua University, No. 101, Section 2, Kuang Fu Road, Hsinchu 30013, Taiwan (China); Division of Cardiology, Department of Medicine, China Medical University Hospital, No. 2, Yuh-Der Road, Taichung 40447, Taiwan (China); Lo, Wan-Yu [Department of Medical Research, China Medical University Hospital, No. 2, Yuh-Der Road, Taichung 40447, Taiwan (China); Graduate Integration of Chinese and Western Medicine, China Medical University, No. 91, Hsueh-Shih Road, Taichung 40402, Taiwan (China); Lu, Te-Ling [School of Pharmacy, China Medical University, No. 91, Hsueh-Shih Road, Taichung 40402, Taiwan (China); Huang, Haimei, E-mail: hmhuang@life.nthu.edu.tw [Institute of Biotechnology, National Tsing Hua University, No. 101, Section 2, Kuang Fu Road, Hsinchu 30013, Taiwan (China)

    2010-01-01

    Patients with paclitaxel-eluting stents are concerned with stent thrombosis caused by premature discontinuation of dual antiplatelet therapy or clopidogrel resistance. This study investigates the effect of (-)-epigallocatechin-3-gallate (EGCG) on the expression of thrombin/paclitaxel-induced endothelial tissue factor (TF) expressions in human aortic endothelial cells (HAECs). EGCG was nontoxic to HAECs at 6 h up to a concentration of 25 {mu}mol/L. At a concentration of 25 {mu}mol/L, EGCG pretreatment potently inhibited both thrombin-stimulated and thrombin/paclitaxel-stimulated endothelial TF protein expression. Thrombin and thrombin/paclitaxel-induced 2.6-fold and 2.9-fold increases in TF activity compared with the control. EGCG pretreatment caused a 29% and 38% decrease in TF activity on thrombin and thrombin/paclitaxel treatment, respectively. Real-time polymerase chain reaction (PCR) showed that thrombin and thrombin/paclitaxel-induced 3.0-fold and 4.6-fold TF mRNA expressions compared with the control. EGCG pretreatment caused an 82% and 72% decrease in TF mRNA expression on thrombin and thrombin/paclitaxel treatment, respectively. The c-Jun terminal NH2 kinase (JNK) inhibitor SP600125 reduced thrombin/paclitaxel-induced TF expression. Furthermore, EGCG significantly inhibited the phosphorylation of JNK to 49% of thrombin/paclitaxel-stimulated HAECs at 60 min. Immunofluorescence assay did not show an inhibitory effect of EGCG on P65 NF-{kappa}B nuclear translocation in the thrombin/paclitaxel-stimulated endothelial cells. In conclusion, EGCG can inhibit TF expression in thrombin/paclitaxel-stimulated endothelial cells via the inhibition of JNK phosphorylation. The unique property of EGCG may be used to develop a new drug-eluting stent by co-coating EGCG and paclitaxel.

  10. UV Damage-Induced Phosphorylation of HBO1 Triggers CRL4DDB2-Mediated Degradation To Regulate Cell Proliferation.

    Science.gov (United States)

    Matsunuma, Ryoichi; Niida, Hiroyuki; Ohhata, Tatsuya; Kitagawa, Kyoko; Sakai, Satoshi; Uchida, Chiharu; Shiotani, Bunsyo; Matsumoto, Masaki; Nakayama, Keiichi I; Ogura, Hiroyuki; Shiiya, Norihiko; Kitagawa, Masatoshi

    2015-11-16

    Histone acetyltransferase binding to ORC-1 (HBO1) is a critically important histone acetyltransferase for forming the prereplicative complex (pre-RC) at the replication origin. Pre-RC formation is completed by loading of the MCM2-7 heterohexameric complex, which functions as a helicase in DNA replication. HBO1 recruited to the replication origin by CDT1 acetylates histone H4 to relax the chromatin conformation and facilitates loading of the MCM complex onto replication origins. However, the acetylation status and mechanism of regulation of histone H3 at replication origins remain elusive. HBO1 positively regulates cell proliferation under normal cell growth conditions. Whether HBO1 regulates proliferation in response to DNA damage is poorly understood. In this study, we demonstrated that HBO1 was degraded after DNA damage to suppress cell proliferation. Ser50 and Ser53 of HBO1 were phosphorylated in an ATM/ATR DNA damage sensor-dependent manner after UV treatment. ATM/ATR-dependently phosphorylated HBO1 preferentially interacted with DDB2 and was ubiquitylated by CRL4(DDB2). Replacement of endogenous HBO1 in Ser50/53Ala mutants maintained acetylation of histone H3K14 and impaired cell cycle regulation in response to UV irradiation. Our findings demonstrate that HBO1 is one of the targets in the DNA damage checkpoint. These results show that ubiquitin-dependent control of the HBO1 protein contributes to cell survival during UV irradiation.

  11. The use of quartz crystal microbalance with dissipation (QCM-D) for studying nanoparticle-induced platelet aggregation

    Science.gov (United States)

    Santos-Martinez, Maria Jose; Inkielewicz-Stepniak, Iwona; Medina, Carlos; Rahme, Kamil; D’Arcy, Deirdre M; Fox, Daniel; Holmes, Justin D; Zhang, Hongzhou; Radomski, Marek Witold

    2012-01-01

    Interactions between blood platelets and nanoparticles have both pharmacological and toxicological significance and may lead to platelet activation and aggregation. Platelet aggregation is usually studied using light aggregometer that neither mimics the conditions found in human microvasculature nor detects microaggregates. A new method for the measurement of platelet microaggregation under flow conditions using a commercially available quartz crystal microbalance with dissipation (QCM-D) has recently been developed. The aim of the current study was to investigate if QCM-D could be used for the measurement of nanoparticle-platelet interactions. Silica, polystyrene, and gold nanoparticles were tested. The interactions were also studied using light aggregometry and flow cytometry, which measured surface abundance of platelet receptors. Platelet activation was imaged using phase contrast and scanning helium ion microscopy. QCM-D was able to measure nanoparticle-induced platelet microaggregation for all nanoparticles tested at concentrations that were undetectable by light aggregometry and flow cytometry. Microaggregates were measured by changes in frequency and dissipation, and the presence of platelets on the sensor surface was confirmed and imaged by phase contrast and scanning helium ion microscopy. PMID:22275839

  12. Single chain variable fragment antibodies block aggregation and toxicity induced by familial ALS-linked mutant forms of SOD1

    Science.gov (United States)

    Ghadge, Ghanashyam D.; Pavlovic, John; Koduvayur, Sujatha P.; Kay, Brian K.; Roos, Raymond P.

    2013-01-01

    Approximately 10% of amyotrophic lateral sclerosis (ALS) cases are familial (known as FALS) with an autosomal dominant inheritance pattern, and ~25% of FALS cases are caused by mutations in Cu/Zn superoxide dismutase (SOD1). There is convincing evidence that mutant SOD1 (mtSOD1) kills motor neurons (MNs) because of a gain-of-function toxicity, most likely related to aggregation of mtSOD1. A number of recent reports have suggested that antibodies can be used to treat mtSOD1-induced FALS. To follow up on the use of antibodies as potential therapeutics, we generated single chain fragments of variable region antibodies (scFvs) against SOD1, and then expressed them as ‘intrabodies’ within a motor neuron cell line. In the present study, we describe isolation of human scFvs that interfere with mtSOD1 in vitro aggregation and toxicity. These scFvs may have therapeutic potential in sporadic ALS, as well as FALS, given that sporadic ALS may also involve abnormalities in the SOD1 protein or activity. PMID:23607939

  13. Effect of dextran-induced changes in refractive index and aggregation on optical properties of whole blood

    Energy Technology Data Exchange (ETDEWEB)

    Xu Xiangqun [Institute of Bioscience and Technology, Cranfield University, Silsoe, Bedfordshire MK45 4DT (United Kingdom); Wang, Ruikang K [Institute of Bioscience and Technology, Cranfield University, Silsoe, Bedfordshire MK45 4DT (United Kingdom); Elder, James B [School of Medicine, Keele University, Stoke-on-Trent, ST4 7QB (United Kingdom); Tuchin, Valery V [Department of Optics, Saratov State University, 155 Moskovskaya Str., 410026 Saratov (Russian Federation)

    2003-05-07

    The purpose of the present study is to investigate systematically the mechanisms of alterations in the optical properties of whole blood immersed in the biocompatible agent dextran, and to define the optimal concentration of dextrans required for blood optical clearing in order to enhance the capability of light penetration depth for optical imaging applications. In the experiments, dextrans with different molecular weights and various concentrations were employed and investigated by the use of the optical coherence tomography technique. Changes in light attenuation, refractive index and aggregation properties of blood immersed in dextrans were studied. It was concluded from the results that the mechanisms for blood optical clearing are characteristic of the types of dextrans employed, their concentrations and the application stages. Among the substances applied, Dx500 at a concentration at 0.5 g dl{sup -1} gives the best result in improving light penetration depth through the blood. The increase of light transmission at the beginning of the addition of dextrans is mainly attributed to refractive index matching between the scattering centres and the ground matter. Thereafter, the transmission change is probably due to a dextran-induced aggregation-disaggregation effect. Overall, light scattering in the blood could be effectively reduced by the application of dextrans. It represents a promising approach to increasing the imaging depth for in vivo optical imaging of biological tissue, for example optical coherence tomography.

  14. Drosophila UNC-45 prevents heat-induced aggregation of skeletal muscle myosin and facilitates refolding of citrate synthase

    Energy Technology Data Exchange (ETDEWEB)

    Melkani, Girish C.; Lee, Chi F.; Cammarato, Anthony [Department of Biology and the Molecular Biology Institute, San Diego State University, San Diego, CA 92182-4614 (United States); Bernstein, Sanford I., E-mail: sbernst@sciences.sdsu.edu [Department of Biology and the Molecular Biology Institute, San Diego State University, San Diego, CA 92182-4614 (United States)

    2010-05-28

    UNC-45 belongs to the UCS (UNC-45, CRO1, She4p) domain protein family, whose members interact with various classes of myosin. Here we provide structural and biochemical evidence that Escherichia coli-expressed Drosophila UNC-45 (DUNC-45) maintains the integrity of several substrates during heat-induced stress in vitro. DUNC-45 displays chaperone function in suppressing aggregation of the muscle myosin heavy meromyosin fragment, the myosin S-1 motor domain, {alpha}-lactalbumin and citrate synthase. Biochemical evidence is supported by electron microscopy, which reveals the first structural evidence that DUNC-45 prevents inter- or intra-molecular aggregates of skeletal muscle heavy meromyosin caused by elevated temperatures. We also demonstrate for the first time that UNC-45 is able to refold a denatured substrate, urea-unfolded citrate synthase. Overall, this in vitro study provides insight into the fate of muscle myosin under stress conditions and suggests that UNC-45 protects and maintains the contractile machinery during in vivo stress.

  15. Calcium absorption by Cav1.3 induces terminal web myosin II phosphorylation and apical GLUT2 insertion in rat intestine.

    Science.gov (United States)

    Mace, Oliver J; Morgan, Emma L; Affleck, Julie A; Lister, Norma; Kellett, George L

    2007-04-15

    Glucose absorption in rat jejunum involves Ca(2+)- and PKC betaII-dependent insertion of GLUT2 into the apical membrane. Ca(2+)-induced rearrangement of the enterocyte cytoskeleton is thought to enhance paracellular flow. We have therefore investigated the relationships between myosin II regulatory light chain phosphorylation (RLC(20)), absorption of glucose, water and calcium, and mannitol clearance. ML-7, an inhibitor of myosin light chain kinase, diminished the phloretin-sensitive apical GLUT2 but not the phloretin-insensitive SGLT1 component of glucose absorption in rat jejunum perfused with 75 mM glucose. Western blotting and immunocytochemistry revealed marked decreases in RLC(20) phosphorylation in the terminal web and in the levels of apical GLUT2 and PKC betaII, but not SGLT1. Perfusion with phloridzin or 75 mM mannitol, removal of luminal Ca(2+), or inhibition of unidirectional (45)Ca(2+) absorption by nifedipine exerted similar effects. ML-7 had no effect on the absorption of 10 mM Ca(2+), nor clearance of [(14)C]-mannitol, which was less than 0.7% of the rate of glucose absorption. Water absorption did not correlate with (45)Ca(2+) absorption or mannitol clearance. We conclude that the Ca(2+) necessary for contraction of myosin II in the terminal web enters via an L-type channel, most likely Ca(v)1.3, and is dependent on SGLT1. Moreover, terminal web RLC(20) phosphorylation is necessary for apical GLUT2 insertion. The data confirm that glucose absorption by paracellular flow is negligible, and show further that paracellular flow makes no more than a minimal contribution to jejunal Ca(2+) absorption at luminal concentrations prevailing after a meal.

  16. Melatonin Attenuates Aortic Endothelial Permeability and Arteriosclerosis in Streptozotocin-Induced Diabetic Rats: Possible Role of MLCK- and MLCP-Dependent MLC Phosphorylation.

    Science.gov (United States)

    Tang, Song-tao; Su, Huan; Zhang, Qiu; Tang, Hai-qin; Wang, Chang-jiang; Zhou, Qing; Wei, Wei; Zhu, Hua-qing; Wang, Yuan

    2016-01-01

    The development of diabetic macrovascular complications is a multifactorial process, and melatonin may possess cardiovascular protective properties. This study was designed to evaluate whether melatonin attenuates arteriosclerosis and endothelial permeability by suppressing the myosin light-chain kinase (MLCK)/myosin light-chain phosphorylation (p-MLC) system via the mitogen-activated protein kinase (MAPK) signaling pathway or by suppressing the myosin phosphatase-targeting subunit phosphorylation (p-MYPT)/p-MLC system in diabetes mellitus (DM). Rats were randomly divided into 4 groups, including control, high-fat diet, DM, and DM + melatonin groups. Melatonin was administered (10 mg/kg/d) by gavage for 12 weeks. The DM significantly increased the serum fasting blood glucose and lipid levels, as well as insulin resistance and endothelial dysfunction, which were attenuated by melatonin therapy to various extents. Importantly, the aortic endothelial permeability was significantly increased in DM rats but was dramatically reversed following treatment with melatonin. Our findings further indicated that hyperglycemia and hyperlipidemia enhanced the expressions of MLCK, p-MYPT, and p-MLC, which were partly associated with decreased membrane type 1 expression, increased extracellular signal-regulated kinase (ERK) phosphorylation, and increased p38 expression. However, these changes in protein expression were also significantly reversed by melatonin. Thus, our results are the first to demonstrate that the endothelial hyperpermeability induced by DM is associated with increased expressions of MLCK, p-MYPT, and p-MLC, which can be attenuated by melatonin at least partly through the ERK/p38 signaling pathway.

  17. Melatonin Attenuates Aortic Endothelial Permeability and Arteriosclerosis in Streptozotocin-Induced Diabetic Rats: Possible Role of MLCK- and MLCP-Dependent MLC Phosphorylation.

    Science.gov (United States)

    Tang, Song-tao; Su, Huan; Zhang, Qiu; Tang, Hai-qin; Wang, Chang-jiang; Zhou, Qing; Wei, Wei; Zhu, Hua-qing; Wang, Yuan

    2016-01-01

    The development of diabetic macrovascular complications is a multifactorial process, and melatonin may possess cardiovascular protective properties. This study was designed to evaluate whether melatonin attenuates arteriosclerosis and endothelial permeability by suppressing the myosin light-chain kinase (MLCK)/myosin light-chain phosphorylation (p-MLC) system via the mitogen-activated protein kinase (MAPK) signaling pathway or by suppressing the myosin phosphatase-targeting subunit phosphorylation (p-MYPT)/p-MLC system in diabetes mellitus (DM). Rats were randomly divided into 4 groups, including control, high-fat diet, DM, and DM + melatonin groups. Melatonin was administered (10 mg/kg/d) by gavage for 12 weeks. The DM significantly increased the serum fasting blood glucose and lipid levels, as well as insulin resistance and endothelial dysfunction, which were attenuated by melatonin therapy to various extents. Importantly, the aortic endothelial permeability was significantly increased in DM rats but was dramatically reversed following treatment with melatonin. Our findings further indicated that hyperglycemia and hyperlipidemia enhanced the expressions of MLCK, p-MYPT, and p-MLC, which were partly associated with decreased membrane type 1 expression, increased extracellular signal-regulated kinase (ERK) phosphorylation, and increased p38 expression. However, these changes in protein expression were also significantly reversed by melatonin. Thus, our results are the first to demonstrate that the endothelial hyperpermeability induced by DM is associated with increased expressions of MLCK, p-MYPT, and p-MLC, which can be attenuated by melatonin at least partly through the ERK/p38 signaling pathway. PMID:25944844

  18. Inhibitory Effects of Cytosolic Ca2+ Concentration by Ginsenoside Ro Are Dependent on Phosphorylation of IP3RI and Dephosphorylation of ERK in Human Platelets

    Directory of Open Access Journals (Sweden)

    Hyuk-Woo Kwon

    2015-01-01

    Full Text Available Intracellular Ca2+ ([Ca2+]i is platelet aggregation-inducing molecule and is involved in activation of aggregation associated molecules. This study was carried out to understand the Ca2+-antagonistic effect of ginsenoside Ro (G-Ro, an oleanane-type saponin in Panax ginseng. G-Ro, without affecting leakage of lactate dehydrogenase, dose-dependently inhibited thrombin-induced platelet aggregation, and the half maximal inhibitory concentration was approximately 155 μM. G-Ro inhibited strongly thrombin-elevated [Ca2+]i, which was strongly increased by A-kinase inhibitor Rp-8-Br-cAMPS compared to G-kinase inhibitor Rp-8-Br-cGMPS. G-Ro increased the level of cAMP and subsequently elevated the phosphorylation of inositol 1, 4, 5-triphosphate receptor I (IP3RI (Ser1756 to inhibit [Ca2+]i mobilization in thrombin-induced platelet aggregation. Phosphorylation of IP3RI (Ser1756 by G-Ro was decreased by PKA inhibitor Rp-8-Br-cAMPS. In addition, G-Ro inhibited thrombin-induced phosphorylation of ERK 2 (42 kDa, indicating inhibition of Ca2+ influx across plasma membrane. We demonstrate that G-Ro upregulates cAMP-dependent IP3RI (Ser1756 phosphorylation and downregulates phosphorylation of ERK 2 (42 kDa to decrease thrombin-elevated [Ca2+]i, which contributes to inhibition of ATP and serotonin release, and p-selectin expression. These results indicate that G-Ro in Panax ginseng is a beneficial novel Ca2+-antagonistic compound and may prevent platelet aggregation-mediated thrombotic disease.

  19. Chitosan-functionalized gold nanoparticles for colorimetric detection of mercury ions based on chelation-induced aggregation

    International Nuclear Information System (INIS)

    We are presenting a colorimetric assay for mercury (II) ions. It is based on citosan-functionalized gold nanoparticles (AuNPs) that act as a signaling probe. Hg (II) induces the aggregation of the chitosan-AuNPs through a chelation reaction that occurs between chitosan and Hg (II). This results in a strong decrease of the absorbance of the modified AuNPs and a color change from red to blue. This sensing system displays excellent selectivity over other metal ions and a detection limit as low as 1.35 μM which is lower than the allowed level of Hg (II) in drinking water (30 μM) as defined by World Health Organization. The method is inexpensive, facile, sensitive, and does not require the addition of other reagents in order to improving sensitivity. (author)

  20. Hybridization-Induced Aggregation Technology for Practical Clinical Testing: KRAS Mutation Detection in Lung and Colorectal Tumors.

    Science.gov (United States)

    Sloane, Hillary S; Landers, James P; Kelly, Kimberly A

    2016-07-01

    KRAS mutations have emerged as powerful predictors of response to targeted therapies in the treatment of lung and colorectal cancers; thus, prospective KRAS genotyping is essential for appropriate treatment stratification. Conventional mutation testing technologies are not ideal for routine clinical screening, as they often involve complex, time-consuming processes and/or costly instrumentation. In response, we recently introduced a unique analytical strategy for revealing KRAS mutations, based on the allele-specific hybridization-induced aggregation (HIA) of oligonucleotide probe-conjugated microbeads. Using simple, inexpensive instrumentation, this approach allows for the detection of any common KRAS mutation in platform may involve the detection of mutations in other key oncogenic pathways. PMID:27289420

  1. Fabrication of aggregation-induced emission based fluorescent nanoparticles and their biological imaging application: recent progress and perspectives

    Directory of Open Access Journals (Sweden)

    Bin Yang

    2016-06-01

    Full Text Available Aggregation-induced emission (AIE dyes have received wide-spread concern since their inception. Several types of AIE-based fluorescent nanoparticle (FNP have been developed, and the potential applications of these FNPs have also been explored. Recent studies of AIE-based FNPs in biological areas have suggested that they show promise as bio-materials for cell imaging and other biomedical applications. This article reviews recent progress in the synthesis of AIE-based FNPs via non-covalent, covalent and novel one-pot strategies, and the subsequent cell-imaging of those AIE-based FNPs. Many successes have been achieved, and there is still plenty of space for the development of AIE-based FNPs as new bio-materials.

  2. Detection of adenosine triphosphate through polymerization-induced aggregation of actin-conjugated gold/silver nanorods

    Science.gov (United States)

    Liao, Yu-Ju; Shiang, Yen-Chun; Chen, Li-Yi; Hsu, Chia-Lun; Huang, Chih-Ching; Chang, Huan-Tsung

    2013-11-01

    We have developed a simple and selective nanosensor for the optical detection of adenosine triphosphate (ATP) using globular actin-conjugated gold/silver nanorods (G-actin-Au/Ag NRs). By simply mixing G-actin and Au/Ag NRs (length ˜56 nm and diameter ˜12 nm), G-actin-Au/Ag NRs were prepared which were stable in physiological solutions (25 mM Tris-HCl, 150 mM NaCl, 5.0 mM KCl, 3.0 mM MgCl2 and 1.0 mM CaCl2; pH 7.4). Introduction of ATP into the G-actin-Au/Ag NR solutions in the presence of excess G-actin induced the formation of filamentous actin-conjugated Au/Ag NR aggregates through ATP-induced polymerization of G-actin. When compared to G-actin-modified spherical Au nanoparticles having a size of 13 nm or 56 nm, G-actin-Au/Ag NRs provided better sensitivity for ATP, mainly because the longitudinal surface plasmon absorbance of the Au/Ag NR has a more sensitive response to aggregation. This G-actin-Au/Ag NR probe provided high sensitivity (limit of detection 25 nM) for ATP with remarkable selectivity (>10-fold) over other adenine nucleotides (adenosine, adenosine monophosphate and adenosine diphosphate) and nucleoside triphosphates (guanosine triphosphate, cytidine triphosphate and uridine triphosphate). It also allowed the determination of ATP concentrations in plasma samples without conducting tedious sample pretreatments; the only necessary step was simple dilution. Our experimental results are in good agreement with those obtained from a commercial luciferin-luciferase bioluminescence assay. Our simple, sensitive and selective approach appears to have a practical potential for the clinical diagnosis of diseases (e.g. cystic fibrosis) associated with changes in ATP concentrations.

  3. Vitamin B₂ Sensitizes Cancer Cells to Vitamin-C-Induced Cell Death via Modulation of Akt and Bad Phosphorylation.

    Science.gov (United States)

    Chen, Ni; Yin, Shutao; Song, Xinhua; Fan, Lihong; Hu, Hongbo

    2015-08-01

    Vitamin C is an essential dietary nutrient that has a variety of biological functions. Recent studies have provided promising evidence for its additional health benefits, including anticancer activity. Vitamin B2, another essential dietary nutrient, often coexists with vitamin C in some fruits, vegetables, or dietary supplements. The objective of the present study is to determine whether the combination of vitamin C and B2 can achieve a synergistic anticancer activity. MDA-MB-231, MCF-7, and A549 cells were employed to evaluate the combinatory effects of vitamin C and B2. We found that the combination of vitamin C and B2 resulted in a synergistic cell death induction in all cell lines tested. Further mechanistic investigations revealed that vitamin B2 sensitized cancer cells to vitamin C through inhibition of Akt and Bad phosphorylation. Our findings identified vitamin B2 as a promising sensitizer for improving the efficacy of vitamin-C-based cancer chemoprevention and chemotherapy.

  4. Alpha-synuclein-induced aggregation of cytoplasmic vesicles in Saccharomyces cerevisiae.

    Science.gov (United States)

    Soper, James H; Roy, Subhojit; Stieber, Anna; Lee, Eliza; Wilson, Robert B; Trojanowski, John Q; Burd, Christopher G; Lee, Virginia M-Y

    2008-03-01

    Aggregated alpha-synuclein (alpha-syn) fibrils form Lewy bodies (LBs), the signature lesions of Parkinson's disease (PD) and related synucleinopathies, but the pathogenesis and neurodegenerative effects of LBs remain enigmatic. Recent studies have shown that when overexpressed in Saccharomyces cerevisiae, alpha-syn localizes to plasma membranes and forms cytoplasmic accumulations similar to human alpha-syn inclusions. However, the exact nature, composition, temporal evolution, and underlying mechanisms of yeast alpha-syn accumulations and their relevance to human synucleinopathies are unknown. Here we provide ultrastructural evidence that alpha-syn accumulations are not comprised of LB-like fibrils, but are associated with clusters of vesicles. Live-cell imaging showed alpha-syn initially localized to the plasma membrane and subsequently formed accumulations in association with vesicles. Imaging of truncated and mutant forms of alpha-syn revealed the molecular determinants and vesicular trafficking pathways underlying this pathological process. Because vesicular clustering is also found in LB-containing neurons of PD brains, alpha-syn-mediated vesicular accumulation in yeast represents a model system to study specific aspects of neurodegeneration in PD and related synucleinopathies.

  5. Organic solvents induce the formation of oil-in-ionic liquid microemulsion aggregations.

    Science.gov (United States)

    Gao, Yanan; Li, Na; Zhang, Shaohua; Zheng, Liqiang; Li, Xinwei; Dong, Bin; Yu, Li

    2009-02-01

    The role of four organic solvents in the formation process of 1-butyl-3-methylimidazolium tetrafluoroborate (bmimBF4) based ionic liquid (IL) microemulsions is investigated. The results showed that the addition of Triton X-100 remarkably decreased the conductivity of bmimBF4. The added organic solvents provided a strong apolar environment for the hydrophobic tails of Triton X-100 and caused the surfactant molecules to aggregate into the interfacial film of oil-in-bmimBF4 (O/IL) microemulsions. As a result, the conductivities of the solutions were initially increased because the insulative Triton X-100 molecules were assembled, which corresponded to increasing the concentration of continuous bmimBF4 solutions. The hydrophobic interaction between the dispersed organic solvents and the hydrophobic tails of Triton X-100 may be the driving force for the formation of O/IL microemulsions. The droplets of O/IL microemulsions were successively swollen by organic solvents, and a bicontinuous IL-containing microemulsion was observed by freeze-fracture transmission electron microscopy for the first time. The current study can help in further understanding the ILs-containing microemulsions and thereby improve microemulsion theory. PMID:19138136

  6. Tooth discoloration induced by a novel mineral trioxide aggregate-based root canal sealer

    Science.gov (United States)

    Lee, Dae-Sung; Lim, Myung-Jin; Choi, Yoorina; Rosa, Vinicius; Hong, Chan-Ui; Min, Kyung-San

    2016-01-01

    Objectives: The aim of this study was to evaluate tooth discoloration caused by contact with a novel injectable mineral trioxide aggregate (MTA)-based root canal sealer (Endoseal; Maruchi, Wonju, Korea) compared with a widely used resin-based root canal sealer (AHplus; Dentsply De Trey, Konstanz, Germany) and conventional MTA (ProRoot; Dentsply, Tulsa, OK, USA). Materials and Methods: Forty standardized bovine tooth samples were instrumented and divided into three experimental groups and one control group (n = 10/group). Each material was inserted into the cavity, and all specimens were sealed with a self-adhesive resin. Based on CIE Lab system, brightness change (ΔL) and total color change (ΔE) of each specimen between baseline and 1, 2, 4, and 8 weeks were obtained. Results: At all time points, Endoseal showed no significant difference in ΔL and ΔE compared to AHplus and control group (P > 0.05), whereas the ProRoot group showed significantly higher ΔL and ΔE values than the Endoseal group at 2, 4, and 8 weeks (P MTA and a similar color change to AHplus. Conclusions: Within the limitations of this study, our data indicate that the MTA-based sealer produces a similar amount of tooth discoloration as AHplus which is considered to be acceptable. PMID:27403062

  7. Latent heat induced rotation limited aggregation in 2D ice nanocrystals

    CERN Document Server

    Bampoulis, Pantelis; Kooij, E Stefan; Lohse, Detlef; Zandvliet, Harold J W; Poelsema, Bene

    2016-01-01

    The basic science responsible for the fascinating shapes of ice crystals and snowflakes is still not understood. Insufficient knowledge of the interaction potentials and the lack of relevant experimental access to the growth process are to blame for this failure. Here, we study the growth of fractal nanostructures in a two-dimensional (2D) system, intercalated between mica and graphene. Based on our Scanning Tunneling Spectroscopy (STS) data we provide compelling evidence that these fractals are 2D ice. They grow while they are in material contact with the atmosphere at 20 $^{\\circ}$C and without significant thermal contact to the ambient. The growth is studied in-situ, in real time and space at the nanoscale. We find that the growing 2D ice nanocrystals assume a fractal shape, which is conventionally attributed to Diffusion Limited Aggregation (DLA). However, DLA requires a low mass density mother phase, in contrast to the actual currently present high mass density mother phase. Latent heat effects and conse...

  8. Duration of streptozotocin-induced diabetes differentially affects p38-mitogen-activated protein kinase (MAPK phosphorylation in renal and vascular dysfunction

    Directory of Open Access Journals (Sweden)

    Gupta Akanksha

    2005-03-01

    Full Text Available Abstract Background In the present study we tested the hypothesis that progression of streptozotocin (STZ-induced diabetes (14-days to 28-days would produce renal and vascular dysfunction that correlate with altered p38- mitogen-activated protein kinase (p38-MAPK phosphorylation in kidneys and thoracic aorta. Methods Male Sprague Dawley rats (350–400 g were randomized into three groups: sham (N = 6, 14-days diabetic (N = 6 and 28-days diabetic rats (N = 6. Diabetes was induced using a single tail vein injection of STZ (60 mg/kg, I.V. on the first day. Rats were monitored for 28 days and food, water intake and plasma glucose levels were noted. At both 14-days and 28-days post diabetes blood samples were collected and kidney cortex, medulla and aorta were harvested from each rat. Results The diabetic rats lost body weight at both 14-days (-10% and 28-days (-13% more significantly as compared to sham (+10% group. Glucose levels were significantly elevated in the diabetic rats at both 14-days and 28-days post-STZ administration. Renal dysfunction as evidenced by renal hypertrophy, increased plasma creatinine concentration and reduced renal blood flow was observed in 14-days and 28-days diabetes. Vascular dysfunction as evidenced by decreased carotid blood flow was observed in 14-days and 28-days diabetes. We observed an up-regulation of inducible nitric oxide synthase (iNOS, prepro endothelin-1 (preproET-1 and phosphorylated p38-MAPK in thoracic aorta and kidney cortex but not in kidney medulla in 28-days diabetes group. Conclusion The study provides evidence that diabetes produces vascular and renal dysfunction with a profound effect on signaling mechanisms at later stage of diabetes.

  9. Diphenylarsinic Acid Induced Activation of Cultured Rat Cerebellar Astrocytes: Phosphorylation of Mitogen-Activated Protein Kinases, Upregulation of Transcription Factors, and Release of Brain-Active Cytokines.

    Science.gov (United States)

    Negishi, Takayuki; Matsumoto, Mami; Kojima, Mikiya; Asai, Ryota; Kanehira, Tomoko; Sakaguchi, Fumika; Takahata, Kazuaki; Arakaki, Rina; Aoyama, Yohei; Yoshida, Hikari; Yoshida, Kenji; Yukawa, Kazunori; Tashiro, Tomoko; Hirano, Seishiro

    2016-03-01

    Diphenylarsinic acid (DPAA) was detected as the primary compound responsible for the arsenic poisoning that occurred in Kamisu, Ibaraki, Japan, where people using water from a well that was contaminated with a high level of arsenic developed neurological (mostly cerebellar) symptoms and dysregulation of regional cerebral blood flow. To understand the underlying molecular mechanism of DPAA-induced cerebellar symptoms, we focused on astrocytes, which have a brain-protective function. Incubation with 10 µM DPAA for 96 h promoted cell proliferation, increased the expression of antioxidative stress proteins (heme oxygenase-1 and heat shock protein 70), and induced the release of cytokines (MCP-1, adrenomedullin, FGF2, CXCL1, and IL-6). Furthermore, DPAA overpoweringly increased the phosphorylation of three major mitogen-activated protein kinases (MAPKs) (ERK1/2, p38MAPK, and SAPK/JNK), which indicated MAPK activation, and subsequently induced expression and/or phosphorylation of transcription factors (Nrf2, CREB, c-Jun, and c-Fos) in cultured rat cerebellar astrocytes. Structure-activity relationship analyses of DPAA and other related pentavalent organic arsenicals revealed that DPAA at 10 µM activated astrocytes most effective among organic arsenicals tested at the same dose. These results suggest that in a cerebellum exposed to DPAA, abnormal activation of the MAPK-transcription factor pathway and irregular secretion of these neuroactive, glioactive, and/or vasoactive cytokines in astrocytes can be the direct/indirect cause of functional abnormalities in surrounding neurons, glial cells, and vascular cells: This in turn might lead to the onset of cerebellar symptoms and disruption of cerebral blood flow.

  10. Diphenylarsinic Acid Induced Activation of Cultured Rat Cerebellar Astrocytes: Phosphorylation of Mitogen-Activated Protein Kinases, Upregulation of Transcription Factors, and Release of Brain-Active Cytokines.

    Science.gov (United States)

    Negishi, Takayuki; Matsumoto, Mami; Kojima, Mikiya; Asai, Ryota; Kanehira, Tomoko; Sakaguchi, Fumika; Takahata, Kazuaki; Arakaki, Rina; Aoyama, Yohei; Yoshida, Hikari; Yoshida, Kenji; Yukawa, Kazunori; Tashiro, Tomoko; Hirano, Seishiro

    2016-03-01

    Diphenylarsinic acid (DPAA) was detected as the primary compound responsible for the arsenic poisoning that occurred in Kamisu, Ibaraki, Japan, where people using water from a well that was contaminated with a high level of arsenic developed neurological (mostly cerebellar) symptoms and dysregulation of regional cerebral blood flow. To understand the underlying molecular mechanism of DPAA-induced cerebellar symptoms, we focused on astrocytes, which have a brain-protective function. Incubation with 10 µM DPAA for 96 h promoted cell proliferation, increased the expression of antioxidative stress proteins (heme oxygenase-1 and heat shock protein 70), and induced the release of cytokines (MCP-1, adrenomedullin, FGF2, CXCL1, and IL-6). Furthermore, DPAA overpoweringly increased the phosphorylation of three major mitogen-activated protein kinases (MAPKs) (ERK1/2, p38MAPK, and SAPK/JNK), which indicated MAPK activation, and subsequently induced expression and/or phosphorylation of transcription factors (Nrf2, CREB, c-Jun, and c-Fos) in cultured rat cerebellar astrocytes. Structure-activity relationship analyses of DPAA and other related pentavalent organic arsenicals revealed that DPAA at 10 µM activated astrocytes most effective among organic arsenicals tested at the same dose. These results suggest that in a cerebellum exposed to DPAA, abnormal activation of the MAPK-transcription factor pathway and irregular secretion of these neuroactive, glioactive, and/or vasoactive cytokines in astrocytes can be the direct/indirect cause of functional abnormalities in surrounding neurons, glial cells, and vascular cells: This in turn might lead to the onset of cerebellar symptoms and disruption of cerebral blood flow. PMID:26645585

  11. Hydrogen sulfide donor NaHS induces death of alveolar epithelial L2 cells that is associated with cellular shrinkage, transgelin expression and myosin phosphorylation.

    Science.gov (United States)

    Fujii, Yusuke; Funakoshi, Takeshi; Unuma, Kana; Noritake, Kanako; Aki, Toshihiko; Uemura, Koichi

    2016-01-01

    Hydrogen sulfide (H2S) is a highly toxic gaseous molecule that causes death to humans exposed to high concentrations. H2S is absorbed into the body through the alveolar epithelium and other tissues. The aim of this study is to evaluate the molecular mechanism underling acute lung injury caused by the inhalation of high concentrations of H2S. Rat lung epithelium-derived L2 cells were exposed to a H2S donor, NaHS, at concentrations of 2-4 mM for 1-6 hr. NaHS caused shrinkage and death of the cells without caspase activation. An actin-binding protein, transgelin, was identified as one of the NaHS-inducible proteins in the cells. NaHS increased myosin light chain (MLC) phosphorylation, indicating that actomyosin-mediated cellular contractility and/or motility could be increased after NaHS exposure. The administration of ML-7, a myosin light chain kinase (MLCK) inhibitor, accelerated cell death after NaHS exposure. Based on these data, we conclude that the increase in MLC phosphorylation in response to NaHS exposure is a cellular protective reaction against NaHS toxicity. Enhancements in smooth muscle cell properties such as transgelin expression and actomyosin-mediated contractility/motility might be involved in cell survival after NaHS exposure. PMID:27665774

  12. Constraint-induced movement therapy promotes motor function recovery and downregulates phosphorylated extracellular regulated protein kinase expression in ischemic brain tissue of rats.

    Science.gov (United States)

    Zhang, Bei; He, Qiang; Li, Ying-Ying; Li, Ce; Bai, Yu-Long; Hu, Yong-Shan; Zhang, Feng

    2015-12-01

    Motor function impairment is a common outcome of stroke. Constraint-induced movement therapy (CIMT) involving intensive use of the impaired limb while restraining the unaffected limb is widely used to overcome the effects of 'learned non-use' and improve limb function after stroke. However, the underlying mechanism of CIMT remains unclear. In the present study, rats were randomly divided into a middle cerebral artery occlusion (model) group, a CIMT + model (CIMT) group, or a sham group. Restriction of the affected limb by plaster cast was performed in the CIMT and sham groups. Compared with the model group, CIMT significantly improved the forelimb functional performance in rats. By western blot assay, the expression of phosphorylated extracellular regulated protein kinase in the bilateral cortex and hippocampi of cerebral ischemic rats in the CIMT group was significantly lower than that in the model group, and was similar to sham group levels. These data suggest that functional recovery after CIMT may be related to decreased expression of phosphorylated extracellular regulated protein kinase in the bilateral cortex and hippocampi.

  13. Constraint-induced movement therapy promotes motor function recovery and downregulates phosphorylated extracellular regulated protein kinase expression in ischemic brain tissue of rats

    Directory of Open Access Journals (Sweden)

    Bei Zhang

    2015-01-01

    Full Text Available Motor function impairment is a common outcome of stroke. Constraint-induced movement therapy (CIMT involving intensive use of the impaired limb while restraining the unaffected limb is widely used to overcome the effects of ′learned non-use′ and improve limb function after stroke. However, the underlying mechanism of CIMT remains unclear. In the present study, rats were randomly divided into a middle cerebral artery occlusion (model group, a CIMT + model (CIMT group, or a sham group. Restriction of the affected limb by plaster cast was performed in the CIMT and sham groups. Compared with the model group, CIMT significantly improved the forelimb functional performance in rats. By western blot assay, the expression of phosphorylated extracellular regulated protein kinase in the bilateral cortex and hippocampi of cerebral ischemic rats in the CIMT group was significantly lower than that in the model group, and was similar to sham group levels. These data suggest that functional recovery after CIMT may be related to decreased expression of phosphorylated extracellular regulated protein kinase in the bilateral cortex and hippocampi.

  14. Nesfatin-1 induces the phosphorylation levels of cAMP response element-binding protein for intracellular signaling in a neural cell line.

    Directory of Open Access Journals (Sweden)

    Emi Ishida

    Full Text Available Nesfatin-1 is a novel anorexic peptide that reduces the food intake of rodents when administered either intraventricularly or intraperitoneally. However, the molecular mechanism of intracellular signaling via Nesfatin-1 is yet to be resolved. In the current study, we investigated the ability of different neuronal cell lines to respond to Nesfatin-1 and further elucidated the signal transduction pathway of Nesfatin-1. To achieve this, we transfected several cell lines with various combinations of reporter vectors containing different kinds of response elements and performed reporter assays with Nesfatin-1, its active midsegment encoding 30 amino acid residues (M30 and M30-derived mutants. Notably, we found that both Nesfatin-1 as well as M30, significantly increased cAMP response element (CRE reporter activity in a mouse neuroblastoma cell line, NB41A3. An antagonist of Melanocortin 3/4 receptor, SHU9119, aborted the promoter activity, and a mutant M30, which exerts no anorexic effect in vivo did not induce the CRE reporter activity in NB41A3 cells. Western blotting analyses revealed that Nesfatin-1 and M30 significantly increased the phosphorylation levels of CRE-binding protein (CREB, without altering the intracellular cAMP levels. Further, our study showed that a mitogen-activated protein kinase (MAPK kinase inhibitor and an L-type Calcium (Ca(2+ channel blocker abolished the M30-induced CREB phosphorylation. Furthermore, the radio-receptor assay revealed that (125I-Nesfatin-1 binds in a saturable fashion to the membrane fractions of the mouse hypothalamus and NB41A3 cells, with Kd values of 0.79 nM and 0.17 nM, respectively. Collectively, our findings indicate the presence of a Nesfatin-1-specific receptor on the cell surface of NB41A3 cells and mouse hypothalamus. Our study highlights that Nesfatin-1, via its receptor, induces the phosphorylation of CREB, thus activating the intracellular signaling cascade in neurons.

  15. Translation of the radioresistance kinase TLK1B is induced by γ-irradiation through activation of mTOR and phosphorylation of 4E-BP1

    Directory of Open Access Journals (Sweden)

    De Benedetti Arrigo

    2004-04-01

    Full Text Available Abstract Background The mammalian protein kinase TLK1 is a homologue of Tousled, a gene involved in flower development in Arabidopsis thaliana. The function of TLK1 is not well known, although knockout of the gene in Drosophila, or expression of a dominant negative mutant in mouse cells causes loss of nuclear divisions and chromosome missegregation probably due to alterations in chromatin remodeling capacity. Overexpression of TLK1B, a spliced variant of the TLK1 mRNA, in a model mouse cell line increases their resistance to ionizing radiation, also likely through changes in chromatin remodeling. The TLK1B mRNA is translationally repressed by its 5'UTR and is regulated by the availability of eIF4E. We now report that radiation or doxorubicin result in an increase in the translation of TLK1B, and we have uncovered the likely mechanism for this effect. Results Radiation causes a shift in the polysomal distribution of TLK1B mRNA, from the untranslated region and small polysomes to the large polysomes, concomitant with an increase in the expression of TLK1B protein. This change is preceded by an increase in phosphorylation of the eIF4E inhibitory protein 4E-BP1, which releases eIF4E when it is phosphorylated. The phosphorylation of 4E-BP1 depends on mTOR, since rapamycin blocked the increase in phosphorylation induced by radiation, and prevented the increase in TLK1B protein expression. The activation of mTOR was likely due to the rapid activation of Akt following radiation. The activation of Akt could be inhibited with wortmannin, an inhibitor of PI3 kinase, hence placing PI3 kinase upstream of Akt as a very early event following radiation. Wortmannin also inhibited translation of TLK1B mRNA following activation by IR. This was shown both by western blot and by measuring the initiation capacity of the mRNA, as indicated by its distribution on polysomes. Conclusions The translational upregulation of TLK1B elicited by DNA double strand breaks

  16. UV-inducible cellular aggregation of the hyperthermophilic archaeon Sulfolobus solfataricus is mediated by pili formation

    NARCIS (Netherlands)

    Froels, Sabrina; Ajon, Malgorzata; Wagner, Michaela; Teichmann, Daniela; Zolghadr, Behnam; Folea, Mihaela; Boekema, Egbert J.; Driessen, Arnold J. M.; Schleper, Christa; Albers, Sonja-Verena

    2008-01-01

    The hyperthermophilic archaeon Sulfolobus solfataricus has been shown to exhibit a complex transcriptional response to UV irradiation involving 55 genes. Among the strongest UV-induced genes was a putative pili biogenesis operon encoding a potential secretion ATPase, two pre-pilins, a putative trans

  17. PKCη/Rdx-driven phosphorylation of PDK1: a novel mechanism promoting cancer cell survival and permissiveness for parvovirus-induced lysis.

    Directory of Open Access Journals (Sweden)

    Séverine Bär

    2015-03-01

    Full Text Available The intrinsic oncotropism and oncosuppressive activities of rodent protoparvoviruses (PVs are opening new prospects for cancer virotherapy. Virus propagation, cytolytic activity, and spread are tightly connected to activation of the PDK1 signaling cascade, which delays stress-induced cell death and sustains functioning of the parvoviral protein NS1 through PKC(η-driven modifications. Here we reveal a new PV-induced intracellular loop-back mechanism whereby PKCη/Rdx phosphorylates mouse PDK1:S138 and activates it independently of PI3-kinase signaling. The corresponding human PDK1phosphoS135 appears as a hallmark of highly aggressive brain tumors and may contribute to the very effective targeting of human gliomas by H-1PV. Strikingly, although H-1PV does not trigger PDK1 activation in normal human cells, such cells show enhanced viral DNA amplification and NS1-induced death upon expression of a constitutively active PDK1 mimicking PDK1phosphoS135. This modification thus appears as a marker of human glioma malignant progression and sensitivity to H-1PV-induced tumor cell killing.

  18. Genistein inhibits phorbol ester-induced NF-κB transcriptional activity and COX-2 expression by blocking the phosphorylation of p65/RelA in human mammary epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Chung, Myung-Hoon; Kim, Do-Hee [Research Institute for Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul (Korea, Republic of); Na, Hye-Kyung [Department of Food and Nutrition, Sungshin Women' s University, Seoul (Korea, Republic of); Kim, Jung-Hwan; Kim, Ha-Na [Research Institute for Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul (Korea, Republic of); Haegeman, Guy [LEGEST, University of Gent (Belgium); Surh, Young-Joon, E-mail: surh@snu.ac.kr [Research Institute for Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul (Korea, Republic of); Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, Seoul (Korea, Republic of); Cancer Research Institute, Seoul National University, Seoul (Korea, Republic of)

    2014-10-15

    Genistein, an isoflavone present in soy products, has chemopreventive effects on mammary carcinogenesis. In the present study, we have investigated the effects of genistein on phorbol ester-induced expression of cyclooxygenase-2 (COX-2) that plays an important role in the pathophysiology of inflammation-associated carcinogenesis. Pretreatment of cultured human breast epithelial (MCF10A) cells with genistein reduced COX-2 expression induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). There are multiple lines of evidence supporting that the induction of COX-2 is regulated by the eukaryotic transcription factor NF-κB. Genistein failed to inhibit TPA-induced nuclear translocation and DNA binding of NF-κB as well as degradation of IκB. However, genistein abrogated the TPA-induced transcriptional activity of NF-κB as determined by the luciferase reporter gene assay. Genistein inhibited phosphorylation of the p65 subunit of NF-κB and its interaction with cAMP regulatory element-binding protein-binding protein (CBP)/p300 and TATA-binding protein (TBP). TPA-induced NF-κB phosphorylation was abolished by pharmacological inhibition of extracellular signal-regulated kinase (ERK). Likewise, pharmacologic inhibition or dominant negative mutation of ERK suppressed phosphorylation of p65. The above findings, taken together, suggest that genistein inhibits TPA-induced COX-2 expression in MCF10A cells by blocking ERK-mediated phosphorylation of p65 and its subsequent interaction with CBP and TBP.

  19. Mannosylerythritol lipid induces granulocytic differentiation and inhibits the tyrosine phosphorylation of human myelogenous leukemia cell line K562

    OpenAIRE

    Isoda, Hiroko; Nakahara, Tadaatsu

    1997-01-01

    Mannosylerythritol lipid (MEL), which induced granulocytic differentiation of human promyelocytic leukemia cell line HL60, also induced differentiation of human myelogenous leukemia cell line K562. MEL inhibited insulin-dependent cell proliferation and induced leukocyte esterase activity of K562 cells. MEL markedly increased the differentiation-associated characteristics in granulocytes, such as nitroblue tetrazolium (NBT) reducing ability, expression of Fc receptors, and phagocytic activity ...

  20. Heat Shock Proteins Regulate Activation-induced Proteasomal Degradation of the Mature Phosphorylated Form of Protein Kinase C*

    OpenAIRE

    Lum, Michelle A.; Balaburski, Gregor M; Murphy, Maureen E.; Black, Adrian R.; Black, Jennifer D.

    2013-01-01

    Background: Mechanisms of activation-induced PKC down-regulation are poorly understood. A characterized pathway involves priming site dephosphorylation and degradation of the dephosphorylated species.

  1. Suppressing Akt phosphorylation and activating Fas by safrole oxide inhibited angiogenesis and induced vascular endothelial cell apoptosis in the presence of fibroblast growth factor-2 and serum.

    Science.gov (United States)

    Zhao, Jing; Miao, Junying; Zhao, Baoxiang; Zhang, Shangli; Yin, Deling

    2006-01-01

    At present, vascular endothelial cell (VEC) apoptosis induced by deprivation of fibroblast growth factor-2 (FGF-2) and serum has been well studied. But how to trigger VEC apoptosis in the presence of FGF-2 and serum is not well known. To address this question, in this study, the effects of safrole oxide on angiogenesis and VEC growth stimulated by FGF-2 were investigated. The results showed that safrole oxide inhibited angiogenesis and induced VEC apoptosis in the presence of FGF-2 and serum. To understand the possible mechanism of safrole oxide acting, we first examined the phosphorylation of Akt and the activity of nitric oxide synthase (NOS); secondly, we analyzed the expressions and distributions of Fas and P53; then we measured the activity of phosphatidylcholine specific phospholipase C (PC-PLC) in the VECs treated with and without safrole oxide. The results showed that this small molecule obviously suppressed Akt phosphorylation and the activity of NOS, and promoted the expressions of Fas and P53 markedly. Simultaneously, Fas protein clumped on cell membrane, instead of homogenously distributed. The activity of PC-PLC was not changed obviously. The data suggested that safrole oxide effectively inhibited angiogenesis and triggered VEC apoptosis in the presence of FGF-2 and serum, and it might perform its functions by suppressing Akt/NOS signal pathway, upregulating the expressions of Fas and P53 and modifying the distributing pattern of Fas in VEC. This finding provided a powerful chemical probe for promoting VEC apoptosis during angiogenesis stimulated by FGF-2.

  2. Flavonoids targeting of IκB phosphorylation abrogates carcinogen-induced MMP-9 and COX-2 expression in human brain endothelial cells

    Directory of Open Access Journals (Sweden)

    Tahanian E

    2011-05-01

    Full Text Available Elizabeth Tahanian¹, Luis Arguello Sanchez¹, Tze Chieh Shiao², René Roy², Borhane Annabi¹¹Centre de Recherche BioMED, ²Centre de Recherche PharmaQAM, Département de chimie, Université du Québec à Montréal, QC, CanadaAbstract: Brain endothelial cells play an essential role as structural and functional components of the blood–brain barrier (BBB. Increased BBB breakdown and brain injury are associated with neuroinflammation and are thought to trigger mechanisms involving matrix metalloproteinase upregulation. Emerging evidence also indicates that cyclooxygenase (COX inhibition limits BBB disruption, but the mechanisms linking metalloproteinase to COX remain unknown. In this study, we sought to investigate the nuclear factor-kappa B (NF-κB signaling pathway, a common pathway in both the regulation of matrix metalloproteinase-9 (MMP-9 and COX-2 expression, and the inhibitory properties of several chemopreventive flavonoids. Human brain microvascular endothelial cells were treated with a combination of phorbol 12-myristate 13-acetate (PMA, a carcinogen documented to increase MMP-9 and COX-2 through NF-κB, and several naturally occurring flavonoids. Among the molecules tested, we found that fisetin, apigenin, and luteolin specifically and dose-dependently antagonized PMA-induced COX-2 and MMP-9 gene and protein expressions as assessed by qRT-PCR, immunoblotting, and zymography respectively. We further demonstrate that flavonoids impact on IκK-mediated phosphorylation activity as demonstrated by the inhibition of PMA-induced IκB phosphorylation levels. Our results suggest that BBB disruption during neuroinflammation could be pharmacologically reduced by a specific class of flavonoids acting as NF-κB signal transduction inhibitors.Keywords: blood–brain barrier, flavonoids, neuroinflammation, NF-κB signal transduction inhibitors

  3. BAFF induces spleen CD4{sup +} T cell proliferation by down-regulating phosphorylation of FOXO3A and activates cyclin D2 and D3 expression

    Energy Technology Data Exchange (ETDEWEB)

    Ji, Fang; Chen, Rongjing [Department of Orthodontics, Ninth People' s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai (China); Liu, Baojun [Laboratory of Lung, Inflammation and Cancers, Huashan Hospital, Fudan University, Shanghai (China); Zhang, Xiaoping [Department of Nuclear Medicine, Shanghai 10th People' s Hospital, Tongji University School of Medicine, Shanghai 200072 (China); Han, Junli; Wang, Haining [Department of General Dentistry, Ninth People' s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai (China); Shen, Gang [Department of Orthodontics, Ninth People' s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai (China); Tao, Jiang, E-mail: taojiang2012@yahoo.cn [Department of General Dentistry, Ninth People' s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai (China)

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer Firstly analyze the mechanism of BAFF and anti-CD3 co-stimulation on purified mouse splenic CD4{sup +} T cells. Black-Right-Pointing-Pointer Carrying out siRNA technology to study FOXO3A protein function. Black-Right-Pointing-Pointer Helpful to understand the T cell especially CD4{sup +} T cell's role in immunological reaction. -- Abstract: The TNF ligand family member 'B cell-activating factor belonging to the TNF family' (BAFF, also called BLyS, TALL-1, zTNF-4, and THANK) is an important survival factor for B and T cells. In this study, we show that BAFF is able to induce CD4{sup +} spleen T cell proliferation when co-stimulated with anti-CD3. Expression of phosphorylated FOXO3A was notably down-regulated and cyclins D2 and D3 were up-regulated and higher in the CD4{sup +} T cells when treated with BAFF and anti-CD3, as assessed by Western blotting. Furthermore, after FOXO3A was knocked down, expression of cyclin D1 was unchanged, compared with control group levels, but the expression of cyclins D2 and D3 increased, compared with the control group. In conclusion, our results suggest that BAFF induced CD4{sup +} spleen T cell proliferation by down-regulating the phosphorylation of FOXO3A and then activating cyclin D2 and D3 expression, leading to CD4{sup +} T cell proliferation.

  4. Transferases for alkylation, glycosylation and phosphorylation

    NARCIS (Netherlands)

    D. Auriol; R. ter Halle; F. Lefèvre; D.F. Visser; G.E.R. Gordon; M.L. Bode; K. Mathiba; D. Brady; K. De Winter; T. Desmet; A. Cerdobbel; W. Soetaert; T. van Herk; A.F. Hartog; R. Wever; M. Brzezińska-rodak; M. Klimek-Ochab; E. Żymańczyk-Duda; J. Mukherjee; M.N. Gupta; W.B. Yin; S.M. Li; M. Gruber-Khadjawi

    2012-01-01

    This chapter contains sections titled: Industrial Production of Caffeic Acid-α-D-O-Glucoside Enzymatic Synthesis of 5-Methyluridine by Transglycosylation of Guanosine and Thymine Preparation and Use of Sucrose Phosphorylase as Cross-Linked Enzyme Aggregate (CLEA) Enzymatic Synthesis of Phosphorylate

  5. Interactions of Divalent and Trivalent Metal Counterions with Anionic Sulfonate Gemini Surfactant and Induced Aggregate Transitions in Aqueous Solution.

    Science.gov (United States)

    Liu, Zhang; Cao, Meiwen; Chen, Yao; Fan, Yaxun; Wang, Dong; Xu, Hai; Wang, Yilin

    2016-05-01

    Interactions of multivalent metal counterions with anionic sulfonate gemini surfactant 1,3-bis(N-dodecyl-N-propanesulfonate sodium)-propane (C12C3C12(SO3)2) and the induced aggregate transitions in aqueous solution have been studied. Divalent metal ions Ca(2+), Mg(2+), Cu(2+), Zn(2+), Mn(2+), Co(2+), and Ni(2+) and trivalent metal ions Al(3+), Fe(3+), and Cr(3+) were chosen. The results indicate that the critical micelle concentration (CMC) of C12C3C12(SO3)2 is greatly reduced by the ions, and the aggregate morphologies of C12C3C12(SO3)2 are adjusted by changing the nature and molar ratio of the metal ions. These metal ions can be classified into four groups because the ions in each group have very similar interaction mechanisms with C12C3C12(SO3)2: (I) Cu(2+) and Zn(2+); (II) Ca(2+), Mn(2+) and Mg(2+); (III) Ni(2+) and Co(2+); and (IV) Cr(3+), Al(3+) and Fe(3+). Cu(2+), Mg(2+), Ni(2+), and Al(3+) then were selected as representatives for each group to further study their interaction with C12C3C12(SO3)2. C12C3C12(SO3)2 interacts with the multivalent metal ions by electrostatic interaction and coordination interaction. C12C3C12(SO3)2 forms prolate micelles and plate-like micelles with Cu(2+), vesicles and wormlike micelles with Al(3+) or Ni(2+), and viscous three-dimensional network structure with Mg(2+). Moreover, precipitation does not take place in aqueous solution even at a high ion/surfactant ratio. The related mechanisms have been discussed. The present work provides guidance on how to apply the anionic surfactant into the solutions containing the multivalent metal ions, and those aggregates may have potential usage in separating heavy metal ions from aqueous solutions. PMID:27096262

  6. Live Cell MicroRNA Imaging Using Exonuclease III-Aided Recycling Amplification Based on Aggregation-Induced Emission Luminogens.

    Science.gov (United States)

    Min, Xuehong; Zhang, Mengshi; Huang, Fujian; Lou, Xiaoding; Xia, Fan

    2016-04-13

    Enzyme-assisted detection strategies of microRNAs (miRNAs) in vitro have accomplished both great sensitivity and specificity. However, low expression of miRNAs and a complex environment in cells induces big challenges for monitoring and tracking miRNAs in vivo. The work reports the attempt to carry miRNA imaging into live cells, by enzyme-aided recycling amplification. We utilize facile probes based yellow aggregation-induced emission luminogens (AIEgens) with super photostable property but without quencher, which are applied to monitor miRNAs not only from urine sample extracts (in vitro) but also in live cells (in vivo). The assay could distinguish the cancer patients' urine samples from the healthy urine due to the good specificity. Moreover, the probe showed much higher fluorescence intensity in breast cancer cells (MCF-7) (miR-21 in high expression) than that in cervical cancer cells (HeLa) and human lung fibroblast cells (HLF) (miR-21 in low expression) in more than 60 min, which showed the good performance and super photostability for the probe in vivo. As controls, another two probes with FAM/Cy3 and corresponding quenchers, respectively, could perform miRNAs detections in vitro and parts of in vivo tests but were not suitable for the long-term cell tracking due to the photobleach phenomena, which also demonstrates that the probe with AIEgens is a potential candidate for the accurate identification of cancer biomarkers.

  7. Enhanced aggregation of androgen receptor in induced pluripotent stem cell-derived neurons from spinal and bulbar muscular atrophy.

    Science.gov (United States)

    Nihei, Yoshihiro; Ito, Daisuke; Okada, Yohei; Akamatsu, Wado; Yagi, Takuya; Yoshizaki, Takahito; Okano, Hideyuki; Suzuki, Norihiro

    2013-03-22

    Spinal and bulbar muscular atrophy (SBMA) is an X-linked motor neuron disease caused by a CAG repeat expansion in the androgen receptor (AR) gene. Ligand-dependent nuclear accumulation of mutant AR protein is a critical characteristic of the pathogenesis of SBMA. SBMA has been modeled in AR-overexpressing animals, but precisely how the polyglutamine (polyQ) expansion leads to neurodegeneration is unclear. Induced pluripotent stem cells (iPSCs) are a new technology that can be used to model human diseases, study pathogenic mechanisms, and develop novel drugs. We established SBMA patient-derived iPSCs, investigated their cellular biochemical characteristics, and found that SBMA-iPSCs can differentiate into motor neurons. The CAG repeat numbers in the AR gene of SBMA-iPSCs and also in the atrophin-1 gene of iPSCs derived from another polyQ disease, dentato-rubro-pallido-luysian atrophy (DRPLA), remain unchanged during reprogramming, long term passage, and differentiation, indicating that polyQ disease-associated CAG repeats are stable during maintenance of iPSCs. The level of AR expression is up-regulated by neuronal differentiation and treatment with the AR ligand dihydrotestosterone. Filter retardation assays indicated that aggregation of ARs following dihydrotestosterone treatment in neurons derived from SBMA-iPSCs increases significantly compared with neurological control iPSCs, easily recapitulating the pathological feature of mutant ARs in SBMA-iPSCs. This phenomenon was not observed in iPSCs and fibroblasts, thereby showing the neuron-dominant phenotype of this disease. Furthermore, the HSP90 inhibitor 17-allylaminogeldanamycin sharply decreased the level of aggregated AR in neurons derived from SBMA-iPSCs, indicating a potential for discovery and validation of candidate drugs. We found that SBMA-iPSCs possess disease-specific biochemical features and could thus open new avenues of research into not only SBMA, but also other polyglutamine diseases.

  8. Hexanol-induced order-disorder transitions in lamellar self-assembling aggregates of bacteriochlorophyll c in Chlorobium tepidum chlorosomes.

    Science.gov (United States)

    Arellano, Juan B; Torkkeli, Mika; Tuma, Roman; Laurinmäki, Pasi; Melø, Thor B; Ikonen, Teemu P; Butcher, Sarah J; Serimaa, Ritva E; Psencík, Jakub

    2008-03-01

    Chlorosomes are light-harvesting complexes of green photosynthetic bacteria. Chlorosomes contain bacteriochlorophyll (BChl) c, d, or e aggregates that exhibit strong excitonic coupling. The short-range order, which is responsible for the coupling, has been proposed to be augmented by pigment arrangement into undulated lamellar structures with spacing between 2 and 3 nm. Treatment of chlorosomes with hexanol reversibly converts the aggregated chlorosome chlorophylls into a form with spectral properties very similar to that of the monomer. Although this transition has been extensively studied, the structural basis remains unclear due to variability in the obtained morphologies. Here we investigated hexanol-induced structural changes in the lamellar organization of BChl c in chlorosomes from Chlorobium tepidum by a combination of X-ray scattering, electron cryomicroscopy, and optical spectroscopy. At a low hexanol/pigment ratio, the lamellae persisted in the presence of hexanol while the short-range order and exciton interactions between chlorin rings were effectively eliminated, producing a monomer-like absorption. The result suggested that hexanol hydroxyls solvated the chlorin rings while the aliphatic tail partitioned into the hydrophobic part of the lamellar structure. This partitioning extended the chlorosome along its long axis. Further increase of the hexanol/pigment ratio produced round pigment-hexanol droplets, which lost all lamellar order. After hexanol removal the spectral properties were restored. In the samples treated under the high hexanol/pigment ratio, lamellae reassembled in small domains after hexanol removal while the shape and long-range order were irreversibly lost. Thus, all the interactions required for establishing the short-range order by self-assembly are provided by BChl c molecules alone. However, the long-range order and overall shape are imposed by an external structure, e.g., the proteinaceous chlorosome baseplate. PMID:18197717

  9. Real-Time Monitoring of Heat-Induced Aggregation of β-Lactoglobulin in Aqueous Solutions Using High-Resolution Ultrasonic Spectroscopy

    Science.gov (United States)

    Ochenduszko, Agnieszka; Buckin, Vitaly

    2010-01-01

    High-resolution ultrasonic spectroscopy was applied for real-time monitoring of the heat-induced denaturation and aggregation processes in aqueous solutions of β-lactoglobulin. The temperature profiles for the ultrasonic velocity and attenuation in the frequency range from 4MHz to 16MHz were measured during heating and cooling cycles, 35°C to 120°C to 35°C, with different heating and cooling rates. Two processes were identified in the heating profiles: transition to the molten globular state followed by formation of protein aggregates. Both processes are accompanied by a decrease in the ultrasonic velocity and an increase in compressibility. The ultrasonic attenuation did not show a significant change during the transition to the molten globule but increased significantly during aggregation. The diameter of the aggregates (calculated from ultrasonic attenuation) was of the order of 100nm and depended on the pH and the heating rate. Variation of pH from 6.0 to 7.5 had a pronounced effect on the size of protein aggregates. Some effect of pH on the intrinsic properties of aggregates was also detected.

  10. Small-angle X-ray scattering studies of metastable intermediates of beta-lactoglobulin isolated after heat-induced aggregation

    DEFF Research Database (Denmark)

    Carrotta, R.; Arleth, L.; Pedersen, J.S.;

    2003-01-01

    Small-angle x-ray scattering was used for studying intermediate species, isolated after heat-induced aggregation of the A variant of bovine P-lactoglobulin. The intermediates were separated in two fractions, the heated metastable dimer and heated metastable oligomers larger than the dimer. The pa...

  11. Effect of extraction pH on heat-induced aggregation, gelation and microstructure of protein isolate from quinoa (Chenopodium quinoa Willd)

    NARCIS (Netherlands)

    Ruiz, Geraldine Avila; Xiao, Wukai; Boekel, van Tiny; Minor, Marcel; Stieger, Markus

    2016-01-01

    The aim of this study was to determine the influence of extraction pH on heat-induced aggregation, gelation and microstructure of suspensions of protein isolates extracted from quinoa (Chenopodium quinoa Willd). Quinoa seed protein was extracted by alkaline treatment at various pH values (pH 8 (E

  12. Incorporation of an aggregation-induced-emissive tetraphenylethene derivative into cationic gene delivery vehicles manifested the nuclear translocation of uncomplexed DNA.

    Science.gov (United States)

    Han, Xiongqi; Chen, Qixian; Lu, Hongguang; Guo, Pan; Li, Wei; Wu, Guolin; Ma, Jianbiao; Gao, Hui

    2016-03-11

    A fluorophore displaying aggregation-induced emission was introduced at the terminus of branched polyethylenimine (PEI). The formulated polyplex not only demonstrated an improved safety profile and preserved transfection activity but also importantly indicated that the uncomplexed naked DNA rather than the polyplexes translocated into the nucleus.

  13. CysLT1 receptor-induced human airway smooth muscle cells proliferation requires ROS generation, EGF receptor transactivation and ERK1/2 phosphorylation

    Directory of Open Access Journals (Sweden)

    Capra Valérie

    2006-03-01

    Full Text Available Abstract Background Cysteine-containing leukotrienes (cysteinyl-LTs are pivotal inflammatory mediators that play important roles in the pathophysiology of asthma, allergic rhinitis, and other inflammatory conditions. In particular, cysteinyl-LTs exert a variety of effects with relevance to the aetiology of asthma such as smooth muscle contraction, eosinophil recruitment, increased microvascular permeability, enhanced mucus secretion and decreased mucus transport and, finally, airway smooth muscle cells (ASMC proliferation. We used human ASMC (HASMC to identify the signal transduction pathway(s of the leukotriene D4 (LTD4-induced DNA synthesis. Methods Proliferation of primary HASMC was measured by [3H]thymidine incorporation. Phosphorylation of EGF receptor (EGF-R and ERK1/2 was assessed with a polyclonal anti-EGF-R or anti-phosphoERKl/2 monoclonal antibody. A Ras pull-down assay kit was used to evaluate Ras activation. The production of reactive oxygen species (ROS was estimated by measuring dichlorodihydrofluorescein (DCF oxidation. Results We demonstrate that in HASMC LTD4-stimulated thymidine incorporation and potentiation of EGF-induced mitogenic signaling mostly depends upon EGF-R transactivation through the stimulation of CysLT1-R. Accordingly, we found that LTD4 stimulation was able to trigger the increase of Ras-GTP and, in turn, to activate ERK1/2. We show here that EGF-R transactivation was sensitive to pertussis toxin (PTX and phosphoinositide 3-kinase (PI3K inhibitors and that it occurred independently from Src activity, despite the observation of a strong impairment of LTD4-induced DNA synthesis following Src inhibition. More interestingly, CysLT1-R stimulation increased the production of ROS and N-acetylcysteine (NAC abolished LTD4-induced EGF-R phosphorylation and thymidine incorporation. Conclusion Collectively, our data demonstrate that in HASMC LTD4 stimulation of a Gi/o coupled CysLT1-R triggers the transactivation of the EGF

  14. Multifunctional 8-hydroxyquinoline-appended cyclodextrins as new inhibitors of metal-induced protein aggregation.

    Science.gov (United States)

    Oliveri, Valentina; Attanasio, Francesco; Puglisi, Antonino; Spencer, John; Sgarlata, Carmelo; Vecchio, Graziella

    2014-07-14

    Mounting evidence suggests a pivotal role of metal imbalances in protein misfolding and amyloid diseases. As such, metal ions represent a promising therapeutic target. In this context, the synthesis of chelators that also contain complementary functionalities to combat the multifactorial nature of neurodegenerative diseases is a highly topical issue. We report two new 8-hydroxyquinoline-appended cyclodextrins and highlight their multifunctional properties, including their Cu(II) and Zn(II) binding abilities, and capacity to act as antioxidants and metal-induced antiaggregants. In particular, the latter property has been applied in the development of an effective assay that exploits the formation of amyloid fibrils when β-lactoglobulin A is heated in the presence of metal ions.

  15. Individual aggregates of amyloid beta induce temporary calcium influx through the cell membrane of neuronal cells

    Science.gov (United States)

    Drews, Anna; Flint, Jennie; Shivji, Nadia; Jönsson, Peter; Wirthensohn, David; De Genst, Erwin; Vincke, Cécile; Muyldermans, Serge; Dobson, Chris; Klenerman, David

    2016-01-01

    Local delivery of amyloid beta oligomers from the tip of a nanopipette, controlled over the cell surface, has been used to deliver physiological picomolar oligomer concentrations to primary astrocytes or neurons. Calcium influx was observed when as few as 2000 oligomers were delivered to the cell surface. When the dosing of oligomers was stopped the intracellular calcium returned to basal levels or below. Calcium influx was prevented by the presence in the pipette of the extracellular chaperone clusterin, which is known to selectively bind oligomers, and by the presence a specific nanobody to amyloid beta. These data are consistent with individual oligomers larger than trimers inducing calcium entry as they cross the cell membrane, a result supported by imaging experiments in bilayers, and suggest that the initial molecular event that leads to neuronal damage does not involve any cellular receptors, in contrast to work performed at much higher oligomer concentrations. PMID:27553885

  16. Ischemia in tumors induces early and sustained phosphorylation changes in stress kinase pathways but does not affect global protein levels

    Energy Technology Data Exchange (ETDEWEB)

    Mertins, Philipp; Yang, Feng; Liu, Tao; Mani, DR; Petyuk, Vladislav A.; Gillette, Michael; Clauser, Karl; Qiao, Jana; Gritsenko, Marina A.; Moore, Ronald J.; Levine, Douglas; Townsend, Reid; Erdmann-Gilmore, Petra; Snider, Jacqueline E.; Davies, Sherri; Ruggles, Kelly; Fenyo, David; Kitchens, R. T.; Li, Shunqiang; Olvera, Narcisco; Dao, Fanny; Rodriguez, Henry; Chan, Daniel W.; Liebler, Daniel; White, Forest; Rodland, Karin D.; Mills, Gordon; Smith, Richard D.; Paulovich, Amanda G.; Ellis, Matthew; Carr, Steven A.

    2014-07-01

    Advances in quantitative mass spectrometry (MS)-based proteomics have sparked efforts to characterize the proteomes of tumor samples to provide complementary and unique information inaccessible by genomics. Tumor samples are usually not accrued with proteomic characterization in mind, raising concerns regarding effects of undocumented sample ischemia on protein abundance and phosphosite stoichiometry. Here we report the effects of cold ischemia time on clinical ovarian cancer samples and patient-derived basal and luminal breast cancer xenografts. Tumor tissues were excised and collected prior to vascular ligation, subjected to accurately defined ischemia times up to 60 min, and analyzed by quantitative proteomics and phosphoproteomics using isobaric tags and high-performance, multidimensional LC-MS/MS. No significant changes were detected at the protein level in each tumor type after 60 minutes of ischemia, and the majority of the >25,000 phosphosites detected were also stable. However, large, reproducible increases and decreases in protein phosphorylation at specific sites were observed in up to 24% of the phosphoproteome starting as early as 5 minutes post-excision. Early and sustained activation of stress response, transcriptional regulation and cell death pathways were observed in common across tumor types. Tissue-specific changes in phosphosite stability were also observed suggesting idiosyncratic effects of ischemia in particular lineages. Our study provides insights into the information that may be obtained by proteomic characterization of tumor samples after undocumented periods of ischemia, and suggests caution especially in interpreting activation of stress pathways in such samples as they may reflect sample handling rather than tumor physiology.

  17. The CB1 cannabinoid receptor agonist reduces L-DOPA-induced motor fluctuation and ERK1/2 phosphorylation in 6-OHDA-lesioned rats.

    Science.gov (United States)

    Song, Lu; Yang, Xinxin; Ma, Yaping; Wu, Na; Liu, Zhenguo

    2014-01-01

    The dopamine precursor L-3,4-dihydroxyphenylalanine (L-DOPA) has been used as an effective drug for treating dopamine depletion-induced Parkinson's disease (PD). However, long-term administration of L-DOPA produces motor complications. L-DOPA has also been found to modify the two key signaling cascades, protein kinase A/dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) and extracellular signal-regulated kinases 1 and 2 (ERK1/2), in striatal neurons, which are thought to play a pivotal role in forming motor complications. In the present study, we tested the possible effect of a CB1 cannabinoid receptor agonist on L-DOPA-stimulated abnormal behavioral and signaling responses in vivo. Intermittent L-DOPA administration for 3 weeks induced motor fluctuation in a rat model of PD induced by intrastriatal infusion of dopamine-depleting neurotoxin 6-hydroxydopamine (6-OHDA). A single injection of a CB1 cannabinoid receptor agonist WIN-55,212-2 had no effect on L-DOPA-induced motor fluctuation. However, chronic injections of WIN-55,212-2 significantly attenuated abnormal behavioral responses to L-DOPA in 6-OHDA-lesioned rats. Similarly, chronic injections of WIN-55,212-2 influence the L-DOPA-induced alteration of DARPP-32 and ERK1/2 phosphorylation status in striatal neurons. These data provide evidence for the active involvement of CB1 cannabinoid receptors in the regulation of L-DOPA action during PD therapy.

  18. β淀粉样蛋白1-42促进α-突触核蛋白在Ser129位点磷酸化和聚集%Amyloid-β1-42 Promotes the Phosphorylation of α-synuclein and Its Aggregation at Serine 129 Site

    Institute of Scientific and Technical Information of China (English)

    喻哲明; 丁正同; 任惠民; 邬剑军; 王坚; 陈嬿

    2012-01-01

    Aim: To explore the effect of amyloid-β1-42 (AP,.42) on the phosphorylation and aggregation of α-synuclein(a-syn) at serine 129 site in A53T-PC12 cells, which were made from the PC12 cells transfected with human A53T mutant SNCA gene. Methods: Reactive oxygen species(ROS) in A53T-PC12 cells treated by H2O2, PBS, Aβ1-42, Aβ1-42 + vitamin C were detected, and the changes of α-syn aggregation were also detected by the double immunofluorescence dyeing, respectively. Then phosphorylated α-syn at serine 129 (Pα-syn), the aggregation of a-syn were analyzed by Western blot. Results: The amount of ROS increased in A53T-PC12 cells treated respectively with H2O2 and Aβ1-42, while decreased in the cells treated with Aβ1-42 + vitamin C. Immunofluorescence showed that Aβ1-42 promoted the aggregation of a-syn in A53T-PC12 cells. Compared with the PBS group, Aβ1-42 could significantly increase the relative amount of Pa-syn (p<0.001) and a-syn aggregations (P<0.05). Conclusion: Aβ1-42 could enhance a-syn phosphorylation at serine 129 and promote aggregation of a-syn in A53T-PC12 cells.%目的:通过转染人α-突触核蛋白(α-syn)A53T突变基因的PC12细胞(A53T-PC12细胞)模型,探讨β-淀粉样蛋白1-42(Aβ1-42)对A53T-PC12细胞在Ser29位点α-syn磷酸化(Pα-syn)水平、聚集程度的影响.方法:分别予H2O2、PBS、Aβ1-42干预A53T-PC12细胞(将A53T-PC12细胞分为:PBS干预组; 5μmol·L-1 Aβ1-42干预组; 5μmol·L-1 Aβ1-42+300 μmol·L-1维生素C干预组;200μmol·L-1 H2O2干预组),观察细胞内活性氧自由基(ROS)水平变化.并通过双重免疫荧光染色观察A53T-PC12细胞内α-syn聚集情况,并通过Western blot半定量分析A53T-PC12细胞内Ser129位点磷酸化Pα-syn水平.结果:Aβ1-42增加细胞内ROS.免疫荧光染色提示Aβ1-42干预后促进A53T-PC12细胞内α-syn聚集;Western blot半定量分析显示Pα-syn较PBS干预组升高(P<0.001,P<0.05).结论:Aβ1-42促进A53T-PC12细胞内α-syn 在Ser129位点磷酸化和聚集.

  19. Multisite light-induced phosphorylation of the transcription factor PIF3 is necessary for both its rapid degradation and concomitant negative feedback modulation of photoreceptor phyB levels in Arabidopsis.

    Science.gov (United States)

    Ni, Weimin; Xu, Shou-Ling; Chalkley, Robert J; Pham, Thao Nguyen D; Guan, Shenheng; Maltby, Dave A; Burlingame, Alma L; Wang, Zhi-Yong; Quail, Peter H

    2013-07-01

    Plants constantly monitor informational light signals using sensory photoreceptors, which include the phytochrome (phy) family (phyA to phyE), and adjust their growth and development accordingly. Following light-induced nuclear translocation, photoactivated phy molecules bind to and induce rapid phosphorylation and degradation of phy-interacting basic Helix Loop Helix (bHLH) transcription factors (PIFs), such as PIF3, thereby regulating the expression of target genes. However, the mechanisms underlying the signal-relay process are still not fully understood. Here, using mass spectrometry, we identify multiple, in vivo, light-induced Ser/Thr phosphorylation sites in PIF3. Using transgenic expression of site-directed mutants of PIF3, we provide evidence that a set of these phosphorylation events acts collectively to trigger rapid degradation of the PIF3 protein in response to initial exposure of dark-grown seedlings to light. In addition, we show that phyB-induced PIF3 phosphorylation is also required for the known negative feedback modulation of phyB levels in prolonged light, potentially through codegradation of phyB and PIF3. This mutually regulatory intermolecular transaction thus provides a mechanism with the dual capacity to promote early, graded, or threshold regulation of the primary, PIF3-controlled transcriptional network in response to initial light exposure, and later, to attenuate global sensitivity to the light signal through reductions in photoreceptor levels upon prolonged exposure. PMID:23903316

  20. Phosphorylation of Thr18 and Ser20 of p53 in Ad-p53 – induced apoptosis

    OpenAIRE

    Nakamizo, Akira; Amano, Toshiyuko; Zhang, Wei; Zhang, Xin-qiao; Ramdas, Latha; Liu, Ta-Jen; Bekele, B. Nebiyou; Shono, Tadahisa; Sasaki, Tomio; Benedict, William F.; Sawaya, Raymond; Lang, Frederick F.

    2008-01-01

    The p53 protein plays a critical role in inducing cell cycle arrest or apoptosis. Because p53 is inactivated in human gliomas, restoring p53 function is a major focus of glioma therapy. The most clinically tested strategy for replacing p53 has been adenoviral-mediated p53 gene therapy (Ad-p53). In addition to their therapeutic implications, investigations into Ad-p53 provide model systems for understanding p53’s ability to induce cell cycle arrest versus apoptosis, particularly because wild-t...

  1. Modification in hydrophobic packing of HAMP domain induces a destabilization of the auto-phosphorylation site in the histidine kinase CpxA.

    Science.gov (United States)

    Martinez, Marlet; Duclert-Savatier, Nathalie; Betton, Jean-Michel; Alzari, Pedro M; Nilges, Michael; Malliavin, Thérèse E

    2016-10-01

    The histidine kinases belong to the family of two-component systems, which serves in bacteria to couple environmental stimuli to adaptive responses. Most of the histidine kinases are homodimers, in which the HAMP and DHp domains assemble into an elongated helical region flanked by two CA domains. Recently, X-ray crystallographic structures of the cytoplasmic region of the Escherichia coli histidine kinase CpxA were determined and a phosphotransferase-defective mutant, M228V, located in HAMP, was identified. In the present study, we recorded 1 μs molecular dynamics trajectories to compare the behavior of the WT and M228V protein dimers. The M228V modification locally induces the appearance of larger voids within HAMP as well as a perturbation of the number of voids within DHp, thus destabilizing the HAMP and DHp hydrophobic packing. In addition, a disruption of the stacking interaction between F403 located in the lid of the CA domain involved in the auto-phosphorylation and R296 located in the interacting DHp region, is more often observed in the presence of the M228V modification. Experimental modifications R296A and R296D of CpxA have been observed to reduce also the CpxA activity. These observations agree with the destabilization of the R296/F403 stacking, and could be the sign of the transmission of a conformational event taking place in HAMP to the auto-phosphorylation site of histidine kinase. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 670-682, 2016. PMID:27124288

  2. Injection of bradykinin or/and cyclosporine A to hippocampus induces Alzheimer-like phosphorylation of tau and abnormal behavior in rats

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Bradykinin (BK) is a calcium/calmodulin dependent protein kinase Ⅱ (CaMKⅡ) specific activator, and Cyclosporin A (CSA) is reported to suppress protein phosphotase (PP)-2B activity. In vitro studies have shown that CaMKⅡ and PP-2B play an important role in Alzheimer-like phosphorylation of microtube-associated protein tau. To reconstitute an animal model based on the imbalance of protein kinase (s) and protein phosphatase (s) seen in Alzheimer brain, we injected BK and/or CSA into rat hippocampus. The results from behavioral study showed that an obvious disturbance in learning and memory was seen with BK or BK plus CSA injected rats. Moreover, the behavior abnormality appeared earlier in aged rats than young adults of the same kind after the injection. On the other hand, no obvious dysfunction in living and behavior was observed with CSA alone injected rats. The results obtained by immunohistochemical assay indicated that the staining for M4\\, 12E8\\, PHF-1 and CaMKⅡ was stronger, and for Tau-1 was weaker in BK injected rats compared with Control group. It was also found that the binding of M4 and PHF-1 but not 12E8 to tau was significantly increased in CSA injected rats. As the same as BK injection, binding of Tau-1 to tau was decreased after CSA injection. The immunostaining for 12E8\\,PHF-1 and CaMKⅡ was increased, whereas for Tau-1\\, M4\\, and GSK-3 was decreased after combination injection of BK and CSA. In addition, the staining of PP-2B decreased in all the three models. To our knowledge, this is the first data shown in vivo that the activation of CaMKⅡ induces both Alzheimer-like tau phosphorylation and behavioral disturbance.

  3. Triptolide (PG-490) induces apoptosis of dendritic cells through sequential p38 MAP kinase phosphorylation and caspase 3 activation

    Institute of Scientific and Technical Information of China (English)

    Liu Q; Chen T; Chen H; Zhang M; Li N; Lu Z; Ma P; Cao X

    2004-01-01

    Dendritic cells (DCs) are the most potent antigen-presenting cells that play crucial roles in the regulation of immune response. Triptolide, an active component purified from the medicinal plant Tripterygium wilfordii Hook F. , has been demonstrated to act as a potent immunosuppressive drug capable of inhibiting T cell activation and proliferation. However, little is known about the effects of triptolide on DCs. The present study shows that triptolide does not affect phenotypic differentiation and LPS-induced maturation of murine DCs. But triptolide can dramatically reduce cell recovery by inducing apoptosis of DCs at concentration as low as 10 ng/ml, as demonstrated by phosphatidylserine exposure, mitochondria potential decrease, and nuclear DNA condensation. Triptolide induces activation of p38 in DCs, which precedes the activation of caspase 3. SB203580, a specific kinase inhibitor for p38, can block the activation of caspase 3 and inhibit the resultant apoptosis of DCs. Our results suggest that the anti-inflammatory and immunosuppressive activities of triptolide may be due, in part,to its apoptosis-inducing effects on DCs.

  4. Validation of the Antidiabetic and Hypolipidemic Effects of Clitocybe nuda by Assessment of Glucose Transporter 4 and Gluconeogenesis and AMPK Phosphorylation in Streptozotocin-Induced Mice

    Directory of Open Access Journals (Sweden)

    Chun-Ching Shih

    2014-01-01

    Full Text Available The study was designed to investigate the effects of extract of Clitocybe nuda (CNE on type 1 diabetes mellitus and dyslipidemia in streptozotocin- (STZ- induced diabetic mice. Diabetes was induced by injection of STZ. Diabetic mice were randomly divided into five groups and given orally CNE (C1: 0.2, C2: 0.5, and C3: 1.0 g/kg body weight or metformin (Metf or vehicle for 4 weeks. STZ induction decreased in the levels of insulin, body weight, and the weight of skeletal muscle, whereas the levels of blood glucose, hemoglobin nonenzymatically (percent HbA1c, and circulating triglyceride (P < 0.001, P < 0.001, and P < 0.01, resp. were increased. CNE decreased the levels of blood glucose, HbA1c, and triglyceride levels, whereas it increased the levels of insulin and leptin compared with the vehicle-treated STZ group. STZ induction caused a decrease in the protein contents of skeletal muscular and hepatic phosphorylation of AMP-activated protein kinase (phospho-AMPK and muscular glucose transporter 4 (GLUT4. Muscular phospho-AMPK contents were increased in C2-, C3-, and Metf-treated groups. CNE and Metf significantly increased the muscular proteins of GLUT4. Liver phospho-AMPK showed an increase in all CNE- and Metf-treated groups combined with the decreased hepatic glucose production by decreasing phosphenolpyruvate carboxykinase (PEPCK, glucose-6-phosphatase (G6Pase, and 11beta hydroxysteroid dehydroxygenase (11β-HSD1 gene, which contributed to attenuating diabetic state. The study indicated that the hypoglycemic properties of CNE were related to both the increased muscular glucose uptake and the reduction in hepatic gluconeogenesis. CNE exerts hypolipidemic effect by increasing gene expressions of peroxisome proliferator-activated receptor α (PPARα and decreasing expressions of fatty acid synthesis, including acyl-coenzyme A: diacylglycerol acyltransferase (DGAT 2. Therefore, amelioration of diabetic and dyslipidemic state by CNE in STZ-induced

  5. Amyloid beta dimers/trimers potently induce cofilin-actin rods that are inhibited by maintaining cofilin-phosphorylation

    Directory of Open Access Journals (Sweden)

    Podlisny Marcia

    2011-01-01

    Full Text Available Abstract Background Previously we reported 1 μM synthetic human amyloid beta1-42 oligomers induced cofilin dephosphorylation (activation and formation of cofilin-actin rods within rat hippocampal neurons primarily localized to the dentate gyrus. Results Here we demonstrate that a gel filtration fraction of 7PA2 cell-secreted SDS-stable human Aβ dimers and trimers (Aβd/t induces maximal neuronal rod response at ~250 pM. This is 4,000-fold more active than traditionally prepared human Aβ oligomers, which contain SDS-stable trimers and tetramers, but are devoid of dimers. When incubated under tyrosine oxidizing conditions, synthetic human but not rodent Aβ1-42, the latter lacking tyrosine, acquires a marked increase (620 fold for EC50 in rod-inducing activity. Gel filtration of this preparation yielded two fractions containing SDS-stable dimers, trimers and tetramers. One, eluting at a similar volume to 7PA2 Aβd/t, had maximum activity at ~5 nM, whereas the other, eluting at the void volume (high-n state, lacked rod inducing activity at the same concentration. Fractions from 7PA2 medium containing Aβ monomers are not active, suggesting oxidized SDS-stable Aβ1-42 dimers in a low-n state are the most active rod-inducing species. Aβd/t-induced rods are predominantly localized to the dentate gyrus and mossy fiber tract, reach significance over controls within 2 h of treatment, and are reversible, disappearing by 24 h after Aβd/t washout. Overexpression of cofilin phosphatases increase rod formation when expressed alone and exacerbate rod formation when coupled with Aβd/t, whereas overexpression of a cofilin kinase inhibits Aβd/t-induced rod formation. Conclusions Together these data support a mechanism by which Aβd/t alters the actin cytoskeleton via effects on cofilin in neurons critical to learning and memory.

  6. Gadolinium-functionalized aggregation-induced emission dots as dual-modality probes for cancer metastasis study.

    Science.gov (United States)

    Li, Kai; Ding, Dan; Prashant, Chandrasekharan; Qin, Wei; Yang, Chang-Tong; Tang, Ben Zhong; Liu, Bin

    2013-12-01

    Understanding the localization and engraftment of tumor cells at postintravasation stage of metastasis is of high importance in cancer diagnosis and treatment. Advanced fluorescent probes and facile methodologies for cell tracing play a key role in metastasis studies. In this work, we design and synthesize a dual-modality imaging dots with both optical and magnetic contrast through integration of a magnetic resonance imaging reagent, gadolinium(III), into a novel long-term cell tracing probe with aggregation-induced emission (AIE) in far-red/near-infrared region. The obtained fluorescent-magnetic AIE dots have both high fluorescence quantum yield (25%) and T1 relaxivity (7.91 mM(-1) s(-1) ) in aqueous suspension. After further conjugation with a cell membrane penetrating peptide, the dual-modality dots can be efficiently internalized into living cells. The gadolinium(III) allows accurate quantification of biodistribution of cancer cells via intraveneous injection, while the high fluorescence provides engraftment information of cells at single cellular level. The dual-modality AIE dots show obvious synergistic advantages over either single imaging modality and hold great promises in advanced biomedical studies.

  7. Purine twisted-intercalating nucleic acids: a new class of anti-gene molecules resistant to potassium-induced aggregation.

    Science.gov (United States)

    Paramasivam, Manikandan; Cogoi, Susanna; Filichev, Vyacheslav V; Bomholt, Niels; Pedersen, Erik B; Xodo, Luigi E

    2008-06-01

    Sequence-specific targeting of genomic DNA by triplex-forming oligonucleotides (TFOs) is a promising strategy to modulate in vivo gene expression. Triplex formation involving G-rich oligonucleotides as third strand is, however, strongly inhibited by potassium-induced TFO self-association into G-quartet structures. We report here that G-rich TFOs with bulge insertions of (R)-1-O-[4-(1-pyrenylethynyl)-phenylmethyl] glycerol (called twisted intercalating nucleic acids, TINA) show a much lower tendency to aggregate in potassium than wild-type analogues do. We designed purine-motif TINA-TFOs for binding to a regulatory polypurine-polypyrimidine (pur/pyr) motif present in the promoter of the KRAS proto-oncogene. The binding of TINA-TFOs to the KRAS target has been analysed by electrophoresis mobility shift assays and DNase I footprinting experiments. We discovered that in the presence of potassium the wild-type TFOs did not bind to the KRAS target, differently from the TINA analogues, whose binding was observed up to 140 mM KCl. The designed TINA-TFOs were found to abrogate the formation of a DNA-protein complex at the pur/pyr site and to down-regulate the transcription of CAT driven by the murine KRAS promoter. Molecular modelling of the DNA/TINA-TFO triplexes are also reported. This study provides a new and promising approach to create TFOs to target in vivo the genome. PMID:18456705

  8. Aggregation-induced emission of diarylamino-π-carborane triads: effects of charge transfer and π-conjugation.

    Science.gov (United States)

    Cho, Yang-Jin; Kim, So-Yoen; Cho, Minji; Han, Won-Sik; Son, Ho-Jin; Cho, Dae Won; Kang, Sang Ook

    2016-04-14

    Carborane-based donor-π-acceptor triads (D-π-A-π-D) bearing triarylamine moieties were synthesised. All the monomeric triads showed a blue-green emission in a dilute solution, which was assigned as an intramolecular charge-transfer (CT) emission. The intramolecular CT emission showed large Stokes shifts at a higher solvent polarity. The intramolecular CT emission further shifted to a longer wavelength with the increase in π-conjugation. Interestingly, a strong red emission was observed in highly concentrated solutions or in the solid state, which was assigned as an aggregation-induced emission (AIE). Moreover, the AIE strongly depended on solvent polarity. A large Stokes shift in AIE was attributed to the strong CT character. The changes in the dipole moment for the AIE state and monomer emission were evaluated using the Lippert-Mataga relationship. The density functional theory calculations showed that the change in electron distribution between the aryl amino group (highest occupied molecular orbital, HOMO) and the carborane moiety (lowest unoccupied molecular orbital, LUMO) indicates the intramolecular CT character, and the emission colour changes were attributed to the HOMO-LUMO energy gap controlled by the π-extension of the phenylene linker. The electrochemical properties such as oxidation and reduction potentials were consistent with theoretical calculation results. The emission properties were affected by two main factors: solvent polarity and solubility. PMID:26996491

  9. cAMP elevators inhibit LPS-induced IL-12 p40 expression by interfering with phosphorylation of p38 MAPK in Murine Peritoneal Macrophages

    Institute of Scientific and Technical Information of China (English)

    WEI; GUO; FENG; YI; BING; WANG; JIN; SONG; ZHANG; XING; YU; WANG; CHANG; LIN; LI; ZONG; LIANG; CHANG

    2002-01-01

    cAMP mediated signaling may play a suppressive role in immune response. We previously found thatthe cAMP-elevators (CTx and 8-Br-cAMP) inhibited IL-12, IL-la, IL-6 gene expression, but increasedthe transcriptional levels of IL-10 and IL-1Ra in LPS-treated murine peritoneal macrophages. The presentstudy examined a possible molecular mechanism involved in cAMP elevators-induced inhibition of IL-12 p40expression in response to LPS. Our data demonstrated that cAMP elevators downregulated IL-12 p40 mRNAexpression and IL-12 p70 production in murine peritoneal macrophages. Subsequent studies revealed thatcAMP-elevators blocked phosphorylation of p38 MAPK, but did not affect the activity of NF-κB bindingto IL-12 promoter (-136/-112). This is the first report that cAMP elevators inhibit LPS-induced IL-12production by a mechanism that is associated, at least in part, with p38-dependent inhibition by cAMPsignaling pathways.

  10. Phosphorylation of the GluN1 subunit in dorsal horn neurons by remifentanil: a mechanism for opioid-induced hyperalgesia.

    Science.gov (United States)

    Zhang, C; Li, S S; Zhao, N; Yu, C

    2015-03-13

    Remifentanil (an ultra-short acting μ-opioid receptor agonist) use has been associated with acute opioid tolerance and hyperalgesia. Previous electrophysiological studies have shown that remifentanil elicits rapid and prolonged upregulation of N-methyl-D-aspartate receptor (NMDAR) currents. However, the effect of remifentanil on the levels of the GluN1 subunit of the NMDAR in dorsal horn neurons (DHNs) has not been reported. We investigated the effect of remifentanil, along with ketamine (NMDAR antagonist) and naloxone (μ-opioid receptor antagonist), on GluN1 mRNA levels and the amount of phosphorylated GluN1 in primary cultures of embryonic rat DHNs. DHNs were isolated from 18-19-day rat embryos and treated with remifentanil or vehicle for 1 h. GluN1 mRNA and protein levels, determined by real time reverse transcription polymerase chain reaction (RT-PCR) and Western blot, respectively, were significantly and persistently increased by remifentanil exposure compared with the control group (P < 0.05). These results may partially account for the mechanism of remifentanil-induced hyperalgesia. This increase was prevented by ketamine (NMDAR antagonist) and naloxone (μ-opioid receptors antagonist), thus providing a potential therapeutic mechanism for the prevention of opioid-induced hyperalgesia.

  11. Allicin inhibits SDF-1alpha-induced T cell interactions with fibronectin and endothelial cells by down-regulating cytoskeleton rearrangement, Pyk-2 phosphorylation and VLA-4 expression.

    Science.gov (United States)

    Sela, Uri; Ganor, Sharon; Hecht, Iris; Brill, Alexander; Miron, Talia; Rabinkov, Aharon; Wilchek, Meir; Mirelman, David; Lider, Ofer; Hershkoviz, Rami

    2004-04-01

    Allicin, a major ingredient of fresh garlic extract that is produced during the crushing of garlic cloves, exerts various beneficial biological effects, including a broad spectrum of antimicrobial activity, antihyperlipidaemic and antihypertensive effects. However, how allicin affects the immune system is less well known, and its effect on human T cells has never been studied. Here, we examined the in-vitro effects of allicin on the functioning of T cells related to their entry to inflamed extravascular sites. We found that allicin (20-100 microm) inhibits the SDF-1alpha (CXCL12)-induced T cell migration through fibronectin (FN), and that this inhibition is mediated by the down-regulation of (i) the reorganization of cortical actin and the subsequent T cell polarization, and (ii) T cell adhesion to FN. Moreover, allicin also inhibited T cell adhesion to endothelial cells and transendothelial migration. The mechanisms underlying these inhibitory effects of allicin are associated with its ability to down-regulate the phosphorylation of Pyk2, an intracellular member of the focal adhesion kinases, and to reduce the expression of the VCAM-1- and FN-specific alpha4beta1-integrin (VLA-4). The ability of allicin to down-regulate these chemokine-induced and VLA-4-mediated T cell functions explains its beneficial biological effects in processes where T cells play an important role and suggests that allicin may be used therapeutically with chronic inflammatory diseases. PMID:15056375

  12. Deletion of dopamine D1 and D3 receptors differentially affects spontaneous behaviour and cocaine-induced locomotor activity, reward and CREB phosphorylation.

    Science.gov (United States)

    Karasinska, Joanna M; George, Susan R; Cheng, Regina; O'Dowd, Brian F

    2005-10-01

    Co-localization of dopamine D1 and D3 receptors in striatal neurons suggests that these two receptors interact at a cellular level in mediating dopaminergic function including psychostimulant-induced behaviour. To study D1 and D3 receptor interactions in cocaine-mediated effects, cocaine-induced locomotion and reward in mice lacking either D1, D3 or both receptors were analysed. Spontaneous locomotor activity was increased in D1-/- and D1-/-D3-/- mice and D1-/-D3-/- mice did not exhibit habituation of spontaneous rearing activity. Cocaine (20 mg/kg) increased locomotor activity in wild-type and D3-/- mice, failed to stimulate activity in D1-/- mice and reduced activity in D1-/-D3-/- mice. In the conditioned place preference, all groups exhibited reward at 5, 10 and 20 mg/kg of cocaine. D1-/-D3-/- mice did not demonstrate preference at 2.5 mg/kg of cocaine although preference was observed in wild-type, D1-/- and D3-/- mice. The transcription factor cAMP-responsive element binding protein (CREB) is activated by phosphorylation in striatal regions following dopamine receptor activation. Striatal pCREB levels following acute cocaine were increased in wild-type and D3-/- mice and decreased in D1-/- and D1-/-D3-/- mice. After repeated administration of 2.5 mg/kg of cocaine, D1-/- mice had lower pCREB levels in caudate-putamen and nucleus accumbens. Our findings suggest that, although spontaneous and cocaine-induced horizontal activity depended mainly on the presence of the D1 receptor, there may be crosstalk between D1 and D3 receptors in rearing habituation and the perception of cocaine reward at low doses of the drug. Furthermore, alterations in pCREB levels were associated with changes in cocaine-induced locomotor activity but not reward. PMID:16197514

  13. Effect of Sodium Tanshinone Ⅱ A Sulfonate on Phosphorylation of Extracellular Signal-regulated Kinase1/2 in Angiotensin Ⅱ-induced Hypertrophy of Myocardial Cells

    Institute of Scientific and Technical Information of China (English)

    LI Shu-sheng; FENG Jun; ZHENG Zhi; LIANG Qian-sheng

    2008-01-01

    Objective:To observe the effects of sodium tanshinone Ⅱ A sulfonate(STS)on angiotensin Ⅱ(Ang Ⅱ)-induced hypertrophy of myocardial cells through the expression of phosphorylated extracellular signal-regulated kinase(P-ERK1/2).Methods:In the primary culture of neonatal rat myocardial cells.the total protein content in myocardial cells was determined by coomassie brilliant blue and the protein synthesis rate was measured by[3H]-Leucine incorporation as indexes for hypertrophy of myocardial cells.The expression of p-ERK1/2 was determined using Western blot and immunofluorescence Iabeling.Results:(1)The totaI protein and protein synthesis rate increased significantly in contrast to the control group after the myocardial cells were stimulated by Ang Ⅱ (1 μmol/L)for 24 h;STS markedly inhibited the increment of the total protein level induced by Ang Ⅱ and the syntheses of protein.(2)After pretreatment of myocardial cells with Ang Ⅱ(1 μ mol/L)for 5 min,the p-ERK1/2 protein expression was increased,with the most obvious effect shown at about 10 min;pretreatment of myocardial cells with STS at different doses(2,10,50 μ mol/L)for 30 min resulted in obvious inhibition of the expression of p-ERK1/2 stimulated by Ang Ⅱ in a dose-dependent manner.(3)After the myocardial cells were stimulated by Ang Ⅱ(1 μ mol/L),the immunofluorescence of ERK1/2 rapidly appeared in the nucleus.The activation and translocation process of ERK1/2 induced by Ang Ⅱ was blocked distinctly by STS.Conclusion:STS inhibited the myocardial cell hypertrophy induced by Ang Ⅱ,and the mechanism may be associated with the inhibition of p-ERK1/2 expression.

  14. Aggregation of Trp > Glu point mutants of human gamma-D crystallin provides a model for hereditary or UV-induced cataract.

    Science.gov (United States)

    Serebryany, Eugene; Takata, Takumi; Erickson, Erika; Schafheimer, Nathaniel; Wang, Yongting; King, Jonathan A

    2016-06-01

    Numerous mutations and covalent modifications of the highly abundant, long-lived crystallins of the eye lens cause their aggregation leading to progressive opacification of the lens, cataract. The nature and biochemical mechanisms of the aggregation process are poorly understood, as neither amyloid nor native-state polymers are commonly found in opaque lenses. The βγ-crystallin fold contains four highly conserved buried tryptophans, which can be oxidized to more hydrophilic products, such as kynurenine, upon UV-B irradiation. We mimicked this class of oxidative damage using Trp→Glu point mutants of human γD-crystallin. Such substitutions may represent a model of UV-induced photodamage-introduction of a charged group into the hydrophobic core generating "denaturation from within." The effects of Trp→Glu substitutions were highly position dependent. While each was destabilizing, only the two located in the bottom of the double Greek key fold-W42E and W130E-yielded robust aggregation of partially unfolded intermediates at 37°C and pH 7. The αB-crystallin chaperone suppressed aggregation of W130E, but not W42E, indicating distinct aggregation pathways from damage in the N-terminal vs C-terminal domain. The W130E aggregates had loosely fibrillar morphology, yet were nonamyloid, noncovalent, showed little surface hydrophobicity, and formed at least 20°C below the melting temperature of the native β-sheets. These features are most consistent with domain-swapped polymerization. Aggregation of partially destabilized crystallins under physiological conditions, as occurs in this class of point mutants, could provide a simple in vitro model system for drug discovery and optimization. PMID:26991007

  15. Acid-Induced Cold Gelation of Globular Proteins: Effects of Protein Aggregate Characteristics and Disulfide Bonding on Rheological Properties

    NARCIS (Netherlands)

    Alting, A.C.; Weijers, M.; Hoog, E.H.A. de; Pijpekamp, A.M. van de; Cohen Stuart, M.A.; Hamer, R.J.; Kruif, C.G. de; Visschers, R.W.

    2004-01-01

    The process of cold gelation of ovalbumin and the properties of the resulting cold-set gels were compared to those of whey protein isolate. Under the chosen heating conditions, most protein was organized in aggregates. For both protein preparations, the aggregates consisted of covalently linked mono

  16. Bimetallic Au2 Cu6 Nanoclusters: Strong Luminescence Induced by the Aggregation of Copper(I) Complexes with Gold(0) Species.

    Science.gov (United States)

    Kang, Xi; Wang, Shuxin; Song, Yongbo; Jin, Shan; Sun, Guodong; Yu, Haizhu; Zhu, Manzhou

    2016-03-01

    The concept of aggregation-induced emission (AIE) has been exploited to render non-luminescent Cu(I) SR complexes strongly luminescent. The Cu(I) SR complexes underwent controlled aggregation with Au(0) . Unlike previous AIE methods, our strategy does not require insoluble solutions or cations. X-ray crystallography validated the structure of this highly fluorescent nanocluster: Six thiolated Cu atoms are aggregated by two Au atoms (Au2 Cu6 nanoclusters). The quantum yield of this nanocluster is 11.7 %. DFT calculations imply that the fluorescence originates from ligand (aryl groups on the phosphine) to metal (Cu(I) ) charge transfer (LMCT). Furthermore, the aggregation is affected by the restriction of intramolecular rotation (RIR), and the high rigidity of the outer ligands enhances the fluorescence of the Au2 Cu6 nanoclusters. This study thus presents a novel strategy for enhancing the luminescence of metal nanoclusters (by the aggregation of active metal complexes with inert metal atoms), and also provides fundamental insights into the controllable synthesis of highly luminescent metal nanoclusters. PMID:26890334

  17. Functional activation of the T-cell antigen receptor induces tyrosine phosphorylation of phospholipase C-gamma 1.

    OpenAIRE

    Weiss, A; Koretzky, G; Schatzman, R C; Kadlecek, T

    1991-01-01

    Stimulation of the T-cell antigen receptor (TCR), which itself is not a protein-tyrosine kinase (PTK), activates a PTK and phospholipase C (PLC). Using the human T-cell leukemic line Jurkat and normal peripheral blood lymphocytes, we demonstrate that stimulation of the TCR specifically induces the recovery of PLC activity in eluates from anti-phosphotyrosine immunoprecipitates. Stimulation of the human muscarinic receptor, subtype 1, when expressed in Jurkat activates PLC through a guanine nu...

  18. Propofol directly increases tau phosphorylation.

    Directory of Open Access Journals (Sweden)

    Robert A Whittington

    Full Text Available In Alzheimer's disease (AD and other tauopathies, the microtubule-associated protein tau can undergo aberrant hyperphosphorylation potentially leading to the development of neurofibrillary pathology. Anesthetics have been previously shown to induce tau hyperphosphorylation through a mechanism involving hypothermia-induced inhibition of protein phosphatase 2A (PP2A activity. However, the effects of propofol, a common clinically used intravenous anesthetic, on tau phosphorylation under normothermic conditions are unknown. We investigated the effects of a general anesthetic dose of propofol on levels of phosphorylated tau in the mouse hippocampus and cortex under normothermic conditions. Thirty min following the administration of propofol 250 mg/kg i.p., significant increases in tau phosphorylation were observed at the AT8, CP13, and PHF-1 phosphoepitopes in the hippocampus, as well as at AT8, PHF-1, MC6, pS262, and pS422 epitopes in the cortex. However, we did not detect somatodendritic relocalization of tau. In both brain regions, tau hyperphosphorylation persisted at the AT8 epitope 2 h following propofol, although the sedative effects of the drug were no longer evident at this time point. By 6 h following propofol, levels of phosphorylated tau at AT8 returned to control levels. An initial decrease in the activity and expression of PP2A were observed, suggesting that PP2A inhibition is at least partly responsible for the hyperphosphorylation of tau at multiple sites following 30 min of propofol exposure. We also examined tau phosphorylation in SH-SY5Y cells transfected to overexpress human tau. A 1 h exposure to a clinically relevant concentration of propofol in vitro was also associated with tau hyperphosphorylation. These findings suggest that propofol increases tau phosphorylation both in vivo and in vitro under normothermic conditions, and further studies are warranted to determine the impact of this anesthetic on the acceleration of

  19. Molecular Level Insights into Thermally Induced [alpha]-Chymotrypsinogen A Amyloid Aggregation Mechanism and Semiflexible Protofibril Morphology

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Aming; Jordan, Jacob L.; Ivanova, Magdalena I.; Weiss, IV., William F.; Roberts, Christopher J.; Fernandez, Erik J. (UCLA); (Delaware); (UV)

    2010-12-07

    Understanding nonnative protein aggregation is critical not only to a number of amyloidosis disorders but also for the development of effective and safe biopharmaceuticals. In a series of previous studies [Weiss et al. (2007) Biophys. J. 93, 4392-4403; Andrews et al. (2007) Biochemistry 46, 7558-7571; Andrews et al. (2008) Biochemistry 47, 2397-2403], {alpha}-chymotrypsinogen A (aCgn) and bovine granulocyte colony stimulating factor (bG-CSF) have been shown to exhibit the kinetic and morphological features of other nonnative aggregating proteins at low pH and ionic strength. In this study, we investigated the structural mechanism of aCgn aggregation. The resultant aCgn aggregates were found to be soluble and exhibited semiflexible filamentous aggregate morphology under transmission electron microscopy. In addition, the filamentous aggregates were demonstrated to possess amyloid characteristics by both Congo red binding and X-ray diffraction. Peptide level hydrogen exchange (HX) analysis suggested that a buried native {beta}-sheet comprised of three peptide segments (39-46, 51-64, and 106-114) reorganizes into the cross-{beta} amyloid core of aCgn aggregates and that at least 50% of the sequence adopts a disordered structure in the aggregates. Furthermore, the equimolar, bimodal HX labeling distribution observed for three reported peptides (65-102, 160-180, and 229-245) suggested a heterogeneous assembly of two molecular conformations in aCgn aggregates. This demonstrates that extended {beta}-sheet interactions typical of the amyloid are sufficiently strong that a relatively small fraction of polypeptide sequence can drive formation of filamentous aggregates even under conditions favoring colloidal stability.

  20. Dexmedetomidine Increases Tau Phosphorylation Under Normothermic Conditions In Vivo and In Vitro

    Science.gov (United States)

    Whittington, Robert A.; Virág, László; Gratuze, Maud; Petry, Franck R.; Noël, Anastasia; Poitras, Isabelle; Truchetti, Geoffrey; Marcouiller, François; Papon, Marie-Amélie; Khoury, Noura El; Wong, Kevin; Bretteville, Alexis; Morin, Françoise; Planel, Emmanuel

    2015-01-01

    There is developing interest in the potential association between anesthesia and the onset and progression of Alzheimer's disease. Several anesthetics have thus been demonstrated to induce tau hyperphosphorylation, an effect mostly mediated by anesthesia-induced hypothermia. Here, we tested the hypothesis that acute normothermic administration of dexmedetomidine, an intravenous sedative used in intensive care units, would result in tau hyperphosphorylation in vivo and in vitro. When administered to non-transgenic mice, dexmedetomidine induced tau hyperphosphorylation persisting up to 6h in the hippocampus for the AT8 epitope. Pretreatment with atipamezole, a highly specific α2-adrenergic receptor (α2-AR) antagonist, blocked dexmedetomidine-induced tau hyperphosphorylation. Furthermore, dexmedetomidine dose-dependently increased tau phosphorylation at AT8 in SH-SY5Y cells, impaired mice spatial memory in the Barnes maze, and promoted tau hyperphosphorylation and aggregation in transgenic hTau mice. These findings suggest that dexmedetomidine: i) increases tau phosphorylation, in vivo and in vitro, in the absence of anesthetic-induced hypothermia and through α2-AR activation, ii) promotes tau aggregation in a mouse model of tauopathy, and iii) impacts spatial reference memory. PMID:26058840

  1. Thrombin induces Egr-1 expression in fibroblasts involving elevation of the intracellular Ca2+ concentration, phosphorylation of ERK and activation of ternary complex factor

    Directory of Open Access Journals (Sweden)

    Thiel Gerald

    2009-05-01

    Full Text Available Abstract Background The serine protease thrombin catalyzes fibrin clot formation by converting fibrinogen into fibrin. Additionally, thrombin stimulation leads to an activation of stimulus-responsive transcription factors in different cell types, indicating that the gene expression pattern is changed in thrombin-stimulated cells. The objective of this study was to analyze the signaling cascade leading to the expression of the zinc finger transcription factor Egr-1 in thrombin-stimulated lung fibroblasts. Results Stimulation of 39M1-81 fibroblasts with thrombin induced a robust and transient biosynthesis of Egr-1. Reporter gene analysis revealed that the newly synthesized Egr-1 was biologically active. The signaling cascade connecting thrombin stimulation with Egr-1 gene expression required elevated levels of cytosolic Ca2+, the activation of diacylgycerol-dependent protein kinase C isoenzymes, and the activation of extracellular signal-regulated protein kinase (ERK. Stimulation of the cells with thrombin triggered the phosphorylation of the transcription factor Elk-1. Expression of a dominant-negative mutant of Elk-1 completely prevented Egr-1 expression in stimulated 39M1-81 cells, indicating that Elk-1 or related ternary complex factors connect the intracellular signaling cascade elicited by activation of protease-activated receptors with transcription of the Egr-1 gene. Lentiviral-mediated expression of MAP kinase phosphatase-1, a dual-specific phosphatase that dephosphorylates and inactivates ERK in the nucleus, prevented Elk-1 phosphorylation and Egr-1 biosynthesis in thrombin stimulated 39M1-81 cells, confirming the importance of nuclear ERK and Elk-1 for the upregulation of Egr-1 expression in thrombin-stimulated lung fibroblasts. 39M1-81 cells additionally express M1 muscarinic acetylcholine receptors. A comparison between the signaling cascades induced by thrombin or carbachol showed no differences, except that signal transduction via M

  2. Phosphorylation of CRTC3 by the salt-inducible kinases controls the interconversion of classically activated and regulatory macrophages.

    Science.gov (United States)

    Clark, Kristopher; MacKenzie, Kirsty F; Petkevicius, Kasparas; Kristariyanto, Yosua; Zhang, Jiazhen; Choi, Hwan Geun; Peggie, Mark; Plater, Lorna; Pedrioli, Patrick G A; McIver, Ed; Gray, Nathanael S; Arthur, J Simon C; Cohen, Philip

    2012-10-16

    Macrophages acquire strikingly different properties that enable them to play key roles during the initiation, propagation, and resolution of inflammation. Classically activated (M1) macrophages produce proinflammatory mediators to combat invading pathogens and respond to tissue damage in the host, whereas regulatory macrophages (M2b) produce high levels of anti-inflammatory molecules, such as IL-10, and low levels of proinflammatory cytokines, like IL-12, and are important for the resolution of inflammatory responses. A central problem in this area is to understand how the formation of regulatory macrophages can be promoted at sites of inflammation to prevent and/or alleviate chronic inflammatory and autoimmune diseases. Here, we demonstrate that the salt-inducible kinases (SIKs) restrict the formation of regulatory macrophages and that their inhibition induces striking increases in many of the characteristic markers of regulatory macrophages, greatly stimulating the production of IL-10 and other anti-inflammatory molecules. We show that SIK inhibitors elevate IL-10 production by inducing the dephosphorylation of cAMP response element-binding protein (CREB)-regulated transcriptional coactivator (CRTC) 3, its dissociation from 14-3-3 proteins and its translocation to the nucleus where it enhances a gene transcription program controlled by CREB. Importantly, the effects of SIK inhibitors on IL-10 production are lost in macrophages that express a drug-resistant mutant of SIK2. These findings identify SIKs as a key molecular switch whose inhibition reprograms macrophages to an anti-inflammatory phenotype. The remarkable effects of SIK inhibitors on macrophage function suggest that drugs that target these protein kinases may have therapeutic potential for the treatment of inflammatory and autoimmune diseases.

  3. A critical role for the regulation of Syk from agglutination to aggregation in human platelets.

    Science.gov (United States)

    Shih, Chun-Ho; Chiang, Tin-Bin; Wang, Wen-Jeng

    2014-01-10

    Agglucetin, a tetrameric glycoprotein (GP) Ibα agonist from Formosan Agkistrodon acutus venom, has been characterized as an agglutination inducer in human washed platelets (WPs). In platelet-rich plasma (PRP), agglucetin dramatically elicits a biphasic response of agglutination and subsequent aggregation. For clarifying the intracellular signaling events from agglutination to aggregation in human platelets, we examined the essential signaling molecules involved through the detection of protein tyrosine phosphorylation (PTP). In WPs, an anti-GPIbα monoclonal antibody (mAb) AP1, but not a Src kinase inhibitor PP1, completely inhibited agglucetin-induced agglutination. However, PP1 but not AP1 had a potent suppression on platelet aggregation by a GPVI activator convulxin. The PTP analyses showed agglucetin alone can cause a weak pattern involving sequential phosphorylation of Lyn/Fyn, Syk, SLP-76 and phospholipase Cγ2 (PLCγ2). Furthermore, a Syk-selective kinase inhibitor, piceatannol, significantly suppressed the aggregating response in agglucetin-activated PRP. Analyzed by flow cytometry, the binding capacity of fluorophore-conjugated PAC-1, a mAb recognizing activated integrin αIIbβ3, was shown to increase in agglucetin-stimulated platelets. Again, piceatannol but not PP1 had a concentration-dependent suppression on agglucetin-induced αIIbβ3 exposure. Moreover, the formation of signalosome, including Syk, SLP-76, VAV, adhesion and degranulation promoting adapter protein (ADAP) and PLCγ2, are required for platelet aggregation in agglucetin/fibrinogen-activated platelets. In addition, GPIbα-ligation via agglucetin can substantially promote the interactions between αIIbβ3 and fibrinogen. Therefore, the signal pathway of Lyn/Fyn/Syk/SLP-76/ADAP/VAV/PLCγ2/PKC is sufficient to trigger platelet aggregation in agglucetin/fibrinogen-pretreated platelets. Importantly, Syk may function as a major regulator for the response from GPIbα-initiated agglutination to

  4. Augmenter of liver regeneration causes different kinetics of ERK1/2 and Akt/PKB phosphorylation than EGF and induces hepatocyte proliferation in an EGF receptor independent and liver specific manner

    Energy Technology Data Exchange (ETDEWEB)

    Ilowski, Maren; Putz, Christine [Department of Surgery, Ludwig-Maximilians-University of Munich Hospital Grosshadern, Munich (Germany); Weiss, Thomas S. [Department of Surgery, University of Regensburg Hospital, Regensburg (Germany); Brand, Stephan [Department of Internal Medicine II, Ludwig-Maximilians-University of Munich Hospital Grosshadern, Munich (Germany); Jauch, Karl-Walter [Department of Surgery, Ludwig-Maximilians-University of Munich Hospital Grosshadern, Munich (Germany); Hengstler, Jan G. [Leibniz Research Centre for Working Environment and Human Factors, TU Dortmund University, Dortmund (Germany); Thasler, Wolfgang Erwin, E-mail: wolfgang.thasler@med.uni-muenchen.de [Department of Surgery, Ludwig-Maximilians-University of Munich Hospital Grosshadern, Munich (Germany)

    2010-04-16

    Background/Aim: Augmenter of liver regeneration (ALR) is a potent growth factor which supports liver regeneration in experimental animals. The aim of this study was to compare proliferation as well as the kinetics of ERK1/2 and Akt/PKB phosphorylation by recombinant human ALR (rhALR) and EGF in human hepatocytes and extrahepatic cells. Methods: Kinetics of ERK1/2 and Akt/PKB phosphorylation were determined in primary human hepatocytes (phh) after stimulation with rhALR and EGF. Induction of proliferation was analyzed in phh and several cell lines of hepatic and extrahepatic origin by the MTT and [{sup 3}H]-thymidine assay. Results: The kinetics of ERK phosphorylation showed clear differences, whereby rhALR caused a transient and EGF a permanent increase during the observation period of 60 min. For both, Akt and ERK phosphorylation, EGF caused a faster effect with maximal levels observed already after 2 min, whereas rhALR caused maximal phosphorylation between 10 and 15 min. Using the EGF receptor inhibitor AG1478 we provide evidence of an EGF receptor independent induction of proliferation by rhALR. Furthermore, rhALR induced proliferation only in phh and the human liver derived cell lines HepG2 and Chang. In contrast, EGF enhanced proliferation in all analyzed cell types including cell lines of colon, bronchial, pancreatic and gastric origin (SW480, BC1, L36PL and GC1). Conclusion: rhALR and EGF induce different kinetics of ERK and Akt phosphorylation in human hepatocytes. The mitogenic effect of rhALR is liver specific and seems to be at least partially independent from EGF receptor mediated signaling.

  5. Influence of calcium-induced aggregation on the sensitivity of aminobis(methylenephosphonate)-containing potential MRI contrast agents.

    Science.gov (United States)

    Henig, Jörg; Mamedov, Ilgar; Fouskova, Petra; Tóth, Éva; Logothetis, Nikos K; Angelovski, Goran; Mayer, Hermann A

    2011-07-18

    A novel class of 1,4,7,10-tetraazacyclododecane-1,4,7-tris(methylenecarboxylic) acid (DO3A)-based lanthanide complexes with relaxometric response to Ca(2+) was synthesized, and their physicochemical properties were investigated. Four macrocyclic ligands containing an alkyl-aminobis(methylenephosphonate) side chain for Ca(2+)-chelation have been studied (alkyl is propyl, butyl, pentyl, and hexyl for L(1), L(2), L(3), and L(4), respectively). Upon addition of Ca(2+), the r(1) relaxivity of their Gd(3+) complexes decreased up to 61% of the initial value for the best compounds GdL(3) and GdL(4). The relaxivity of the complexes was concentration dependent (it decreases with increasing concentration). Diffusion NMR studies on the Y(3+) analogues evidenced the formation of agglomerates at higher concentrations; the aggregation becomes even more important in the presence of Ca(2+). (31)P NMR experiments on EuL(1) and EuL(4) indicated the coordination of a phosphonate to the Ln(3+) for the ligand with a propyl chain, while phosphonate coordination was not observed for the analogue bearing a hexyl linker. Potentiometric titrations yielded protonation constants of the Gd(3+) complexes. log K(H1) values for all complexes lie between 6.12 and 7.11 whereas log K(H2) values are between 4.61 and 5.87. Luminescence emission spectra recorded on the Eu(3+) complexes confirmed the coordination of a phosphonate group to the Ln(3+) center in EuL(1). Luminescence lifetime measurements showed that Ca-induced agglomeration reduces the hydration number which is the main cause for the change in r(1). Variable temperature (17)O NMR experiments evidenced high water exchange rates on GdL(1), GdL(2), and GdL(3) comparable to that of the aqua ion. PMID:21671565

  6. PF573,228 inhibits vascular tumor cell growth, migration as well as angiogenesis, induces apoptosis and abrogates PRAS40 and S6RP phosphorylation.

    Science.gov (United States)

    Mabeta, Peace

    2016-09-01

    PF573,228 is a compound that targets focal adhesion kinase (FAK), a non-receptor protein kinase, which is over-expressed in various tumors. The aim of this study was to evaluate the effects of PF573,228 on the cells derived from mouse vascular tumors, namely, endothelioma cells. The treatment of endothelioma cells with PF573,228 reduced their growth with an IC50 of approximately 4.6 μmol L-1 and inhibited cell migration with an IC50 of about 0.01 μmol L-1. Microscopic studies revealed morphological attributes of apoptosis. These observations were confirmed by ELISA, which showed increased caspase-3 activity. PF573,228 also inhibited angiogenesis in a dose-dependent manner, with an IC50 of approximately 3.7 μmol L-1, and abrogated the phosphorylation of cell survival proteins, proline-rich Akt substrate (PRAS40) and S6 ribosomal protein (S6RP). Array data further revealed that PF573,228 induced caspase-3 activation, thus promoting apoptosis. Since all the processes inhibited by PF573,228 provide important support to tumor survival and progression, the drug may have a potential role in the treatment of vascular tumors. PMID:27383888

  7. Endostar, a novel recombinant human endostatin, exerts antiangiogenic effect via blocking VEGF-induced tyrosine phosphorylation of KDR/Flk-1 of endothelial cells

    International Nuclear Information System (INIS)

    Endostar, a novel recombinant human endostatin expressed and purified in Escherichia coli with an additional nine-amino acid sequence and forming another his-tag structure, was approved by the SFDA in 2005 for the treatment of non-small-cell lung cancer. But its mechanism of action has not been illustrated before. In this study, we examined the antiangiogenic activities of endostar in vitro and in vivo. The results showed that endostar suppressed the VEGF-stimulated proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs) in vitro. Endostar blocked microvessel sprouting from rat aortic rings in vitro. Moreover, it could inhibit the formation of new capillaries from pre-existing vessels in the chicken chorioallantoic membrane (CAM) assay and affect the growth of vessels in tumor. We further found the antiangiogenic effects of endostar were correlated with the VEGF-triggered signaling. Endostar suppressed the VEGF-induced tyrosine phosphorylation of KDR/Flk-1(VEGFR-2) as well as the overall VEGFR-2 expression and the activation of ERK, p38 MAPK, and AKT in HUVECs. Collectively, these data indicated the relationship between endostar and VEGF signal pathways and provided a molecular basis for the antiangiogenic effects of endostar

  8. The role of Rab6a and phosphorylation of non-muscle myosin IIA tailpiece in alcohol-induced Golgi disorganization

    Science.gov (United States)

    Petrosyan, Armen; Casey, Carol A.; Cheng, Pi-Wan

    2016-01-01

    Abnormalities in the Golgi apparatus function are important to the development of alcoholic liver injury. We recently reported that Golgi disorganization in ethanol (EtOH)-treated hepatocytes is caused by impaired dimerization of the largest Golgi matrix protein, giantin. However, little is known about the mechanism which forces fragmentation. Here, in both HepG2 cells overexpressing alcohol dehydrogenase and in rat hepatocytes, we found that EtOH administration reduces the complex between giantin and Rab6a GTPase and results in the S1943 phosphorylation of non-muscle Myosin IIA (NMIIA) heavy chain, thus facilitating NMIIA association with Golgi enzymes, as detected by biochemical approaches and 3D Structured Illumination Microscopy. We revealed that NMIIA-P-S1943 competes with giantin for the Rab6a dimer, which was converted to monomer after Golgi fragmentation. Therefore, Rab6a plays a dual role in the Golgi, serving as master regulator of Golgi organization and disorganization, and that NMIIA and giantin engage in a “tug-of-war”. However, the inhibition of F-actin and downregulation of NMIIA or overexpression of NMHC-IIAΔtailpiece, as well the overexpression of dominant negative Rab6a(T27N), preserved a compact Golgi phenotype. Thus, the actomyosin complex forces EtOH-induced Golgi disorganization, and the targeting of NMIIA-P-S1943 may be important for preventing the damaging effects of alcohol metabolism on the cell. PMID:27535804

  9. Fucoidan induces G1 arrest of the cell cycle in EJ human bladder cancer cells through down-regulation of pRB phosphorylation

    Directory of Open Access Journals (Sweden)

    Hye Young Park

    2015-06-01

    Full Text Available AbstractFucoidan, a sulfated polysaccharide found in marine algae and brown seaweeds, has been shown to inhibit the in vitro growth of human cancer cells. This study was conducted in cultured human bladder cancer EJ cells to elucidate the possible mechanisms by which fucoidan exerts its anti-proliferative activity, which until now has remained poorly understood. Fucoidan treatment of EJ cells resulted in dose-dependent inhibition of cell growth and induced apoptotic cell death. Flow cytometric analysis revealed that fucoidan led to G1 arrest in cell cycle progression. It was associated with down-regulation of cyclin D1, cyclin E, and cyclin-dependent-kinases (Cdks in a concentration-dependent manner, without any change in Cdk inhibitors, such as p21 and p27. Furthermore, dephosphorylation of retinoblastoma protein (pRB by this compound was associated with enhanced binding of pRB with the transcription factors E2F-1 and E2F-4. Overall, our results demonstrate that fucoidan possesses anticancer activity potential against bladder cancer cells by inhibiting pRB phosphorylation.

  10. The role of Rab6a and phosphorylation of non-muscle myosin IIA tailpiece in alcohol-induced Golgi disorganization.

    Science.gov (United States)

    Petrosyan, Armen; Casey, Carol A; Cheng, Pi-Wan

    2016-01-01

    Abnormalities in the Golgi apparatus function are important to the development of alcoholic liver injury. We recently reported that Golgi disorganization in ethanol (EtOH)-treated hepatocytes is caused by impaired dimerization of the largest Golgi matrix protein, giantin. However, little is known about the mechanism which forces fragmentation. Here, in both HepG2 cells overexpressing alcohol dehydrogenase and in rat hepatocytes, we found that EtOH administration reduces the complex between giantin and Rab6a GTPase and results in the S1943 phosphorylation of non-muscle Myosin IIA (NMIIA) heavy chain, thus facilitating NMIIA association with Golgi enzymes, as detected by biochemical approaches and 3D Structured Illumination Microscopy. We revealed that NMIIA-P-S1943 competes with giantin for the Rab6a dimer, which was converted to monomer after Golgi fragmentation. Therefore, Rab6a plays a dual role in the Golgi, serving as master regulator of Golgi organization and disorganization, and that NMIIA and giantin engage in a "tug-of-war". However, the inhibition of F-actin and downregulation of NMIIA or overexpression of NMHC-IIAΔtailpiece, as well the overexpression of dominant negative Rab6a(T27N), preserved a compact Golgi phenotype. Thus, the actomyosin complex forces EtOH-induced Golgi disorganization, and the targeting of NMIIA-P-S1943 may be important for preventing the damaging effects of alcohol metabolism on the cell. PMID:27535804

  11. pERK 1/2 inhibit Caspase-8 induced apoptosis in cancer cells by phosphorylating it in a cell cycle specific manner.

    Science.gov (United States)

    Mandal, Ranadip; Raab, Monika; Matthess, Yves; Becker, Sven; Knecht, Rainald; Strebhardt, Klaus

    2014-03-01

    ERK 1/2 are found to be hyperactive in many cancers. Active ERK 1/2 (pERK 1/2) are known to protect cancer cells from undergoing death receptor-mediated apoptosis, although the mechanism(s) behind this is poorly understood. Through in vitro kinase assays and mass-spectrometry we demonstrate that pERK 1/2 can phosphorylate pro-Caspase-8 at S387. Also, in EGFR-overexpressing Type I and II ovarian and breast cancer cell lines respectively, ERK 1/2 remain active only during the interphase. During this period, pERK 1/2 could inhibit Trail-induced apoptosis, most effectively during the G1/S phase. By knocking-down the endogenous pro-Caspase-8 using RNAi and replacing it with its non-phosphorylatable counterpart (S387A), a significant increase in Caspase-8 activity upon Trail stimulation was observed, even in the presence of pERK 1/2. Taken together, we propose that a combination of Trail and an inhibitor of ERK 1/2 activities could potentially enhance of Trail's effectiveness as an anti-cancer agent in ERK 1/2 hyperactive cancer cells.

  12. Fluorescence characterization of co-immobilization-induced multi-enzyme aggregation in a polymer matrix using Förster resonance energy transfer (FRET): toward the metabolon biomimic.

    Science.gov (United States)

    Wu, Fei; Minteer, Shelley D

    2013-08-12

    Sequential metabolic enzymes can form supramolecular complexes named metabolons in vivo through enzyme-enzyme association or aggregation to facilitate efficient substrate channeling. By separately labeling enzymes with lysine-targeting carboxylic acid succinimidyl ester fluorophores of distinct excitation wavelengths, this research presents a quantitative study of polymer-entrapment-induced in vitro multi-enzyme aggregation from three Krebs cycle enzymes using Förster resonance energy transfer (FRET) to find potential polymer materials for immobilizing enzyme cascades and inducing the metabolon biomimic formation on electrodes. The effect of hydrophobic modification of linear polyethylenimine, Nafion, and chitosan polymers on metabolon formation has been investigated through photobleaching FRET imaging in addition to traditional steady-state fluorescence spectroscopy. By partially destroying FRET acceptors of longer excitation wavelength, increased fluorescence from dequenched donors of shorter excitation wavelength was measured and enzyme interactions in terms of energy-transfer efficiencies were mapped point by point. Results show that trimethyloctadecylammonium-modified Nafion works best in inducing multi-enzyme aggregation and exhibits a promising future in immobilized metabolon biomimics with the most uniform enzyme organization, as indicated by the protein distance distribution.

  13. Dynamic alteration in H3 serine 10 phosphorylation is G1-phase specific during ionization radiation induced DNA damage response in human cells

    International Nuclear Information System (INIS)

    Highlights: • Loss of H3S10P in response to DNA damage is a universal phenomenon from G1 cells. • The loss happens predominantly from histone H3.3, a transcription activation mark. • Compaction of chromatin occurs during repair stage of DDR. • The alteration of H3S10P shows an inverse correlation with γH2AX. - Abstract: Chromatin acts as a natural barrier in DNA-damage recognition and repair. Histones undergo differential post-translational modification(s) to facilitate DNA damage response (DDR). Importance of modifications like phosphorylation of histone variant H2A.X in DNA repair is very well understood, however, ambiguous results exist in literature regarding the levels of certain histone modifications and their possible role in repair. In the present study, we have investigated in depth the alteration in the level of the highly dynamic histone mark H3S10P as it plays a dual role in different phases of the cell cycle. We show here that H3S10P decreases specifically from irradiated G1-enriched cells irrespective of the damaging agent or the cell line used in the study. Interestingly, the loss occurs predominantly from H3.3 variant which is a transcription activation mark like H3S10P itself, suggesting that the alteration might be implicated in transcription repression. The decrease in other transcription marks like H3K9Ac, H3K14Ac, H3K56Ac and H3S28P along with the occurrence of chromatin condensation in response to DNA damage in G1 phase strengthens the hypothesis. In addition, the alteration in the level of H3S10P shows an inverse correlation with that of γH2AX in a dose-dependent manner and probably occurs from the same mononucleosome. We propose that the drop in the levels of histone H3S10 phosphorylation is a universal phenomenon in response to DNA damage and is a trigger to induce transcription repressive state to facilitate repair

  14. Dynamic alteration in H3 serine 10 phosphorylation is G1-phase specific during ionization radiation induced DNA damage response in human cells

    Energy Technology Data Exchange (ETDEWEB)

    Sharma, Ajit K.; Bhattacharya, Saikat; Khan, Shafqat A.; Khade, Bharat; Gupta, Sanjay, E-mail: sgupta@actrec.gov.in

    2015-03-15

    Highlights: • Loss of H3S10P in response to DNA damage is a universal phenomenon from G1 cells. • The loss happens predominantly from histone H3.3, a transcription activation mark. • Compaction of chromatin occurs during repair stage of DDR. • The alteration of H3S10P shows an inverse correlation with γH2AX. - Abstract: Chromatin acts as a natural barrier in DNA-damage recognition and repair. Histones undergo differential post-translational modification(s) to facilitate DNA damage response (DDR). Importance of modifications like phosphorylation of histone variant H2A.X in DNA repair is very well understood, however, ambiguous results exist in literature regarding the levels of certain histone modifications and their possible role in repair. In the present study, we have investigated in depth the alteration in the level of the highly dynamic histone mark H3S10P as it plays a dual role in different phases of the cell cycle. We show here that H3S10P decreases specifically from irradiated G1-enriched cells irrespective of the damaging agent or the cell line used in the study. Interestingly, the loss occurs predominantly from H3.3 variant which is a transcription activation mark like H3S10P itself, suggesting that the alteration might be implicated in transcription repression. The decrease in other transcription marks like H3K9Ac, H3K14Ac, H3K56Ac and H3S28P along with the occurrence of chromatin condensation in response to DNA damage in G1 phase strengthens the hypothesis. In addition, the alteration in the level of H3S10P shows an inverse correlation with that of γH2AX in a dose-dependent manner and probably occurs from the same mononucleosome. We propose that the drop in the levels of histone H3S10 phosphorylation is a universal phenomenon in response to DNA damage and is a trigger to induce transcription repressive state to facilitate repair.

  15. Corepressor SMRT promotes oxidative phosphorylation in adipose tissue and protects against diet-induced obesity and insulin resistance.

    Science.gov (United States)

    Fang, Sungsoon; Suh, Jae Myoung; Atkins, Annette R; Hong, Suk-Hyun; Leblanc, Mathias; Nofsinger, Russell R; Yu, Ruth T; Downes, Michael; Evans, Ronald M

    2011-02-22

    The ligand-dependent competing actions of nuclear receptor (NR)-associated transcriptional corepressor and coactivator complexes allow for the precise regulation of NR-dependent gene expression in response to both temporal and environmental cues. Here we report the mouse model termed silencing mediator of retinoid and thyroid hormone receptors (SMRT)(mRID1) in which targeted disruption of the first receptor interaction domain (RID) of the nuclear corepressor SMRT disrupts interactions with a subset of NRs and leads to diet-induced superobesity associated with a depressed respiratory exchange ratio, decreased ambulatory activity, and insulin resistance. Although apparently normal when chow fed, SMRT(mRID1) mice develop multiple metabolic dysfunctions when challenged by a high-fat diet, manifested by marked lipid accumulation in white and brown adipose tissue and the liver. The increased weight gain of SMRT(mRID1) mice on a high-fat diet occurs predominantly in fat with adipocyte hypertrophy evident in both visceral and s.c. depots. Importantly, increased inflammatory gene expression was detected only in the visceral depots. SMRT(mRID1) mice are both insulin-insensitive and refractory to the glucose-lowering effects of TZD and AICAR. Increased serum cholesterol and triglyceride levels were observed, accompanied by increased leptin and decreased adiponectin levels. Aberrant storage of lipids in the liver occurred as triglycerides and cholesterol significantly compromised hepatic function. Lipid accumulation in brown adipose tissue was associated with reduced thermogenic capacity and mitochondrial biogenesis. Collectively, these studies highlight the essential role of NR corepressors in maintaining metabolic homeostasis and describe an essential role for SMRT in regulating the progression, severity, and therapeutic outcome of metabolic diseases.

  16. Identification of an RNA-binding protein that is phosphorylated by PTH and potentially mediates PTH-induced destabilization of Npt2a mRNA.

    Science.gov (United States)

    Murray, Rebecca D; Merchant, Michael L; Hardin, Ericka; Clark, Barbara; Khundmiri, Syed J; Lederer, Eleanor D

    2016-02-01

    Parathyroid hormone (PTH) is a key regulator of the expression and function of the type IIa sodium-phosphate cotransporter (Npt2a), the protein responsible for regulated renal phosphate reabsorption. We previously showed that PTH induces rapid decay of Npt2a mRNA through posttranscriptional mechanisms. We hypothesized that PTH-induced changes in RNA-binding protein (RBP) activity mediate the degradation of Npt2a mRNA. To address this aim, we treated opossum kidney (OK) cells, a PTH-sensitive proximal tubule cell culture model, with 100 nM PTH for 30 min and 2 h, followed by mass spectrometry characterization of the PTH-stimulated phosphoproteome. We identified 1,182 proteins differentially phosphorylated in response to PTH, including 68 RBPs. Preliminary analysis identified a phospho-RBP, hnRNPK-homology-type-splicing regulatory protein (KSRP), with predicted binding sites for the 3'-untranslated region (UTR) of Npt2a mRNA. Western blot analysis confirmed expression of KSRP in OK cells and showed PTH-dependent translocation to the nucleus. Immunoprecipitation of KSRP from control and PTH-treated cells followed by RNA isolation and RT-quantitative PCR analysis identified Npt2a mRNA from both control and PTH-treated KSRP pulldowns. Knockdown of KSRP followed by PTH treatment showed that KSRP is required for mediating PTH-stimulated reduction in sodium/hydrogen exchanger 3 mRNA, but not Npt2a mRNA. We conclude that 1) PTH is a major regulator of both transcription and translation, and 2) KSRP binds Npt2a mRNA but its role in PTH regulation of Npt2a mRNA is not clear.

  17. Poor efficacy of the phosphorylated high-molecular-weight neurofilament heavy subunit serum level, a biomarker of axonal damage, as a marker of chemotherapy-induced peripheral neuropathy

    Science.gov (United States)

    SUMITANI, MASAHIKO; OGATA, TORU; NATORI, AKINA; HOZUMI, JUN; SHIMOJO, NOBUTAKE; KIDA, KUMIKO; YAMAUCHI, HIDEKO; YAMAUCHI, TERUO

    2016-01-01

    The phosphorylated form of the high-molecular-weight neurofilament heavy subunit (pNF-H) is a major structural protein in axons. The pNF-H level is elevated in the serum of certain patients with central nervous disorders, including chemotherapy-induced cognitive impairment. The present study was conducted to elucidate the potential role of pNF-H as a marker of chemotherapy-induced peripheral neuropathy (CIPN). A total of 71 patients with early breast cancer in various stages of treatment (following 1, 3 or 7 cycles of chemotherapy, or a previous history of breast cancer chemotherapy) were assessed with a self-administered PainDETECT questionnaire [pain location, pain intensity on an 11-point numeric rating scale (NRS), and various pain qualities] and a single serum pNF-H measurement. Patients were divided into two groups based on the presence or absence of bilateral symmetric pain in the distal portions of the extremities [CIPN(+) or CIPN(−)]. The χ2 and Mann-Whitney tests were used for statistical analyses. Among the participants, only 8 patients complained of CIPN. Their pain intensity was 3.5±1.9 (mean ± standard deviation) compared with 1.5±1.8 in the CIPN(−) group (P<0.01). The NRS of numbness in the CIPN(+) group was significantly higher (2.4±1.4) than that of the CIPN(−) group (1.0±1.0). Increased pNF-H levels were observed in 37.5% of the CIPN(+) patients and in 23.8% of CIPN(−) patients (P=0.40). In conclusion, CIPN is observed in the most distal portions of the peripheral nerves that are composed of dendrites but not axons. Although serum pNF-H is a biomarker of axonal damage, it is not useful as a marker of CIPN. PMID:27284419

  18. A combination of indol-3-carbinol and genistein synergistically induces apoptosis in human colon cancer HT-29 cells by inhibiting Akt phosphorylation and progression of autophagy

    Directory of Open Access Journals (Sweden)

    Watanabe Hirotsuna

    2009-11-01

    Full Text Available Abstract Background The chemopreventive effects of dietary phytochemicals on malignant tumors have been studied extensively because of a relative lack of toxicity. To achieve desirable effects, however, treatment with a single agent mostly requires high doses. Therefore, studies on effective combinations of phytochemicals at relatively low concentrations might contribute to chemopreventive strategies. Results Here we found for the first time that co-treatment with I3C and genistein, derived from cruciferous vegetables and soy, respectively, synergistically suppressed the viability of human colon cancer HT-29 cells at concentrations at which each agent alone was ineffective. The suppression of cell viability was due to the induction of a caspase-dependent apoptosis. Moreover, the combination effectively inhibited phosphorylation of Akt followed by dephosphorylation of caspase-9 or down-regulation of XIAP and survivin, which contribute to the induction of apoptosis. In addition, the co-treatment also enhanced the induction of autophagy mediated by the dephosphorylation of mTOR, one of the downstream targets of Akt, whereas the maturation of autophagosomes was inhibited. These results give rise to the possibility that co-treatment with I3C and genistein induces apoptosis through the simultaneous inhibition of Akt activity and progression of the autophagic process. This possibility was examined using inhibitors of Akt combined with inhibitors of autophagy. The combination effectively induced apoptosis, whereas the Akt inhibitor alone did not. Conclusion Although in vivo study is further required to evaluate physiological efficacies and toxicity of the combination treatment, our findings might provide a new insight into the development of novel combination therapies/chemoprevention against malignant tumors using dietary phytochemicals.

  19. A mobile system for a comprehensive online-characterization of nanoparticle aggregates based on wide-angle light scattering and laser-induced incandescence

    Science.gov (United States)

    Huber, Franz J. T.; Altenhoff, Michael; Will, Stefan

    2016-05-01

    A mobile demonstrator for the comprehensive online-characterization of gas-borne nanoparticle aggregates is presented. Two optical measurement techniques are combined, both utilizing a pulsed Nd:YAG laser as light source. Aggregate size and fractal dimension are measured by Wide-Angle Light Scattering (WALS). An ellipsoidal mirror images elastically scattered light from scattering angles between 10° and 165° onto a CCD-camera chip resulting in an almost complete scattering diagram with high angular resolution. Primary particle size and volume fraction are measured by time-resolved Laser-Induced Incandescence (TiRe-LII). Here, particles are heated up to about 3000 K by the short laser pulse, the enhanced thermal radiation signal is detected with gated photomultiplier tubes. Analysis of the signal decay time and maximum LII-signal allows for the determination of primary particle diameter and volume fraction. The performance of the system is demonstrated by combined measurements on soot nanoparticle aggregates from a soot aerosol generator. Particle and aggregate sizes are varied by using different equivalence ratios of the combustion in the generator. Soot volume fraction can be adjusted by different levels of dilution with air. Online-measurements were carried out demonstrating the favorable performance of the system and the potential for industrial applications such as process control and product development. The particle properties obtained are confirmed through transmission electron microscopy analysis on representative samples.

  20. Angiotensin II-induced hypertension blunts thick ascending limb NO production by reducing NO synthase 3 expression and enhancing threonine 495 phosphorylation.

    Science.gov (United States)

    Ramseyer, Vanesa D; Gonzalez-Vicente, Agustin; Carretero, Oscar A; Garvin, Jeffrey L

    2015-01-15

    Thick ascending limbs reabsorb 30% of the filtered NaCl load. Nitric oxide (NO) produced by NO synthase 3 (NOS3) inhibits NaCl transport by this segment. In contrast, chronic angiotensin II (ANG II) infusion increases net thick ascending limb transport. NOS3 activity is regulated by changes in expression and phosphorylation at threonine 495 (T495) and serine 1177 (S1177), inhibitory and stimulatory sites, respectively. We hypothesized that NO production by thick ascending limbs is impaired by chronic ANG II infusion, due to reduced NOS3 expression, increased phosphorylation of T495, and decreased phosphorylation of S1177. Rats were infused with 200 ng·kg(-1)·min(-1) ANG II or vehicle for 1 and 5 days. ANG II infusion for 5 days decreased NOS3 expression by 40 ± 12% (P hypertension NO production by thick ascending limbs is impaired due to decreased NOS3 expression and altered phosphorylation. PMID:25377910

  1. A novel fusion protein domain III-capsid from dengue-2, in a highly aggregated form, induces a functional immune response and protection in mice

    International Nuclear Information System (INIS)

    Based on the immunogenicity of domain III from the Envelope protein of dengue virus as well as the proven protective capacity of the capsid antigen, we have designed a novel domain III-capsid chimeric protein with the goal of obtaining a molecule potentially able to induce both humoral and cell-mediated immunity (CMI). After expression of the recombinant gene in Escherichia coli, the domain III moiety retained its antigenicity as evaluated with anti-dengue sera. In order to explore alternatives for modulating the immunogenicity of the protein, it was mixed with oligodeoxynucleotides in order to obtain particulated aggregates and then immunologically evaluated in mice in comparison with non-aggregated controls. Although the humoral immune response induced by both forms of the protein was equivalent, the aggregated variant resulted in a much stronger CMI as measured by in vitro IFN-γ secretion and protection experiments, mediated by CD4+ and CD8+ cells. The present work provides additional evidence in support for a crucial role of CMI in protection against dengue virus and describes a novel vaccine candidate against the disease based on a recombinant protein that can stimulate both arms of the acquired immune system.

  2. Quantitative analysis of the calorimetric parameters associated with the temperature induced aggregation of aqueous solutions of polyoxypropylene

    International Nuclear Information System (INIS)

    High sensitivity differential scanning calorimetry (HSDSC)--coupled with the application of a previously outlined thermodynamic model [Patterson et al., Langmuir 13 (1997) 2219]--has been used to the obtain thermodynamic parameters that characterise thermal aggregation in aqueous solutions of polyoxypropylene (POP) of molecular mass 1000 g mol-1 over a range of concentrations (2.5-51.5 g dm-3). An important aspect of the derived thermodynamic values, which complements previously reported HSDSC data [Armstrong et al., J. Phys. Chem. 99 (1995) 4590], is the elaboration of heat capacity changes which accompany the aggregation transition. The concentration dependence of the POP thermodynamic data, obtained in this investigation, has been established. These observations provide the means for establishing functional relationships between enthalpy and temperature as well as heat capacity and temperature. The parameters describing the quadratic relationship between enthalpy change associated with aggregation and temperature are in close agreement with those describing the linear relationship between heat capacity change and temperature

  3. 2-Methoxy-4-vinylphenol can induce cell cycle arrest by blocking the hyper-phosphorylation of retinoblastoma protein in benzo[a]pyrene-treated NIH3T3 cells

    Energy Technology Data Exchange (ETDEWEB)

    Jeong, Jin Boo [Bioresource Sciences, Andong National University, Andong 760749 (Korea, Republic of); Jeong, Hyung Jin, E-mail: jhj@andong.ac.kr [Bioresource Sciences, Andong National University, Andong 760749 (Korea, Republic of)

    2010-10-01

    Research highlights: {yields} 2M4VP activated the expression of p21 and p15 protein, and down-regulated the expression of cyclin D1 and cyclin E. {yields} 2M4VP inhibited hyper-phosphorylation of Rb protein. {yields} 2M4VP induced cell cycle arrest from G1 to S. {yields} 2M4VP inhibited hyper-proliferation of the cells in BaP-treated cells. {yields} 2M4VP induces growth arrest of BaP-treated cells by blocking hyper-phosphorylation of Rb via regulating the expression of cell cycle-related proteins. -- Abstract: Benzo[a]pyrene (BaP) is an environment carcinogen that can enhance cell proliferation by disturbing the signal transduction pathways in cell cycle regulation. In this study, the effects of 2M4VP on cell proliferation, cell cycle and cell cycle regulatory proteins were studied in BaP-treated NIH 3T3 cells to establish the molecular mechanisms of 2M4VP as anti-proliferative agents. 2M4VP exerted a dose-dependent inhibitory effect on cell growth correlated with a G1 arrest. Analysis of G1 cell cycle regulators expression revealed 2M4VP increased expression of CDK inhibitor, p21Waf1/Cip1 and p15 INK4b, decreased expression of cyclin D1 and cyclin E, and inhibited kinase activities of CDK4 and CDK2. However, 2M4VP did not affect the expression of CDK4 and CDK2. Also, 2M4VP inhibited the hyper-phosphorylation of Rb induced by BaP. Our results suggest that 2M4VP induce growth arrest of BaP-treated NIH 3T3 cells by blocking the hyper-phosphorylation of Rb via regulating the expression of cell cycle-related proteins.

  4. Signal Transducer and Activator of Transcription (Stat)-Induced Stat Inhibitor 1 (Ssi-1)/Suppressor of Cytokine Signaling 1 (Socs1) Inhibits Insulin Signal Transduction Pathway through Modulating Insulin Receptor Substrate 1 (Irs-1) Phosphorylation

    OpenAIRE

    Kawazoe, Yoshinori; Naka, Tetsuji; Fujimoto, Minoru; Kohzaki, Hidetsugu; Morita, Yoshiaki; Narazaki, Masashi; Okumura, Kohichi; Saitoh, Hiroshi; Nakagawa, Reiko; Uchiyama, Yasuo; Akira, Shizuo; Kishimoto, Tadamitsu

    2001-01-01

    Signal transducer and activator of transcription (STAT)-induced STAT inhibitor 1 (SSI-1) is known to function as a negative feedback regulator of cytokine signaling, but it is unclear whether it is involved in other biological events. Here, we show that SSI-1 participates and plays an important role in the insulin signal transduction pathway. SSI-1–deficient mice showed a significantly low level of blood sugar. While the forced expression of SSI-1 reduced the phosphorylation level of insulin ...

  5. Engineering a Dual-Layer Chitosan-Lactide Hydrogel To Create Endothelial Cell Aggregate-Induced Microvascular Networks In Vitro and Increase Blood Perfusion In Vivo.

    Science.gov (United States)

    Kim, Sungwoo; Kawai, Toshiyuki; Wang, Derek; Yang, Yunzhi

    2016-08-01

    Here, we report the use of chemically cross-linked and photo-cross-linked hydrogels to engineer human umbilical vein endothelial cell (HUVEC) aggregate-induced microvascular networks to increase blood perfusion in vivo. First, we studied the effect of chemically cross-linked and photo-cross-linked chitosan-lactide hydrogels on stiffness, degradation rates, and HUVEC behaviors. The photo-cross-linked hydrogel was relatively stiff (E = ∼15 kPa) and possessed more compact networks, denser surface texture, and lower enzymatic degradation rates than the relatively soft, chemically cross-linked hydrogel (E = ∼2 kPa). While both hydrogels exhibited nontoxicity, the soft chemically cross-linked hydrogels expedited the formation of cell aggregates compared to the photo-cross-linked hydrogels. Cells on the less stiff, chemically cross-linked hydrogels expressed more matrix metalloproteinase (MMP) activity than the stiffer, photo-cross-linked hydrogel. This difference in MMP activity resulted in a more dramatic decrease in mechanical stiffness after 3 days of incubation for the chemically cross-linked hydrogel, as compared to the photo-cross-linked one. After determining the physical and biological properties of each hydrogel, we accordingly engineered a dual-layer hydrogel construct consisting of the relatively soft, chemically cross-linked hydrogel layer for HUVEC encapsulation, and the relatively stiff, acellular, photo-cross-linked hydrogel for retention of cell-laden microvasculature above. This dual-layer hydrogel construct enabled a lasting HUVEC aggregate-induced microvascular network due to the combination of stable substrate, enriched cell adhesion molecules, and extracellular matrix proteins. We tested the dual-layer hydrogel construct in a mouse model of hind-limb ischemia, where the HUVEC aggregate-induced microvascular networks significantly enhanced blood perfusion rate to ischemic legs and decreased tissue necrosis compared with both no treatment and

  6. Hydrogen peroxide inhibits transforming growth factor-β1-induced cell cycle arrest by promoting Smad3 linker phosphorylation through activation of Akt-ERK1/2-linked signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Jiyeon; Park, Seong Ji; Jo, Eun Ji [Department of Biochemistry, College of Natural Sciences, Kangwon National University, Chuncheon 200-701 (Korea, Republic of); Lee, Hui-Young [Department of Internal Medicine, Kangwon National University, Chuncheon 200-701 (Korea, Republic of); Hong, Suntaek [Laboratory of Cancer Cell Biology, Lee Gil Ya Cancer and Diabetes Institute, Gachon University, Incheon 406-840 (Korea, Republic of); Kim, Seong-Jin [CHA Cancer Institute, CHA University of Medicine and Science, Seoul 135-081 (Korea, Republic of); Kim, Byung-Chul, E-mail: bckim@kangwon.ac.kr [Department of Biochemistry, College of Natural Sciences, Kangwon National University, Chuncheon 200-701 (Korea, Republic of)

    2013-06-14

    Highlights: •H{sub 2}O{sub 2} inhibits TGF-β1-induced cell cycle arrest. •H{sub 2}O{sub 2} induces Smad3 linker phosphorylation through Akt-ERK1/2 pathway. •H{sub 2}O{sub 2}-mediated suppression of TGF-β signal requires Smad3 linker phosphorylation. •This is a first report about interplay between H{sub 2}O{sub 2} and growth inhibition pathway. -- Abstract: Hydrogen peroxide (H{sub 2}O{sub 2}) functions as a second messenger in growth factor receptor-mediated intracellular signaling cascade and is tumorigenic by virtue of its ability to promote cell proliferation; however, the mechanisms underlying the growth stimulatory action of H{sub 2}O{sub 2} are less understood. Here we report an important mechanism for antagonistic effects of H{sub 2}O{sub 2} on growth inhibitory response to transforming growth factor-β1 (TGF-β1). In Mv1Lu and HepG2 cells, pretreatment of H{sub 2}O{sub 2} (0.05–0.2 mM) completely blocked TGF-β1-mediated induction of p15{sup INK4B} expression and increase of its promoter activity. Interestingly, H{sub 2}O{sub 2} selectively suppressed the transcriptional activation potential of Smad3, not Smad2, in the absence of effects on TGF-β1-induced phosphorylation of the COOH-tail SSXS motif of Smad3 and its nuclear translocation. Mechanism studies showed that H{sub 2}O{sub 2} increases the phosphorylation of Smad3 at the middle linker region in a concentration- and time-dependent manner and this effect is mediated by activation of extracellular signal-activated kinase 1/2 through Akt. Furthermore, expression of a mutant Smad3 in which linker phosphorylation sites were ablated significantly abrogated the inhibitory effects of H{sub 2}O{sub 2} on TGF-β1-induced increase of p15{sup INK4B}-Luc reporter activity and blockade of cell cycle progression from G1 to S phase. These findings for the first time define H{sub 2}O{sub 2} as a signaling molecule that modulate Smad3 linker phosphorylation and its transcriptional activity, thus providing

  7. Computational Study of Pseudo-phosphorylation of the Microtubule associated Protein Tau

    Science.gov (United States)

    Prokopovich, Dmitriy; Larini, Luca

    This computational study focuses on the effect of pseudo-phosphorylation on the aggregation of the microtubule associated protein tau. In the axon of the neuron, tau regulates the assembly of microtubules in the cytoskeleton. This is important for both stabilization of and transport across the microtubules. One of the hallmarks of the Alzheimer's disease is that tau is hyper-phosphorylated and aggregates into neurofibrillary tangles that lay waste to the neurons. It is not known if hyper-phosphorylation directly causes the aggregation of tau into tangles. Experimentally, pseudo-phosphorylation mimics the effects of phosphorylation by mutating certain residues of the protein chain into charged residues. In this study, we will consider the fragment called PHF43 that belongs to the microtubule binding region and has been shown to readily aggregate.

  8. Investigating the feasibility of scale up and automation of human induced pluripotent stem cells cultured in aggregates in feeder free conditions.

    Science.gov (United States)

    Soares, Filipa A C; Chandra, Amit; Thomas, Robert J; Pedersen, Roger A; Vallier, Ludovic; Williams, David J

    2014-03-10

    The transfer of a laboratory process into a manufacturing facility is one of the most critical steps required for the large scale production of cell-based therapy products. This study describes the first published protocol for scalable automated expansion of human induced pluripotent stem cell lines growing in aggregates in feeder-free and chemically defined medium. Cells were successfully transferred between different sites representative of research and manufacturing settings; and passaged manually and using the CompacT SelecT automation platform. Modified protocols were developed for the automated system and the management of cells aggregates (clumps) was identified as the critical step. Cellular morphology, pluripotency gene expression and differentiation into the three germ layers have been used compare the outcomes of manual and automated processes.

  9. Mechanical stimulation of cyclic tensile strain induces reduction of pluripotent related gene expressions via activation of Rho/ROCK and subsequent decreasing of AKT phosphorylation in human induced pluripotent stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Teramura, Takeshi, E-mail: teramura@med.kindai.ac.jp [Institute of Advanced Clinical Medicine, Kinki University, Faculty of Medicine, Osaka (Japan); Takehara, Toshiyuki; Onodera, Yuta [Institute of Advanced Clinical Medicine, Kinki University, Faculty of Medicine, Osaka (Japan); Nakagawa, Koichi; Hamanishi, Chiaki [Department of Orthopaedic Surgery, Kinki University, Faculty of Medicine, Osaka (Japan); Fukuda, Kanji [Institute of Advanced Clinical Medicine, Kinki University, Faculty of Medicine, Osaka (Japan); Department of Orthopaedic Surgery, Kinki University, Faculty of Medicine, Osaka (Japan)

    2012-01-13

    Highlights: Black-Right-Pointing-Pointer Mechanical stimulation is an important factor for regulation of stem cell fate. Black-Right-Pointing-Pointer Cyclic stretch to human induced pluripotent stem cells activated small GTPase Rho. Black-Right-Pointing-Pointer Rho-kinase activation attenuated pluripotency via inhibition of AKT activation. Black-Right-Pointing-Pointer This reaction could be reproduced only by transfection of dominant active Rho. Black-Right-Pointing-Pointer Rho/ROCK are important molecules in mechanotransduction and control of stemness. -- Abstract: Mechanical stimulation has been shown to regulate the proliferation and differentiation of stem cells. However, the effects of the mechanical stress on the stemness or related molecular mechanisms have not been well determined. Pluripotent stem cells such as embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are used as good materials for cell transplantation therapy and research of mammalian development, since they can self-renew infinitely and differentiate into various cell lineages. Here we demonstrated that the mechanical stimulation to human iPS cells altered alignment of actin fibers and expressions of the pluripotent related genes Nanog, POU5f1 and Sox2. In the mechanically stimulated iPS cells, small GTPase Rho was activated and interestingly, AKT phosphorylation was decreased. Inhibition of Rho-associated kinase ROCK recovered the AKT phosphorylation and the gene expressions. These results clearly suggested that the Rho/ROCK is a potent primary effector of mechanical stress in the pluripotent stem cells and it participates to pluripotency-related signaling cascades as an upper stream regulator.

  10. Salt-inducible kinase 2 links transcriptional coactivator p300 phosphorylation to the prevention of ChREBP-dependent hepatic steatosis in mice.

    Science.gov (United States)

    Bricambert, Julien; Miranda, Jonatan; Benhamed, Fadila; Girard, Jean; Postic, Catherine; Dentin, Renaud

    2010-12-01

    Obesity and type 2 diabetes are associated with increased lipogenesis in the liver. This results in fat accumulation in hepatocytes, a condition known as hepatic steatosis, which is a form of nonalcoholic fatty liver disease (NAFLD), the most common cause of liver dysfunction in the United States. Carbohydrate-responsive element-binding protein (ChREBP), a transcriptional activator of glycolytic and lipogenic genes, has emerged as a major player in the development of hepatic steatosis in mice. However, the molecular mechanisms enhancing its transcriptional activity remain largely unknown. In this study, we have identified the histone acetyltransferase (HAT) coactivator p300 and serine/threonine kinase salt-inducible kinase 2 (SIK2) as key upstream regulators of ChREBP activity. In cultured mouse hepatocytes, we showed that glucose-activated p300 acetylated ChREBP on Lys672 and increased its transcriptional activity by enhancing its recruitment to its target gene promoters. SIK2 inhibited p300 HAT activity by direct phosphorylation on Ser89, which in turn decreased ChREBP-mediated lipogenesis in hepatocytes and mice overexpressing SIK2. Moreover, both liver-specific SIK2 knockdown and p300 overexpression resulted in hepatic steatosis, insulin resistance, and inflammation, phenotypes reversed by SIK2/p300 co-overexpression. Finally, in mouse models of type 2 diabetes and obesity, low SIK2 activity was associated with increased p300 HAT activity, ChREBP hyperacetylation, and hepatic steatosis. Our findings suggest that inhibition of hepatic p300 activity may be beneficial for treating hepatic steatosis in obesity and type 2 diabetes and identify SIK2 activators and specific p300 inhibitors as potential targets for pharmaceutical intervention.

  11. Tumor necrosis factor-α-mediated threonine 435 phosphorylation of p65 nuclear factor-κB subunit in endothelial cells induces vasogenic edema and neutrophil infiltration in the rat piriform cortex following status epilepticus

    Directory of Open Access Journals (Sweden)

    Kim Ji-Eun

    2012-01-01

    Full Text Available Abstract Background Status epilepticus (SE induces severe vasogenic edema in the piriform cortex (PC accompanied by neuronal and astroglial damages. To elucidate the mechanism of SE-induced vasogenic edema, we investigated the roles of tumor necrosis factor (TNF-α in blood-brain barrier (BBB disruption during vasogenic edema and its related events in rat epilepsy models provoked by pilocarpine-induced SE. Methods SE was induced by pilocarpine in rats that were intracerebroventricularly infused with saline-, and soluble TNF p55 receptor (sTNFp55R prior to SE induction. Thereafter, we performed Fluoro-Jade B staining and immunohistochemical studies for TNF-α and NF-κB subunits. Results Following SE, most activated microglia showed strong TNF-α immunoreactivity. In addition, TNF p75 receptor expression was detected in endothelial cells as well as astrocytes. In addition, only p65-Thr435 phosphorylation was increased in endothelial cells accompanied by SMI-71 expression (an endothelial barrier antigen. Neutralization of TNF-α by soluble TNF p55 receptor (sTNFp55R infusion attenuated SE-induced vasogenic edema and neuronal damages via inhibition of p65-Thr435 phosphorylation in endothelial cells. Furthermore, sTNFp55R infusion reduced SE-induced neutrophil infiltration in the PC. Conclusion These findings suggest that impairments of endothelial cell functions via TNF-α-mediated p65-Thr 485 NF-κB phosphorylation may be involved in SE-induced vasogenic edema. Subsequently, vasogenic edema results in extensive neutrophil infiltration and neuronal-astroglial loss.

  12. Diallyl trisulfide-induced apoptosis in human prostate cancer cells involves c-Jun N-terminal kinase and extracellular-signal regulated kinase-mediated phosphorylation of Bcl-2.

    Science.gov (United States)

    Xiao, Dong; Choi, Sunga; Johnson, Daniel E; Vogel, Victor G; Johnson, Candace S; Trump, Donald L; Lee, Yong J; Singh, Shivendra V

    2004-07-22

    Garlic-derived organosulfides (OSCs) including diallyl trisulfide (DATS) are highly effective in affording protection against chemically induced cancer in animals. Evidence is also mounting to indicate that some naturally occurring OSCs can suppress proliferation of cancer cells by causing apoptosis, but the sequence of events leading to proapoptotic effect of OSCs is poorly defined. Using PC-3 and DU145 human prostate cancer cells as a model, we now demonstrate that DATS is a significantly more potent apoptosis inducer than diallyl sulfide (DAS) or diallyl disulfide (DADS). DATS-induced apoptosis in PC-3 cells was associated with phosphorylation of Bcl-2, reduced Bcl-2 : Bax interaction, and cleavage of procaspase-9 and -3. Bcl-2 overexpressing PC-3 cells were significantly more resistant to apoptosis induction by DATS compared with vector-transfected control cells. DATS treatment resulted in activation of extracellular-signal regulated kinase 1/2 (ERK1/2) and c-jun N-terminal kinase 1 (JNK1) and/or JNK2, but not p38 mitogen-activated protein kinase. Phosphorylation of Bcl-2 in DATS-treated PC-3 cells was fully blocked in the presence of JNK-specific inhibitor SP600125. Moreover, JNK inhibitor afforded significant protection against DATS-induced apoptosis in both cells. DATS-induced Bcl-2 phosphorylation and apoptosis were partially attenuated by pharmacological inhibition of ERK1/2 using PD98059 or U0126. Overexpression of catalase inhibited DATS-mediated activation of JNK1/2, but not ERK1/2, and apoptosis induction in DU145 cells suggesting involvement of hydrogen peroxide as a second messenger in DATS-induced apoptosis. In conclusion, our data point towards important roles for Bcl-2, JNK and ERK in DATS-induced apoptosis in human prostate cancer cells.

  13. Protein kinase C-α signals P115RhoGEF phosphorylation and RhoA activation in TNF-α-induced mouse brain microvascular endothelial cell barrier dysfunction

    Directory of Open Access Journals (Sweden)

    Deng Xiaolu

    2011-04-01

    Full Text Available Abstract Background Tumor necrosis factor-α (TNF-α, a proinflammatory cytokine, is capable of activating the small GTPase RhoA, which in turn contributes to endothelial barrier dysfunction. However, the underlying signaling mechanisms remained undefined. Therefore, we aimed to determine the role of protein kinase C (PKC isozymes in the mechanism of RhoA activation and in signaling TNF-α-induced mouse brain microvascular endothelial cell (BMEC barrier dysfunction. Methods Bend.3 cells, an immortalized mouse brain endothelial cell line, were exposed to TNF-α (10 ng/mL. RhoA activity was assessed by pull down assay. PKC-α activity was measured using enzyme assasy. BMEC barrier function was measured by transendothelial electrical resistance (TER. p115RhoGEF phosphorylation was detected by autoradiography followed by western blotting. F-actin organization was observed by rhodamine-phalloidin staining. Both pharmacological inhibitors and knockdown approaches were employed to investigate the role of PKC and p115RhoGEF in TNF-α-induced RhoA activation and BMEC permeability. Results We observed that TNF-α induces a rapid phosphorylation of p115RhoGEF, activation of PKC and RhoA in BMECs. Inhibition of conventional PKC by Gö6976 mitigated the TNF-α-induced p115RhoGEF phosphorylation and RhoA activation. Subsequently, we found that these events are regulated by PKC-α rather than PKC-β by using shRNA. In addition, P115-shRNA and n19RhoA (dominant negative mutant of RhoA transfections had no effect on mediating TNF-α-induced PKC-α activation. These data suggest that PKC-α but not PKC-β acts as an upstream regulator of p115RhoGEF phosphorylation and RhoA activation in response to TNF-α. Moreover, depletion of PKC-α, of p115RhoGEF, and inhibition of RhoA activation also prevented TNF-α-induced stress fiber formation and a decrease in TER. Conclusions Taken together, our results show that PKC-α phosphorylation of p115RhoGEF mediates TNF

  14. Aggregate productivity and aggregate technology

    OpenAIRE

    Susanto Basu; John G. Fernald

    1997-01-01

    Aggregate productivity and aggregate technology are meaningful but distinct concepts. We show that a slightly-modified Solow productivity residual measures changes in economic welfare, even when productivity and technology differ because of distortions such as imperfect competition. We then present a general accounting framework that identifies several new non-technological gaps between productivity and technology, gaps reflecting imperfections and frictions in output and factor markets. Empi...

  15. Nanoparticle-Induced Ellipse-to-Vesicle Morphology Transition of Rod-Coil-Rod Triblock Copolymer Aggregates.

    Science.gov (United States)

    Yang, Chaoying; Li, Qing; Cai, Chunhua; Lin, Jiaping

    2016-07-12

    Cooperative self-assembly behavior of rod-coil-rod poly(γ-benzyl-l-glutamate)-block-poly(ethylene glycol)-block-poly(γ-benzyl-l-glutamate) (PBLG-b-PEG-b-PBLG) amphiphilic triblock copolymers and hydrophobic gold nanoparticles (AuNPs) was investigated by both experiments and dissipative particle dynamics (DPD) simulations. It was discovered that pure PBLG-b-PEG-b-PBLG copolymers self-assemble into ellipse-like aggregates, and the morphology transforms into vesicles as AuNPs are introduced. When the hydrophobicity of AuNPs is close to that of the copolymers, AuNPs are homogeneously distributed in the vesicle wall. While for the AuNPs with higher hydrophobicity, they are embedded in the vesicle wall as clusters. In addition to the experimental observations, DPD simulations were performed on the self-assembly behavior of triblock copolymer/nanoparticle mixtures. Simulations well reproduced the morphology transition observed in the experiments and provided additional information such as chain packing mode in aggregates. It is deduced that the main reason for the ellipse-to-vesicle transition of the aggregates is attributed to the breakage of ordered and dense packing of PBLG rods in the aggregate core by encapsulating AuNPs. This study deepens our understanding of the self-assembly behavior of rod-coil copolymer/nanoparticle mixtures and provides strategy for designing hybrid polypeptide nanostructures. PMID:27314970

  16. Temperature-induced aggregation in aqueous solutions of pluronic F68 triblock copolymer containing small amount of o-xylene

    DEFF Research Database (Denmark)

    Borbely, S.; Pedersen, J.S.

    2000-01-01

    The temperature- and concentration-dependent aggregation of EO(78)PO(30)EO(78) triblock copolymer in aqueous solutions has been studied in the concentration range from 5 to 200 g/dm(3) at 25 degrees C, 45 degrees C and 60 degrees C. The influence of o-xylene on the micellization was measured at v...

  17. Phosphorylation of Recombinant Tristetraprolin in Vitro

    OpenAIRE

    Cao, Heping; Lin, Rui

    2008-01-01

    Tristetraprolin/zinc finger protein 36 (TTP/ZFP36) binds and destabilizes some proinflammatory cytokine mRNAs. TTP-deficient mice develop a profound inflammatory syndrome due to excessive production of proinflammatory cytokines. TTP gene expression is induced by various factors including insulin, cinnamon, and green tea extracts. Previous studies have shown that TTP is highly phosphorylated in vivo and multiple phosphorylation sites are identified in human TTP. This study evaluated the potent...

  18. Viral infection increases glucocorticoid-induced interleukin-10 production through ERK-mediated phosphorylation of the glucocorticoid receptor in dendritic cells: potential clinical implications.

    Directory of Open Access Journals (Sweden)

    Sinnie Sin Man Ng

    Full Text Available The hypothalamic-pituitary-adrenal axis plays a central role in the adaptive response to stress including infection of pathogens through glucocorticoids. Physical and/or mental stress alter susceptibility to viral infection possibly by affecting this regulatory system, thus we explored potential cellular targets and mechanisms that underlie this phenomenon in key immune components dendritic cells (DCs. Dexamethasone (DEX treatment and subsequent Newcastle disease virus (NDV infection most significantly and cooperatively stimulated mRNA expression of the interleukin (IL-10 in murine bone marrow-derived DCs among 89 genes involved in the Toll-like receptor signaling pathways. NDV increased DEX-induced IL-10 mRNA and protein expression by 7- and 3-fold, respectively, which was observed from 3 hours after infection. Conventional DCs (cDCs, but not plasmacytoid DCs (pDCs were major sources of IL-10 in bone marrow-derived DCs treated with DEX and/or infected with NDV. Murine cytomegalovirus and DEX increased serum IL-10 cooperatively in female mice. Pre-treatment of DCs with the extracellular signal-regulated kinase (ERK inhibitor U0126 abolished cooperative induction of IL-10 by DEX and NDV. Further, ERK overexpression increased IL-10 promoter activity stimulated by wild-type human GR but not by its mutant defective in serine 203, whereas ERK knockdown abolished NDV/DEX cooperation on IL-10 mRNA and phosphorylation of the mouse GR at serine 213. NDV also increased DEX-induced mRNA expression of three known glucocorticoid-responsive genes unrelated to the Toll-like receptor signaling pathways in DCs. These results indicate that virus and glucocorticoids cooperatively increase production of anti-inflammatory cytokine IL-10 by potentiating the transcriptional activity of GR in DCs, through which virus appears to facilitate its own propagation in infected hosts. The results may further underlie in part known exacerbation of IL-10/T helper-2-related

  19. Aggregation and structural changes of α(S1)-, β- and κ-caseins induced by homocysteinylation.

    Science.gov (United States)

    Stroylova, Yulia Y; Zimny, Jaroslaw; Yousefi, Reza; Chobert, Jean-Marc; Jakubowski, Hieronim; Muronetz, Vladimir I; Haertlé, Thomas

    2011-10-01

    Elevated homocysteine levels are resulting in N-homocysteinylation of lysyl residues in proteins and they correlate with a number of human pathologies. However, the role of homocysteinylation of lysyl residues is still poorly known. In order to study the features of homocysteinylation of intrinsically unstructured proteins (IUP) bovine caseins were used as a model. α(S1)-, β- and κ-caseins, showing different aggregations and micelle formation, were modified with homocysteine-thiolactone and their physico-chemical properties were studied. Efficiency of homocysteine incorporation was estimated to be about 1.5, 2.1 and 1.3 homocysteyl residues per one β-, α(S1)-, and κ-casein molecule, respectively. Use of intrinsic and extrinsic fluorescent markers such as Trp, thioflavin T and ANS, reveal structural changes of casein structures after homocysteinylation reflected by an increase in beta-sheet content, which in some cases may be characteristic of amyloid-like transformations. CD spectra also show an increase in beta-sheet content of homocysteinylated caseins. Casein homocysteinylation leads in all cases to aggregation. The sizes of aggregates and aggregation rates were dependent on homocysteine thiolactone concentration and temperature. DLS and microscopic studies have revealed the formation of large aggregates of about 1-3μm. Homocysteinylation of α(S1)- and β-caseins results in formation of regular spheres. Homocysteinylated κ-casein forms thin unbranched fibrils about 400-800nm long. In case of κ-casein amyloidogenic effect of homocysteinylation was confirmed by Congo red spectra. Taken together, data indicate that N-homocysteinylation provokes significant changes in properties of native caseins. A comparison of amyloidogenic transformation of 3 different casein types, belonging to the IUP protein family, shows that the efficiency of amyloidogenic transformation upon homocysteinylation depends on micellization capacity, additional disulphide bonds and

  20. Bypassing AMPK Phosphorylation

    OpenAIRE

    Viollet, Benoit; Foretz, Marc; Schlattner, Uwe

    2014-01-01

    AMP-activated protein kinase (AMPK) functions as a signaling hub to balance energy supply with demand. Phosphorylation of activation loop Thr172 has been considered as an essential step in AMPK activation. In this issue of Chemistry & Biology, Scott and colleagues show that the small molecule direct AMPK activator, A-769662, bypasses this phosphorylation event, and acts synergistically with AMP on naive AMPK.

  1. Jaridonin-induced G2/M phase arrest in human esophageal cancer cells is caused by reactive oxygen species-dependent Cdc2-tyr15 phosphorylation via ATM–Chk1/2–Cdc25C pathway

    Energy Technology Data Exchange (ETDEWEB)

    Ma, Yong-Cheng [Clinical Pharmacology Laboratory, Henan Province People' s Hospital, No. 7, Wei Wu Road, Zhengzhou, Henan (China); Su, Nan [Department of Quality Detection and Management, Henan University of Animal Husbandry and Economy, Zhengzhou, Henan (China); Shi, Xiao-Jing; Zhao, Wen; Ke, Yu [School of Pharmaceutical Sciences, Zhengzhou University, No. 100, Science Avenue, Zhengzhou, Henan (China); Zi, Xiaolin [Department of Urology, University of California, Irvine, Orange, CA (United States); Department of Pharmacology, University of California, Irvine, Orange, CA (United States); Department of Pharmaceutical Sciences, University of California, Irvine, Orange, CA (United States); Zhao, Ning-Min; Qin, Yu-Hua; Zhao, Hong-Wei [Clinical Pharmacology Laboratory, Henan Province People' s Hospital, No. 7, Wei Wu Road, Zhengzhou, Henan (China); Liu, Hong-Min, E-mail: liuhm@zzu.edu.cn [School of Pharmaceutical Sciences, Zhengzhou University, No. 100, Science Avenue, Zhengzhou, Henan (China)

    2015-01-15

    Jaridonin, a novel diterpenoid from Isodon rubescens, has been shown previously to inhibit proliferation of esophageal squamous cancer cells (ESCC) through G2/M phase cell cycle arrest. However, the involved mechanism is not fully understood. In this study, we found that the cell cycle arrest by Jaridonin was associated with the increased expression of phosphorylation of ATM at Ser1981 and Cdc2 at Tyr15. Jaridonin also resulted in enhanced phosphorylation of Cdc25C via the activation of checkpoint kinases Chk1 and Chk2, as well as in increased phospho-H2A.X (Ser139), which is known to be phosphorylated by ATM in response to DNA damage. Furthermore, Jaridonin-mediated alterations in cell cycle arrest were significantly attenuated in the presence of NAC, implicating the involvement of ROS in Jaridonin's effects. On the other hand, addition of ATM inhibitors reversed Jaridonin-related activation of ATM and Chk1/2 as well as phosphorylation of Cdc25C, Cdc2 and H2A.X and G2/M phase arrest. In conclusion, these findings identified that Jaridonin-induced cell cycle arrest in human esophageal cancer cells is associated with ROS-mediated activation of ATM–Chk1/2–Cdc25C pathway. - Highlights: • Jaridonin induced G2/M phase arrest through induction of redox imbalance. • Jaridonin increased the level of ROS through depleting glutathione in cell. • ATM–Chk1/2–Cdc25C were involved in Jaridonin-induced cell cycle arrest. • Jaridonin selectively inhibited cancer cell viability and cell cycle progression.

  2. PKA regulates calcineurin function through the phosphorylation of RCAN1: Identification of a novel phosphorylation site

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Seon Sook; Lee, Eun Hye [Department of Molecular Bioscience, College of Biomedical Science, Institute of Bioscience & Biotechnology, Kangwon National University, Chuncheon 200-701 (Korea, Republic of); Lee, Kooyeon [Department of Bio-Health Technology, College of Biomedical Science, Institute of Bioscience & Biotechnology, Kangwon National University, Chuncheon 200-701 (Korea, Republic of); Jo, Su-Hyun, E-mail: suhyunjo@kangwon.ac.kr [Department of Physiology, BK21 Plus Graduate Program, Institute of Bioscience & Biotechnology, Kangwon National University, Chuncheon 200-701 (Korea, Republic of); Seo, Su Ryeon, E-mail: suryeonseo@kangwon.ac.kr [Department of Molecular Bioscience, College of Biomedical Science, Institute of Bioscience & Biotechnology, Kangwon National University, Chuncheon 200-701 (Korea, Republic of)

    2015-04-17

    Calcineurin is a calcium/calmodulin-dependent phosphatase that has been implicated in T cell activation through the induction of nuclear factors of activated T cells (NFAT). We have previously suggested that endogenous regulator of calcineurin (RCAN1, also known as DSCR1) is targeted by protein kinase A (PKA) for the control of calcineurin activity. In the present study, we characterized the PKA-mediated phosphorylation site in RCAN1 by mass spectrometric analysis and revealed that PKA directly phosphorylated RCAN1 at the Ser 93. PKA-induced phosphorylation and the increase in the half-life of the RCAN1 protein were prevented by the substitution of Ser 93 with Ala (S93A). Furthermore, the PKA-mediated phosphorylation of RCAN1 at Ser 93 potentiated the inhibition of calcineurin-dependent pro-inflammatory cytokine gene expression by RCAN1. Our results suggest the presence of a novel phosphorylation site in RCAN1 and that its phosphorylation influences calcineurin-dependent inflammatory target gene expression. - Highlights: • We identify novel phosphorylation sites in RCAN1 by LC-MS/MS analysis. • PKA-dependent phosphorylation of RCAN1 at Ser 93 inhibits calcineurin-mediated intracellular signaling. • We show the immunosuppressive function of RCAN1 phosphorylation at Ser 93 in suppressing cytokine expression.

  3. Role of protein kinase D2 phosphorylation on Tyr in modulation by ghrelin of Helicobacter pylori-induced up-regulation in gastric mucosal matrix metalloproteinase-9 (MMP-9) secretion.

    Science.gov (United States)

    Slomiany, B L; Slomiany, A

    2016-06-01

    Matrix metalloproteinas-9 (MMP-9) is a glycosylated endopeptidase associated with host reaction to microbial endotoxins and also characterizes gastric mucosal inflammatory response to H. pylori infection. Here, we report on the factors involved in gastric mucosal MMP-9 secretion in response to H. pylori LPS, and the effect of hormone, ghrelin. We show that both the LPS-elicited induction in MMP-9 secretion and also the modulatory influence of ghrelin occur at the level of MMP-9 processing between the endoplasmic reticulum (ER) and Golgi. Further, we demonstrate that the LPS effect is associated with up-regulation in the activation of Arf1, a small GTPase of the ADP-ribosylation factor family, and the recruitment and phosphorylation of protein kinase D2 (PKD2), involved in the secretory cargo processing in the Golgi. Moreover, we reveal that the LPS-induced up-regulation in MMP-9 secretion is reflected in a marked increase in PKCδ-mediated PKD2 phosphorylation on Ser, while the modulatory effect of ghrelin is manifested by the SFK-PTKs-dependent phosphorylation of PKD2 on Tyr. Thus, our findings demonstrate the role of Arf1/PKD2 in mediation of H. pylori LPS-induced up-regulation in gastric mucosal MMP-9 secretion and suggest the modulatory mechanism of ghrelin action. PMID:27209313

  4. Effect of extraction pH on heat-induced aggregation, gelation and microstructure of protein isolate from quinoa (Chenopodium quinoa Willd).

    Science.gov (United States)

    Ruiz, Geraldine Avila; Xiao, Wukai; van Boekel, Martinus; Minor, Marcel; Stieger, Markus

    2016-10-15

    The aim of this study was to determine the influence of extraction pH on heat-induced aggregation, gelation and microstructure of suspensions of protein isolates extracted from quinoa (Chenopodium quinoa Willd). Quinoa seed protein was extracted by alkaline treatment at various pH values (pH 8 (E8), 9 (E9), 10 (E10) and 11 (E11)), followed by acid precipitation. The obtained protein isolates were freeze dried. The protein isolates E8 and E9 resulted in a lower protein yield as well as less protein denaturation. These isolates also had a higher protein purity, more protein bands at higher molecular weights, and a higher protein solubility in the pH range of 3-4.5, compared to the isolates E10 and E11. Heating the 10%w/w protein isolate suspensions E8 and E9 led to increased aggregation, and semi-solid gels with a dense microstructure were formed. The isolate suspensions E10 and E11, on the other hand, aggregated less, did not form self-supporting gels and had loose particle arrangements. We conclude that extraction pH plays an important role in determining the functionality of quinoa protein isolates. PMID:27173553

  5. Stable and Size-Tunable Aggregation-Induced Emission Nanoparticles Encapsulated with Nanographene Oxide and Applications in Three-Photon Fluorescence Bioimaging.

    Science.gov (United States)

    Zhu, Zhenfeng; Qian, Jun; Zhao, Xinyuan; Qin, Wei; Hu, Rongrong; Zhang, Hequn; Li, Dongyu; Xu, Zhengping; Tang, Ben Zhong; He, Sailing

    2016-01-26

    Organic fluorescent dyes with high quantum yield are widely applied in bioimaging and biosensing. However, most of them suffer from a severe effect called aggregation-caused quenching (ACQ), which means that their fluorescence is quenched at high molecular concentrations or in the aggregation state. Aggregation-induced emission (AIE) is a diametrically opposite phenomenon to ACQ, and luminogens with this feature can effectively solve this problem. Graphene oxide has been utilized as a quencher for many fluorescent dyes, based on which biosensing can be achieved. However, using graphene oxide as a surface modification agent of fluorescent nanoparticles is seldom reported. In this article, we used nanographene oxide (NGO) to encapsulate fluorescent nanoparticles, which consisted of a type of AIE dye named TPE-TPA-FN (TTF). NGO significantly improved the stability of nanoparticles in aqueous dispersion. In addition, this method could control the size of nanoparticles' flexibly as well as increase their emission efficiency. We then used the NGO-modified TTF nanoparticles to achieve three-photon fluorescence bioimaging. The architecture of ear blood vessels in mice and the distribution of nanoparticles in zebrafish could be observed clearly. Furthermore, we extended this method to other AIE luminogens and showed it was widely feasible.

  6. Design of small molecules that target metal-A{beta} species and regulate metal-induced A{beta} aggregation and neurotoxicity.

    Science.gov (United States)

    Choi, Jung-Suk; Braymer, Joseph J; Nanga, Ravi P R; Ramamoorthy, Ayyalusamy; Lim, Mi Hee

    2010-12-21

    The accumulation of metal ions and amyloid-β (Aβ) aggregates found in the brain of patients with Alzheimer's disease (AD) has been suggested to be involved in AD pathogenesis. To investigate metal-Aβ-associated pathways in AD, development of chemical tools to target metal-Aβ species is desired. Only a few efforts, however, have been reported. Here, we report bifunctional small molecules, N-(pyridin-2-ylmethyl)aniline (L2-a) and N(1),N(1)-dimethyl-N(4)-(pyridin-2-ylmethyl)benzene-1,4-diamine (L2-b) that can interact with both metal ions and Aβ species, as determined by spectroscopic methods including high-resolution NMR spectroscopy. Using the bifunctional compound L2-b, metal-inducedaggregation and neurotoxicity were modulated in vitro as well as in human neuroblastoma cells. Furthermore, treatment of human AD brain tissue homogenates containing metal ions and Aβ species with L2-b showed disassembly of Aβ aggregates. Therefore, our studies presented herein demonstrate the value of bifunctional compounds as chemical tools for investigating metal-Aβ-associated events and their mechanisms in the development and pathogenesis of AD and as potential therapeutics.

  7. Sulindac Sulfide Induces the Formation of Large Oligomeric Aggregates of the Alzheimer's Disease Amyloid-β Peptide Which Exhibit Reduced Neurotoxicity.

    Science.gov (United States)

    Prade, Elke; Barucker, Christian; Sarkar, Riddhiman; Althoff-Ospelt, Gerhard; Lopez del Amo, Juan Miguel; Hossain, Shireen; Zhong, Yifei; Multhaup, Gerd; Reif, Bernd

    2016-03-29

    Alzheimer's disease is characterized by deposition of the amyloid β-peptide (Aβ) in brain tissue of affected individuals. In recent years, many potential lead structures have been suggested that can potentially be used for diagnosis and therapy. However, the mode of action of these compounds is so far not understood. Among these small molecules, the nonsteroidal anti-inflammatory drug (NSAID) sulindac sulfide received a lot of attention. In this manuscript, we characterize the interaction between the monomeric Aβ peptide and the NSAID sulindac sulfide. We find that sulindac sulfide efficiently depletes the pool of toxic oligomers by enhancing the rate of fibril formation. In vitro, sulindac sulfide forms colloidal particles which catalyze the formation of fibrils. Aggregation is immediate, presumably by perturbing the supersaturated Aβ solution. We find that sulindac sulfide inducedaggregates are structurally homogeneous. The C-terminal part of the peptide adopts a β-sheet structure, whereas the N-terminus is disordered. The salt bridge between D23 and K28 is present, similar as in wild type fibril structures. (13)C-(19)F transferred echo double resonance experiments suggest that sulindac sulfide colocalizes with the Aβ peptide in the aggregate. PMID:26900939

  8. Pulsed Laser-Driven Molecular Self-assembly of Cephalexin: Aggregation-Induced Fluorescence and Its Utility as a Mercury Ion Sensor.

    Science.gov (United States)

    Singh, Pradeep Kumar; Prabhune, Asmita; Ogale, Satishchandra

    2015-11-01

    A fluorescent self-assembly of cephalexin is obtained by pulsed laser irradiation process. An intense fluorescence emission is found in the self-assembled form due to occurrence of a typical aggregation-induced emission in cephalexin molecules. It is observed that fluorescence quenching of the self-assembled fluorescent nanostructures occurs in the presence of extremely low Hg(++) ions concentrations (10(-7) m) as compared to other heavy metal ions e.g. Ferrous (Fe(++) ), Manganese (Mn(++) ), Magnesium (Mg(++) ), Cobalt (Co(++) ), Nickel (Ni(++) ) and Zinc (Zn(++) ) at the same concentrations. PMID:26333412

  9. Antigen 43 from Escherichia coli induces inter- and intraspecies cell aggregation and changes in colony morphology of Pseudomonas fluorescens

    DEFF Research Database (Denmark)

    Kjærgaard, Kristian; Schembri, Mark; Hasman, Henrik;

    2000-01-01

    demonstrate that Ag43 can be used as a tool to provide interspecies cell aggregation between E. coli and P. fluorescens. Furthermore, Ag43 expression enhances biofilm formation in P. fluorescens to glass surfaces. The versatility of this protein was also reflected in Ag43 surface display in a variety of other...... with a distinct frizzy colony morphology in E. coli. Here we show that Ag43 can be expressed in a functional form on the surface of the environmentally important Pseudomonas fluorescens strain SBW25 with ensuing cell aggregation and frizzy colony types. Using green fluorescence protein-tagged cells, we...... gram-negative bacteria, Display of heterologous Ag43 in selected bacteria might offer opportunities for rational design of multispecies consortia where the concerted action of several bacterial species is required, e.g., waste treatment and degradation of pollutants....

  10. DNA base-stacking assay utilizing catalytic hairpin assembly-induced gold nanoparticle aggregation for colorimetric protein sensing.

    Science.gov (United States)

    Chang, Chia-Chen; Chen, Chie-Pein; Chen, Chen-Yu; Lin, Chii-Wann

    2016-03-18

    A label-free and enzyme-free colorimetric sensing platform for the amplified detection of fibronectin was developed based on an ingenious combination of catalytic hairpin assembly and a base stacking hybridization-based gold nanoparticle aggregation strategy. The detection limit of 2.3 pM is at least one order of magnitude lower than that of established fibronectin biosensors. PMID:26906691

  11. Hydrogen peroxide inhibits transforming growth factor-β1-induced cell cycle arrest by promoting Smad3 linker phosphorylation through activation of Akt-ERK1/2-linked signaling pathway.

    Science.gov (United States)

    Choi, Jiyeon; Park, Seong Ji; Jo, Eun Ji; Lee, Hui-Young; Hong, Suntaek; Kim, Seong-Jin; Kim, Byung-Chul

    2013-06-14

    Hydrogen peroxide (H2O2) functions as a second messenger in growth factor receptor-mediated intracellular signaling cascade and is tumorigenic by virtue of its ability to promote cell proliferation; however, the mechanisms underlying the growth stimulatory action of H2O2 are less understood. Here we report an important mechanism for antagonistic effects of H2O2 on growth inhibitory response to transforming growth factor-β1 (TGF-β1). In Mv1Lu and HepG2 cells, pretreatment of H2O2 (0.05-0.2 mM) completely blocked TGF-β1-mediated induction of p15(INK4B) expression and increase of its promoter activity. Interestingly, H2O2 selectively suppressed the transcriptional activation potential of Smad3, not Smad2, in the absence of effects on TGF-β1-induced phosphorylation of the COOH-tail SSXS motif of Smad3 and its nuclear translocation. Mechanism studies showed that H2O2 increases the phosphorylation of Smad3 at the middle linker region in a concentration- and time-dependent manner and this effect is mediated by activation of extracellular signal-activated kinase 1/2 through Akt. Furthermore, expression of a mutant Smad3 in which linker phosphorylation sites were ablated significantly abrogated the inhibitory effects of H2O2 on TGF-β1-induced increase of p15(INK4B)-Luc reporter activity and blockade of cell cycle progression from G1 to S phase. These findings for the first time define H2O2 as a signaling molecule that modulate Smad3 linker phosphorylation and its transcriptional activity, thus providing a potential mechanism whereby H2O2 antagonizes the cytostatic function of TGF-β1.

  12. Erection capability is potentiated by long-term sildenafil treatment: role of blood flow-induced endothelial nitric-oxide synthase phosphorylation.

    Science.gov (United States)

    Musicki, Biljana; Champion, Hunter C; Becker, Robyn E; Liu, Tongyun; Kramer, Melissa F; Burnett, Arthur L

    2005-07-01

    Despite demonstrated clinical efficacy of sildenafil for the temporary treatment of erectile dysfunction, the possibility that sildenafil used long-term durably augments erectile ability remains unclear. We investigated whether continuous long-term administration of sildenafil at clinically relevant levels to aged rats "primes" the penis for improved erectile ability and involves nitric oxide (NO) or RhoA/Rho-kinase signaling pathways. In aged, but not young rats, sildenafil prolonged erection and increased the protein expressions of phosphorylated endothelial NO synthase (eNOS) at serine-1177 and phosphorylated Akt at serine-473 in penes. Only in the young rat penis, protein expressions of phosphodiesterase-5 and phosphomyosin phosphatase target subunit 1, a marker of Rho-kinase activity, were increased by sildenafil. Sildenafil inhibited phosphodiesterase-5 activity in penes of young and aged rats coincident with assayed free plasma levels of the drug equivalent to clinically therapeutic measurements. We conclude that erectile ability can be enhanced under preconditions of erectile impairment by long-term inhibition of phosphodiesterase-5 and that the effect is mediated by Akt-dependent eNOS phosphorylation. The lack of erectile ability enhancement in young rats by long-term phosphodiesterase-5 inhibition may relate to restrained NO signaling by phosphodiesterase-5 up-regulation, lack of incremental Akt and eNOS phosphorylation, and heightened Rho-kinase signaling in the penis. PMID:15851653

  13. Interleukin-6-induced STAT3 transactivation and Ser(727) phosphorylation involves Vav, Rac-1 and the kinase SEK-1/MKK-4 as signal transduction components

    NARCIS (Netherlands)

    Schuringa, JJ; Jonk, LJC; Dokter, WHA; Vellenga, E; Kruijer, W

    2000-01-01

    In the present study, signal transducer and activator of transcription 3 (STAT3) Ser(727) phosphorylation and transactivation was investigated in relation to activation of mitogen-activated protein (MAP) kinase family members including extracellular-signal-regulated protein kinase (ERK)-1, c-Jun N-t

  14. Integrin-linked Kinase Modulates Lipopolysaccharide- and Helicobacter pylori-induced Nuclear Factor κB-activated Tumor Necrosis Factor-α Production via Regulation of p65 Serine 536 Phosphorylation*

    Science.gov (United States)

    Ahmed, Afsar U.; Sarvestani, Soroush T.; Gantier, Michael P.; Williams, Bryan R. G.; Hannigan, Gregory E.

    2014-01-01

    Integrin-linked kinase (ILK) is a ubiquitously expressed and highly conserved serine-threonine protein kinase that regulates cellular responses to a wide variety of extracellular stimuli. ILK is involved in cell-matrix interactions, cytoskeletal organization, and cell signaling. ILK signaling has also been implicated in oncogenesis and progression of cancers. However, its role in the innate immune system remains unknown. Here, we show that ILK mediates pro-inflammatory signaling in response to lipopolysaccharide (LPS). Pharmacological or genetic inhibition of ILK in mouse embryonic fibroblasts and macrophages selectively blocks LPS-indu