WorldWideScience

Sample records for total pkc activity

  1. Regulation of CCK-induced ERK1/2 activation by PKC epsilon in rat pancreatic acinar cells

    Directory of Open Access Journals (Sweden)

    Chenwei Li

    2017-11-01

    Full Text Available The extracellular signal-regulated kinase ERK1/2 is activated in pancreatic acinar cells by cholecystokinin (CCK and other secretagogues with this activation mediated primarily by protein kinase C (PKC. To identify the responsible PKC isoform, we utilized chemical inhibitors, cell permeant inhibitory peptides and overexpression of individual PKC dominant negative variants by means of adenoviral vectors. While the broad-spectrum PKC inhibitor GF109203X strongly inhibited ERK1/2 activation induced by 100 pM CCK, Go6976 which inhibits the classical PKC isoforms (alpha, beta and gamma, as well as Rottlerin, a specific PKC delta inhibitor, had no inhibitory effect. To test the role of PKC epsilon, we used specific cell permeant peptide inhibitors which block PKC interaction with their intracellular receptors or RACKs. Only PP93 (PKC epsilon peptide inhibitor inhibited CCK-induced ERK1/2 activation, while PP95, PP101 and PP98, which are PKC alpha, delta and zeta peptide inhibitors respectively, had no effect. We also utilized adenovirus to express dominant negative PKC isoforms in pancreatic acini. Only PKC epsilon dominant negative inhibited CCK-induced ERK1/2 activation. Dominant negative PKC epsilon expression similarly blocked the effect of carbachol and bombesin to activate ERK1/2. Immunoprecipitation results demonstrated that CCK can induce an interaction of c-Raf-1 and PKC epsilon, but not that of other isoforms of Raf or PKC. We conclude that PKC epsilon is the isoform of PKC primarily involved with CCK-induced ERK1/2 activation in pancreatic acinar cells.

  2. A novel mouse PKC{delta} splice variant, PKC{delta}IX, inhibits etoposide-induced apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jung D. [School of Biological Sciences, University of Ulsan, Ulsan (Korea, Republic of); Seo, Kwang W. [Department of Internal Medicines, Ulsan University Hospital and School of Medicine, University of Ulsan, Ulsan (Korea, Republic of); Lee, Eun A.; Quang, Nguyen N. [School of Biological Sciences, University of Ulsan, Ulsan (Korea, Republic of); Cho, Hong R. [Department of Surgery, Ulsan University Hospital and School of Medicine, University of Ulsan, Ulsan (Korea, Republic of); Biomedical Research Center, Ulsan University Hospital and School of Medicine, University of Ulsan, Ulsan (Korea, Republic of); Kwon, Byungsuk, E-mail: bskwon@mail.ulsan.as.kr [School of Biological Sciences, University of Ulsan, Ulsan (Korea, Republic of); Biomedical Research Center, Ulsan University Hospital and School of Medicine, University of Ulsan, Ulsan (Korea, Republic of)

    2011-07-01

    Highlights: {yields} A novel PKC{delta} isoform, named PKC{delta}IX, that lacks the C1 domain and the ATP-binding site is ubiquitously expressed. {yields} PKC{delta}IX inhibits etoposide-induced apoptosis. {yields} PKC{delta}IX may function as an endogenous dominant negative isoform for PKC{delta}. -- Abstract: Protein kinase C (PKC) {delta} plays an important role in cellular proliferation and apoptosis. The catalytic fragment of PKC{delta} generated by caspase-dependent cleavage is essential for the initiation of etoposide-induced apoptosis. In this study, we identified a novel mouse PKC{delta} isoform named PKC{delta}IX (Genebank Accession No. (HQ840432)). PKC{delta}IX is generated by alternative splicing and is ubiquitously expressed, as seen in its full-length PKC{delta}. PKC{delta}IX lacks the C1 domain, the caspase 3 cleavage site, and the ATP binding site but preserves an almost intact c-terminal catalytic domain and a nuclear localization signal (NLS). The structural characteristics of PKC{delta}IX provided a possibility that this PKC{delta} isozyme functions as a novel dominant-negative form for PKC{delta} due to its lack of the ATP-binding domain that is required for the kinase activity of PKC{delta}. Indeed, overexpression of PKC{delta}IX significantly inhibited etoposide-induced apoptosis in NIH3T3 cells. In addition, an in vitro kinase assay showed that recombinant PKC{delta}IX protein could competitively inhibit the kinase activity of PKC{delta}. We conclude that PKC{delta}IX can function as a natural dominant-negative inhibitor of PKC{delta}in vivo.

  3. Oncogenic PKCactivates Vimentin during epithelial-mesenchymal transition in melanoma; a study based on PKC-ι and PKC-ζ specific inhibitors.

    Science.gov (United States)

    Ratnayake, Wishrawana S; Apostolatos, Christopher A; Apostolatos, André H; Schutte, Ryan J; Huynh, Monica A; Ostrov, David A; Acevedo-Duncan, Mildred

    2018-05-21

    Melanoma is one of the fastest growing cancers in the United States and is accompanied with a poor prognosis owing to tumors being resistant to most therapies. Atypical protein kinase Cs (aPKC) are involved in malignancy in many cancers. We previously reported that aPKCs play a key role in melanoma's cell motility by regulating cell signaling pathways which induce epithelial-mesenchymal Transition (EMT). We tested three novel inhibitors; [4-(5-amino-4-carbamoylimidazol-1-yl)-2,3-dihydroxycyclopentyl] methyl dihydrogen phosphate (ICA-1T) along with its nucleoside analog 5-amino-1-((1R,2S,3S,4R)-2,3-dihydroxy-4-methylcyclopentyl)-1H-imidazole-4-carboxamide (ICA-1S) which are specific to protein kinase C-iota (PKC-ι) and 8-hydroxy-1,3,6-naphthalenetrisulfonic acid (ζ-Stat) which is specific to PKC-zeta (PKC-ζ) on cell proliferation, apoptosis, migration and invasion of two malignant melanoma cell lines compared to normal melanocytes. Molecular modeling was used to identify potential binding sites for the inhibitors and to predict selectivity. Kinase assay showed >50% inhibition for specified targets beyond 5 μM for all inhibitors. Both ICA-1 and ζ-Stat significantly reduced cell proliferation and induced apoptosis, while ICA-1 also significantly reduced migration and melanoma cell invasion. PKC-ι stimulated EMT via TGFβ/Par6/RhoA pathway and activated Vimentin by phosphorylation at S39. Both ICA-1 and ζ-Stat downregulate TNF-α induced NF-κB translocation to the nucleus there by inducing apoptosis. Results suggest that PKC-ι is involved in melanoma malignancy than PKC-ζ. Inhibitors proved to be effective under in-vitro conditions and need to be tested in-vivo for the validity as effective therapeutics. Overall, results show that aPKCs are essential for melanoma progression and metastasis and that they could be used as effective therapeutic targets for malignant melanoma.

  4. NMDA modulates oligodendrocyte differentiation of subventricular zone cells through PKC activation

    Directory of Open Access Journals (Sweden)

    Fabio eCavaliere

    2013-12-01

    Full Text Available Multipotent cells from the juvenile subventricular zone (SVZ possess the ability to differentiate into new neural cells. Depending on local signals, SVZ can generate new neurons, astrocytes or oligodendrocytes. We previously demonstrated that activation of NMDA receptors in SVZ progenitors increases the rate of oligodendrocyte differentiation. Here we investigated the mechanisms involved in NMDA receptor-dependent differentiation. Using functional studies performed with the reporter gene luciferase we found that activation of NMDA receptor stimulates PKC. In turn, stimulation of PKC precedes the activation of NADPH oxidase (NOX as demonstrated by translocation of the p67phox subunit to the cellular membrane. We propose that NOX2 is involved in the transduction of the signal from NMDA receptors through PKC activation as the inhibitor gp91 reduced their pro-differentiation effect. In addition, our data and that from other groups suggest that signaling through the NMDA receptor/PKC/NOX2 cascade generates ROS that activate the PI3/mTOR pathway and finally leads to the generation of new oligodendrocytes.

  5. Atypical PKC, PKCλ/ι, activates β-secretase and increases Aβ1-40/42 and phospho-tau in mouse brain and isolated neuronal cells, and may link hyperinsulinemia and other aPKC activators to development of pathological and memory abnormalities in Alzheimer's disease.

    Science.gov (United States)

    Sajan, Mini P; Hansen, Barbara C; Higgs, Margaret G; Kahn, C Ron; Braun, Ursula; Leitges, Michael; Park, Collin R; Diamond, David M; Farese, Robert V

    2018-01-01

    Hyperinsulinemia activates brain Akt and PKC-λ/ι and increases Aβ 1-40/42 and phospho-tau in insulin-resistant animals. Here, we examined underlying mechanisms in mice, neuronal cells, and mouse hippocampal slices. Like Aβ 1-40/42 , β-secretase activity was increased in insulin-resistant mice and monkeys. In insulin-resistant mice, inhibition of hepatic PKC-λ/ι sufficient to correct hepatic abnormalities and hyperinsulinemia simultaneously reversed increases in Akt, atypical protein kinase C (aPKC), β-secretase, and Aβ 1-40/42 , and restored acute Akt activation. However, 2 aPKC inhibitors additionally blocked insulin's ability to activate brain PKC-λ/ι and thereby increase β-secretase and Aβ 1-40/42 . Furthermore, direct blockade of brain aPKC simultaneously corrected an impairment in novel object recognition in high-fat-fed insulin-resistant mice. In neuronal cells and/or mouse hippocampal slices, PKC-ι/λ activation by insulin, metformin, or expression of constitutive PKC-ι provoked increases in β-secretase, Aβ 1-40/42 , and phospho-thr-231-tau that were blocked by various PKC-λ/ι inhibitors, but not by an Akt inhibitor. PKC-λ/ι provokes increases in brain β-secretase, Aβ 1-40/42 , and phospho-thr-231-tau. Excessive signaling via PKC-λ/ι may link hyperinsulinemia and other PKC-λ/ι activators to pathological and functional abnormalities in Alzheimer's disease. Published by Elsevier Inc.

  6. Investigating the Potential Signaling Pathways That Regulate Activation of the Novel PKC Downstream of Serotonin in Aplysia.

    Directory of Open Access Journals (Sweden)

    Carole A Farah

    Full Text Available Activation of the novel PKC Apl II in sensory neurons by serotonin (5HT underlies the ability of 5HT to reverse synaptic depression, but the pathway from 5HT to PKC Apl II activation remains unclear. Here we find no evidence for the Aplysia-specific B receptors, or for adenylate cyclase activation, to translocate fluorescently-tagged PKC Apl II. Using an anti-PKC Apl II antibody, we monitor translocation of endogenous PKC Apl II and determine the dose response for PKC Apl II translocation, both in isolated sensory neurons and sensory neurons coupled with motor neurons. Using this assay, we confirm an important role for tyrosine kinase activation in 5HT mediated PKC Apl II translocation, but rule out roles for intracellular tyrosine kinases, epidermal growth factor (EGF receptors and Trk kinases in this response. A partial inhibition of translocation by a fibroblast growth factor (FGF-receptor inhibitor led us to clone the Aplysia FGF receptor. Since a number of related receptors have been recently characterized, we use bioinformatics to define the relationship between these receptors and find a single FGF receptor orthologue in Aplysia. However, expression of the FGF receptor did not affect translocation or allow it in motor neurons where 5HT does not normally cause PKC Apl II translocation. These results suggest that additional receptor tyrosine kinases (RTKs or other molecules must also be involved in translocation of PKC Apl II.

  7. Natural Product Vibsanin A Induces Differentiation of Myeloid Leukemia Cells through PKC Activation.

    Science.gov (United States)

    Yu, Zu-Yin; Xiao, He; Wang, Li-Mei; Shen, Xing; Jing, Yu; Wang, Lin; Sun, Wen-Feng; Zhang, Yan-Feng; Cui, Yu; Shan, Ya-Jun; Zhou, Wen-Bing; Xing, Shuang; Xiong, Guo-Lin; Liu, Xiao-Lan; Dong, Bo; Feng, Jian-Nan; Wang, Li-Sheng; Luo, Qing-Liang; Zhao, Qin-Shi; Cong, Yu-Wen

    2016-05-01

    All-trans retinoic acid (ATRA)-based cell differentiation therapy has been successful in treating acute promyelocytic leukemia, a unique subtype of acute myeloid leukemia (AML). However, other subtypes of AML display resistance to ATRA-based treatment. In this study, we screened natural, plant-derived vibsane-type diterpenoids for their ability to induce differentiation of myeloid leukemia cells, discovering that vibsanin A potently induced differentiation of AML cell lines and primary blasts. The differentiation-inducing activity of vibsanin A was mediated through direct interaction with and activation of protein kinase C (PKC). Consistent with these findings, pharmacological blockade of PKC activity suppressed vibsanin A-induced differentiation. Mechanistically, vibsanin A-mediated activation of PKC led to induction of the ERK pathway and decreased c-Myc expression. In mouse xenograft models of AML, vibsanin A administration prolonged host survival and inhibited PKC-mediated inflammatory responses correlated with promotion of skin tumors in mice. Collectively, our results offer a preclinical proof of concept for vibsanin A as a myeloid differentiation-inducing compound, with potential application as an antileukemic agent. Cancer Res; 76(9); 2698-709. ©2016 AACR. ©2016 American Association for Cancer Research.

  8. Black Ink of Activated Carbon Derived From Palm Kernel Cake (PKC)

    Science.gov (United States)

    Selamat, M. H.; Ahmad, A. H.

    2009-06-01

    Recycling the waste from natural plant to produce useful end products will benefit many industries and help preserve the environment. The research reported in this paper is an investigation on the use of the natural waste of palm kernel cake (PKC) to produce carbon residue as a black carbon for pigment source by using pyrolysis process. The activated carbons (AC) is produced in powder form using ball milling process. Rheological spectra in ink is one of quality control process in determining its performance properties. Findings from this study will help expand the scientific knowledge-base for black ink production and formulation base on PKC. Various inks with different weight percentage compositions of AC will be made and tested against its respective rheological properties in order to determine ideal ink printing system. The items in the formulation used comprised of organic and bio-waste materials with added additive to improve the quality of the black ink. Modified Polyurethane was used as binder. The binder's properties highlighted an ideal vehicle to be applied for good black ink opacity performance. The rheological behaviour is a general foundation for ink characterization where the wt% of AC-PKC resulted in different pseudoplastic behaviors, including the Newtonian behavior. The result found that Newtonian field was located in between 2 wt% and 10 wt% of AC-PKC composition with binder. Mass spectroscopy results shown that the carbon content in PKC is high and very suitable for black performance. In the ageing test, the pigment of PKC perform fairly according to the standard pigment of Black carbon (CB) of ferum oxide pigment. The contact angle for substrate's wettability of the ink system shown a good angle proven to be a water resistive coating on paper subtrates; an advantage of the PKC ink pigment performance.

  9. Apoptosis of murine melanoma B16-BL6 cells induced by quercetin targeting mitochondria, inhibiting expression of PKC-alpha and translocating PKC-delta.

    Science.gov (United States)

    Zhang, Xian-Ming; Chen, Jia; Xia, Yu-Gui; Xu, Qiang

    2005-03-01

    In our previous study, quercetin was found to induce apoptosis of murine melanoma B16-BL6 cells. The cellular and molecular mechanism of quercetin-induced apoptosis was investigated in the present study. Nuclear morphology was determined by fluorescence microscopy. DNA fragmentation was analyzed by electrophoresis and quantified by the diphenylamine method. The transmembrane potential of mitochondria was measured by flow cytometry. Bcl-2, Bcl-X(L), PKC-alpha, PKC-beta, and PKC-delta were detected by Western blotting. Caspase activity was determined spectrophotometrically. Quercetin induced the condensation of nuclei of B16-BL6 cells in a dose-dependent pattern as visualized by Hoechst 33258 and propidium iodide dying. Phorbol 12-myristate 13-acetate (PMA), a PKC activator, significantly enhanced apoptosis induced by quercetin, while doxorubicin, a PKC inhibitor, markedly decreased it. Both PMA and doxorubicin showed a consistent effect on the fragmentation of nuclear DNA caused by various dosages of quercetin. Quercetin dose-dependently led to loss of the mitochondrial membrane potential, which was also significantly reinforced or antagonized by PMA and doxorubicin, respectively. Moreover, PMA showed reinforcement, while doxorubicin showed significant antagonization, of the quercetin-mediated decrease in the expression of Bcl-2. Quercetin promoted caspase-3 activity in a dose-dependent manner, which was also regulated by PMA and doxorubicin with a pattern similar to that seen in their effect on apoptosis, mitochondrial membrane potential and Bcl-2 expression, but none of these were directly affected by PMA and doxorubicin. Free fatty acid and chlorpromazine, a PKC activator and inhibitor, respectively, did not interfere with these effects of quercetin. B16-BL6 cells expressed PKC-alpha, PKC-beta, and PKC-delta. Quercetin dose-dependently inhibited the expression of PKC-alpha but not that of PKC-beta and PKC-delta. Doxorubicin almost completely blocked the effect of

  10. Angiotensin II inhibits the Na+-K+ pump via PKC-dependent activation of NADPH oxidase.

    Science.gov (United States)

    White, Caroline N; Figtree, Gemma A; Liu, Chia-Chi; Garcia, Alvaro; Hamilton, Elisha J; Chia, Karin K M; Rasmussen, Helge H

    2009-04-01

    The sarcolemmal Na(+)-K(+) pump, pivotal in cardiac myocyte function, is inhibited by angiotensin II (ANG II). Since ANG II activates NADPH oxidase, we tested the hypothesis that NADPH oxidase mediates the pump inhibition. Exposure to 100 nmol/l ANG II increased superoxide-sensitive fluorescence of isolated rabbit ventricular myocytes. The increase was abolished by pegylated superoxide dismutase (SOD), by the NADPH oxidase inhibitor apocynin, and by myristolated inhibitory peptide to epsilon-protein kinase C (epsilonPKC), previously implicated in ANG II-induced Na(+)-K(+) pump inhibition. A role for epsilonPKC was also supported by an ANG II-induced increase in coimmunoprecipitation of epsilonPKC with the receptor for the activated kinase and with the cytosolic p47(phox) subunit of NADPH oxidase. ANG II decreased electrogenic Na(+)-K(+) pump current in voltage-clamped myocytes. The decrease was abolished by SOD, by the gp91ds inhibitory peptide that blocks assembly and activation of NADPH oxidase, and by epsilonPKC inhibitory peptide. Since colocalization should facilitate NADPH oxidase-dependent regulation of the Na(+)-K(+) pump, we examined whether there is physical association between the pump subunits and NADPH oxidase. The alpha(1)-subunit coimmunoprecipitated with caveolin 3 and with membrane-associated p22(phox) and cytosolic p47(phox) NADPH oxidase subunits at baseline. ANG II had no effect on alpha(1)/caveolin 3 or alpha(1)/p22(phox) interaction, but it increased alpha(1)/p47(phox) coimmunoprecipitation. We conclude that ANG II inhibits the Na(+)-K(+) pump via PKC-dependent NADPH oxidase activation.

  11. Brassica juncea nitric oxide synthase like activity is stimulated by PKC activators and calcium suggesting modulation by PKC-like kinase.

    Science.gov (United States)

    Talwar, Pooja Saigal; Gupta, Ravi; Maurya, Arun Kumar; Deswal, Renu

    2012-11-01

    Nitric oxide (NO) is an important signaling molecule having varied physiological and regulatory roles in biological systems. The fact that nitric oxide synthase (NOS) is responsible for NO generation in animals, prompted major search for a similar enzyme in plants. Arginine dependent NOS like activity (BjNOSla) was detected in Brassica juncea seedlings using oxyhemoglobin and citrulline assays. BjNOSla showed 25% activation by NADPH (0.4 mM) and 40% by calcium (0.4 mM) but the activity was flavin mononucleotide (FMN), flavin dinucleotide (FAD) and calmodulin (CaM) independent. Pharmacological approach using mammalian NOS inhibitors, NBT (300 μM) and l-NAME (5 mM), showed significant inhibition (100% and 67% respectively) supporting that the BjNOSla operates via the oxidative pathway. Most of the BjNOSla activity (80%) was confined to shoot while root showed only 20% activity. Localization studies by NADPH-diaphorase and DAF-2DA staining showed the presence of BjNOSla in guard cells. Kinetic analysis showed positive cooperativity with calcium as reflected by a decreased K(m) (∼13%) and almost two fold increase in V(max). PMA (438 nM), a kinase activator, activated BjNOSla ∼1.9 fold while its inactive analog 4αPDD was ineffective. Calcium and PMA activated the enzyme to ∼3 folds. Interestingly, 1,2-DG6 (2.5 μM) and PS (1 μM) with calcium activated the enzyme activity to ∼7 fold. A significant inhibition of BjNOSla by PKC inhibitors-staurosporine (∼90%) and calphostin-C (∼40%), further supports involvement of PKC-like kinase. The activity was also enhanced by abiotic stress conditions (7-46%). All these findings suggest that BjNOSla generates NO via oxidative pathway and is probably regulated by phosphorylation. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  12. Astrocytic connexin hemichannels are regulated by PKC phosphorylation in an isoform-specific manner

    DEFF Research Database (Denmark)

    MacAulay, N.; Alstrom, J. S.; Hansen, D. B.

    2017-01-01

    /activation of PKC and by mutational disruption of the proposed PKC-phosphorylation sites. Cx30 hemichannel activity, in contrast, was down-regulated by PKC activation, in a manner suggesting PKC-mediated channel closure. No single PKC consensus site could be assigned to this regulatory property by mutational...

  13. PKC activation induces inflammatory response and cell death in human bronchial epithelial cells.

    Directory of Open Access Journals (Sweden)

    Hyunhee Kim

    Full Text Available A variety of airborne pathogens can induce inflammatory responses in airway epithelial cells, which is a crucial component of host defence. However, excessive inflammatory responses and chronic inflammation also contribute to different diseases of the respiratory system. We hypothesized that the activation of protein kinase C (PKC is one of the essential mechanisms of inflammatory response in airway epithelial cells. In the present study, we stimulated human bronchial lung epithelial (BEAS-2B cells with the phorbol ester Phorbol 12, 13-dibutyrate (PDBu, and examined gene expression profile using microarrays. Microarray analysis suggests that PKC activation induced dramatic changes in gene expression related to multiple cellular functions. The top two interaction networks generated from these changes were centered on NFκB and TNF-α, which are two commonly known pathways for cell death and inflammation. Subsequent tests confirmed the decrease in cell viability and an increase in the production of various cytokines. Interestingly, each of the increased cytokines was differentially regulated at mRNA and/or protein levels by different sub-classes of PKC isozymes. We conclude that pathological cell death and cytokine production in airway epithelial cells in various situations may be mediated through PKC related signaling pathways. These findings suggest that PKCs can be new targets for treatment of lung diseases.

  14. Protein kinase C (PKC) isoforms in cancer, tumor promotion and tumor suppression.

    Science.gov (United States)

    Isakov, Noah

    2018-02-01

    The AGC family of serine/threonine kinases (PKA, PKG, PKC) includes more than 60 members that are critical regulators of numerous cellular functions, including cell cycle and differentiation, morphogenesis, and cell survival and death. Mutation and/or dysregulation of AGC kinases can lead to malignant cell transformation and contribute to the pathogenesis of many human diseases. Members of one subgroup of AGC kinases, the protein kinase C (PKC), have been singled out as critical players in carcinogenesis, following their identification as the intracellular receptors of phorbol esters, which exhibit tumor-promoting activities. This observation attracted the attention of researchers worldwide and led to intense investigations on the role of PKC in cell transformation and the potential use of PKC as therapeutic drug targets in cancer diseases. Studies demonstrated that many cancers had altered expression and/or mutation of specific PKC genes. However, the causal relationships between the changes in PKC gene expression and/or mutation and the direct cause of cancer remain elusive. Independent studies in normal cells demonstrated that activation of PKC is essential for the induction of cell activation and proliferation, differentiation, motility, and survival. Based on these observations and the general assumption that PKC isoforms play a positive role in cell transformation and/or cancer progression, many PKC inhibitors have entered clinical trials but the numerous attempts to target PKC in cancer has so far yielded only very limited success. More recent studies demonstrated that PKC function as tumor suppressors, and suggested that future clinical efforts should focus on restoring, rather than inhibiting, PKC activity. The present manuscript provides some historical perspectives on the tumor promoting function of PKC, reviewing some of the observations linking PKC to cancer progression, and discusses the role of PKC in the pathogenesis of cancer diseases and its

  15. Apoptosis by [Pt(O,O'-acac)(γ-acac)(DMS)] requires PKC-δ mediated p53 activation in malignant pleural mesothelioma.

    Science.gov (United States)

    Muscella, Antonella; Vetrugno, Carla; Cossa, Luca Giulio; Antonaci, Giovanna; Barca, Amilcare; De Pascali, Sandra Angelica; Fanizzi, Francesco Paolo; Marsigliante, Santo

    2017-01-01

    Mesothelioma cancer cells have epithelioid or sarcomatoid morphology. The worst prognosis is associated with sarcomatoid phenotype and resistance to therapy is affected by cells heterogeneity. We recently showed that in ZL55 mesothelioma cell line of epithelioid origin [Pt(O,O'-acac)(γ-acac)(DMS)] (Ptac2S) has an antiproliferative effect in vitro and in vivo. Aim of this work was to extend the study on the effects of Ptac2S on ZL34 cell line, representative of sarcomatoid mesothelioma. ZL34 cells were used to assay the antitumor activity of Ptac2S in a mouse xenograft model in vivo. Then, both ZL34 and ZL55 cells were used in order to assess the involvement of p53 protein in (a) the processes underlying the sensitivity to chemotherapy and (b) the activation of various transduction proteins involved in apoptosis/survival processes. Ptac2S increases ZL34 cell death in vivo compared with cisplatin and, in vitro, Ptac2S was more efficacious than cisplatin in inducing apoptosis. In Ptac2S-treated ZL34 and ZL55 cells, p53 regulated gene products of apoptotic BAX and anti-apoptotic Bcl-2 proteins via transcriptional activation. Ptac2S activated PKC-δ and PKC-ε; their inhibition by PKC-siRNA decreased the apoptotic death of cells. PKC-δ was responsible for JNK1/2 activation that has a role in p53 activation. In addition, PKCactivation provoked phosphorylation of p38MAPK, concurring to apoptosis. In ZL34 cells, Ptac2S also activated PKC-α thus provoking ERK1/2 activation; inhibition of PKC-α, or ERK1/2, increased Ptac2S cytotoxicity. Results confirm that Ptac2S is a promising therapeutic agent for malignant mesothelioma, giving a substantial starting point for its further validation.

  16. Correlation between AQP4 mRNA and PKC activity after gamma knife radiosurgery in rat brain

    International Nuclear Information System (INIS)

    Shen Guangjian; Xu Minhui; Gen Mingying; Tang Wenyuan; Sun Shanquan

    2009-01-01

    Objective: To explore the change of AQP4 mRNA expression and the correlation with PKC in rat brain irradiated by γ knife radiosurgery (GKS). Methods: 30 Wistar rats were used in the study. The experimental radiosurgery model was established by radiating rat left rotral caudate nucleus with GKS(one target, 100 Gy in isocenter dose and 4 mm in collimator), and was examined at 1,3,7,15,30 and 45 d post-irradiation. AQP4 mRNA expression, PKC activity and free intracellular calcium ion concentration ([Ca 2+ ] i ) of brain tissue were determined by RT-PCR, liquid scintillation counter and Fura-2/AM, respectively. Results: AQP4 mRNA expression increased gradually from 0.99 ± 0.05 in control group to 2.32 ± 0.10 at 30 d post-irradiation, and decreased to 2.21 ± 0.08 at 45 d post-irradiation. The PKC activity and the free [Ca 2+ ] i decreased gradually from 0.5896 ± 0.2101 and 455.82 ± 20.13 in control group to 0.0404 ± 0.0294 and 196.72 ± 9.87 at 30 d post- irradiation, and increased to 0.1050 ± 0.0607 and 219.26 ± 10.43 at 45 d post-irradiation, respectively. The significant differences were found between experimental group and control group except at 1 d post-irradiation (P 2+ ] i and the PKC activity was positive (P=0.001, r=0.959). Conclusions: The increased expression of AQP4 mRNA might result from the inhibition of PKC activity due to the reduction of free [Ca 2+ ] i after GKS. (authors)

  17. Dopaminergic neurotoxicant 6-OHDA induces oxidative damage through proteolytic activation of PKC{delta} in cell culture and animal models of Parkinson's disease

    Energy Technology Data Exchange (ETDEWEB)

    Latchoumycandane, Calivarathan; Anantharam, Vellareddy; Jin, Huajun; Kanthasamy, Anumantha; Kanthasamy, Arthi, E-mail: arthik@iastate.edu

    2011-11-15

    The neurotoxicant 6-hydroxydopamine (6-OHDA) is used to investigate the cellular and molecular mechanisms underlying selective degeneration of dopaminergic neurons in Parkinson's disease (PD). Oxidative stress and caspase activation contribute to the 6-OHDA-induced apoptotic cell death of dopaminergic neurons. In the present study, we sought to systematically characterize the key downstream signaling molecule involved in 6-OHDA-induced dopaminergic degeneration in cell culture and animal models of PD. Treatment of mesencephalic dopaminergic neuronal N27 cells with 6-OHDA (100 {mu}M) for 24 h significantly reduced mitochondrial activity and increased cytosolic cytochrome c, followed by sequential activation of caspase-9 and caspase-3. Co-treatment with the free radical scavenger MnTBAP (10 {mu}M) significantly attenuated 6-OHDA-induced caspase activities. Interestingly, 6-OHDA induced proteolytic cleavage and activation of protein kinase C delta (PKC{delta}) was completely suppressed by treatment with a caspase-3-specific inhibitor, Z-DEVD-FMK (50 {mu}M). Furthermore, expression of caspase-3 cleavage site-resistant mutant PKC{delta}{sup D327A} and kinase dead PKC{delta}{sup K376R} or siRNA-mediated knockdown of PKC{delta} protected against 6-OHDA-induced neuronal cell death, suggesting that caspase-3-dependent PKC{delta} promotes oxidative stress-induced dopaminergic degeneration. Suppression of PKC{delta} expression by siRNA also effectively protected N27 cells from 6-OHDA-induced apoptotic cell death. PKC{delta} cleavage was also observed in the substantia nigra of 6-OHDA-injected C57 black mice but not in control animals. Viral-mediated delivery of PKC{delta}{sup D327A} protein protected against 6-OHDA-induced PKC{delta} activation in mouse substantia nigra. Collectively, these results strongly suggest that proteolytic activation of PKC{delta} is a key downstream event in dopaminergic degeneration, and these results may have important translational value for

  18. Protease-activated receptor-2 stimulates intestinal epithelial chloride transport through activation of PLC and selective PKC isoforms.

    Science.gov (United States)

    van der Merwe, Jacques Q; Moreau, France; MacNaughton, Wallace K

    2009-06-01

    Serine proteases play important physiological roles through their activity at G protein-coupled protease-activated receptors (PARs). We examined the roles that specific phospholipase (PL) C and protein kinase (PK) C (PKC) isoforms play in the regulation of PAR(2)-stimulated chloride secretion in intestinal epithelial cells. Confluent SCBN epithelial monolayers were grown on Snapwell supports and mounted in modified Ussing chambers. Short-circuit current (I(sc)) responses to basolateral application of the selective PAR(2) activating peptide, SLIGRL-NH(2), were monitored as a measure of net electrogenic ion transport caused by PAR(2) activation. SLIGRL-NH(2) induced a transient I(sc) response that was significantly reduced by inhibitors of PLC (U73122), phosphoinositol-PLC (ET-18), phosphatidylcholine-PLC (D609), and phosphatidylinositol 3-kinase (PI3K; LY294002). Immunoblot analysis revealed the phosphorylation of both PLCbeta and PLCgamma following PAR(2) activation. Pretreatment of the cells with inhibitors of PKC (GF 109203X), PKCalpha/betaI (Gö6976), and PKCdelta (rottlerin), but not PKCzeta (selective pseudosubstrate inhibitor), also attenuated this response. Cellular fractionation and immunoblot analysis, as well as confocal immunocytochemistry, revealed increases of PKCbetaI, PKCdelta, and PKCepsilon, but not PKCalpha or PKCzeta, in membrane fractions following PAR(2) activation. Pretreatment of the cells with U73122, ET-18, or D609 inhibited PKC activation. Inhibition of PI3K activity only prevented PKCdelta translocation. Immunoblots revealed that PAR(2) activation induced phosphorylation of both cRaf and ERK1/2 via PKCdelta. Inhibition of PKCbetaI and PI3K had only a partial effect on this response. We conclude that basolateral PAR(2)-induced chloride secretion involves activation of PKCbetaI and PKCdelta via a PLC-dependent mechanism resulting in the stimulation of cRaf and ERK1/2 signaling.

  19. Amarogentin, a Secoiridoid Glycoside, Abrogates Platelet Activation through PLCγ2-PKC and MAPK Pathways

    Directory of Open Access Journals (Sweden)

    Ting-Lin Yen

    2014-01-01

    Full Text Available Amarogentin, an active principle of Gentiana lutea, possess antitumorigenic, antidiabetic, and antioxidative properties. Activation of platelets is associated with intravascular thrombosis and cardiovascular diseases. The present study examined the effects of amarogentin on platelet activation. Amarogentin treatment (15~60 μM inhibited platelet aggregation induced by collagen, but not thrombin, arachidonic acid, and U46619. Amarogentin inhibited collagen-induced phosphorylation of phospholipase C (PLCγ2, protein kinase C (PKC, and mitogen-activated protein kinases (MAPKs. It also inhibits in vivo thrombus formation in mice. In addition, neither the guanylate cyclase inhibitor ODQ nor the adenylate cyclase inhibitor SQ22536 affected the amarogentin-mediated inhibition of platelet aggregation, which suggests that amarogentin does not regulate the levels of cyclic AMP and cyclic GMP. In conclusion, amarogentin prevents platelet activation through the inhibition of PLCγ2-PKC cascade and MAPK pathway. Our findings suggest that amarogentin may offer therapeutic potential for preventing or treating thromboembolic disorders.

  20. Kibra and aPKC regulate starvation-induced autophagy in Drosophila

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Ahrum [Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 291 Daehak-ro, Yuseong-gu, Daejeon 34141 (Korea, Republic of); Neufeld, Thomas P. [Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455 (United States); Choe, Joonho, E-mail: jchoe@kaist.ac.kr [Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 291 Daehak-ro, Yuseong-gu, Daejeon 34141 (Korea, Republic of)

    2015-12-04

    Autophagy is a bulk degradation system that functions in response to cellular stresses such as metabolic stress, endoplasmic reticulum stress, oxidative stress, and developmental processes. During autophagy, cytoplasmic components are captured in double-membrane vesicles called autophagosomes. The autophagosome fuses with the lysosome, producing a vacuole known as an autolysosome. The cellular components are degraded by lysosomal proteases and recycled. Autophagy is important for maintaining cellular homeostasis, and the process is evolutionarily conserved. Kibra is an upstream regulator of the hippo signaling pathway, which controls organ size by affecting cell growth, proliferation, and apoptosis. Kibra is mainly localized in the apical membrane domain of epithelial cells and acts as a scaffold protein. We found that Kibra is required for autophagy to function properly. The absence of Kibra caused defects in the formation of autophagic vesicles and autophagic degradation. We also found that the well-known cell polarity protein aPKC interacts with Kibra, and its activity affects autophagy upstream of Kibra. Constitutively active aPKC decreased autophagic vesicle formation and autophagic degradation. We confirmed the interaction between aPKC and Kibra in S2 cells and Drosophila larva. Taken together, our data suggest that Kibra and aPKC are essential for regulating starvation-induced autophagy. - Highlights: • Loss of Kibra causes defects in autophagosome formation and autophagic degradation. • Constitutively-active aPKCs negatively regulate autophagy. • Kibra interacts with aPKC in vitro and in vivo. • Kibra regulates autophagy downstream of aPKC.

  1. Amarogentin, a secoiridoid glycoside, abrogates platelet activation through PLC γ 2-PKC and MAPK pathways.

    Science.gov (United States)

    Yen, Ting-Lin; Lu, Wan-Jung; Lien, Li-Ming; Thomas, Philip Aloysius; Lee, Tzu-Yin; Chiu, Hou-Chang; Sheu, Joen-Rong; Lin, Kuan-Hung

    2014-01-01

    Amarogentin, an active principle of Gentiana lutea, possess antitumorigenic, antidiabetic, and antioxidative properties. Activation of platelets is associated with intravascular thrombosis and cardiovascular diseases. The present study examined the effects of amarogentin on platelet activation. Amarogentin treatment (15~60  μM) inhibited platelet aggregation induced by collagen, but not thrombin, arachidonic acid, and U46619. Amarogentin inhibited collagen-induced phosphorylation of phospholipase C (PLC) γ2, protein kinase C (PKC), and mitogen-activated protein kinases (MAPKs). It also inhibits in vivo thrombus formation in mice. In addition, neither the guanylate cyclase inhibitor ODQ nor the adenylate cyclase inhibitor SQ22536 affected the amarogentin-mediated inhibition of platelet aggregation, which suggests that amarogentin does not regulate the levels of cyclic AMP and cyclic GMP. In conclusion, amarogentin prevents platelet activation through the inhibition of PLC γ2-PKC cascade and MAPK pathway. Our findings suggest that amarogentin may offer therapeutic potential for preventing or treating thromboembolic disorders.

  2. PKC{eta} is a negative regulator of AKT inhibiting the IGF-I induced proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Shahaf, Galit; Rotem-Dai, Noa; Koifman, Gabriela; Raveh-Amit, Hadas; Frost, Sigal A.; Livneh, Etta, E-mail: etta@bgu.ac.il

    2012-04-15

    The PI3K-AKT pathway is frequently activated in human cancers, including breast cancer, and its activation appears to be critical for tumor maintenance. Some malignant cells are dependent on activated AKT for their survival; tumors exhibiting elevated AKT activity show sensitivity to its inhibition, providing an Achilles heel for their treatment. Here we show that the PKC{eta} isoform is a negative regulator of the AKT signaling pathway. The IGF-I induced phosphorylation on Ser473 of AKT was inhibited by the PKC{eta}-induced expression in MCF-7 breast adenocarcinoma cancer cells. This was further confirmed in shRNA PKC{eta}-knocked-down MCF-7 cells, demonstrating elevated phosphorylation on AKT Ser473. While PKC{eta} exhibited negative regulation on AKT phosphorylation it did not alter the IGF-I induced ERK phosphorylation. However, it enhanced ERK phosphorylation when stimulated by PDGF. Moreover, its effects on IGF-I/AKT and PDGF/ERK pathways were in correlation with cell proliferation. We further show that both PKC{eta} and IGF-I confer protection against UV-induced apoptosis and cell death having additive effects. Although the protective effect of IGF-I involved activation of AKT, it was not affected by PKC{eta} expression, suggesting that PKC{eta} acts through a different route to increase cell survival. Hence, our studies show that PKC{eta} provides negative control on AKT pathway leading to reduced cell proliferation, and further suggest that its presence/absence in breast cancer cells will affect cell death, which could be of therapeutic value.

  3. The role of laserpuncture exposure on gonad maturation mechanism of catfish (Clarias sp. through Ca2+, PKC and GABA neurotransmitter

    Directory of Open Access Journals (Sweden)

    Pungky Slamet Wisnu Kusuma

    2017-12-01

    Full Text Available Laser puncture exposure at reproduction acupoint is proven to increase cellular activity like Ca2+ in the skin tissues. The aim of the study is to determine the role of laserpuncture exposure on gonad maturation by evaluating Ca2+ stimulation and PKC activity in skin tissue and the release of GABA from GABAergic neurons of the brain tissue of catfish (Clarias sp.. A total of 36 females and 36 males of 8–9-month old of F1 catfish broodstock Sangkuriang (female and Paiton (male. This study used Completely Randomized Design (CDR experimental method. Expression analysis was conducted using immunohistochemical staining with a streptavidinbiotin method with calcineurin kit, PKC kit, and GABA kit. The results showed that laserpuncture can stimulate calcineurin and PKC expression in skin tissue, and GABA expression in the brain tissue on the condition pre-spawn, spawn, and post-spawn (P < .05. It can be concluded that laserpuncture stimulates gonad maturation through Ca2+, PKC, and GABA neurotransmitter.

  4. Functional divergence of platelet protein kinase C (PKC) isoforms in thrombus formation on collagen.

    Science.gov (United States)

    Gilio, Karen; Harper, Matthew T; Cosemans, Judith M E M; Konopatskaya, Olga; Munnix, Imke C A; Prinzen, Lenneke; Leitges, Michael; Liu, Qinghang; Molkentin, Jeffery D; Heemskerk, Johan W M; Poole, Alastair W

    2010-07-23

    Arterial thrombosis, a major cause of myocardial infarction and stroke, is initiated by activation of blood platelets by subendothelial collagen. The protein kinase C (PKC) family centrally regulates platelet activation, and it is becoming clear that the individual PKC isoforms play distinct roles, some of which oppose each other. Here, for the first time, we address all four of the major platelet-expressed PKC isoforms, determining their comparative roles in regulating platelet adhesion to collagen and their subsequent activation under physiological flow conditions. Using mouse gene knock-out and pharmacological approaches in human platelets, we show that collagen-dependent alpha-granule secretion and thrombus formation are mediated by the conventional PKC isoforms, PKCalpha and PKCbeta, whereas the novel isoform, PKC, negatively regulates these events. PKCdelta also negatively regulates thrombus formation but not alpha-granule secretion. In addition, we demonstrate for the first time that individual PKC isoforms differentially regulate platelet calcium signaling and exposure of phosphatidylserine under flow. Although platelet deficient in PKCalpha or PKCbeta showed reduced calcium signaling and phosphatidylserine exposure, these responses were enhanced in the absence of PKC. In summary therefore, this direct comparison between individual subtypes of PKC, by standardized methodology under flow conditions, reveals that the four major PKCs expressed in platelets play distinct non-redundant roles, where conventional PKCs promote and novel PKCs inhibit thrombus formation on collagen.

  5. Palmitic acid mediates hypothalamic insulin resistance by altering PKC-θ subcellular localization in rodents

    Science.gov (United States)

    Benoit, Stephen C.; Kemp, Christopher J.; Elias, Carol F.; Abplanalp, William; Herman, James P.; Migrenne, Stephanie; Lefevre, Anne-Laure; Cruciani-Guglielmacci, Céline; Magnan, Christophe; Yu, Fang; Niswender, Kevin; Irani, Boman G.; Holland, William L.; Clegg, Deborah J.

    2009-01-01

    Insulin signaling can be modulated by several isoforms of PKC in peripheral tissues. Here, we assessed whether one specific isoform, PKC-θ, was expressed in critical CNS regions that regulate energy balance and whether it mediated the deleterious effects of diets high in fat, specifically palmitic acid, on hypothalamic insulin activity in rats and mice. Using a combination of in situ hybridization and immunohistochemistry, we found that PKC-θ was expressed in discrete neuronal populations of the arcuate nucleus, specifically the neuropeptide Y/agouti-related protein neurons and the dorsal medial nucleus in the hypothalamus. CNS exposure to palmitic acid via direct infusion or by oral gavage increased the localization of PKC-θ to cell membranes in the hypothalamus, which was associated with impaired hypothalamic insulin and leptin signaling. This finding was specific for palmitic acid, as the monounsaturated fatty acid, oleic acid, neither increased membrane localization of PKC-θ nor induced insulin resistance. Finally, arcuate-specific knockdown of PKC-θ attenuated diet-induced obesity and improved insulin signaling. These results suggest that many of the deleterious effects of high-fat diets, specifically those enriched with palmitic acid, are CNS mediated via PKCactivation, resulting in reduced insulin activity. PMID:19726875

  6. Palmitic acid mediates hypothalamic insulin resistance by altering PKC-theta subcellular localization in rodents.

    Science.gov (United States)

    Benoit, Stephen C; Kemp, Christopher J; Elias, Carol F; Abplanalp, William; Herman, James P; Migrenne, Stephanie; Lefevre, Anne-Laure; Cruciani-Guglielmacci, Céline; Magnan, Christophe; Yu, Fang; Niswender, Kevin; Irani, Boman G; Holland, William L; Clegg, Deborah J

    2009-09-01

    Insulin signaling can be modulated by several isoforms of PKC in peripheral tissues. Here, we assessed whether one specific isoform, PKC-theta, was expressed in critical CNS regions that regulate energy balance and whether it mediated the deleterious effects of diets high in fat, specifically palmitic acid, on hypothalamic insulin activity in rats and mice. Using a combination of in situ hybridization and immunohistochemistry, we found that PKC-theta was expressed in discrete neuronal populations of the arcuate nucleus, specifically the neuropeptide Y/agouti-related protein neurons and the dorsal medial nucleus in the hypothalamus. CNS exposure to palmitic acid via direct infusion or by oral gavage increased the localization of PKC-theta to cell membranes in the hypothalamus, which was associated with impaired hypothalamic insulin and leptin signaling. This finding was specific for palmitic acid, as the monounsaturated fatty acid, oleic acid, neither increased membrane localization of PKC-theta nor induced insulin resistance. Finally, arcuate-specific knockdown of PKC-theta attenuated diet-induced obesity and improved insulin signaling. These results suggest that many of the deleterious effects of high-fat diets, specifically those enriched with palmitic acid, are CNS mediated via PKC-theta activation, resulting in reduced insulin activity.

  7. Phosphorylation of synaptotagmin-1 controls a post-priming step in PKC-dependent presynaptic plasticity

    DEFF Research Database (Denmark)

    de Jong, Arthur P H; Meijer, Marieke; Saarloos, Ingrid

    2016-01-01

    Presynaptic activation of the diacylglycerol (DAG)/protein kinase C (PKC) pathway is a central event in short-term synaptic plasticity. Two substrates, Munc13-1 and Munc18-1, are essential for DAG-induced potentiation of vesicle priming, but the role of most presynaptic PKC substrates is not unde......Presynaptic activation of the diacylglycerol (DAG)/protein kinase C (PKC) pathway is a central event in short-term synaptic plasticity. Two substrates, Munc13-1 and Munc18-1, are essential for DAG-induced potentiation of vesicle priming, but the role of most presynaptic PKC substrates...... is not understood. Here, we show that a mutation in synaptotagmin-1 (Syt1(T112A)), which prevents its PKC-dependent phosphorylation, abolishes DAG-induced potentiation of synaptic transmission in hippocampal neurons. This mutant also reduces potentiation of spontaneous release, but only if alternative Ca(2+)sensors...

  8. Age-related deficits in synaptic plasticity rescued by activating PKA or PKC in sensory neurons of Aplysia californica

    Directory of Open Access Journals (Sweden)

    Andrew T Kempsell

    2015-09-01

    Full Text Available Brain aging is associated with declines in synaptic function that contribute to memory loss, including reduced postsynaptic response to neurotransmitters and decreased neuronal excitability. To understand how aging affects memory in a simple neural circuit, we studied neuronal proxies of memory for sensitization in mature versus advanced age Aplysia. Glutamate- (L-Glu- evoked excitatory currents were facilitated by the neuromodulator serotonin (5-HT in sensory neurons (SN isolated from mature but not aged animals. Activation of PKA and PKC signaling rescued facilitation of L-Glu currents in aged SN. Similarly, PKA and PKC activators restored increased excitability in aged tail SN. These results suggest that altered synaptic plasticity during aging involves defects in second messenger systems

  9. ETV6-NTRK3 as a therapeutic target of small molecule inhibitor PKC412

    Energy Technology Data Exchange (ETDEWEB)

    Chi, Hoang Thanh, E-mail: kk086406@mgs.k.u-tokyo.ac.jp [Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Tokyo 108-8639 (Japan); Ly, Bui Thi Kim [Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Tokyo 108-8639 (Japan); Kano, Yasuhiko [Division of Hematology and Medical Oncology, Tochigi Cancer Center, Tochigi 321-0293 (Japan); Tojo, Arinobu [Division of Molecular Therapy, Department of Hematology/Oncology, Research Hospital, The Institute of Medical Science, The University of Tokyo, Tokyo (Japan); Watanabe, Toshiki [Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Tokyo 108-8639 (Japan); Sato, Yuko [Musashimurayama Hospital, Musashimurayama, Tokyo 208-0011 (Japan)

    2012-12-07

    Highlights: Black-Right-Pointing-Pointer ETV6-NTRK3 is an oncogene with transformation activity in multiple cell lineages. Black-Right-Pointing-Pointer PKC412 could block ETV6-NTRK3 activation. Black-Right-Pointing-Pointer Loss of ETV6-NTRK3 phosphorylation leads to inactivation of its downstream signaling pathway. Black-Right-Pointing-Pointer Inhibition of ETV6-NTRK3 activation by PKC412 could be a novel strategy for the treatment. -- Abstract: The ETV6-NTRK3 (EN) fusion gene which encodes a chimeric tyrosine kinase was first identified by cloning of the t(12;15)(p13;q25) translocation in congenital fibrosarcoma (CFS). Since then, EN has been also found in congenital mesoblastic nephroma (CMN), secretory breast carcinoma (SBC) and acute myelogenous leukemia (AML). Using IMS-M2 and M0-91 cell lines harboring the EN fusion gene, and Ba/F3 cells stably transfected with EN, we demonstrated that PKC412, also known as midostaurin, is an inhibitor of EN. Inhibition of EN activity by PKC412 suppressed the activity of it downstream molecules leading to inhibition of cell proliferation and induction of apoptosis. Our data for the first time suggested that PKC412 could serve as therapeutic drug for treatment of patients with this fusion.

  10. Age-related deficits in synaptic plasticity rescued by activating PKA or PKC in sensory neurons of Aplysia californica.

    Science.gov (United States)

    Kempsell, Andrew T; Fieber, Lynne A

    2015-01-01

    Brain aging is associated with declines in synaptic function that contribute to memory loss, including reduced postsynaptic response to neurotransmitters and decreased neuronal excitability. To understand how aging affects memory in a simple neural circuit, we studied neuronal proxies of memory for sensitization in mature vs. advanced age Aplysia californica (Aplysia). L-Glutamate- (L-Glu-) evoked excitatory currents were facilitated by the neuromodulator serotonin (5-HT) in sensory neurons (SN) isolated from mature but not aged animals. Activation of protein kinase A (PKA) and protein kinase C (PKC) signaling rescued facilitation of L-Glu currents in aged SN. Similarly, PKA and PKC activators restored increased excitability in aged tail SN. These results suggest that altered synaptic plasticity during aging involves defects in second messenger systems.

  11. Functional Divergence of Platelet Protein Kinase C (PKC) Isoforms in Thrombus Formation on Collagen*

    Science.gov (United States)

    Gilio, Karen; Harper, Matthew T.; Cosemans, Judith M. E. M.; Konopatskaya, Olga; Munnix, Imke C. A.; Prinzen, Lenneke; Leitges, Michael; Liu, Qinghang; Molkentin, Jeffery D.; Heemskerk, Johan W. M.; Poole, Alastair W.

    2010-01-01

    Arterial thrombosis, a major cause of myocardial infarction and stroke, is initiated by activation of blood platelets by subendothelial collagen. The protein kinase C (PKC) family centrally regulates platelet activation, and it is becoming clear that the individual PKC isoforms play distinct roles, some of which oppose each other. Here, for the first time, we address all four of the major platelet-expressed PKC isoforms, determining their comparative roles in regulating platelet adhesion to collagen and their subsequent activation under physiological flow conditions. Using mouse gene knock-out and pharmacological approaches in human platelets, we show that collagen-dependent α-granule secretion and thrombus formation are mediated by the conventional PKC isoforms, PKCα and PKCβ, whereas the novel isoform, PKCθ, negatively regulates these events. PKCδ also negatively regulates thrombus formation but not α-granule secretion. In addition, we demonstrate for the first time that individual PKC isoforms differentially regulate platelet calcium signaling and exposure of phosphatidylserine under flow. Although platelet deficient in PKCα or PKCβ showed reduced calcium signaling and phosphatidylserine exposure, these responses were enhanced in the absence of PKCθ. In summary therefore, this direct comparison between individual subtypes of PKC, by standardized methodology under flow conditions, reveals that the four major PKCs expressed in platelets play distinct non-redundant roles, where conventional PKCs promote and novel PKCs inhibit thrombus formation on collagen. PMID:20479008

  12. The interrelation between aPKC and glucose uptake in the skeletal muscle during contraction and insulin stimulation.

    Science.gov (United States)

    Santos, J M; Benite-Ribeiro, S A; Queiroz, G; Duarte, J A

    2014-12-01

    Contraction and insulin increase glucose uptake in skeletal muscle. While the insulin pathway, better characterized, requires activation of phosphoinositide 3-kinase (PI3K) and atypical protein kinase (aPKC), muscle contraction seems to share insulin-activated components to increase glucose uptake. This study aimed to investigate the interrelation between the pathway involved in glucose uptake evoked by insulin and muscle contraction. Isolated muscle of rats was treated with solvent (control), insulin, wortmannin (PI3K inhibitor) and the combination of insulin plus wortmannin. After treatment, muscles were electrically stimulated (contracted) or remained at rest. Glucose transporter 4 (GLUT4) localization, glucose uptake and phospho-aPKC (aPKC activated form) were assessed. Muscle contraction and insulin increased glucose uptake in all conditions when compared with controls not stimulating an effect that was accompanied by an increase in GLUT4 and of phospho-aPKC at the muscle membrane. Contracted muscles treated with insulin did not show additive effects on glucose uptake or aPKC activity compared with the response when these stimuli were applied alone. Inhibition of PI3K blocked insulin effect on glucose uptake and aPKC but not in the contractile response. Thus, muscle contraction seems to stimulate aPKC and glucose uptake independently of PI3K. Therefore, aPKC may be a convergence point and a rate limit step in the pathway by which, insulin and contraction, increase glucose uptake in skeletal muscle. Copyright © 2014 John Wiley & Sons, Ltd.

  13. PKC-theta in regulatory and effector T-cell functions

    Directory of Open Access Journals (Sweden)

    Vedran eBrezar

    2015-10-01

    Full Text Available One of the major goals in immunology research is to understand the regulatory mechanisms that underpin the rapid switch on/off of robust and efficient effector (Teff or regulatory (Tregs T-cell responses. Understanding the molecular mechanisms underlying the regulation of such responses is critical for the development of effective therapies. T-cell activation involves the engagement of T-cell receptor and co-stimulatory signals, but the subsequent recruitment of serine/threonine-specific protein Kinase C-theta (PKC-θ to the immunological synapse is instrumental for the formation of signalling complexes, that ultimately lead to a transcriptional network in T cells. Recent studies demonstrated that major differences between Teffs and Tregs occurred at the immunological synapse where its formation induces altered signalling pathways in Tregs. These pathways are characterized by reduced recruitment of PKC-θ, suggesting that PKC-θ inhibits Tregs suppressive function in a negative feedback loop. As the balance of Teffs and Tregs has been shown to be central in several diseases, it was not surprising that some studies revealed that PKC-θ plays a major role in the regulation of this balance.This review will examine recent knowledge on the role of PKC-θ in T-cell transcriptional responses and how this protein can impact on the function of both Tregs and Teffs.

  14. The aPKC-CBP Pathway Regulates Adult Hippocampal Neurogenesis in an Age-Dependent Manner

    Directory of Open Access Journals (Sweden)

    Ayden Gouveia

    2016-10-01

    Full Text Available While epigenetic modifications have emerged as attractive substrates to integrate environmental changes into the determination of cell identity and function, specific signals that directly activate these epigenetic modifications remain unknown. Here, we examine the role of atypical protein kinase C (aPKC-mediated Ser436 phosphorylation of CBP, a histone acetyltransferase, in adult hippocampal neurogenesis and memory. Using a knockin mouse strain (CbpS436A in which the aPKC-CBP pathway is deficient, we observe impaired hippocampal neuronal differentiation, maturation, and memory and diminished binding of CBP to CREB in 6-month-old CbpS436A mice, but not at 3 months of age. Importantly, elevation of CREB activity rescues these deficits, and CREB activity is reduced whereas aPKC activity is increased in the murine hippocampus as they age from 3 to 6 months regardless of genotype. Thus, the aPKC-CBP pathway is a homeostatic compensatory mechanism that modulates hippocampal neurogenesis and memory in an age-dependent manner in response to reduced CREB activity.

  15. Runx-dependent expression of PKC is critical for cell survival in the sea urchin embryo

    Directory of Open Access Journals (Sweden)

    McCarthy John J

    2005-08-01

    Full Text Available Abstract Background Runx transcription factors play critical roles in the developmental control of cell fate and contribute variously as oncoproteins and tumor suppressors to leukemia and other cancers. To discover fundamental Runx functions in the cell biology of animal development, we have employed morpholino antisense-mediated knockdown of the sea urchin Runx protein SpRunt-1. Previously we showed that embryos depleted of SpRunt-1 arrest development at early gastrula stage and underexpress the conventional protein kinase C SpPKC1. Results We report here that SpRunt-1 deficiency leads to ectopic cell proliferation and extensive apoptosis. Suppression of the apoptosis by pharmacological inhibition of caspase-3 prevents the ectopic proliferation and rescues gastrulation, indicating that many of the overt defects obtained by knockdown of SpRunt-1 are secondary to the apoptosis. Inhibition or knockdown of SpPKC1 also causes apoptosis, while cell survival is rescued in SpRunt-1 morphant embryos coinjected with SpPKC1 mRNA, suggesting that the apoptosis associated with SpRunt-1 deficiency is caused by the deficit in SpPKC1 expression. Chromatin immunoprecipitation indicates that SpRunt-1 interacts physically with SpPKC1 in vivo, and cis-regulatory analysis shows that this interaction activates SpPKC1 transcription. Conclusions Our results show that Runx-dependent activation of SpPKC1 is essential for maintaining protein kinase C activity at levels conducive to cell survival during embryogenesis.

  16. Regulation of Kv1.4 potassium channels by PKC and AMPK kinases

    DEFF Research Database (Denmark)

    Andersen, Martin Nybo; Skibsbye, Lasse; Saljic, Arnela

    2018-01-01

    around the ubiquitin ligase Nedd4-2. In the present study we examined whether Kv1.4, constituting the cardiac Ito,s current, is subject to similar regulation. In the epithelial Madin-Darby Canine Kidney (MDCK) cell line, which constitutes a highly reproducible model system for addressing membrane...... targeting, we find, by confocal microscopy, that Kv1.4 cell surface expression is downregulated by activation of protein kinase C (PKC) and AMP-activated protein kinase (AMPK). In contrast, manipulating the activities of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and serum and glucocorticoid......-regulated kinase 1 (SGK1) were without effect on channel localization. The PKC and AMPK-mediated downregulation of Kv1.4 membrane surface localization was confirmed by two-electrode voltage clamp in Xenopus laevis oocytes, where pharmacological activation of PKC and AMPK reduced Kv1.4 current levels. We further...

  17. PKA and PKC Are Required for Long-Term but Not Short-Term in Vivo Operant Memory in "Aplysia"

    Science.gov (United States)

    Michel, Maximilian; Green, Charity L.; Lyons, Lisa C.

    2011-01-01

    We investigated the involvement of PKA and PKC signaling in a negatively reinforced operant learning paradigm in "Aplysia", learning that food is inedible (LFI). In vivo injection of PKA or PKC inhibitors blocked long-term LFI memory formation. Moreover, a persistent phase of PKA activity, although not PKC activity, was necessary for long-term…

  18. Knockout of the predominant conventional PKC isoform, PKCalpha, in mouse skeletal muscle does not affect contraction-stimulated glucose uptake

    DEFF Research Database (Denmark)

    Jensen, Thomas E; Maarbjerg, Stine J; Rose, Adam J

    2009-01-01

    Conventional (c) protein kinase C (PKC) activity has been shown to increase with skeletal muscle contraction, and numerous studies using primarily pharmacological inhibitors have implicated cPKCs in contraction-stimulated glucose uptake. Here, to confirm that cPKC activity is required for contrac...... working on other parts of contraction-induced signaling or the remaining cPKC isoforms are sufficient for stimulating glucose uptake during contractions.......Conventional (c) protein kinase C (PKC) activity has been shown to increase with skeletal muscle contraction, and numerous studies using primarily pharmacological inhibitors have implicated cPKCs in contraction-stimulated glucose uptake. Here, to confirm that cPKC activity is required...... for contraction-stimulated glucose uptake in mouse muscles, contraction-stimulated glucose uptake ex vivo was first evaluated in the presence of three commonly used cPKC inhibitors (calphostin C, Gö-6976, and Gö-6983) in incubated mouse soleus and extensor digitorum longus (EDL) muscles. All potently inhibited...

  19. ETV6–NTRK3 as a therapeutic target of small molecule inhibitor PKC412

    International Nuclear Information System (INIS)

    Chi, Hoang Thanh; Ly, Bui Thi Kim; Kano, Yasuhiko; Tojo, Arinobu; Watanabe, Toshiki; Sato, Yuko

    2012-01-01

    Highlights: ► ETV6–NTRK3 is an oncogene with transformation activity in multiple cell lineages. ► PKC412 could block ETV6–NTRK3 activation. ► Loss of ETV6–NTRK3 phosphorylation leads to inactivation of its downstream signaling pathway. ► Inhibition of ETV6–NTRK3 activation by PKC412 could be a novel strategy for the treatment. -- Abstract: The ETV6–NTRK3 (EN) fusion gene which encodes a chimeric tyrosine kinase was first identified by cloning of the t(12;15)(p13;q25) translocation in congenital fibrosarcoma (CFS). Since then, EN has been also found in congenital mesoblastic nephroma (CMN), secretory breast carcinoma (SBC) and acute myelogenous leukemia (AML). Using IMS-M2 and M0–91 cell lines harboring the EN fusion gene, and Ba/F3 cells stably transfected with EN, we demonstrated that PKC412, also known as midostaurin, is an inhibitor of EN. Inhibition of EN activity by PKC412 suppressed the activity of it downstream molecules leading to inhibition of cell proliferation and induction of apoptosis. Our data for the first time suggested that PKC412 could serve as therapeutic drug for treatment of patients with this fusion.

  20. The Role of miR-330-3p/PKC-α Signaling Pathway in Low-Dose Endothelial-Monocyte Activating Polypeptide-II Increasing the Permeability of Blood-Tumor Barrier

    Directory of Open Access Journals (Sweden)

    Jiahui Liu

    2017-12-01

    Full Text Available This study was performed to determine whether EMAP II increases the permeability of the blood-tumor barrier (BTB by affecting the expression of miR-330-3p as well as its possible mechanisms. We determined the over-expression of miR-330-3p in glioma microvascular endothelial cells (GECs by Real-time PCR. Endothelial monocyte-activating polypeptide-II (EMAP-II significantly decreased the expression of miR-330-3p in GECs. Pre-miR-330-3p markedly decreased the permeability of BTB and increased the expression of tight junction (TJ related proteins ZO-1, occludin and claudin-5, however, anti-miR-330-3p had the opposite effects. Anti-miR-330-3p could enhance the effect of EMAP-II on increasing the permeability of BTB, however, pre-miR-330-3p partly reversed the effect of EMAP-II on that. Similarly, anti-miR-330-3p improved the effects of EMAP-II on increasing the expression levels of PKC-α and p-PKC-α in GECs and pre-miR-330-3p partly reversed the effects. MiR-330-3p could target bind to the 3′UTR of PKC-α. The results of in vivo experiments were similar to those of in vitro experiments. These suggested that EMAP-II could increase the permeability of BTB through inhibiting miR-330-3p which target negative regulation of PKC-α. Pre-miR-330-3p and PKC-α inhibitor decreased the BTB permeability and up-regulated the expression levels of ZO-1, occludin and claudin-5 while anti-miR-330-3p and PKCactivator brought the reverse effects. Compared with EMAP-II, anti-miR-330-3p and PKCactivator alone, the combination of the three combinations significantly increased the BTB permeability. EMAP-II combined with anti-miR-330-3p and PKCα activator could enhance the DOX’s effects on inhibiting the cell viabilities and increasing the apoptosis of U87 glioma cells. Our studies suggest that low-dose EMAP-II up-regulates the expression of PKC-α and increases the activity of PKC-α by inhibiting the expression of miR-330-3p, reduces the expression of ZO-1

  1. PKC-η-MARCKS Signaling Promotes Intracellular Survival of Unopsonized Burkholderia thailandensis.

    Science.gov (United States)

    Micheva-Viteva, Sofiya N; Shou, Yulin; Ganguly, Kumkum; Wu, Terry H; Hong-Geller, Elizabeth

    2017-01-01

    Pathogenic Burkholderia rely on host factors for efficient intracellular replication and are highly refractory to antibiotic treatment. To identify host genes that are required by Burkholderia spp. during infection, we performed a RNA interference (RNAi) screen of the human kinome and identified 35 host kinases that facilitated Burkholderia thailandensis intracellular survival in human monocytic THP-1 cells. We validated a selection of host kinases using imaging flow cytometry to assess efficiency of B. thailandensis survival in the host upon siRNA-mediated knockdown. We focused on the role of the novel protein kinase C isoform, PKC-η, in Burkholderia infection and characterized PKC-η/MARCKS signaling as a key event that promotes the survival of unopsonized B. thailandensis CDC2721121 within host cells. While infection of lung epithelial cells with unopsonized Gram-negative bacteria stimulated phosphorylation of Ser175/160 in the MARCKS effector domain, siRNA-mediated knockdown of PKC-η expression reduced the levels of phosphorylated MARCKS by >3-fold in response to infection with Bt CDC2721121. We compared the effect of the conventional PKC-α and novel PKC-η isoforms on the growth of B. thailandensis CDC2721121 within monocytic THP-1 cells and found that ≥75% knock-down of PRKCH transcript levels reduced intracellular bacterial load 100% more efficiently when compared to growth in cells siRNA-depleted of the classical PKC-α, suggesting that the PKC-η isoform can specifically mediate Burkholderia intracellular survival. Based on imaging studies of intracellular B. thailandensis , we found that PKC-η function stimulates phagocytic pathways that promote B. thailandensis escape into the cytoplasm leading to activation of autophagosome flux. Identification of host kinases that are targeted by Burkholderia during infection provides valuable molecular insights in understanding Burkholderia pathogenesis, and ultimately, in designing effective host

  2. Palmitic acid mediates hypothalamic insulin resistance by altering PKC-θ subcellular localization in rodents

    OpenAIRE

    Benoit, Stephen C.; Kemp, Christopher J.; Elias, Carol F.; Abplanalp, William; Herman, James P.; Migrenne, Stephanie; Lefevre, Anne-Laure; Cruciani-Guglielmacci, Céline; Magnan, Christophe; Yu, Fang; Niswender, Kevin; Irani, Boman G.; Holland, William L.; Clegg, Deborah J.

    2009-01-01

    Insulin signaling can be modulated by several isoforms of PKC in peripheral tissues. Here, we assessed whether one specific isoform, PKC-θ, was expressed in critical CNS regions that regulate energy balance and whether it mediated the deleterious effects of diets high in fat, specifically palmitic acid, on hypothalamic insulin activity in rats and mice. Using a combination of in situ hybridization and immunohistochemistry, we found that PKC-θ was expressed in discrete neuronal populations of ...

  3. PKC 412 sensitizes U1810 non-small cell lung cancer cells to DNA damage

    International Nuclear Information System (INIS)

    Hemstroem, Therese H.; Joseph, Bertrand; Schulte, Gunnar; Lewensohn, Rolf; Zhivotovsky, Boris

    2005-01-01

    Non-small cell lung carcinoma (NSCLC) is characterized by resistance to drug-induced apoptosis, which might explain the survival of lung cancer cells following treatment. Recently we have shown that the broad-range kinase inhibitor staurosporine (STS) reactivates the apoptotic machinery in U1810 NSCLC cells [Joseph et al., Oncogene 21 (2002) 65]. Lately, several STS analogs that are more specific in kinase inhibition have been suggested for tumor treatment. In this study the apoptosis-inducing ability of the STS analogs PKC 412 and Ro 31-8220 used alone or in combination with DNA-damaging agents in U1810 cells was investigated. In these cells Ro 31-8220 neither induced apoptosis when used alone, nor sensitized cells to etoposide treatment. PKC 412 as a single agent induced death of a small number of U1810 cells, whereas it efficiently triggered a dose- and time-dependent apoptosis in U1285 small cell lung carcinoma cells. In both cell types PKC 412 triggered release of mitochondrial proteins followed by caspase activation. However, concomitant activation of a caspase-independent pathway was essential to kill NSCLC cells. Importantly, PKC 412 was able to sensitize etoposide- and radiation-induced death of U1810 cells. The best sensitization was achieved when PKC 412 was administered 24 h after treatments. In U1810 cells, Ro 31-8220 decreased PMA-induced ERK phosphorylation as efficiently as PKC 412, indicating that the failure of Ro 31-8220 to induce apoptosis was not due to weaker inhibition of conventional and novel PKC isoforms. However, Ro 31-8220 increased the basal level of ERK and Akt phosphorylation in both cell lines, whereas Akt phosphorylation was suppressed in the U1810 cells, which might influence apoptosis. These results suggest that PKC 412 could be a useful tool in increasing the efficiency of therapy of NSCLC

  4. Role for PKC-ε in neuronal death induced by oxidative stress

    International Nuclear Information System (INIS)

    Jung, Yi-Sook; Ryu, Bo Rum; Lee, Bo Kyung; Mook-Jung, Inhee; Kim, Seung Up; Lee, Soo Hwan; Baik, Eun Joo; Moon, Chang-Hyun

    2004-01-01

    We investigated which isoforms of PKCs can be modulated and what their roles are during L-buthionine-S,R-sulfoximine (BSO)-induced neuronal death. We observed the isoform specific translocation of PKC-ε from the soluble fraction to the particulate in cortical neurons treated with 10 mM BSO. The translocation of PKC-ε by BSO was blocked by antioxidant trolox, suggesting the PKC-ε as a downstream of reactive oxygen species (ROS) elevated by BSO. Trolox inhibited the ROS elevation and the neuronal death in BSO-treated cortical cells. The BSO-induced neuronal death was remarkably inhibited by both the pharmacological inhibition of PKC-ε with εV1-2 and the functional blockade for PKC-ε through overexpression of PKC-ε V1 region, suggesting the detrimental role of PKC-ε. These results suggest that PKC-ε is the major PKC isoform involved in the pathways triggered by ROS, leading to neuronal death in BSO-treated cortical neurons

  5. Intermittent reductions in respiratory neural activity elicit spinal TNF-α-independent, atypical PKC-dependent inactivity-induced phrenic motor facilitation.

    Science.gov (United States)

    Baertsch, Nathan A; Baker-Herman, Tracy L

    2015-04-15

    In many neural networks, mechanisms of compensatory plasticity respond to prolonged reductions in neural activity by increasing cellular excitability or synaptic strength. In the respiratory control system, a prolonged reduction in synaptic inputs to the phrenic motor pool elicits a TNF-α- and atypical PKC-dependent form of spinal plasticity known as inactivity-induced phrenic motor facilitation (iPMF). Although iPMF may be elicited by a prolonged reduction in respiratory neural activity, iPMF is more efficiently induced when reduced respiratory neural activity (neural apnea) occurs intermittently. Mechanisms giving rise to iPMF following intermittent neural apnea are unknown. The purpose of this study was to test the hypothesis that iPMF following intermittent reductions in respiratory neural activity requires spinal TNF-α and aPKC. Phrenic motor output was recorded in anesthetized and ventilated rats exposed to brief intermittent (5, ∼1.25 min), brief sustained (∼6.25 min), or prolonged sustained (30 min) neural apnea. iPMF was elicited following brief intermittent and prolonged sustained neural apnea, but not following brief sustained neural apnea. Unlike iPMF following prolonged neural apnea, spinal TNF-α was not required to initiate iPMF during intermittent neural apnea; however, aPKC was still required for its stabilization. These results suggest that different patterns of respiratory neural activity induce iPMF through distinct cellular mechanisms but ultimately converge on a similar downstream pathway. Understanding the diverse cellular mechanisms that give rise to inactivity-induced respiratory plasticity may lead to development of novel therapeutic strategies to treat devastating respiratory control disorders when endogenous compensatory mechanisms fail. Copyright © 2015 the American Physiological Society.

  6. Nup358 interacts with Dishevelled and aPKC to regulate neuronal polarity

    Directory of Open Access Journals (Sweden)

    Pankhuri Vyas

    2013-10-01

    Par polarity complex, consisting of Par3, Par6, and aPKC, plays a conserved role in the establishment and maintenance of polarization in diverse cellular contexts. Recent reports suggest that Dishevelled (Dvl, a cytoplasmic mediator of Wnt signalling, interacts with atypical protein kinase C and regulates its activity during neuronal differentiation and directed cell migration. Here we show that Nup358 (also called RanBP2, a nucleoporin previously implicated in polarity during directed cell migration, interacts with Dishevelled and aPKC through its N-terminal region (BPN and regulates axon–dendrite differentiation of cultured hippocampal neurons. Depletion of endogenous Nup358 leads to generation of multiple axons, whereas overexpression of BPN abrogates the process of axon formation. Moreover, siRNA-mediated knockdown of Dvl or inhibition of aPKC by a pseudosubstrate inhibitor significantly reverses the multiple axon phenotype produced by Nup358 depletion. Collectively, these data suggest that Nup358 plays an important role in regulating neuronal polarization upstream to Dvl and aPKC.

  7. Inhibition of MAPK and PKC pathways by 60Co γ-radiation in cultured vascular smooth muscle cells

    International Nuclear Information System (INIS)

    Jia Guanghong; Ma Yexin; Xiao Jianming

    2002-01-01

    Objective: To investigate the signal transduction pathways inhibited by 60 Co γ-radiation in cultured vascular smooth muscle cells (VSMC). Methods: The cultured VSMC were irradiated with 60 Co γ-radiation of 3.5, 7.0 and 14 Gy respectively. VSMC proliferation was measured by 3 H-TdR incorporation, while PKC, MAPK activities were determined by radioactivity assay. Results: Proliferation of VSMC was inhibited by 7.0, 14 Gy 60 Co γ-irradiation and the activities of PKC, MAPK were decreased significantly. Conclusion: Inhibitory effect of 7.0, 14 Gy 60 Co γ-irradiation on proliferation of VSMC might be resulted from decrease of the activity of PKC, MAPK

  8. Decreased phosphorylation of δ and ε subunits of the acetylcholine receptor coincides with delayed postsynaptic maturation in PKC θ deficient mouse.

    Science.gov (United States)

    Lanuza, Maria A; Besalduch, Núria; González, Carmen; Santafé, Manel M; Garcia, Neus; Tomàs, Marta; Nelson, Phillip G; Tomàs, Josep

    2010-09-01

    Protein kinase C (PKC) activity is involved in the nicotinic acetylcholine receptor (nAChR) redistribution at the neuromuscular junction in vivo during postnatal maturation. Here we studied, in PKC theta (PKCtheta) deficient mice (KO), how the theta isoform of PKC is involved in the nAChR cluster maturation that is accompanied by the developmental activity-dependent neuromuscular synapse elimination process. We found that axonal elimination and dispersion of nAChR from the postsynaptic plaques and its redistribution to form the mature postsynaptic apparatus were delayed but not totally suppressed in PKCtheta deficient mice. Moreover, the delay in the maturation of the morphology of the nAChR clusters during the early postnatal synapse elimination period in the PKCtheta deficient mice coincides with a reduction in the PKCtheta-mediated phosphorylation on the delta subunit of the nAChR. In addition, we show evidence for PKCtheta regulation of PKA in normally phosphorylating the epsilon subunit of nAChR. We have also found that the theta isoform of PKC is located on the postsynaptic component of the neuromuscular junction but is also expressed by motoneurons in the spinal cord and in the motor nerve terminals. The results allow us to hypothesize that a spatially specific and opposing action of PKCtheta and PKA may result in activity-dependent alterations to synaptic connectivity at both the nerve inputs and the postsynaptic nAChR clusters. Copyright 2010 Elsevier Inc. All rights reserved.

  9. A novel DLX3-PKC integrated signaling network drives keratinocyte differentiation.

    Science.gov (United States)

    Palazzo, Elisabetta; Kellett, Meghan D; Cataisson, Christophe; Bible, Paul W; Bhattacharya, Shreya; Sun, Hong-Wei; Gormley, Anna C; Yuspa, Stuart H; Morasso, Maria I

    2017-04-01

    Epidermal homeostasis relies on a well-defined transcriptional control of keratinocyte proliferation and differentiation, which is critical to prevent skin diseases such as atopic dermatitis, psoriasis or cancer. We have recently shown that the homeobox transcription factor DLX3 and the tumor suppressor p53 co-regulate cell cycle-related signaling and that this mechanism is functionally involved in cutaneous squamous cell carcinoma development. Here we show that DLX3 expression and its downstream signaling depend on protein kinase C α (PKCα) activity in skin. We found that following 12-O-tetradecanoyl-phorbol-13-acetate (TPA) topical treatment, DLX3 expression is significantly upregulated in the epidermis and keratinocytes from mice overexpressing PKCα by transgenic targeting (K5-PKCα), resulting in cell cycle block and terminal differentiation. Epidermis lacking DLX3 (DLX3cKO), which is linked to the development of a DLX3-dependent epidermal hyperplasia with hyperkeratosis and dermal leukocyte recruitment, displays enhanced PKCα activation, suggesting a feedback regulation of DLX3 and PKCα. Of particular significance, transcriptional activation of epidermal barrier, antimicrobial peptide and cytokine genes is significantly increased in DLX3cKO skin and further increased by TPA-dependent PKC activation. Furthermore, when inhibiting PKC activity, we show that epidermal thickness, keratinocyte proliferation and inflammatory cell infiltration are reduced and the PKC-DLX3-dependent gene expression signature is normalized. Independently of PKC, DLX3 expression specifically modulates regulatory networks such as Wnt signaling, phosphatase activity and cell adhesion. Chromatin immunoprecipitation sequencing analysis of primary suprabasal keratinocytes showed binding of DLX3 to the proximal promoter regions of genes associated with cell cycle regulation, and of structural proteins and transcription factors involved in epidermal differentiation. These results indicate

  10. Neurotensin Phosphorylates GSK-3α/β through the Activation of PKC in Human Colon Cancer Cells

    Directory of Open Access Journals (Sweden)

    Qingding Wang

    2006-09-01

    Full Text Available Neurotensin (NT, a gastrointestinal hormone, binds its receptor [neurotensin receptor (NTR] to regulate the growth of normal and neoplastic intestinal cells; molecular mechanisms remain largely undefined. Glycogen synthase kinase-3 (GSK-3 regulates diverse cellular processes, including cell growth and apoptosis. Here, we show that NT induces the phosphorylation of GSK-3α/β in the human colon cancer cell line HT29, HCT116, or SW480, which possesses high-affinity NTR. The effect of NT was blocked by inhibitors of protein kinase C (PKC, but not by inhibitors of mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK1 or phosphatidylinositol-3 kinase, suggesting a predominant role for PKC in GSK-3β phosphorylation by NT. Pretreatment with Gö6976 (which inhibits PKCα and PKCβ1 or downregulation of endogenous PKCα or PKCβ1 blocked NT-mediated GSK-3β (but not GSK-3α phosphorylation. Moreover, a selective PKCβ inhibitor, LY379196, reduced NT-mediated GSK-3β (but not GSK-3α phosphorylation, suggesting a role for PKCbβ in the NT-mediated phosphorylation of GSK-3β and an undefined kinase in the NT-mediated phosphorylation of GSK-3α. Treatment with NT or the GSK-3 inhibitor SB216763 increased the expression of cyclin D1, a downstream effector protein of GSK-3 and a critical protein for the proliferation of various cells. Our results indicate that NT uses PKC-dependent pathways to modulate GSK-3, which may play a role in the NT regulation of intestinal cell growth.

  11. Curcumin inhibits EMMPRIN and MMP-9 expression through AMPK-MAPK and PKC signaling in PMA induced macrophages.

    Science.gov (United States)

    Cao, Jiatian; Han, Zhihua; Tian, Lei; Chen, Kan; Fan, Yuqi; Ye, Bozhi; Huang, Weijian; Wang, Changqian; Huang, Zhouqing

    2014-09-21

    In coronary arteries, plaque disruption, the major acute clinical manifestations of atherosclerosis, leads to a subsequent cardiac event, such as acute myocardial infarction (AMI) and unstable angina pectoris (UA). Numerous reports have shown that high expression of MMP-9 (matrix metalloproteinase-9), MMP-13 (matrix metalloproteinase-13) and EMMPRIN (extracellular matrix metalloproteinase induce) in monocyte/macrophage results in the plaque progression and destabilization. Curcumin exerts well-known anti-inflammatory and antioxidant effects and probably has a protective role in the atherosclerosis. The purpose of our study was to investigate the molecular mechanisms by which curcumin affects MMP-9, MMP13 and EMMPRIN in PMA (phorbol 12-myristate 13-acetate) induced macrophages. Human monocytic cells (THP-1 cells) were pretreated with curcumin or compound C for 1 h, and then induced by PMA for 48 h. Total RNA and proteins were collected for real-time PCR and Western blot analysis, respectively. In the present study, the exposure to curcumin resulted in attenuated JNK, p38, and ERK activation and decreased expression of MMP-9, MMP-13 and EMMPRIN in PMA induced macrophages. Moreover, we demonstrated that AMPK (AMP-activated protein kinase) and PKC (Protein Kinase C) was activated by PMA during monocyte/macrophage differentiation. Furthermore, curcumin reversed PMA stimulated PKC activation and suppressed the chronic activation of AMPK, which in turn reduced the expression of MMP-9, MMP-13 and EMMPRIN. Therefore, it is suggested that curcumin by inhibiting AMPK-MAPK (mitogen activated protein kinase) and PKC pathway may led to down-regulated EMMPRIN, MMP-9 and MMP-13 expression in PMA-induced THP-1 cells.

  12. 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.

  13. PKC-epsilon activation is required for recognition memory in the rat.

    Science.gov (United States)

    Zisopoulou, Styliani; Asimaki, Olga; Leondaritis, George; Vasilaki, Anna; Sakellaridis, Nikos; Pitsikas, Nikolaos; Mangoura, Dimitra

    2013-09-15

    Activation of PKCɛ, an abundant and developmentally regulated PKC isoform in the brain, has been implicated in memory throughout life and across species. Yet, direct evidence for a mechanistic role for PKCɛ in memory is still lacking. Hence, we sought to evaluate this in rats, using short-term treatments with two PKCɛ-selective peptides, the inhibitory ɛV1-2 and the activating ψɛRACK, and the novel object recognition task (NORT). Our results show that the PKCɛ-selective activator ψɛRACK, did not have a significant effect on recognition memory. In the short time frames used, however, inhibition of PKCɛ activation with the peptide inhibitor ɛV1-2 significantly impaired recognition memory. Moreover, when we addressed at the molecular level the immediate proximal signalling events of PKCɛ activation in acutely dissected rat hippocampi, we found that ψɛRACK increased in a time-dependent manner phosphorylation of MARCKS and activation of Src, Raf, and finally ERK1/2, whereas ɛV1-2 inhibited all basal activity of this pathway. Taken together, these findings present the first direct evidence that PKCɛ activation is an essential molecular component of recognition memory and point toward the use of systemically administered PKCɛ-regulating peptides as memory study tools and putative therapeutic agents. Copyright © 2013 Elsevier B.V. All rights reserved.

  14. Platelet activating factor enhances synaptic vesicle exocytosis via PKC, elevated intracellular calcium, and modulation of synapsin 1 dynamics and phosphorylation

    Directory of Open Access Journals (Sweden)

    Jennetta W Hammond

    2016-01-01

    Full Text Available Platelet activating factor (PAF is an inflammatory phospholipid signaling molecule implicated in synaptic plasticity, learning and memory and neurotoxicity during neuroinflammation. However, little is known about the intracellular mechanisms mediating PAF’s physiological or pathological effects on synaptic facilitation. We show here that PAF receptors are localized at the synapse. Using fluorescent reporters of presynaptic activity we show that a non-hydrolysable analogue of PAF (cPAF enhances synaptic vesicle release from individual presynaptic boutons by increasing the size or release of the readily releasable pool and the exocytosis rate of the total recycling pool. cPAF also activates previously silent boutons resulting in vesicle release from a larger number of terminals. The underlying mechanism involves elevated calcium within presynaptic boutons and protein kinase C (PKC activation. Furthermore, cPAF increases synapsin I phosphorylation at sites 1 and 3, and increases dispersion of synapsin I from the presynaptic compartment during stimulation, freeing synaptic vesicles for subsequent release. These findings provide a conceptual framework for how PAF, regardless of its cellular origin, can modulate synapses during normal and pathologic synaptic activity.

  15. A novel DLX3–PKC integrated signaling network drives keratinocyte differentiation

    Science.gov (United States)

    Palazzo, Elisabetta; Kellett, Meghan D; Cataisson, Christophe; Bible, Paul W; Bhattacharya, Shreya; Sun, Hong-wei; Gormley, Anna C; Yuspa, Stuart H; Morasso, Maria I

    2017-01-01

    Epidermal homeostasis relies on a well-defined transcriptional control of keratinocyte proliferation and differentiation, which is critical to prevent skin diseases such as atopic dermatitis, psoriasis or cancer. We have recently shown that the homeobox transcription factor DLX3 and the tumor suppressor p53 co-regulate cell cycle-related signaling and that this mechanism is functionally involved in cutaneous squamous cell carcinoma development. Here we show that DLX3 expression and its downstream signaling depend on protein kinase C α (PKCα) activity in skin. We found that following 12-O-tetradecanoyl-phorbol-13-acetate (TPA) topical treatment, DLX3 expression is significantly upregulated in the epidermis and keratinocytes from mice overexpressing PKCα by transgenic targeting (K5-PKCα), resulting in cell cycle block and terminal differentiation. Epidermis lacking DLX3 (DLX3cKO), which is linked to the development of a DLX3-dependent epidermal hyperplasia with hyperkeratosis and dermal leukocyte recruitment, displays enhanced PKCα activation, suggesting a feedback regulation of DLX3 and PKCα. Of particular significance, transcriptional activation of epidermal barrier, antimicrobial peptide and cytokine genes is significantly increased in DLX3cKO skin and further increased by TPA-dependent PKC activation. Furthermore, when inhibiting PKC activity, we show that epidermal thickness, keratinocyte proliferation and inflammatory cell infiltration are reduced and the PKC-DLX3-dependent gene expression signature is normalized. Independently of PKC, DLX3 expression specifically modulates regulatory networks such as Wnt signaling, phosphatase activity and cell adhesion. Chromatin immunoprecipitation sequencing analysis of primary suprabasal keratinocytes showed binding of DLX3 to the proximal promoter regions of genes associated with cell cycle regulation, and of structural proteins and transcription factors involved in epidermal differentiation. These results indicate

  16. Expression of P-aPKC-iota, E-cadherin, and beta-catenin related to invasion and metastasis in hepatocellular carcinoma.

    Science.gov (United States)

    Du, Guang-Sheng; Wang, Jian-Ming; Lu, Jin-Xi; Li, Qiang; Ma, Chao-Qun; Du, Ji-Tao; Zou, Sheng-Quan

    2009-06-01

    Atypical protein kinase C iota (aPKC-iota) and its associated intracellular molecules, E-cadherin and beta-catenin, are important for cell polarization in tumorigenesis and progression. Expression of aPKC-iota, P-aPKC-iota (activated aPKC-iota), E-cadherin, and beta-catenin in hepatocellular carcinoma (HCC) was measured, and correlation with clinicopathological characteristics of HCC was analyzed. Paraffin-embedded tumor tissue was obtained from patients with HCC after resection without preoperative radiotherapy or chemotherapy. Gene expression was detected by polymerase chain reaction (PCR), and protein expression was detected by immunohistochemistry and Western blot analysis. Expressions of aPKC-iota, P-aPKC-iota, E-cadherin, and beta-catenin were analyzed with relation to the clinicopathological data. The gene and protein expression of aPKC-iota are obviously higher in HCC tissues than that in peritumoral tissues and normal tissues by semiquantitative PCR and immunohistochemistry methods. Accumulation of aPKC-iota in HCC cytoplasm and nucleolus inhibited the later formation of belt-like adherens junctions (AJs) and/or tight junctions (TJs) in cell-cell contact. E-cadherin was reduced and accumulation of cytoplasm beta-catenin was increased in HCC. The expression of aPKC-iota was closely related to pathological differentiation, tumor size, invasion, and metastasis of HCC. Accumulation of cytoplasm aPKC-iota may reflect pathological differentiation, invasion, and metastasis potential of HCC. In this regard, our study on HCC revealed the potential usefulness of aPKC-iota, E-cadherin, and beta-catenin as a prognostic marker, closely related to pathological differentiation, invasion, metastasis, and prognosis of HCC.

  17. Hu-Lu-Ba-Wan Attenuates Diabetic Nephropathy in Type 2 Diabetic Rats through PKC-α/NADPH Oxidase Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Lishan Zhou

    2013-01-01

    Full Text Available Hu-Lu-Ba-Wan (HLBW is a Chinese herbal prescription used to treat kidney deficiency. The aim of this study was to explore the effect and mechanism of HLBW on diabetic nephropathy (DN in type 2 diabetic rats. The rat model of DN was established by being fed a high-fat diet and intravenous injection of streptozotocin. Then, HLBW decoction was administered for 16 weeks. Blood glucose level, lipid profile, renal function, 24-hour total urinary protein, and albumin content were examined. Renal morphology and superoxide anion levels were evaluated. The activity of nicotinamide-adenine dinucleotide phosphate (NADPH and protein kinase C-alpha (PKC-α related genes expression in renal tissue were also determined. Our data demonstrated that HLBW significantly improved hyperglycemia, hyperlipidemia, and proteinuria in diabetic rats compared with those of control group. HLBW also alleviated glomerular expansion and fibrosis, extracellular matrix accumulation and effacement of the foot processes. Additionally, HLBW reduced superoxide anion level, NADPH oxidase activity, the protein and mRNA expressions of p47phox, and the protein expression of phosphorylated PKC-α in renal tissue. These results suggest that HLBW is effective in the treatment of DN in rats. The underlying mechanism may be related to the attenuation of renal oxidative stress via PKC-α/NADPH oxidase signaling pathway.

  18. Quercetin inhibits the invasion of murine melanoma B16-BL6 cells by decreasing pro-MMP-9 via the PKC pathway.

    Science.gov (United States)

    Zhang, Xian-Ming; Huang, Shao-Peng; Xu, Qiang

    2004-01-01

    On the basis of the inhibitory effect of quercetin on the invasion of melanoma B16-BL6 cells previously reported by us, the mechanisms of quercetin-mediated inhibition of invasion were further investigated in the present study. The ability of B16-BL6 cells to invade and migrate was evaluated in terms of the numbers of cells penetrating a reconstituted basement membrane in the Transwell coculture system. The relative levels and activities of matrix metalloproteinase-9 (MMP-9) and MMP-2 were determined by gelatin zymography and quantified using LabWorks 4.0 software. The quercetin-mediated inhibition of invasion was partially blocked by phorbol-12,13-dibutyrate (PDB), a PKC (protein kinase C) activator, and by doxorubicin, a PKC inhibitor. Only the proforms of MMP-9 (92 kDa) and MMP-2 (72 kDa) were detected by gelatin zymography. Quercetin dose-dependently decreased the gelatinolytic activity of pro-MMP-9. Doxorubicin also markedly reversed the quercetin-induced decrease. Quercetin showed a dose-dependent antagonism of increases in gelatinolytic activity of pro-MMP-9 induced by PDB and free fatty acid (another PKC activator). Together with the report that quercetin directly reduces PKC activity, the results reported here suggest that quercetin may inhibit the invasion of B16-BL6 cells by decreasing pro-MMP-9 via the PKC pathway.

  19. PKC signaling regulates drug resistance of the fungal pathogen Candida albicans via circuitry comprised of Mkc1, calcineurin, and Hsp90.

    Directory of Open Access Journals (Sweden)

    Shantelle L LaFayette

    2010-08-01

    Full Text Available Fungal pathogens exploit diverse mechanisms to survive exposure to antifungal drugs. This poses concern given the limited number of clinically useful antifungals and the growing population of immunocompromised individuals vulnerable to life-threatening fungal infection. To identify molecules that abrogate resistance to the most widely deployed class of antifungals, the azoles, we conducted a screen of 1,280 pharmacologically active compounds. Three out of seven hits that abolished azole resistance of a resistant mutant of the model yeast Saccharomyces cerevisiae and a clinical isolate of the leading human fungal pathogen Candida albicans were inhibitors of protein kinase C (PKC, which regulates cell wall integrity during growth, morphogenesis, and response to cell wall stress. Pharmacological or genetic impairment of Pkc1 conferred hypersensitivity to multiple drugs that target synthesis of the key cell membrane sterol ergosterol, including azoles, allylamines, and morpholines. Pkc1 enabled survival of cell membrane stress at least in part via the mitogen activated protein kinase (MAPK cascade in both species, though through distinct downstream effectors. Strikingly, inhibition of Pkc1 phenocopied inhibition of the molecular chaperone Hsp90 or its client protein calcineurin. PKC signaling was required for calcineurin activation in response to drug exposure in S. cerevisiae. In contrast, Pkc1 and calcineurin independently regulate drug resistance via a common target in C. albicans. We identified an additional level of regulatory control in the C. albicans circuitry linking PKC signaling, Hsp90, and calcineurin as genetic reduction of Hsp90 led to depletion of the terminal MAPK, Mkc1. Deletion of C. albicans PKC1 rendered fungistatic ergosterol biosynthesis inhibitors fungicidal and attenuated virulence in a murine model of systemic candidiasis. This work establishes a new role for PKC signaling in drug resistance, novel circuitry through which

  20. Long-term pioglitazone treatment augments insulin sensitivity and PKC-epsilon and PKC-theta activation in skeletal muscles in sucrose fed rats

    Czech Academy of Sciences Publication Activity Database

    Marková, I.; Zídek, Václav; Musilová, Alena; Šimáková, Miroslava; Mlejnek, Petr; Kazdová, L.; Pravenec, Michal

    2010-01-01

    Roč. 59, č. 4 (2010), s. 509-516 ISSN 0862-8408 R&D Projects: GA MŠk(CZ) 1M0520; GA MŠk(CZ) ME08006; GA AV ČR(CZ) IAA500110604; GA MZd(CZ) NR9387; GA MZd(CZ) NR9359; GA MZd(CZ) NS9759 Institutional research plan: CEZ:AV0Z50110509 Keywords : pioglitazone * PKC * insulin resistance Subject RIV: FB - Endocrinology, Diabetology, Metabolism, Nutrition Impact factor: 1.646, year: 2010

  1. Failure of the PTEN/aPKC/Lgl Axis Primes Formation of Adult Brain Tumours in Drosophila

    Directory of Open Access Journals (Sweden)

    Simona Paglia

    2017-01-01

    Full Text Available Different regions in the mammalian adult brain contain immature precursors, reinforcing the concept that brain cancers, such as glioblastoma multiforme (GBM, may originate from cells endowed with stem-like properties. Alterations of the tumour suppressor gene PTEN are very common in primary GBMs. Very recently, PTEN loss was shown to undermine a specific molecular axis, whose failure is associated with the maintenance of the GBM stem cells in mammals. This axis is composed of PTEN, aPKC, and the polarity determinant Lethal giant larvae (Lgl: PTEN loss promotes aPKC activation through the PI3K pathway, which in turn leads to Lgl inhibition, ultimately preventing stem cell differentiation. To find the neural precursors responding to perturbations of this molecular axis, we targeted different neurogenic regions of the Drosophila brain. Here we show that PTEN mutation impacts aPKC and Lgl protein levels also in Drosophila. Moreover, we demonstrate that PI3K activation is not sufficient to trigger tumourigenesis, while aPKC promotes hyperplastic growth of the neuroepithelium and a noticeable expansion of the type II neuroblasts. Finally, we show that these neuroblasts form invasive tumours that persist and keep growing in the adult, leading the affected animals to untimely death, thus displaying frankly malignant behaviours.

  2. Polydatin Attenuates H2O2-Induced Oxidative Stress via PKC Pathway

    Directory of Open Access Journals (Sweden)

    Huilian Qiao

    2016-01-01

    Full Text Available Oxidative stress plays an important role in the pathogenesis of endothelial dysfunction, which is found to precede the development of diverse cardiovascular diseases (CVDs. The aim of this study was to observe the protective effects of PD against H2O2-induced oxidative stress injury (OSI in human umbilical vein endothelial cells (HUVECs and the possible mechanism of PD in OSI treatment. HUVECs were subjected to H2O2 in the absence or presence of PD. It turned out that PD improved cell viability and adhesive and migratory abilities, inhibited the release of lactate dehydrogenase (LDH and reactive oxygen species (ROS, and elevated the content of glutathione peroxidase (GSH-Px and superoxide dismutase (SOD. TUNEL, fluorometric assays, and Western blotting showed that OSI upregulated the apoptosis ratio, the activity of caspase-3 and the level of proapoptotic protein Bax and decreased the level of antiapoptotic protein Bcl-2. However, PD treatment partially reversed these damage effects and Protein Kinase C (PKC activation by thymeleatoxin (THX in turn eliminated the antiapoptotic effect of PD. Furthermore, PD attenuated the H2O2-induced phosphorylation of PKCs α and δ and increased the phosphorylation of PKC ε. Our results indicated that PD might exert protective effects against OSI through various interactions with PKC pathway.

  3. Modulation of transglutaminase 2 activity in H9c2 cells by PKC and PKA signalling: a role for transglutaminase 2 in cytoprotection

    Science.gov (United States)

    Almami, Ibtesam; Dickenson, John M; Hargreaves, Alan J; Bonner, Philip L R

    2014-01-01

    BACKGROUND AND PURPOSE Tissue transglutaminase (TG2) has been shown to mediate cell survival in many cell types. In this study, we investigated whether the role of TG2 in cytoprotection was mediated by the activation of PKA and PKC in cardiomyocyte-like H9c2 cells. EXPERIMENTAL APPROACH H9c2 cells were extracted following stimulation with phorbol-12-myristate-13-acetate (PMA) and forskolin. Transglutaminase activity was determined using an amine incorporating and a protein crosslinking assay. The presence of TG isoforms (TG1, 2, 3) was determined using Western blot analysis. The role of TG2 in PMA- and forskolin-induced cytoprotection was investigated by monitoring H2O2-induced oxidative stress in H9c2 cells. KEY RESULTS Western blotting showed TG2 >> TG1 protein expression but no detectable TG3. The amine incorporating activity of TG2 in H9c2 cells increased in a time and concentration-dependent manner following stimulation with PMA and forskolin. PMA and forskolin-induced TG2 activity was blocked by PKC (Ro 31-8220) and PKA (KT 5720 and Rp-8-Cl-cAMPS) inhibitors respectively. The PMA- and forskolin-induced increases in TG2 activity were attenuated by the TG2 inhibitors Z-DON and R283. Immunocytochemistry revealed TG2-mediated biotin-X-cadaverine incorporation into proteins and proteomic analysis identified known (β-tubulin) and novel (α-actinin) protein substrates for TG2. Pretreatment with PMA and forskolin reversed H2O2-induced decrease in MTT reduction and release of LDH. TG2 inhibitors R283 and Z-DON blocked PMA- and forskolin-induced cytoprotection. CONCLUSIONS AND IMPLICATIONS TG2 activity was stimulated via PKA- and PKC-dependent signalling pathways in H9c2 cells These results suggest a role for TG2 in cytoprotection induced by these kinases. PMID:24821315

  4. A PKC-MARCKS-PI3K regulatory module links Ca2+ and PIP3 signals at the leading edge of polarized macrophages.

    Directory of Open Access Journals (Sweden)

    Brian P Ziemba

    Full Text Available The leukocyte chemosensory pathway detects attractant gradients and directs cell migration to sites of inflammation, infection, tissue damage, and carcinogenesis. Previous studies have revealed that local Ca2+ and PIP3 signals at the leading edge of polarized leukocytes play central roles in positive feedback loop essential to cell polarization and chemotaxis. These prior studies showed that stimulation of the leading edge Ca2+ signal can strongly activate PI3K, thereby triggering a larger PIP3 signal, but did not elucidate the mechanistic link between Ca2+ and PIP3 signaling. A hypothesis explaining this link emerged, postulating that Ca2+-activated PKC displaces the MARCKS protein from plasma membrane PIP2, thereby releasing sequestered PIP2 to serve as the target and substrate lipid of PI3K in PIP3 production. In vitro single molecule studies of the reconstituted pathway on lipid bilayers demonstrated the feasibility of this PKC-MARCKS-PI3K regulatory module linking Ca2+ and PIP3 signals in the reconstituted system. The present study tests the model predictions in live macrophages by quantifying the effects of: (a two pathway activators-PDGF and ATP that stimulate chemoreceptors and Ca2+ influx, respectively; and (b three pathway inhibitors-wortmannin, EGTA, and Go6976 that inhibit PI3K, Ca2+ influx, and PKC, respectively; on (c four leading edge activity sensors-AKT-PH-mRFP, CKAR, MARCKSp-mRFP, and leading edge area that report on PIP3 density, PKC activity, MARCKS membrane binding, and leading edge expansion/contraction, respectively. The results provide additional evidence that PKC and PI3K are both essential elements of the leading edge positive feedback loop, and strongly support the existence of a PKC-MARCKS-PI3K regulatory module linking the leading edge Ca2+ and PIP3 signals. As predicted, activators stimulate leading edge PKC activity, displacement of MARCKS from the leading edge membrane and increased leading edge PIP3 levels, while

  5. A PKC-MARCKS-PI3K regulatory module links Ca2+ and PIP3 signals at the leading edge of polarized macrophages.

    Science.gov (United States)

    Ziemba, Brian P; Falke, Joseph J

    2018-01-01

    The leukocyte chemosensory pathway detects attractant gradients and directs cell migration to sites of inflammation, infection, tissue damage, and carcinogenesis. Previous studies have revealed that local Ca2+ and PIP3 signals at the leading edge of polarized leukocytes play central roles in positive feedback loop essential to cell polarization and chemotaxis. These prior studies showed that stimulation of the leading edge Ca2+ signal can strongly activate PI3K, thereby triggering a larger PIP3 signal, but did not elucidate the mechanistic link between Ca2+ and PIP3 signaling. A hypothesis explaining this link emerged, postulating that Ca2+-activated PKC displaces the MARCKS protein from plasma membrane PIP2, thereby releasing sequestered PIP2 to serve as the target and substrate lipid of PI3K in PIP3 production. In vitro single molecule studies of the reconstituted pathway on lipid bilayers demonstrated the feasibility of this PKC-MARCKS-PI3K regulatory module linking Ca2+ and PIP3 signals in the reconstituted system. The present study tests the model predictions in live macrophages by quantifying the effects of: (a) two pathway activators-PDGF and ATP that stimulate chemoreceptors and Ca2+ influx, respectively; and (b) three pathway inhibitors-wortmannin, EGTA, and Go6976 that inhibit PI3K, Ca2+ influx, and PKC, respectively; on (c) four leading edge activity sensors-AKT-PH-mRFP, CKAR, MARCKSp-mRFP, and leading edge area that report on PIP3 density, PKC activity, MARCKS membrane binding, and leading edge expansion/contraction, respectively. The results provide additional evidence that PKC and PI3K are both essential elements of the leading edge positive feedback loop, and strongly support the existence of a PKC-MARCKS-PI3K regulatory module linking the leading edge Ca2+ and PIP3 signals. As predicted, activators stimulate leading edge PKC activity, displacement of MARCKS from the leading edge membrane and increased leading edge PIP3 levels, while inhibitors

  6. Regulation of taurine transport at the blood-placental barrier by calcium ion, PKC activator and oxidative stress conditions

    Directory of Open Access Journals (Sweden)

    Lee Na-Young

    2010-08-01

    Full Text Available Abstract Background In the present study, we investigated the changes of uptake and efflux transport of taurine under various stress conditions using rat conditionally immortalized syncytiotrophoblast cell line (TR-TBT cells, as in vitro blood-placental barrier (BPB model. Methods The transport of taurine in TR-TBT cells were characterized by cellular uptake study using radiolabeled taurine. The efflux of taurine was measured from the amount of radiolabeled taurine remaining in the cells after the uptake of radiolabeled taurine for 60 min. Results Taurine uptake was significantly decreased by phosphorylation of protein kinase C (PKC activator in TR-TBT cells. Also, calcium ion (Ca2+ was involved in taurine transport in TR-TBT cells. Taurine uptake was inhibited and efflux was enhanced under calcium free conditions in the cells. In addition, oxidative stress induced the change of taurine transport in TR-TBT cells, but the changes were different depending on the types of oxidative stress inducing agents. Tumor necrosis factor-α (TNF-α, lipopolysaccharide (LPS and diethyl maleate (DEM significantly increased taurine uptake, but H2O2 and nitric oxide (NO donor decreased taurine uptake in the cells. Taurine efflux was down-regulated by TNF-α in TR-TBT cells. Conclusion Taurine transport in TR-TBT cells were regulated diversely at extracellular Ca2+ level, PKC activator and oxidative stress conditions. It suggested that variable stresses affected the taurine supplies from maternal blood to fetus and taurine level of fetus.

  7. Modulatory effects of cAMP and PKC activation on gap junctional intercellular communication among thymic epithelial cells

    Directory of Open Access Journals (Sweden)

    Neves-dos-Santos Sandra

    2010-01-01

    Full Text Available Abstract Background We investigated the effects of the signaling molecules, cyclic AMP (cAMP and protein-kinase C (PKC, on gap junctional intercellular communication (GJIC between thymic epithelial cells (TEC. Results Treatment with 8-Br-cAMP, a cAMP analog; or forskolin, which stimulates cAMP production, resulted in an increase in dye transfer between adjacent TEC, inducing a three-fold enhancement in the mean fluorescence of coupled cells, ascertained by flow cytometry after calcein transfer. These treatments also increased Cx43 mRNA expression, and stimulated Cx43 protein accumulation in regions of intercellular contacts. VIP, adenosine, and epinephrine which may also signal through cyclic nucleotides were tested. The first two molecules did not mimic the effects of 8-Br-cAMP, however epinephrine was able to increase GJIC suggesting that this molecule functions as an endogenous inter-TEC GJIC modulators. Stimulation of PKC by phorbol-myristate-acetate inhibited inter-TEC GJIC. Importantly, both the enhancing and the decreasing effects, respectively induced by cAMP and PKC, were observed in both mouse and human TEC preparations. Lastly, experiments using mouse thymocyte/TEC heterocellular co-cultures suggested that the presence of thymocytes does not affect the degree of inter-TEC GJIC. Conclusions Overall, our data indicate that cAMP and PKC intracellular pathways are involved in the homeostatic control of the gap junction-mediated communication in the thymic epithelium, exerting respectively a positive and negative role upon cell coupling. This control is phylogenetically conserved in the thymus, since it was seen in both mouse and human TEC preparations. Lastly, our work provides new clues for a better understanding of how the thymic epithelial network can work as a physiological syncytium.

  8. Bryostatin modulates latent HIV-1 infection via PKC and AMPK signaling but inhibits acute infection in a receptor independent manner.

    Directory of Open Access Journals (Sweden)

    Rajeev Mehla

    2010-06-01

    Full Text Available HIV's ability to establish long-lived latent infection is mainly due to transcriptional silencing in resting memory T lymphocytes and other non dividing cells including monocytes. Despite an undetectable viral load in patients treated with potent antiretrovirals, current therapy is unable to purge the virus from these latent reservoirs. In order to broaden the inhibitory range and effectiveness of current antiretrovirals, the potential of bryostatin was investigated as an HIV inhibitor and latent activator. Bryostatin revealed antiviral activity against R5- and X4-tropic viruses in receptor independent and partly via transient decrease in CD4/CXCR4 expression. Further, bryostatin at low nanomolar concentrations robustly reactivated latent viral infection in monocytic and lymphocytic cells via activation of Protein Kinase C (PKC -alpha and -delta, because PKC inhibitors rottlerin and GF109203X abrogated the bryostatin effect. Bryostatin specifically modulated novel PKC (nPKC involving stress induced AMP Kinase (AMPK inasmuch as an inhibitor of AMPK, compound C partially ablated the viral reactivation effect. Above all, bryostatin was non-toxic in vitro and was unable to provoke T-cell activation. The dual role of bryostatin on HIV life cycle may be a beneficial adjunct to the treatment of HIV especially by purging latent virus from different cellular reservoirs such as brain and lymphoid organs.

  9. PKC-Dependent GlyT1 Ubiquitination Occurs Independent of Phosphorylation: Inespecificity in Lysine Selection for Ubiquitination.

    Directory of Open Access Journals (Sweden)

    Susana P Barrera

    Full Text Available Neurotransmitter transporter ubiquitination is emerging as the main mechanism for endocytosis and sorting of cargo into lysosomes. In this study, we demonstrate PKC-dependent ubiquitination of three different isoforms of the glycine transporter 1 (GlyT1. Incubation of cells expressing transporter with the PKC activator phorbol ester induced a dramatic, time-dependent increase in GlyT1 ubiquitination, followed by accumulation of GlyT1 in EEA1 positive early endosomes. This occurred via a mechanism that was abolished by inhibition of PKC. GlyT1 endocytosis was confirmed in both retinal sections and primary cultures of mouse amacrine neurons. Replacement of only all lysines in the N-and C-termini to arginines prevented ubiquitination and endocytosis, displaying redundancy in the mechanism of ubiquitination. Interestingly, a 40-50% reduction in glycine uptake was detected in phorbol-ester stimulated cells expressing the WT-GlyT1, whereas no significant change was for the mutant protein, demonstrating that endocytosis participates in the reduction of uptake. Consistent with previous findings for the dopamine transporter DAT, ubiquitination of GlyT1 tails functions as sorting signal to deliver transporter into the lysosome and removal of ubiquitination sites dramatically attenuated the rate of GlyT1 degradation. Finally, we showed for the first time that PKC-dependent GlyT1 phosphorylation was not affected by removal of ubiquitination sites, suggesting separate PKC-dependent signaling events for these posttranslational modifications.

  10. Cadmium-induced heme-oxygenase-1 expression plays dual roles in autophagy and apoptosis and is regulated by both PKC-δ and PKB/Akt activation in NRK52E kidney cells

    International Nuclear Information System (INIS)

    So, Keum-Young; Oh, Seon-Hee

    2016-01-01

    Heme oxygenase-1 (HO-1) protects cells against cadmium (Cd)-induced oxidative stress. However, the mechanism underlying this protection is not well understood. In this study, we elucidated the role of HO-1 in Cd-induced cytotoxicity. Exposure of NRK52E cells to Cd induced protein kinase B (PKB)/Akt, protein kinase C (PKC)-δ, and glycogen synthase kinase (GSK) 3αb phosphorylation, and eukaryotic initiation factor (eIF) 2α dephosphorylation. Pharmacological inhibition of Akt resulted in HO-1 suppression and eIF2α activation, which partially suppressed CHOP and PARP-1 cleavage, but promoted autophagy and decreased cell viability. Pharmacological inactivation of PKC-δ markedly suppressed Cd-induced phospho-serine (p-Ser) GSK3αβ, and HO-1, and partially inhibited PARP-1 cleavage, but massively induced autophagy and decreased cell viability. Pharmacological upregulation of p-Ser GSK3αβ enhanced Cd-induced HO-1, CHOP, and PARP-1 cleavage, but decreased autophagy. Genetic deficiency of GSK3β suppressed HO-1 and PARP-1 cleavage and increased autophagy. Genetic suppression of HO-1 reduced Cd-induced PARP-1 cleavage, but increased LC3-II. Cd exposure led to accumulation of p-PKC-δ, p-Ser GSK3αβ, and HO-1 in the nucleus and particulate fractions, suggesting that they have dual functions in response to Cd. N-acetylcysteine treatment suppressed Cd-induced activation of PKC-δ and Akt. These results indicate that HO-1 induced by Cd exposure is regulated by PKC-δ, p-Ser GSK3αβ, and PKB/Akt, which restrain autophagic cell death, but mildly induce apoptosis in NRK52E cells. Together, the results suggest that HO-1 expression in response to Cd maintains cellular homeostasis during oxidative stress.

  11. PKA- and PKC-dependent regulation of angiopoietin 2 mRNA in human granulosa lutein cells.

    Science.gov (United States)

    Witt, P S; Pietrowski, D; Keck, C

    2004-02-01

    New blood vessels develop from preexisting vessels in response to growth factors or hypoxic conditions. Recent studies have shown that angiopoietin 2 (ANGPT-2) plays an important role in the modulation of angiogenesis and vasculogenesis in humans and mice. The signaling pathways that lead to the regulation of ANGPT-2 are largely unclear. Here, we report that protein kinase C and protein kinase A activators (ADMB, 8-Cl-cAMP) increased the mRNA levels of ANGPT-2 in human Granulosa cells, whereas PKC and PKA Inhibitors (Rp-cAMP, GO 6983) decreased markedly the level of ANGPT-2 mRNA. Due to varying specificity of the modulators for certain protein kinases subunits, we conclude that the conventional PKCs, but not PKC alpha and beta1, the atypical PKCs and the PKA I, are involved in the regulation of ANGPT-2. These findings may help to explain the role of both PKA and PKC dependent signaling cascades in the regulation of ANGPT-2 mRNA.

  12. Participation of Antidiuretic Hormone (ADH) in Asthma Exacerbations Induced by Psychological Stress via PKA/PKC Signal Pathway in Airway-Related Vagal Preganglionic Neurons (AVPNs).

    Science.gov (United States)

    Hou, Lili; Zhu, Lei; Zhang, Min; Zhang, Xingyi; Zhang, Guoqing; Liu, Zhenwei; Li, Qiang; Zhou, Xin

    2017-01-01

    Present study was performed to examine whether ADH was implicated in psychological stress asthma and to explore the underlying molecular mechanism. We not only examined ADH levels in the cerebrospinal fluid (CSF) via radioimmunoassay, but also measured ADH receptor (ADHR) expression in airway-related vagal preganglionic neurons (AVPNs) through real-time PCR in all experimental mice. Western blotting was performed to evaluate the relationship between ADH and PKA/PKC in psychological stress asthma. Finally, the role of PKA/PKC in psychological stress asthma was analyzed. Marked asthma exacerbations were noted owing to significantly elevated levels of ADH and ADHR after psychological stress induction as compared to OVA alone (asthma group). ADHR antagonists (SR-49095 or SR-121463A) dramatically lowered higher protein levels of PKAα and PKCα induced by psychological stress as compared to OVA alone, suggesting the correlation between ADH and PKA/PKC in psychological stress asthma. KT-5720 (PKA inhibitor) and Go-7874 (PKC inhibitor) further directly revealed the involvement of PKA/PKC in psychological stress asthma. Some notable changes were also noted after employing PKA and PKC inhibitors in psychological stress asthma, including reduced asthmatic inflammation (lower eosinophil peroxidase (EPO) activity, myeloperoxidase (MPO) activity, immunoglobulin E (IgE) level, and histamine release), substantial decrements in inflammatory cell counts (eosinophils and lymphocytes), and decreased cytokine secretion (IL-6, IL-10, and IFN-γ), indicating the involvement of PKA/PKC in asthma exacerbations induced by psychological stress. Our results strongly suggested that ADH participated in psychological stress-induced asthma exacerbations via PKA/PKC signal pathway in AVPNs. © 2017 The Author(s)Published by S. Karger AG, Basel.

  13. The participation of NMDA receptors, PKC, and MAPK in the formation of memory following operant conditioning in Lymnaea

    Directory of Open Access Journals (Sweden)

    Rosenegger David

    2010-08-01

    Full Text Available Abstract Background Memory is the ability to store, retain, and later retrieve information that has been learned. Intermediate term memory (ITM that persists for up to 3 h requires new protein synthesis. Long term memory (LTM that persists for at least 24 h requires: DNA transcription, RNA translation, and the trafficking of newly synthesized proteins. It has been shown in a number of different model systems that NMDA receptors, protein kinase C (PKC and mitogen activated protein kinase (MAPK are all involved in the memory formation process. Results Here we show that snails trained in control conditions are capable of forming, depending on the training procedure used, either ITM or LTM. However, blockage of NMDA receptors (MK 801, inhibition of PKC (GF109203X hydrochloride and MAPK activity (UO126 prevent the formation of both ITM and LTM. Conclusions The injection of either U0126 or GF109203X, which inhibit MAPK and PKC activity respectively, 1 hour prior to training results in the inhibition of both ITM and LTM formation. We further found that NMDA receptor activity was necessary in order for both ITM and LTM formation.

  14. Niacin activates the PI3K/Akt cascade via PKC- and EGFR-transactivation-dependent pathways through hydroxyl-carboxylic acid receptor 2.

    Directory of Open Access Journals (Sweden)

    Huawang Sun

    Full Text Available Niacin has been demonstrated to activate a PI3K/Akt signaling cascade to prevent brain damage after stroke and UV-induced skin damage; however, the underlying molecular mechanisms for HCA2-induced Akt activation remain to be elucidated. Using CHO-K1 cells stably expressing HCA2 and A431 cells, a human epidermoid cell line with high levels of endogenous expression of functional HCA2 receptors, we first demonstrated that niacin induced a robust Akt phosphorylation at both Thr308 and Ser473 in a time-dependent fashion, with a maximal activation at 5 min and a subsequent reduction to baseline by 30 min through HCA2, and that the activation was significantly blocked by pertussis toxin. The HCA2-mediated activation of Akt was also significantly inhibited by the PKC inhibitors GF109203x and Go6983 in both cell lines, by the PDGFR-selective inhibitor tyrphostin A9 in CHO-HCA2 cells and by the MMP inhibitor GM6001 and EGFR-specific inhibitor AG1478 in A431 cells. These results suggest that the PKC pathway and PDGFR/EGFR transactivation pathway play important roles in HCA2-mediated Akt activation. Further investigation indicated that PI3K and the Gβγ subunit were likely to play an essential role in HCA2-induced Akt activation. Moreover, Immunobloting analyses using an antibody that recognizes p70S6K1 phosphorylated at Thr389 showed that niacin evoked p70S6K1 activation via the PI3K/Akt pathway. The results of our study provide new insight into the signaling pathways involved in HCA2 activation.

  15. Increased aPKC Expression Correlates with Prostatic Adenocarcinoma Gleason Score and Tumor Stage in the Japanese Population

    Directory of Open Access Journals (Sweden)

    Anthony S. Perry

    2014-01-01

    Full Text Available Background. Levels of the protein kinase aPKC have been previously correlated with prostate cancer prognosis in a British cohort. However, prostate cancer incidence and progression rates, as well as genetic changes in this disease, show strong ethnic variance, particularly in Asian populations. Objective. The aim of this study was to validate association of aPKC expression with prostatic adenocarcinoma stages in a Japanese cohort. Methods. Tissue microarrays consisting of 142 malignant prostate cancer cases and 21 benign prostate tissues were subject to immunohistological staining for aPKC. aPKC staining intensity was scored by three independent pathologists and categorized as absent (0, dim (1+, intermediate (2+, and bright (3+. aPKC staining intensities were correlated with Gleason score and tumor stage. Results. Increased aPKC staining was observed in malignant prostate cancer, in comparison to benign tissue. Additionally, aPKC staining levels correlated with Gleason score and tumor stage. Our results extend the association of aPKC with prostate cancer to a Japanese population and establish the suitability of aPKC as a universal prostate cancer biomarker that performs consistently across ethnicities.

  16. "Slow" Voltage-Dependent Inactivation of CaV2.2 Calcium Channels Is Modulated by the PKC Activator Phorbol 12-Myristate 13-Acetate (PMA.

    Directory of Open Access Journals (Sweden)

    Lei Zhu

    Full Text Available CaV2.2 (N-type voltage-gated calcium channels (Ca2+ channels play key roles in neurons and neuroendocrine cells including the control of cellular excitability, neurotransmitter / hormone secretion, and gene expression. Calcium entry is precisely controlled by channel gating properties including multiple forms of inactivation. "Fast" voltage-dependent inactivation is relatively well-characterized and occurs over the tens-to- hundreds of milliseconds timeframe. Superimposed on this is the molecularly distinct, but poorly understood process of "slow" voltage-dependent inactivation, which develops / recovers over seconds-to-minutes. Protein kinases can modulate "slow" inactivation of sodium channels, but little is known about if/how second messengers control "slow" inactivation of Ca2+ channels. We investigated this using recombinant CaV2.2 channels expressed in HEK293 cells and native CaV2 channels endogenously expressed in adrenal chromaffin cells. The PKC activator phorbol 12-myristate 13-acetate (PMA dramatically prolonged recovery from "slow" inactivation, but an inactive control (4α-PMA had no effect. This effect of PMA was prevented by calphostin C, which targets the C1-domain on PKC, but only partially reduced by inhibitors that target the catalytic domain of PKC. The subtype of the channel β-subunit altered the kinetics of inactivation but not the magnitude of slowing produced by PMA. Intracellular GDP-β-S reduced the effect of PMA suggesting a role for G proteins in modulating "slow" inactivation. We postulate that the kinetics of recovery from "slow" inactivation could provide a molecular memory of recent cellular activity and help control CaV2 channel availability, electrical excitability, and neurotransmission in the seconds-to-minutes timeframe.

  17. Role and mechanism of PKC on radiosensitization in pancreatic carcinoma cell line Panc-1

    International Nuclear Information System (INIS)

    Qiao Qiao; Zhang Shuo; Chen Yanzhi; Li Guang

    2008-01-01

    Objective: To explore the effect of PKC on radiosensitization in pancreatic carcinoma cell line Panc-1, and its mediating mechanism. Methods: Panc-1 cells were treated with the specific activator of PKC (phorbol 12-myristate 13-acetate, PMA) and the specific inhibitor of PKC (chelerythrine, CH) to observe the SF2 changes. Cell survival was determined by clonogenic assay. The apoptosis rates of the cells were analyzed by flow cytometry with Annexin V/PI staining. The expression of apoptosis related protein Bcl-2 and Bax after the treatment of CH and/or irradiation was determined by immunocytochemistry. Results: The SF 2 values of radiation group, PMA group and CH group were 0.78 ± 0.02, 0.92 ± 0.11 and 0.19 ± 0.20, respectively. CH can significantly increase the sensitivity of Panc-1 to irradiation. SERs of Panc-1 cells were 1.05, 1.24 and 1.77 after the treatment of 0.5, 2 and 8 μmol/L of CH, respectively. The result of flow cytometry analysis showed that PMA decreased the apoptosis index with irradiation, while CH significantly increased the apoptosis index. Expression of Bax protein was increased significantly (P<0.05) while that of Bcl-2 was not influenced; however, the ratio of Bax/Bcl-2 was increased. Conclusions: PKC regulates the radiosensitivity of Panc-1 by mediating the apoptosis of tumor cells. (authors)

  18. Involvement of HDAC1 and the PI3K/PKC signaling pathways in NF-κB activation by the HDAC inhibitor apicidin

    International Nuclear Information System (INIS)

    Kim, Yong Kee; Seo, Dong-Wan; Kang, Dong-Won; Lee, Hoi Young; Han, Jeung-Whan; Kim, Su-Nam

    2006-01-01

    Histone deacetylase (HDAC) inhibitors are appreciated as one of promising anticancer drugs, but they exert differential responses depending on the cell type. We recently reported the critical role of NF-κB as a modulator in determining cell fate for apoptosis in response to an HDAC inhibitor. In this study, we investigate a possible signaling pathway required for NF-κB activation in response to the HDAC inhibitor apicidin. Treatment of HeLa cells with apicidin leads to an increase in transcriptional activity of NF-κB and the expression of its target genes, IL-8 and TNF-α. TNF-α expression by apicidin is induced at earlier time points than NF-κB activation or IL-8 expression. In addition, our data show that the early expression of TNF-α does not lead to activation of NF-κB, because disruption of TNF-α activity by a neutralizing antibody does not affect nuclear translocation of NF-κB, IκBα degradation or reporter gene activation by apicidin. However, this activation of NF-κB requires the PI3K and PKC signaling pathways, but not ERK or JNK. Furthermore, apicidin activation of NF-κB seems to result from HDAC1 inhibition, as evidenced by the observation that overexpression of HDAC1, but not HDAC2, 3 or 4, dramatically inhibits NF-κB reporter gene activity. Collectively, our results suggest that activation of NF-κB signaling by apicidin requires both the PI3K/PKC signaling pathways and HDAC1, and functions as a critical modulator in determining the cellular effect of apicidin

  19. TNF-alpha stimulates Akt by a distinct aPKC-dependent pathway in premalignant keratinocytes

    DEFF Research Database (Denmark)

    Faurschou, A.; Gniadecki, R.

    2008-01-01

    , ERK1/2 and p38. The specific peptide blocking the activity of the atypical protein kinase C (aPKC) species zeta and iota/lambda abrogated the effects of TNF-alpha on Akt and ERK1/2 but increased the activation of p38. The TNF-alpha-dependent phosphorylation of Akt-ERK1/2 was slightly decreased by NF...

  20. Effect of PKC412, an inhibitor of protein kinase C, on spontaneous metastatic model mice.

    Science.gov (United States)

    Nakamura, Kazuki; Yoshikawa, Noriko; Yamaguchi, Yu; Kagota, Satomi; Shinozuka, Kazumasa; Kunitomo, Masaru

    2003-01-01

    We investigated the anti-metastatic effect of PKC412, a selective inhibitor of protein kinase C (PKC), on a spontaneous metastatic mouse model, which was prepared by inoculation with B16-BL6 mouse melanoma cells into the footpad of the right hind leg. At two weeks after inoculation, the primary tumor was amputated completely. PKC412 (200 mg/kg) administered orally for four weeks after the tumor inoculation, significantly prolonged survival compared with the control. Furthermore, to elucidate the mechanism of the anti-metastatic effect of PKC412, we examined the growth rate of B16-BL6 cells premixed with Matrigel in vivo and the invasiveness of B16-BL6 cells using a chemo-invasion chamber in vitro. PKC412 significantly reduced the growth rate of cells in vivo (100 and 200 mg/kg) and the invading cells in vitro (10, 30 and 100 nM) in a dose-dependent manner. Thus, PKC412 exerts an anti-metastatic action through inhibition of the invasiveness of melanoma cells in the extracellular matrix.

  1. Localization of aPKC lambda/iota and its interacting protein, Lgl2, is significantly associated with lung adenocarcinoma progression.

    Science.gov (United States)

    Imamura, Naoko; Horikoshi, Yosuke; Matsuzaki, Tomohiko; Toriumi, Kentaro; Kitatani, Kanae; Ogura, Go; Masuda, Ryota; Nakamura, Naoya; Takekoshi, Susumu; Iwazaki, Masayuki

    2013-12-20

    Atypical protein kinase C lambda/iota (aPKC λ/ι) is expressed in several human cancers; however, the correlation between aPKC λ/ι localization and cancer progression in human lung adenocarcinoma (LAC) remains to be clarified. We found that patients with a high level of aPKC λ/ι expression in LAC had significantly shorter overall survival than those with a low level of aPKC λ/ι expression. In addition, localization of aPKC λ/ι in the apical membrane or at the cell-cell contact was associated with both lymphatic invasion and metastasis. The intercellular adhesion molecule, E-cadherin, was decreased in LACs with highly expressed aPKC λ/ι at the invasion site of tumor cells. This result suggested that the expression levels of aPKC λ/ι and E-cadherin reflect the progression of LAC. On double-immunohistochemical analysis, aPKC λ/ι and Lgl2, a protein that interacts with aPKC λ/ι, were co-localized within LACs. Furthermore, we found that Lgl2 bound the aPKC λ/ι-Par6 complex in tumor tissue by immune-cosedimentation analysis. Apical membrane localization of Lgl2 was correlated with lymphatic invasion and lymph node metastasis. These results thus indicate that aPKC λ/ι expression is altered upon the progression of LAC. This is also the first evidence to show aPKC λ/ι overexpression in LAC and demonstrates that aPKC λ/ι localization at the apical membrane or cell-cell contact is associated with lymphatic invasion and metastasis of the tumor.

  2. Sodium Phenylbutyrate Enhances Astrocytic Neurotrophin Synthesis via Protein Kinase C (PKC)-mediated Activation of cAMP-response Element-binding Protein (CREB)

    Science.gov (United States)

    Corbett, Grant T.; Roy, Avik; Pahan, Kalipada

    2013-01-01

    Neurotrophins, such as brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), are believed to be genuine molecular mediators of neuronal growth and homeostatic synapse activity. However, levels of these neurotrophic factors decrease in different brain regions of patients with Alzheimer disease (AD). Induction of astrocytic neurotrophin synthesis is a poorly understood phenomenon but represents a plausible therapeutic target because neuronal neurotrophin production is aberrant in AD and other neurodegenerative diseases. Here, we delineate that sodium phenylbutyrate (NaPB), a Food and Drug Administration-approved oral medication for hyperammonemia, induces astrocytic BDNF and NT-3 expression via the protein kinase C (PKC)-cAMP-response element-binding protein (CREB) pathway. NaPB treatment increased the direct association between PKC and CREB followed by phosphorylation of CREB (Ser133) and induction of DNA binding and transcriptional activation of CREB. Up-regulation of markers for synaptic function and plasticity in cultured hippocampal neurons by NaPB-treated astroglial supernatants and its abrogation by anti-TrkB blocking antibody suggest that NaPB-induced astroglial neurotrophins are functionally active. Moreover, oral administration of NaPB increased the levels of BDNF and NT-3 in the CNS and improved spatial learning and memory in a mouse model of AD. Our results highlight a novel neurotrophic property of NaPB that may be used to augment neurotrophins in the CNS and improve synaptic function in disease states such as AD. PMID:23404502

  3. A PKM Generated by Calpain Cleavage of a Classical PKC Is Required for Activity-Dependent Intermediate-Term Facilitation in the Presynaptic Sensory Neuron of "Aplysia"

    Science.gov (United States)

    Farah, Carole A.; Hastings, Margaret H.; Dunn, Tyler W.; Gong, Katrina; Baker-Andresen, Danay; Sossin, Wayne S.

    2017-01-01

    Atypical PKM, a persistently active form of atypical PKC, is proposed to be a molecular memory trace, but there have been few examinations of the role of PKMs generated from other PKCs. We demonstrate that inhibitors used to inhibit PKMs generated from atypical PKCs are also effective inhibitors of other PKMs. In contrast, we demonstrate that…

  4. Complex interactions between GSK3 and aPKC in Drosophila embryonic epithelial morphogenesis.

    Directory of Open Access Journals (Sweden)

    Nicole A Kaplan

    Full Text Available Generally, epithelial cells must organize in three dimensions to form functional tissue sheets. Here we investigate one such sheet, the Drosophila embryonic epidermis, and the morphogenetic processes organizing cells within it. We report that epidermal morphogenesis requires the proper distribution of the apical polarity determinant aPKC. Specifically, we find roles for the kinases GSK3 and aPKC in cellular alignment, asymmetric protein distribution, and adhesion during the development of this polarized tissue. Finally, we propose a model explaining how regulation of aPKC protein levels can reorganize both adhesion and the cytoskeleton.

  5. Interfering RNA against PKC-α Inhibits TNF-α-induced IP3R1 Expression and Improves Glomerular Filtration Rate in Rats with Fulminant Hepatic Failure.

    Science.gov (United States)

    Wang, Dong-Lei; Dai, Wen-Ying; Wang, Wen; Wen, Ying; Zhou, Ying; Zhao, Yi-Tong; Wu, Jian; Liu, Pei

    2018-01-10

    We have reported that tumor necrosis factor- (TNF-α) is critical for reduction of glomerular filtration rate (GFR) in rats with fulminant hepatic failure (FHF). The present study aims to evaluate the underlying mechanisms of decreased GFR during acute hepatic failure. Rats with FHF induced by D-galactosamine plus lipopolysaccharide (GalN/LPS) were injected intravenously with recombinant lentivirus harboring shRNA against the protein kinase C-α (PKC-α) gene (Lenti-shRNA-PKC-α). GFR, serum levels of aminotransferases, creatinine, urea nitrogen, potassium, sodium, chloride, TNF-α and endothelin-1 (ET-1), as well as type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) expression in renal tissue were assessed. The effects of PKC-α silencing on TNF-α-induced IP3R1, specificity protein 1 (SP-1) and c-Jun N-terminal kinase (JNK) expression, as well as cytosolic calcium content were determined in glomerular mesangial cell (GMCs) with RNAi against PKC-α. Renal IP3R1 overexpression was abrogated by pre-treatment with Lenti-shRNA-PKC-α. The PKC- silence significantly improved the compromised GFR, reduced Cr levels, and reversed the decrease in glomerular inulin space and the increase in glomerular calcium content in GalN/LPS-exposed rats. TNF-α treatment increased expression of PKC-α, IP3R1, specificity protein 1 (SP-1), JNK and p-JNK in GMCs, and increased Ca2+ release and binding activity of SP-1 to the IP3R1 promoter. These effects were blocked by transfection of siRNA against the PKC-α gene, and the PKC-α gene silence also restored cytosolic [Ca2+]i. RNAi targeting PKC-α inhibited TNF-α-induced IP3R1 overexpression, and in turn improved compromised GFR in the development of acute kidney injury during FHF in rats.

  6. Protein kinase C δ is activated in mouse ovarian surface epithelial cancer cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

    International Nuclear Information System (INIS)

    Williams, Shalmica R.; Son, Deok-Soo; Terranova, Paul F.

    2004-01-01

    Interactions between the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and protein kinase C (PKC) signaling pathways are governed in cell and tissue-specific manners, albeit the physiological significance of which is unclear. This research sought to define the effects of TCDD on the PKC pathway using a mouse ovarian surface epithelial cancer cell line (ID8). Phorbol-12-myristate-13-acetate (PMA) potentiated (1 nM) TCDD-induced 7-ethoxyresorufin-O-deethylase (EROD) activity after 24 h of treatment, and pre-treatment with (1 μM) of either a general PKC inhibitor (BisI) or PKCδ-specific inhibitor (Rotterlin) abolished the potentiation indicating that activation of PKC enhances TCDD signal transduction. Western blot analysis revealed that unstimulated ID8 cells express PKCα, β, ε, τ, λ and RACK1. PKCγ, η, θ and DGKθ were not detected. TCDD (1 nM) increased PKCδ protein approximately eight-fold after 24 h of treatment and this effect was dose-dependent (0.1-100 nM); other PKC isoforms and related signaling proteins tested were unaffected by TCDD treatment. Immunofluorescent microscopy revealed that TCDD (1 nM) promoted the subcellular redistribution of PKCδ, from the cytoplasm and the nucleus to the perinuclear area after 2 h of treatment, however, after 24 h of treatment PKCδ was observed in nuclear structures that resembled nucleoli. TCDD (1 nM) also increased total PKC and PKCδ-specific kinase activities in biphasic time-responsive manners. Total PKC and PKCδ-specific activities increased after 1-2 h of treatment. Then TCDD increased the total PKC activity again after 12 h of treatment, whereas, PKCδ-specific activity resurged at 24 h and remained elevated at 48 h after treatment. The results indicate that TCDD preferentially induces PKCδ protein expression and phosphotransferase activity, and its membrane translocation, indicating a potential intracellular role for PKCδ as an effector molecule for TCDD-mediated biological events in this ovarian

  7. Tetrandrine, an Activator of Autophagy, Induces Autophagic Cell Death via PKC-α Inhibition and mTOR-Dependent Mechanisms

    Directory of Open Access Journals (Sweden)

    Vincent Kam Wai Wong

    2017-06-01

    Full Text Available Emerging evidence suggests the therapeutic role of autophagic modulators in cancer therapy. This study aims to identify novel traditional Chinese medicinal herbs as potential anti-tumor agents through autophagic induction, which finally lead to autophagy mediated-cell death in apoptosis-resistant cancer cells. Using bioactivity-guided purification, we identified tetrandrine (Tet from herbal plant, Radix stephaniae tetrandrae, as an inducer of autophagy. Across a number of cancer cell lines, we found that breast cancer cells treated with tetrandrine show an increase autophagic flux and formation of autophagosomes. In addition, tetrandrine induces cell death in a panel of apoptosis-resistant cell lines that are deficient for caspase 3, caspase 7, caspase 3 and 7, or Bax-Bak respectively. We also showed that tetrandrine-induced cell death is independent of necrotic cell death. Mechanistically, tetrandrine induces autophagy that depends on mTOR inactivation. Furthermore, tetrandrine induces autophagy in a calcium/calmodulin-dependent protein kinase kinase-β (CaMKK-β, 5′ AMP-activated protein kinase (AMPK independent manner. Finally, by kinase profiling against 300 WT kinases and computational molecular docking analysis, we showed that tetrandrine is a novel PKC-α inhibitor, which lead to autophagic induction through PKC-α inactivation. This study provides detailed insights into the novel cytotoxic mechanism of an anti-tumor compound originated from the herbal plant, which may be useful in promoting autophagy mediated- cell death in cancer cell that is resistant to apoptosis.

  8. Effect of α1-adrenergic stimulation on phosphoinositide metabolism and protein kinase C (PK-C) in rat cardiomyocytes

    International Nuclear Information System (INIS)

    Kaku, T.; Lakatta, E.; Filburn, C.R.

    1986-01-01

    Alpha 1 -adrenergic stimulation is known to enhance membrane phospholipid metabolism resulting in increases in inositol phosphates (IP's) and diacylglycerol (DAG). Cardiomyocytes prelabeled with 3 H-myo-inositol were treated with norepinephrine (NE) for 1-15 min, acid extracted, and IP's separated by ion exchange chromatography. Addition of NE (10 -5 M) in the presence of propranolol (10 -5 M) and LiCl (9 mM) enhanced the accumulation of IP's, linearly with time up to 15 min, and reached 7.3, and 1.5-fold at 15 min for IP 1 , IP 2 , and IP 3 , respectively. KCl at 30 mM had no effect on accumulation of IP's, but augmented the effect of NE. PK-C activity was measured in both cytosol (S) and particulate (P) fractions of treated cells. NE alone had a negligible effect on membrane PK-C, while 30 mM KCl caused a small increase. However, pretreatment with KCl followed by NE produced a significant increase above that seen with KCl alone. Dioctanoylglycerol also stimulated membrane association of PK-C in these cells. These data suggest that α 1 -adrenergic stimulation of membrane association of myocardial PK-C is mediated by DAG but may be dependent on membrane potential and/or the extent of Ca 2+ loading

  9. Metformin and liraglutide ameliorate high glucose-induced oxidative stress via inhibition of PKC-NAD(P)H oxidase pathway in human aortic endothelial cells.

    Science.gov (United States)

    Batchuluun, Battsetseg; Inoguchi, Toyoshi; Sonoda, Noriyuki; Sasaki, Shuji; Inoue, Tomoaki; Fujimura, Yoshinori; Miura, Daisuke; Takayanagi, Ryoichi

    2014-01-01

    Metformin and glucagon like peptide-1 (GLP-1) prevent diabetic cardiovascular complications and atherosclerosis. However, the direct effects on hyperglycemia-induced oxidative stress in endothelial cells are not fully understood. Thus, we aimed to evaluate the effects of metformin and a GLP-1 analog, liraglutide on high glucose-induced oxidative stress. Production of reactive oxygen species (ROS), activation of protein kinase C (PKC) and NAD(P)H oxidase, and changes in signaling molecules in response to high glucose exposure were evaluated in human aortic endothelial cells with and without treatment of metformin and liraglutide, alone or in combination. PKC-NAD(P)H oxidase pathway was assessed by translocation of GFP-fused PKCβ2 isoform and GFP-fused p47phox, a regulatory subunit of NAD(P)H oxidase, in addition to endogenous PKC phosphorylation and NAD(P)H oxidase activity. High glucose-induced ROS overproduction was blunted by metformin or liraglutide treatment, with a further decrease by a combination of these drugs. Exposure to high glucose caused PKCβ2 translocation and a time-dependent phosphorylation of endogenous PKC but failed to induce its translocation and phosphorylation in the cells treated with metformin and liraglutide. Furthermore, both drugs inhibited p47phox translocation and NAD(P)H oxidase activation, and prevented the high glucose-induced changes in intracellulalr diacylglycerol (DAG) level and phosphorylation of AMP-activated protein kinase (AMPK). A combination of these drugs further enhanced all of these effects. Metformin and liraglutide ameliorate high glucose-induced oxidative stress by inhibiting PKC-NAD(P)H oxidase pathway. A combination of these two drugs provides augmented protective effects, suggesting the clinical usefulness in prevention of diabetic vascular complications. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  10. aPKC-ι/P-Sp1/Snail signaling induces epithelial-mesenchymal transition and immunosuppression in cholangiocarcinoma.

    Science.gov (United States)

    Qian, Yawei; Yao, Wei; Yang, Tao; Yang, Yan; Liu, Yan; Shen, Qi; Zhang, Jian; Qi, Weipeng; Wang, Jianming

    2017-10-01

    Cholangiocarcinoma (CCA) is a highly malignant bile duct cancer that tends to invade and metastasize early. The epithelial-mesenchymal transition (EMT) has been implicated in cancer cell invasion and metastasis, as well as in cancer cell evasion of host immunity. In this study, we investigated the interaction between atypical protein kinase C-iota (aPKC-ι) and Snail in the regulation of EMT and its relationship to CCA immunosuppression. Our results demonstrated that aPKC-ι, Snail, and infiltrated immunosuppressive cells were significantly up-regulated in CCA tumor tissues and linked to poor prognosis. aPKC-ι induced EMT and immunosuppression by regulating Snail in vitro and in vivo, although aPKC-ι did not directly interact with Snail in coimmunoprecipitation experiments. To further clarify the molecular interaction between aPKC-ι and Snail in relation to EMT, quantitative iTRAQ-based phosphoproteomic analysis and liquid chromatography-tandem mass spectrometry were conducted to identify the substrates of aPKC-ι-dependent phosphorylation. Combined with coimmunoprecipitation, we showed that specificity protein 1 (Sp1) was directly phosphorylated by aPKC-ι on Ser59 (P-Sp1). Both Sp1 and P-Sp1 were up-regulated in CCA tumor tissues and associated with clinicopathological features and poor prognosis in CCA patients. Moreover, using chromatin immunoprecipitation assays, we found that P-Sp1 regulated Snail expression by increasing Sp1 binding to the Snail promoter. P-Sp1 also regulated aPKC-ι/Snail-induced EMT-like changes and immunosuppression in CCA cells. Our findings further indicated that CCA cells with EMT-like features appear to generate immunosuppressive natural T regulatory-like cluster of differentiation 4-positive (CD4 + )CD25 - cells rather than to increase CD4 + CD25 + natural T regulatory cells, in part by mediating T regulatory-inducible cytokines such as transforming growth factor β1 and interleukin 2. These results demonstrate that aPKC

  11. Anisotropy of Crumbs and aPKC drives myosin cable assembly during tube formation.

    Science.gov (United States)

    Röper, Katja

    2012-11-13

    The formation of tubular structures from epithelial sheets is a key process of organ formation in all animals, but the cytoskeletal rearrangements that cause the cell shape changes that drive tubulogenesis are not well understood. Using live imaging and super-resolution microscopy to analyze the tubulogenesis of the Drosophila salivary glands, I find that an anisotropic plasma membrane distribution of the protein Crumbs, mediated by its large extracellular domain, determines the subcellular localization of a supracellular actomyosin cable in the cells at the placode border, with myosin II accumulating at edges where Crumbs is lowest. Laser ablation shows that the cable is under increased tension, implying an active involvement in the invagination process. Crumbs anisotropy leads to anisotropic distribution of aPKC, which in turn can negatively regulate Rok, thus preventing the formation of a cable where Crumbs and aPKC are localized. Copyright © 2012 Elsevier Inc. All rights reserved.

  12. Notch and PKC are involved in formation of the lateral region of the dorso-ventral axis in Drosophila embryos.

    Science.gov (United States)

    Tremmel, Daniel M; Resad, Sedat; Little, Christopher J; Wesley, Cedric S

    2013-01-01

    The Notch gene encodes an evolutionarily conserved cell surface receptor that generates regulatory signals based on interactions between neighboring cells. In Drosophila embryos it is normally expressed at a low level due to strong negative regulation. When this negative regulation is abrogated neurogenesis in the ventral region is suppressed, the development of lateral epidermis is severely disrupted, and the dorsal aminoserosa is expanded. Of these phenotypes only the anti-neurogenic phenotype could be linked to excess canonical Notch signaling. The other phenotypes were linked to high levels of Notch protein expression at the surface of cells in the lateral regions indicating that a non-canonical Notch signaling activity normally functions in these regions. Results of our studies reported here provide evidence. They show that Notch activities are inextricably linked to that of Pkc98E, the homolog of mammalian PKCδ. Notch and Pkc98E up-regulate the levels of the phosphorylated form of IκBCactus, a negative regulator of Toll signaling, and Mothers against dpp (MAD), an effector of Dpp signaling. Our data suggest that in the lateral regions of the Drosophila embryos Notch activity, in conjunction with Pkc98E activity, is used to form the slopes of the opposing gradients of Toll and Dpp signaling that specify cell fates along the dorso-ventral axis.

  13. Notch and PKC are involved in formation of the lateral region of the dorso-ventral axis in Drosophila embryos.

    Directory of Open Access Journals (Sweden)

    Daniel M Tremmel

    Full Text Available The Notch gene encodes an evolutionarily conserved cell surface receptor that generates regulatory signals based on interactions between neighboring cells. In Drosophila embryos it is normally expressed at a low level due to strong negative regulation. When this negative regulation is abrogated neurogenesis in the ventral region is suppressed, the development of lateral epidermis is severely disrupted, and the dorsal aminoserosa is expanded. Of these phenotypes only the anti-neurogenic phenotype could be linked to excess canonical Notch signaling. The other phenotypes were linked to high levels of Notch protein expression at the surface of cells in the lateral regions indicating that a non-canonical Notch signaling activity normally functions in these regions. Results of our studies reported here provide evidence. They show that Notch activities are inextricably linked to that of Pkc98E, the homolog of mammalian PKCδ. Notch and Pkc98E up-regulate the levels of the phosphorylated form of IκBCactus, a negative regulator of Toll signaling, and Mothers against dpp (MAD, an effector of Dpp signaling. Our data suggest that in the lateral regions of the Drosophila embryos Notch activity, in conjunction with Pkc98E activity, is used to form the slopes of the opposing gradients of Toll and Dpp signaling that specify cell fates along the dorso-ventral axis.

  14. An Asp49 Phospholipase A2 from Snake Venom Induces Cyclooxygenase-2 Expression and Prostaglandin E2 Production via Activation of NF-κB, p38MAPK, and PKC in Macrophages

    Directory of Open Access Journals (Sweden)

    Vanessa Moreira

    2014-01-01

    Full Text Available Phospholipases A2 (PLA2 are key enzymes for production of lipid mediators. We previously demonstrated that a snake venom sPLA2 named MT-III leads to prostaglandin (PGE2 biosynthesis in macrophages by inducing the expression of cyclooxygenase-2 (COX-2. Herein, we explored the molecular mechanisms and signaling pathways leading to these MT-III-induced effects. Results demonstrated that MT-III induced activation of the transcription factor NF-κB in isolated macrophages. By using NF-κB selective inhibitors, the involvement of this factor in MT-III-induced COX-2 expression and PGE2 production was demonstrated. Moreover, MT-III-induced COX-2 protein expression and PGE2 release were attenuated by pretreatment of macrophages with SB202190, and Ly294002, and H-7-dihydro compounds, indicating the involvement of p38MAPK, PI3K, and PKC pathways, respectively. Consistent with this, MT-III triggered early phosphorylation of p38MAPK, PI3K, and PKC. Furthermore, SB202190, H-7-dihydro, but not Ly294002 treatment, abrogated activation of NF-κB induced by MT-III. Altogether, these results show for the first time that the induction of COX-2 protein expression and PGE2 release, which occur via NF-κB activation induced by the sPLA2-MT-III in macrophages, are modulated by p38MAPK and PKC, but not by PI3K signaling proteins.

  15. Differential roles of PKC isoforms (PKCs) in GnRH stimulation of MAPK phosphorylation in gonadotrope derived cells.

    Science.gov (United States)

    Mugami, Shany; Dobkin-Bekman, Masha; Rahamim-Ben Navi, Liat; Naor, Zvi

    2018-03-05

    The role of protein kinase C (PKC) isoforms (PKCs) in GnRH-stimulated MAPK [ERK1/2, JNK1/2 and p38) phosphorylation was examined in gonadotrope derived cells. GnRH induced a protracted activation of ERK1/2 and a slower and more transient activation of JNK1/2 and p38MAPK. Gonadotropes express conventional PKCα and PKCβII, novel PKCδ, PKCε and PKCθ, and atypical PKC-ι/λ. The use of green fluorescent protein (GFP)-PKCs constructs revealed that GnRH induced rapid translocation of PKCα and PKCβII to the plasma membrane, followed by their redistribution to the cytosol. PKCδ and PKCε localized to the cytoplasm and Golgi, followed by the rapid redistribution by GnRH of PKCδ to the perinuclear zone and of PKCε to the plasma membrane. The use of dominant negatives for PKCs and peptide inhibitors for the receptors for activated C kinase (RACKs) has revealed differential role for PKCα, PKCβII, PKCδ and PKCε in ERK1/2, JNK1/2 and p38MAPK phosphorylation in a ligand-and cell context-dependent manner. The paradoxical findings that PKCs activated by GnRH and PMA play a differential role in MAPKs phosphorylation may be explained by persistent vs. transient redistribution of selected PKCs or redistribution of a given PKC to the perinuclear zone vs. the plasma membrane. Thus, we have identified the PKCs involved in GnRH stimulated MAPKs phosphorylation in gonadotrope derived cells. Once activated, the MAPKs will mediate the transcription of the gonadotropin subunits and GnRH receptor genes. Copyright © 2017. Published by Elsevier B.V.

  16. Expression and proliferation profiles of PKC, JNK and p38MAPK in physiologically stretched human bladder smooth muscle cells

    International Nuclear Information System (INIS)

    Wazir, Romel; Luo, De-Yi; Dai, Yi; Yue, Xuan; Tian, Ye; Wang, Kun-Jie

    2013-01-01

    Highlights: •Stretch induces proliferation in human bladder smooth muscle cells (HBSMC). •5% Equibiaxial elongation produces maximum proliferation. •Physiologic stretch decreases apoptotic cell death. •PKC is involved in functional modulation of bladder. •JNK and p38 are not involved in proliferating HBSMC. -- Abstract: Objective: To determine protein kinase C (PKC), c-Jun NH2-Terminal Kinase (JNK) and P38 mitogen-activated protein kinases (p38MAPK) expression levels and effects of their respective inhibitors on proliferation of human bladder smooth muscle cells (HBSMCs) when physiologically stretched in vitro. Materials and methods: HBSMCs were grown on silicone membrane and stretch was applied under varying conditions; (equibiaxial elongation: 2.5%, 5%, 10%, 15%, 20%, 25%), (frequency: 0.05, 0.1, 0.2, 0.5, 1 Hz). Optimal physiological stretch was established by assessing proliferation with 5-Bromo-2-deoxyuridine (BrdU) assay and flow cytometry. PKC, JNK and p38 expression levels were analyzed by Western blot. Specificity was maintained by employing specific inhibitors; (GF109203X for PKC, SP600125 for JNK and SB203580 for p38MAPK), in some experiments. Results: Optimum proliferation was observed at 5% equibiaxial stretch (BrdU: 0.837 ± 0.026 (control) to 1.462 ± 0.023)%, (P 0.05 SP600125) and (1.461 ± 0.01, P > 0.05 SB203580). These findings show that mechanical stretch can promote magnitude-dependent proliferative modulation through PKC and possibly JNK but not via p38MAPK in hBSMCs

  17. Tamoxifen in combination with temozolomide induce a synergistic inhibition of PKC-pan in GBM cell lines.

    Science.gov (United States)

    Balça-Silva, Joana; Matias, Diana; do Carmo, Anália; Girão, Henrique; Moura-Neto, Vivaldo; Sarmento-Ribeiro, Ana Bela; Lopes, Maria Celeste

    2015-04-01

    Glioblastoma (GBM) is a highly proliferative, angiogenic grade IV astrocytoma that develops resistance to the alkylating agents used in chemotherapy, such as temozolomide (TMZ), which is considered the gold standard. The mean survival time for GBM patients is approximately 12 months, increasing to 14.6 months after TMZ treatment. The resistance of GBM to chemotherapy seems to be associated to genetic alterations and to the constitutive activation of several signaling pathways. Therefore, the combination of different drugs with different mechanisms of action may contribute to circumvent the chemoresistance of glioma cells. Here we describe the potential synergistic behavior of the therapeutic combination of tamoxifen (TMX), a known inhibitor of PKC, and TMZ in GBM. We used two GBM cell lines incubated in absence and presence of TMX and/or TMZ and measured cell viability, proliferation, apoptosis, cell cycle, migration ability, cytoskeletal organization and the phosphorylated amount of the p-PKC-pan. The combination of low doses of TMX with increasing doses of TMZ shows an increased antiproliferative and apoptotic effect compared to the effect with TMX alone. The combination of TMX and TMZ seems to potentiate the effect of each other. These alterations seem to be associated to a decrease in the phosphorylation status of PKC. We emphasize that TMX is an inhibitor of the p-PKC-pan and that these combination is more effective in the reduction of proliferation and in the increase of apoptosis than each drug alone, which presents a new therapeutic strategy in GBM treatment. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. [Pt(O,O’-acac)(γ-acac)(DMS)] Alters SH-SY5Y Cell Migration and Invasion by the Inhibition of Na+/H+ Exchanger Isoform 1 Occurring through a PKC-ε/ERK/mTOR Pathway

    Science.gov (United States)

    Muscella, Antonella; Vetrugno, Carla; Calabriso, Nadia; Cossa, Luca Giulio; De Pascali, Sandra Angelica; Fanizzi, Francesco Paolo; Marsigliante, Santo

    2014-01-01

    We previously showed that [Pt(O,O’-acac)(γ-acac)(DMS)] ([Pt(acac)2(DMS)]) exerted substantial cytotoxic effects in SH-SY5Y neuroblastoma cells, and decreased metalloproteases (MMPs) production and cells migration in MCF-7 breast cancer cells. The ubiquitously distributed sodium-hydrogen antiporter 1 (NHE1) is involved in motility and invasion of many solid tumours. The present study focuses on the effects of [Pt(acac)2(DMS)] in SH-SY5Y cell migration and also on the possibility that NHE1 may be involved in such effect. After sublethal [Pt(acac)2(DMS)] treatment cell migration was examined by wounding assay and cell invasion by transwell assay. NHE1 activity was measured in BCECF-loaded SH-SY5Y as the rate of Na+-dependent intracellular pH recovery in response to an acute acid pulse. Gelatin zymography for MMP-2/9 activities, Western blottings of MMPs, MAPKs, mTOR, S6 and PKCs and small interfering RNAs to PKC-ε/-δ mRNA were performed. Sublethal concentrations of [Pt(acac)2(DMS)] decreases NHE1 activity, inhibites cell migration and invasion and decreases expression and activity of MMP-2 and -9. [Pt(acac)2(DMS)] administered to SH-SY5Y cells provokes the increment of ROS, generated by NADPH oxidase, responsible for the PKC-ε and PKCactivation. Whilst PKCactivates p38/MAPK, responsible for the inhibition of MMP-2 and -9 secretion, PKCactivates a pathway made of ERK1/2, mTOR and S6K responsible for the inhibition of NHE1 activity and cell migration. In conclusion, we have shown a drastic impairment in tumour cell metastatization in response to inhibition of NHE1 and MMPs activities by [Pt(acac)2(DMS)] occurring through a novel mechanism mediated by PKC-δ/-ε activation. PMID:25372487

  19. [Pt(O,O'-acac)(γ-acac)(DMS)] alters SH-SY5Y cell migration and invasion by the inhibition of Na+/H+ exchanger isoform 1 occurring through a PKC-ε/ERK/mTOR Pathway.

    Science.gov (United States)

    Muscella, Antonella; Vetrugno, Carla; Calabriso, Nadia; Cossa, Luca Giulio; De Pascali, Sandra Angelica; Fanizzi, Francesco Paolo; Marsigliante, Santo

    2014-01-01

    We previously showed that [Pt(O,O'-acac)(γ-acac)(DMS)] ([Pt(acac)2(DMS)]) exerted substantial cytotoxic effects in SH-SY5Y neuroblastoma cells, and decreased metalloproteases (MMPs) production and cells migration in MCF-7 breast cancer cells. The ubiquitously distributed sodium-hydrogen antiporter 1 (NHE1) is involved in motility and invasion of many solid tumours. The present study focuses on the effects of [Pt(acac)2(DMS)] in SH-SY5Y cell migration and also on the possibility that NHE1 may be involved in such effect. After sublethal [Pt(acac)2(DMS)] treatment cell migration was examined by wounding assay and cell invasion by transwell assay. NHE1 activity was measured in BCECF-loaded SH-SY5Y as the rate of Na+-dependent intracellular pH recovery in response to an acute acid pulse. Gelatin zymography for MMP-2/9 activities, Western blottings of MMPs, MAPKs, mTOR, S6 and PKCs and small interfering RNAs to PKC-ε/-δ mRNA were performed. Sublethal concentrations of [Pt(acac)2(DMS)] decreases NHE1 activity, inhibits cell migration and invasion and decreases expression and activity of MMP-2 and -9. [Pt(acac)2(DMS)] administered to SH-SY5Y cells provokes the increment of ROS, generated by NADPH oxidase, responsible for the PKC-ε and PKCactivation. Whilst PKCactivates p38/MAPK, responsible for the inhibition of MMP-2 and -9 secretion, PKCactivates a pathway made of ERK1/2, mTOR and S6K responsible for the inhibition of NHE1 activity and cell migration. In conclusion, we have shown a drastic impairment in tumour cell metastatization in response to inhibition of NHE1 and MMPs activities by [Pt(acac)2(DMS)] occurring through a novel mechanism mediated by PKC-δ/-ε activation.

  20. NMDAR NR2A and NR2B specific PKC-dependent regulation of mGluR is defective in the Fragile X Syndrome mouse model

    DEFF Research Database (Denmark)

    Banke, Tue G.; Toft, Anna Karina; Lundbye, Camilla Johanne

    The Fragile X Syndrome (FXS) animal model, the Fmr1 knock-out (KO) mouse, has demonstrated an increased mGluR5-mediated long-term depression (LTD). However, surprisingly little information exists about other ion channels/receptors and their effects on FXS, including NMDA receptors (NMDAR). Here we....... Furthermore, in this model it appears that NR2B activation stimulates PKC, while NR2A activation halts or reverses this effect. In addition, in the KO mice, the coupling between specific NMDAR subunits and mGluR-LTD activity through PKC seems defective in an age-dependent manner. These findings suggest strong...

  1. NMDA receptor activation and PKC but not PKA lead to the modification of the long-term potentiation in the insular cortex induced by conditioned taste aversion: differential role of kinases in metaplasticity.

    Science.gov (United States)

    Rodríguez-Durán, Luis F; Escobar, Martha L

    2014-06-01

    It has been reported that training in behavioral tasks modifies the ability to induce long-term potentiation (LTP) in an N-methyl-D-aspartate receptor (NMDAR)-dependent manner. This receptor leads to calcium entry into neuronal cells, promoting the activation of protein kinases as protein kinase A (PKA) and protein kinase C (PKC), which contribute significantly to the formation of different types of memories and play a pivotal role in the expression of LTP. Our previous studies involving the insular cortex (IC) have demonstrated that induction of LTP in the basolateral amygdaloid nucleus (BLA)-IC projection prior to conditioned taste aversion (CTA) training enhances the retention of this task. Recently, we showed that CTA training triggers a persistent impairment in the ability to induce subsequent synaptic plasticity on the BLA-IC pathway in a protein synthesis-dependent manner, but the underlying molecular mechanisms remain unclear. In the present study we investigated whether the blockade of NMDAR, as well as the inhibition of PKC and PKA affects the CTA-dependent impairment of the IC-LTP. Thus, CTA-trained rats received high frequency stimulation in the Bla-IC projection in order to induce LTP 48 h after the aversion test. The NMDAR antagonist CPP and the specific inhibitors for PKC (chelerythrine) and PKA (KT-5720) were intracortically administered during the acquisition session. Our results show that the blockade of NMDAR and the inhibition of PKC activity prevent the CTA memory-formation as well as the IC-LTP impairment. Nevertheless, PKA inhibition prevents the memory formation of taste aversion but produces no interference with the CTA-dependent impairment of the IC-LTP. These findings reveal the differential roles of protein kinases on CTA-dependent modification of IC-LTP enhancing our understanding of the effects of memory-related changes on synaptic function. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Effects of palm kernel cake (PKC on growth performance, blood components and liver histopathology of sex reversed red tilapia (Oreochromis niloticus

    Directory of Open Access Journals (Sweden)

    Sukasem, N.

    2007-09-01

    Full Text Available Effects of Palm Kernel Cake (PKC on growth performance, blood components and liver histopathology of sex- reversed red tilapia Oreochromis niloticus were studied using seven isocaloric diets (3400 kCal/ kg containing different levels of protein and PKC. Diet 1, 2 and 3 contained 20% protein with the supplementation of 15, 30 and 45% PKC, respectively. Diets 4, 5 and 6 contained 24% protein in combinationwith the same PKC supplemention levels mentioned above, and diet 7 was commercial feed containing 20% protein as a control diet. Experimental diets were fed to experimental fish of 48.65 g initial average body weight cultured in floating cages (3 cages/diet for 10 weeks. Fish fed diets containing higher protein (24%; diets 4, 5 and 6 had significantly better growth performance (p<0.05 than those fed lower protein (20%; diets 1, 2 and 3. Considering the effect of PKC, fish fed diet 5 (Prot. 24%, PKC 30% gave the greatest growth performance (p<0.05 and all the PKC-fed groups had significantly higher growth than fish fed control diet. There was evidence that supplementation of PKC in fish feed ranging from 15 to 45% had no effect to the survival rate, blood components, or hepatocytic cells of tilapia. However, liver tissue showed higher numbers of lipid droplets in fish fed diet contained 45% PKC (diets 3 and 6. For the production cost, all test diets with PKC supplementation had significantly higher price (p<0.05 than commercial feed. However, when considering the feeding cost per unit of fish production, fish reared with PKC supplemented diets had significantly lower cost (p<0.05 than fish fed commercial feed.

  3. Role of FAT/CD36 in novel PKC isoform activation in heart of spontaneously hypertensive rats

    Czech Academy of Sciences Publication Activity Database

    Klevstig, M. J.; Marková, I.; Burianová, J.; Kazdová, L.; Pravenec, Michal; Nováková, O.; Novák, F.

    2011-01-01

    Roč. 357, 1-2 (2011), s. 163-169 ISSN 0300-8177 R&D Projects: GA ČR(CZ) GD305/08/H037; GA MŠk(CZ) ME08006 Grant - others:Univerzita Karlova(CZ) SVV33779266 Institutional research plan: CEZ:AV0Z50110509 Keywords : CD36 * novel PKC * spontaneously hypertensive rat * insulin resistance Subject RIV: FB - Endocrinology, Diabetology, Metabolism, Nutrition Impact factor: 2.057, year: 2011

  4. Simultaneous determination of multi-mycotoxins in palm kernel cake (PKC) using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

    Science.gov (United States)

    Yibadatihan, S; Jinap, S; Mahyudin, N A

    2014-01-01

    Palm kernel cake (PKC) is a useful source of protein and energy for livestock. Recently, it has been used as an ingredient in poultry feed. Mycotoxin contamination of PKC due to inappropriate handling during production and storage has increased public concern about economic losses and health risks for poultry and humans. This concern has accentuated the need for the evaluation of mycotoxins in PKC. Furthermore, a method for quantifying mycotoxins in PKC has so far not been established. The aims of this study were therefore (1) to develop a method for the simultaneous determination of mycotoxins in PKC and (2) to validate and verify the method. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using an electrospray ionisation interface (ESI) in both positive- and negative-ion modes was developed for the simultaneous determination of aflatoxins (AFB₁, AFB₂, AFG₁ and AFG₂), ochratoxin A (OTA), zearalenone (ZEA), deoxynivalenol (DON), fumonisins (FB₁ and FB₂), T-2 and HT-2 toxin in PKC. An optimum method using a 0.2 ml min⁻¹ flow rate, 0.2% formic acid in aqueous phase, 10% organic phase at the beginning and 90% organic phase at the end of the gradient was achieved. The extraction of mycotoxins was performed using a solvent mixture of acetonitrile-water-formic acid (79:20:1, v/v) without further clean-up. The mean recoveries of mycotoxins in spiked PKC samples ranged from 81% to 112%. Limits of detection (LODs) and limits of quantification (LOQs) for mycotoxin standards and PKC samples ranged from 0.02 to 17.5 μg kg⁻¹ and from 0.06 to 58.0 μg kg⁻¹, respectively. Finally, the newly developed method was successfully applied to PKC samples. The results illustrated the fact that the method is efficient and accurate for the simultaneous multi-mycotoxin determination in PKC, which can be ideal for routine analysis.

  5. Independence of protein kinase C-delta activity from activation loop phosphorylation: structural basis and altered functions in cells.

    Science.gov (United States)

    Liu, Yin; Belkina, Natalya V; Graham, Caroline; Shaw, Stephen

    2006-04-28

    Activation loop phosphorylation plays critical regulatory roles for many kinases. Unlike other protein kinase Cs (PKC), PKC-delta does not require phosphorylation of its activation loop (Thr-507) for in vitro activity. We investigated the structural basis for this unusual capacity and its relevance to PKC-delta function in intact cells. Mutational analysis demonstrated that activity without Thr-507 phosphorylation depends on 20 residues N-terminal to the kinase domain and a pair of phenylalanines (Phe-500/Phe-527) unique to PKC-delta in/near the activation loop. Molecular modeling demonstrated that these elements stabilize the activation loop by forming a hydrophobic chain of interactions from the C-lobe to activation loop to N-terminal (helical) extension. In cells PKC-delta mediates both apoptosis and transcription regulation. We found that the T507A mutant of the PKC-delta kinase domain resembled the corresponding wild type in mediating apoptosis in transfected HEK293T cells. But the T507A mutant was completely defective in AP-1 and NF-kappaB reporter assays. A novel assay in which the kinase domain of PKC-delta and its substrate (a fusion protein of PKC substrate peptide with green fluorescent protein) were co-targeted to lipid rafts revealed a major substrate-selective defect of the T507A mutant in phosphorylating the substrate in cells. In vitro analysis showed strong product inhibition on the T507A mutant with particular substrates whose characteristics suggest it contributes to the substrate selective defect of the PKC-delta T507A mutant in cells. Thus, activation loop phosphorylation of PKC-delta may regulate its function in cells in a novel way.

  6. 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. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  7. Melatonin potentiates glycine currents through a PLC/PKC signalling pathway in rat retinal ganglion cells.

    Science.gov (United States)

    Zhao, Wen-Jie; Zhang, Min; Miao, Yanying; Yang, Xiong-Li; Wang, Zhongfeng

    2010-07-15

    In vertebrate retina, melatonin regulates various physiological functions. In this work we investigated the mechanisms underlying melatonin-induced potentiation of glycine currents in rat retinal ganglion cells (RGCs). Immunofluorescence double labelling showed that rat RGCs were solely immunoreactive to melatonin MT(2) receptors. Melatonin potentiated glycine currents of RGCs, which was reversed by the MT(2) receptor antagonist 4-P-PDOT. The melatonin effect was blocked by intracellular dialysis of GDP-beta-S. Either preincubation with pertussis toxin or application of the phosphatidylcholine (PC)-specific phospholipase C (PLC) inhibitor D609, but not the phosphatidylinositol (PI)-PLC inhibitor U73122, blocked the melatonin effect. The protein kinase C (PKC) activator PMA potentiated the glycine currents and in the presence of PMA melatonin failed to cause further potentiation of the currents, whereas application of the PKC inhibitor bisindolylmaleimide IV abolished the melatonin-induced potentiation. The melatonin effect persisted when [Ca(2+)](i) was chelated by BAPTA, and melatonin induced no increase in [Ca(2+)](i). Neither cAMP-PKA nor cGMP-PKG signalling pathways seemed to be involved because 8-Br-cAMP or 8-Br-cGMP failed to cause potentiation of the glycine currents and both the PKA inhibitor H-89 and the PKG inhibitor KT5823 did not block the melatonin-induced potentiation. In consequence, a distinct PC-PLC/PKC signalling pathway, following the activation of G(i/o)-coupled MT(2) receptors, is most likely responsible for the melatonin-induced potentiation of glycine currents of rat RGCs. Furthermore, in rat retinal slices melatonin potentiated light-evoked glycine receptor-mediated inhibitory postsynaptic currents in RGCs. These results suggest that melatonin, being at higher levels at night, may help animals to detect positive or negative contrast in night vision by modulating inhibitory signals largely mediated by glycinergic amacrine cells in the inner

  8. Differential acute and chronic response of protein kinase C in cultured neonatal rat heart myocytes to alpha 1-adrenergic and phorbol ester stimulation.

    Science.gov (United States)

    Henrich, C J; Simpson, P C

    1988-12-01

    Both alpha 1-adrenergic agonists (e.g. norepinephrine, NE*) and tumor-promoting phorbol esters (e.g. phorbol myristate acetate, PMA) are known to activate protein kinase C (PKC) (Abdel-Latif, 1986, Niedel and Blackshear, 1986). However, alpha 1 agonists and PMA produce very different effects on cardiac function (see Simpson, 1985; Benfey, 1987; Meidell et al., 1986; Leatherman et al., 1987; Yuan et al., 1987; for examples). PKC activation in heart cells has been studied only for PMA treated perfused heart (Yuan et al., 1987). Therefore, acute activation and chronic regulation of PKC by NE and PMA were compared in cultured neonatal rat heart myocytes. NE acutely and transiently activated PKC, as measured by translocation of PKC activity to the cell particulate fraction (Niedel and Blackshear, 1986). Particulate PKC activity peaked at 23% of total after NE for 30 s, as compared with 8% for control (P less than 0.001). By contrast, acute PKC activation by PMA was more pronounced and persistent, with particulate PKC activity 62% of total at 5 min (P less than 0.001). Calcium/lipid-independent kinase activity increased acutely with PMA, but not with NE. Chronic treatment with NE (24 to 48 h) increased total per cell PKC activity and 3H-phorbol dibutyrate (PDB) binding sites, an index of the number of PKC molecules (Niedel and Blackshear, 1986), by 30 to 60% over control (all P less than 0.05 to 0.01). In contrast with NE, chronic treatment with PMA down-regulated PKC, reducing total per cell PKC activity and 3H-PDB binding sites to 3% and 12% of control, respectively (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

  9. Sodium phenylbutyrate enhances astrocytic neurotrophin synthesis via protein kinase C (PKC)-mediated activation of cAMP-response element-binding protein (CREB): implications for Alzheimer disease therapy.

    Science.gov (United States)

    Corbett, Grant T; Roy, Avik; Pahan, Kalipada

    2013-03-22

    Neurotrophins, such as brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), are believed to be genuine molecular mediators of neuronal growth and homeostatic synapse activity. However, levels of these neurotrophic factors decrease in different brain regions of patients with Alzheimer disease (AD). Induction of astrocytic neurotrophin synthesis is a poorly understood phenomenon but represents a plausible therapeutic target because neuronal neurotrophin production is aberrant in AD and other neurodegenerative diseases. Here, we delineate that sodium phenylbutyrate (NaPB), a Food and Drug Administration-approved oral medication for hyperammonemia, induces astrocytic BDNF and NT-3 expression via the protein kinase C (PKC)-cAMP-response element-binding protein (CREB) pathway. NaPB treatment increased the direct association between PKC and CREB followed by phosphorylation of CREB (Ser(133)) and induction of DNA binding and transcriptional activation of CREB. Up-regulation of markers for synaptic function and plasticity in cultured hippocampal neurons by NaPB-treated astroglial supernatants and its abrogation by anti-TrkB blocking antibody suggest that NaPB-induced astroglial neurotrophins are functionally active. Moreover, oral administration of NaPB increased the levels of BDNF and NT-3 in the CNS and improved spatial learning and memory in a mouse model of AD. Our results highlight a novel neurotrophic property of NaPB that may be used to augment neurotrophins in the CNS and improve synaptic function in disease states such as AD.

  10. Absence of PDGF-induced, PKC-independent c-fos expression in a chemically transformed C3H/10T1/2 cell clone.

    Science.gov (United States)

    Vassbotn, F S; Skar, R; Holmsen, H; Lillehaug, J R

    1992-09-01

    The effect of platelet-derived growth factor (PDGF) on c-fos mRNA transcription was studied in the immortalized mouse embryo fibroblast C3H/10T1/2 Cl 8 (10T1/2) cells and the chemically transformed, tumorigenic subclone C3H/10T1/2 Cl 16 (Cl 16). In the 10T1/2 cells as well as the Cl 16 subclone, the dose-dependent PDGF stimulation of c-fos mRNA synthesis was similar in both logarithmically growing and confluent cultures. c-fos mRNA was induced severalfold by 12-O-tetradecanoylphorbol-13-acetate (TPA) in both 10T1/2 and Cl 16. Down-regulation of protein kinase C (PKC) activity by TPA pretreatment inhibited PDGF-stimulated c-fos mRNA expression in Cl 16 cells but did not affect this induction in the 10T1/2 cells. This inhibition was not a general phenomenon of 3-methylcholanthrene-mediated transformation of 10T1/2 cells since experiments with another transformed 10T1/2 cell clone, C3H/10T1/2 TPA 482, gave qualitatively the same results as the 10T1/2 cells. Receptor binding experiments showed that the nontransformed and transformed cells had a comparable number of PDGF receptors, 1.3 x 10(5) and 0.7 x 10(5) receptors per cell, respectively. Furthermore, cAMP-induced c-fos expression induced by forskolin is formerly shown to be independent of PKC down-regulation. In our experiments, forskolin induced c-fos expression in both clones. However, PKC down-regulation inhibited the forskolin-induced c-fos expression in Cl 16 cells. This apparently demonstrates cross talk between PKC and PKA in the c-fos induction pathway. The present results provide evidence for an impaired mechanism for activating c-fos expression through PKC-independent, PDGF-induced signal transduction in the chemically transformed Cl 16 fibroblasts compared to that in nontransformed 10T1/2 cells.

  11. C-peptide increases Na,K-ATPase expression via PKC- and MAP kinase-dependent activation of transcription factor ZEB in human renal tubular cells.

    Directory of Open Access Journals (Sweden)

    Dana Galuska

    Full Text Available Replacement of proinsulin C-peptide in type 1 diabetes ameliorates nerve and kidney dysfunction, conditions which are associated with a decrease in Na,K-ATPase activity. We determined the molecular mechanism by which long term exposure to C-peptide stimulates Na,K-ATPase expression and activity in primary human renal tubular cells (HRTC in control and hyperglycemic conditions.HRTC were cultured from the outer cortex obtained from patients undergoing elective nephrectomy. Ouabain-sensitive rubidium ((86Rb(+ uptake and Na,K-ATPase activity were determined. Abundance of Na,K-ATPase was determined by Western blotting in intact cells or isolated basolateral membranes (BLM. DNA binding activity was determined by electrical mobility shift assay (EMSA. Culturing of HRTCs for 5 days with 1 nM, but not 10 nM of human C-peptide leads to increase in Na,K-ATPase α(1-subunit protein expression, accompanied with increase in (86Rb(+ uptake, both in normal- and hyperglycemic conditions. Na,K-ATPase α(1-subunit expression and Na,K-ATPase activity were reduced in BLM isolated from cells cultured in presence of high glucose. Exposure to1 nM, but not 10 nM of C-peptide increased PKCε phosphorylation as well as phosphorylation and abundance of nuclear ERK1/2 regardless of glucose concentration. Exposure to 1 nM of C-peptide increased DNA binding activity of transcription factor ZEB (AREB6, concomitant with Na,K-ATPase α(1-subunit mRNA expression. Effects of 1 nM C-peptide on Na,K-ATPase α(1-subunit expression and/or ZEB DNA binding activity in HRTC were abolished by incubation with PKC or MEK1/2 inhibitors and ZEB siRNA silencing.Despite activation of ERK1/2 and PKC by hyperglycemia, a distinct pool of PKCs and ERK1/2 is involved in regulation of Na,K-ATPase expression and activity by C-peptide. Most likely C-peptide stimulates sodium pump expression via activation of ZEB, a transcription factor that has not been previously implicated in C

  12. The diacylglycerol kinase α/atypical PKC/β1 integrin pathway in SDF-1α mammary carcinoma invasiveness.

    Directory of Open Access Journals (Sweden)

    Elena Rainero

    Full Text Available Diacylglycerol kinase α (DGKα, by phosphorylating diacylglycerol into phosphatidic acid, provides a key signal driving cell migration and matrix invasion. We previously demonstrated that in epithelial cells activation of DGKα activity promotes cytoskeletal remodeling and matrix invasion by recruiting atypical PKC at ruffling sites and by promoting RCP-mediated recycling of α5β1 integrin to the tip of pseudopods. In here we investigate the signaling pathway by which DGKα mediates SDF-1α-induced matrix invasion of MDA-MB-231 invasive breast carcinoma cells. Indeed we showed that, following SDF-1α stimulation, DGKα is activated and localized at cell protrusion, thus promoting their elongation and mediating SDF-1α induced MMP-9 metalloproteinase secretion and matrix invasion. Phosphatidic acid generated by DGKα promotes localization at cell protrusions of atypical PKCs which play an essential role downstream of DGKα by promoting Rac-mediated protrusion elongation and localized recruitment of β1 integrin and MMP-9. We finally demonstrate that activation of DGKα, atypical PKCs signaling and β1 integrin are all essential for MDA-MB-231 invasiveness. These data indicates the existence of a SDF-1α induced DGKα - atypical PKC - β1 integrin signaling pathway, which is essential for matrix invasion of carcinoma cells.

  13. PKC α regulates netrin-1/UNC5B-mediated survival pathway in bladder cancer

    International Nuclear Information System (INIS)

    Liu, Jiao; Kong, Chui-ze; Gong, Da-xin; Zhang, Zhe; Zhu, Yu-yan

    2014-01-01

    Netrin-1 and its receptor UNC5B play important roles in angiogenesis, embryonic development, cancer and inflammation. However, their expression patttern and biological roles in bladder cancer have not been well characterized. The present study aims to investigating the clinical significance of PKC α, netrin-1 and UNC5B in bladder cancer as well as their association with malignant biological behavior of cancer cells. Netrin-1 and UNC5B expression was examined in 120 bladder cancer specimens using immunohistochemistry and in 40 fresh cancer tissues by western blot. Immunofluorescence was performed in cancer cell lines. PKC α agonist PMA and PKC siRNA was employed in bladder cancer cells. CCK-8, wound healing assays and flow cytometry analysis were used to examine cell proliferation, migration and cell cycle, respectively. Netrin-1 expression was positively correlated with histological grade, T stage, metastasis and poor prognosis in bladder cancer tissues. Immunofluorescence showed elevated netrin-1 and decreased UNC5B expression in bladder cancer cells compared with normal bladder cell line. Furthermore, cell proliferation, migration and cell cycle progression were promoted with PMA treatment while inhibited by calphostin C. In addition, PMA treatment could induce while calphostin C reduce netrin-1 expression in bladder cancer cells. The present study identified netrin-1/UNC5B, which could be regulated by PKC signaling, was important mediators of bladder cancer progression

  14. The protein kinase C (PKC) inhibitors combined with chemotherapy in the treatment of advanced non-small cell lung cancer: meta-analysis of randomized controlled trials.

    Science.gov (United States)

    Zhang, L L; Cao, F F; Wang, Y; Meng, F L; Zhang, Y; Zhong, D S; Zhou, Q H

    2015-05-01

    The application of newer signaling pathway-targeted agents has become an important addition to chemotherapy in the treatment of advanced non-small cell lung cancer (NSCLC). In this study, we evaluated the efficacy and toxicities of PKC inhibitors combined with chemotherapy versus chemotherapy alone for patients with advanced NSCLC systematically. Literature retrieval, trials selection and assessment, data collection, and statistic analysis were performed according to the Cochrane Handbook 5.1.0. The outcome measures were tumor response rate, disease control rate, progression-free survival (PFS), overall survival (OS), and adverse effects. Five randomized controlled trials, comprising totally 1,005 patients, were included in this study. Meta-analysis showed significantly decreased response rate (RR 0.79; 95 % CI 0.64-0.99) and disease control rate (RR 0.90; 95 % CI 0.82-0.99) in PKC inhibitors-chemotherapy groups versus chemotherapy groups. There was no significant difference between the two treatment groups regarding progression-free survival (PFS, HR 1.05; 95 % CI 0.91-1.22) and overall survival (OS, HR 1.00; 95 % CI 0.86-1.16). The risk of grade 3/4 neutropenia, leucopenia, and thrombosis/embolism increased significantly in PKC inhibitors combination groups as compared with chemotherapy alone groups. The use of PKC inhibitors in addition to chemotherapy was not a valid alternative for patients with advanced NSCLC.

  15. Alterations in protein kinase C activity and processing during zinc-deficiency-induced cell death.

    Science.gov (United States)

    Chou, Susan S; Clegg, Michael S; Momma, Tony Y; Niles, Brad J; Duffy, Jodie Y; Daston, George P; Keen, Carl L

    2004-10-01

    Protein kinases C (PKCs) are a family of serine/threonine kinases that are critical for signal transduction pathways involved in growth, differentiation and cell death. All PKC isoforms have four conserved domains, C1-C4. The C1 domain contains cysteine-rich finger-like motifs, which bind two zinc atoms. The zinc-finger motifs modulate diacylglycerol binding; thus, intracellular zinc concentrations could influence the activity and localization of PKC family members. 3T3 cells were cultured in zinc-deficient or zinc-supplemented medium for up to 32 h. Cells cultured in zinc-deficient medium had decreased zinc content, lowered cytosolic classical PKC activity, increased caspase-3 processing and activity, and reduced cell number. Zinc-deficient cytosols had decreased activity and expression levels of PKC-alpha, whereas PKC-alpha phosphorylation was not altered. Inhibition of PKC-alpha with Gö6976 had no effect on cell number in the zinc-deficient group. Proteolysis of the novel PKC family member, PKC-delta, to its 40-kDa catalytic fragment occurred in cells cultured in the zinc-deficient medium. Occurrence of the PKC-delta fragment in mitochondria was co-incident with caspase-3 activation. Addition of the PKC-delta inhibitor, rottlerin, or zinc to deficient medium reduced or eliminated proteolysis of PKC-delta, activated caspase-3 and restored cell number. Inhibition of caspase-3 processing by Z-DQMD-FMK (Z-Asp-Gln-Met-Asp-fluoromethylketone) did not restore cell number in the zinc-deficient group, but resulted in processing of full-length PKC-delta to a 56-kDa fragment. These results support the concept that intracellular zinc concentrations influence PKC activity and processing, and that zinc-deficiency-induced apoptosis occurs in part through PKC-dependent pathways.

  16. Podocytic PKC-alpha is regulated in murine and human diabetes and mediates nephrin endocytosis.

    Directory of Open Access Journals (Sweden)

    Irini Tossidou

    Full Text Available BACKGROUND: Microalbuminuria is an early lesion during the development of diabetic nephropathy. The loss of high molecular weight proteins in the urine is usually associated with decreased expression of slit diaphragm proteins. Nephrin, is the major component of the glomerular slit diaphragm and loss of nephrin has been well described in rodent models of experimental diabetes as well as in human diabetic nephropathy. METHODOLOGY/PRINCIPAL FINDINGS: In this manuscript we analyzed the role of PKC-alpha (PKCalpha on endocytosis of nephrin in podocytes. We found that treatment of diabetic mice with a PKCalpha-inhibitor (GO6976 leads to preserved nephrin expression and reduced proteinuria. In vitro, we found that high glucose stimulation would induce PKCalpha protein expression in murine and human podocytes. We can demonstrate that PKCalpha mediates nephrin endocytosis in podocytes and that overexpression of PKCalpha leads to an augmented endocytosis response. After PKC-activation, we demonstrate an inducible association of PKCalpha, PICK1 and nephrin in podocytes. Moreover, we can demonstrate a strong induction of PKCalpha in podocytes of patients with diabetic nephropathy. CONCLUSIONS/SIGNIFICANCE: We therefore conclude that activation of PKCalpha is a pathomechanistic key event during the development of diabetic nephropathy. PKCalpha is involved in reduction of nephrin surface expression and therefore PKCalpha inhibition might be a novel target molecule for anti-proteinuric therapy.

  17. Apelin-13 upregulates Egr-1 expression in rat vascular smooth muscle cells through the PI3K/Akt and PKC signaling pathways

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Qi-Feng [Department of Cardiology, The First Affiliated Hospital of Liaoning Medical University, Jinzhou 121001 (China); Yu, Hong-Wei [Department of Cardiology, Jinzhou Central Hospital, Jinzhou 121001 (China); Sun, Li-Li [Department of Ophthalmology, The Third Affiliated Hospital of Liaoning Medical University, Jinzhou 121001 (China); You, Lu; Tao, Gui-Zhou [Department of Cardiology, The First Affiliated Hospital of Liaoning Medical University, Jinzhou 121001 (China); Qu, Bao-Ze, E-mail: qubaoze1971@hotmail.com [Department of Cardiology, The First Affiliated Hospital of Liaoning Medical University, Jinzhou 121001 (China)

    2015-12-25

    Previous studies have shown that Apelin-13 upregulates early growth response factor-1 (Egr-1) via the extracellular signal-regulated protein kinase (ERK) signaling pathway. Apelin-13 induces proliferation and migration of vascular smooth muscle cells (VSMCs) as well as the upregulation of osteopontin (OPN) via the upregulation of Egr-1. This study was designed to further explore the activity of Apelin-13 in VSMCs by investigating members of the mitogen-activated protein kinase (MAPK) family, in particular Jun kinase (JNK) and p38 mitogen-activated protein kinase (P38). We also examined whether the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) and protein kinase C (PKC) signaling pathways were involved in the regulation of Egr-1 by Apelin-13. We treated rat aortic VSMCs with Apelin-13 and examined the expression of JNK, p-JNK, P38, and p-P38 to investigate whether Apelin-13-mediated increases in Egr-1 occurred through the JNK and P38 signaling pathways. We then pretreated VSMCs with the Gi protein inhibitor pertussis toxin (PTX) and the Gq inhibitor YM254890, added Apelin-13 and looked for changes in Egr-1 expression. Finally, we pretreated with the PI3K inhibitor LY294002 and the PKC inhibitor GF109203X, and treated with Apelin-13. Our results showed that JNK and P38 did not participate in Apelin-13-mediated increase in Egr-1. Instead, Apelin-13 upregulation of Egr-1 was mediated by a PTX-sensitive Gi protein. Apelin-13 did increase ERK phosphorylation through the PI3K/Akt and PKC signaling pathways, resulting in changes in Egr-1 expression. These data provide important targets for future studies to modulate vascular remodeling. - Highlights: • Apelin-13 mediates Egr-1 upregulation in vascular smooth muscle cells via ERK1/2. • The underlying mechanisms are unknown, but exclude Jnk or p38 pathway activation. • Apelin-13 binds to Gi, activating the PI3K/Akt and PKC signaling cascades. • Consequent ERK phosphorylation results in increased Egr-1

  18. Apelin-13 upregulates Egr-1 expression in rat vascular smooth muscle cells through the PI3K/Akt and PKC signaling pathways

    International Nuclear Information System (INIS)

    Liu, Qi-Feng; Yu, Hong-Wei; Sun, Li-Li; You, Lu; Tao, Gui-Zhou; Qu, Bao-Ze

    2015-01-01

    Previous studies have shown that Apelin-13 upregulates early growth response factor-1 (Egr-1) via the extracellular signal-regulated protein kinase (ERK) signaling pathway. Apelin-13 induces proliferation and migration of vascular smooth muscle cells (VSMCs) as well as the upregulation of osteopontin (OPN) via the upregulation of Egr-1. This study was designed to further explore the activity of Apelin-13 in VSMCs by investigating members of the mitogen-activated protein kinase (MAPK) family, in particular Jun kinase (JNK) and p38 mitogen-activated protein kinase (P38). We also examined whether the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) and protein kinase C (PKC) signaling pathways were involved in the regulation of Egr-1 by Apelin-13. We treated rat aortic VSMCs with Apelin-13 and examined the expression of JNK, p-JNK, P38, and p-P38 to investigate whether Apelin-13-mediated increases in Egr-1 occurred through the JNK and P38 signaling pathways. We then pretreated VSMCs with the Gi protein inhibitor pertussis toxin (PTX) and the Gq inhibitor YM254890, added Apelin-13 and looked for changes in Egr-1 expression. Finally, we pretreated with the PI3K inhibitor LY294002 and the PKC inhibitor GF109203X, and treated with Apelin-13. Our results showed that JNK and P38 did not participate in Apelin-13-mediated increase in Egr-1. Instead, Apelin-13 upregulation of Egr-1 was mediated by a PTX-sensitive Gi protein. Apelin-13 did increase ERK phosphorylation through the PI3K/Akt and PKC signaling pathways, resulting in changes in Egr-1 expression. These data provide important targets for future studies to modulate vascular remodeling. - Highlights: • Apelin-13 mediates Egr-1 upregulation in vascular smooth muscle cells via ERK1/2. • The underlying mechanisms are unknown, but exclude Jnk or p38 pathway activation. • Apelin-13 binds to Gi, activating the PI3K/Akt and PKC signaling cascades. • Consequent ERK phosphorylation results in increased Egr-1

  19. Protection against Ischemia-Induced Oxidative Stress Conferred by Vagal Stimulation in the Rat Heart: Involvement of the AMPK-PKC Pathway

    Directory of Open Access Journals (Sweden)

    Wei-Jin Zang

    2012-11-01

    Full Text Available Reactive oxygen species (ROS production is an important mechanism in myocardial ischemia and nicotinamide adenine dinucleotide phosphate (NADPH oxidase is one of major sources of ROS in the heart. Previous studies showed that vagus nerve stimulation (VNS is beneficial in treating ischemic heart diseases. However, the effect of VNS on ROS production remains elusive. In this study, we investigated the role of VNS onischemia-induced ROS production. Our results demonstrated that VNS alleviated the myocardial injury, attenuated the cardiac dysfunction, reserved the antioxidant enzyme activity and inhibited the formation of ROS as evidenced by the decreased NADPH oxidase (Nox activity and superoxide fluorescence intensity as well as the expression of p67phox, Rac1 and nitrotyrosine. Furthermore, VNS resulted in the phosphorylation and activation of adenosine monophosphate activated protein kinase (AMPK, which in turn led to an inactivation of Nox by protein kinase C (PKC; however, the phenomena were repressed by the administration of a muscarinic antagonist atropine. Taken together, these data indicate that VNS decreases ROS via AMPK-PKC-Nox pathway; this may have potential importance for the treatment of ischemic heart diseases.

  20. Palm kernel cake extract exerts hepatoprotective activity in heat-induced oxidative stress in chicken hepatocytes.

    Science.gov (United States)

    Oskoueian, Ehsan; Abdullah, Norhani; Idrus, Zulkifli; Ebrahimi, Mahdi; Goh, Yong Meng; Shakeri, Majid; Oskoueian, Armin

    2014-10-02

    Palm kernel cake (PKC), the most abundant by-product of oil palm industry is believed to contain bioactive compounds with hepatoprotective potential. These compounds may serve as hepatoprotective agents which could help the poultry industry to alleviate adverse effects of heat stress on liver function in chickens. This study was performed to evaluate the hepatoprotective potential of PKC extract in heat-induced oxidative stress in chicken hepatocytes. The nature of the active metabolites and elucidation of the possible mechanism involved were also investigated. The PKC extract possessed free radical scavenging activity with values significantly (p < 0.05) lower than silymarin as the reference antioxidant. Heat-induced oxidative stress in chicken hepatocyte impaired the total protein, lipid peroxidation and antioxidant enzymes activity significantly (p < 0.05). Treatment of heat-induced hepatocytes with PKC extract (125 μg/ml) and silymarin as positive control increased these values significantly (p < 0.05). The real time PCR and western blot analyses revealed the significant (p < 0.05) up-regulation of oxidative stress biomarkers including TNF-like, IFN-γ and IL-1β genes; NF-κB, COX-2, iNOS and Hsp70 proteins expression upon heat stress in chicken hepatocytes. The PKC extract and silymarin were able to alleviate the expression of all of these biomarkers in heat-induced chicken hepatocytes. The gas chromatography-mass spectrometry analysis of PKC extract showed the presence of fatty acids, phenolic compounds, sugar derivatives and other organic compounds such as furfural which could be responsible for the observed hepatoprotective activity. Palm kernel cake extract could be a potential agent to protect hepatocytes function under heat induced oxidative stress.

  1. 6-Gingerol inhibits ROS and iNOS through the suppression of PKC-α and NF-κB pathways in lipopolysaccharide-stimulated mouse macrophages

    International Nuclear Information System (INIS)

    Lee, Tzung-Yan; Lee, Ko-Chen; Chen, Shih-Yuan; Chang, Hen-Hong

    2009-01-01

    Inflammation is involved in numerous diseases, including chronic inflammatory diseases and the development of cancer. Many plants possess a variety of biological activities, including antifungal, antibacterial and anti-inflammatory activities. However, our understanding of the anti-inflammatory effects of 6-gingerol is very limited. We used lipopolysaccharide (LPS)-stimulated macrophages as a model of inflammation to investigate the anti-inflammatory effects of 6-gingerol, which contains phenolic structure. We found that 6-gingerol exhibited an anti-inflammatory effect. 6-Gingerol could decrease inducible nitric oxide synthase and TNF-α expression through suppression of I-κBα phosphorylation, NF-κB nuclear activation and PKC-α translocation, which in turn inhibits Ca 2+ mobilization and disruption of mitochondrial membrane potential in LPS-stimulated macrophages. Here, we demonstrate that 6-gingerol acts as an anti-inflammatory agent by blocking NF-κB and PKC signaling, and may be developed as a useful agent for the chemoprevention of cancer or inflammatory diseases.

  2. PKC and AMPK regulation of Kv1.5 potassium channels

    DEFF Research Database (Denmark)

    Andersen, Martin Nybo; Skibsbye, Lasse; Tang, Chuyi

    2015-01-01

    The voltage-gated Kv1.5 potassium channel, conducting the ultra-rapid rectifier K(+) current (IKur), is regulated through several pathways. Here we investigate if Kv1.5 surface expression is controlled by the 2 kinases PKC and AMPK, using Xenopus oocytes, MDCK cells and atrial derived HL-1 cells....

  3. Dioxin modulates expression of receptor for activated C kinase (RACK-1) in developing neurons

    Energy Technology Data Exchange (ETDEWEB)

    Yang, J.H.; Kim, S.Y.; Lee, H.G.; Kim, M.Y.; Lee, J.H.; Chae, W.G. [Catholic Univ. of Daegu, Dept. of Pharmacology/Toxicology, Daegu (Korea)

    2004-09-15

    TCDD is sensitive to the central nerve system of the developing brain. The TCDD-induced neurodevelopmental deficits include the cognitive disability and motor dysfunction. While TCDD may lead to neurodevelopmental and neurobehavioral deficit, it is not known which molecular substances are intracellular targets for TCDD. Since TCDD accumulates in brain and the brain contains the Ah receptor, it is possible that TCDD may act at the target site such as cerebellum, which is responsible for cognitive abilities and motor function. A recent in vitro studies using cerebellar granule cells demonstrated a translocation of PKC-{alpha} and {epsilon} following the TCDD or PCB exposure. One of the most pivotal second messenger molecules involved in neuronal function and development is protein kinase C (PKC). PKC signaling pathways have been implicated as an important factor in learning and memory processes. PKC signaling events are optimized by the adaptor proteins, which organize PKCs near their selective substrates and away from others. RACK-1(receptor for activated C-kinase) is one of adaptor proteins that anchor the activated PKC at the site of translocation 6. RACKs bind PKC only in the presence of PKC activators. RACKs are 30- and 36-kDa proteins located in cytoskeletal compartment and play a key role in PKC activation and in membrane amchoring. Since different PKC isoforms translocate to distinct subcellular sites on activation, it is suggested that isoform-specific RACK may be present. Activation of certain PKC isoforms (PKC-a and {beta}II) is preferentially associated with RACK-1. While TCDD modulates PKC signaling pathway, role of RACK-1 on TCDD-mediated signaling pathway is not known. To identify the intracellular target for TCDD and understand a mechanism of signaling pathway in the developing brain, the present study attempted to analyze effects of RACK-1 in the cerebellar granule cells following TCDD exposure.

  4. The phosphatidylinositol 3-kinase inhibitor, wortmannin, inhibits insulin-induced activation of phosphatidylcholine hydrolysis and associated protein kinase C translocation in rat adipocytes.

    Science.gov (United States)

    Standaert, M L; Avignon, A; Yamada, K; Bandyopadhyay, G; Farese, R V

    1996-02-01

    We questioned whether phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase C (PKC) function as interrelated signalling mechanisms during insulin action in rat adipocytes. Insulin rapidly activated a phospholipase D that hydrolyses phosphatidylcholine (PC), and this activation was accompanied by increases in diacylglycerol and translocative activation of PKC-alpha and PKC-beta in the plasma membrane. Wortmannin, an apparently specific PI 3-kinase inhibitor, inhibited insulin-stimulated, phospholipase D-dependent PC hydrolysis and subsequent translocation of PKC-alpha and PKC-beta to the plasma membrane. Wortmannin did not inhibit PKC directly in vitro, or the PKC-dependent effects of phorbol esters on glucose transport in intact adipocytes. The PKC inhibitor RO 31-8220 did not inhibit PI 3-kinase directly or its activation in situ by insulin, but inhibited both insulin-stimulated and phorbol ester-stimulated glucose transport. Our findings suggest that insulin acts through PI 3-kinase to activate a PC-specific phospholipase D and causes the translocative activation of PKC-alpha and PKC-beta in plasma membranes of rat adipocytes.

  5. PKCθ is required for the activation of human T lymphocytes induced by CD43 engagement

    International Nuclear Information System (INIS)

    Rio, Roxana del; Rincon, Mercedes; Layseca-Espinosa, Esther; Fierro, Nora A.; Rosenstein, Yvonne; Pedraza-Alva, Gustavo

    2004-01-01

    The turnover of phosphoinositides leading to PKC activation constitutes one of the principal axes of intracellular signaling. In T lymphocytes, the enhanced and prolonged PKC activation resulting from the engagement of the TcR and co-receptor molecules ensures a productive T cell response. The CD43 co-receptor promotes activation and proliferation, by inducing IL-2 secretion and CD69 expression. CD43 engagement has been shown to promote phosphoinositide turnover and DAG production. Moreover, PKC activation was found to be required for the activation of the MAP kinase pathway in response to CD43 ligation. Here we show that CD43 engagement led to the membrane translocation and enzymatic activity of specific PKC isoenzymes: cPKC (α/β), nPKC (ε and θ), aPKC (ζ) and PKCμ. We also show that activation of PKCθ resulting from CD43 ligation induced CD69 expression through an ERK-dependent pathway leading to AP-1, NF-κB activation and an ERK independent pathway promoting NFAT activation. Together, these data suggest that PKCθ plays a critical role in the co-stimulatory functions of CD43 in human T cells

  6. Rational design and validation of an anti-protein kinase C active-state specific antibody based on conformational changes.

    Science.gov (United States)

    Pena, Darlene Aparecida; Andrade, Victor Piana de; Silva, Gabriela Ávila Fernandes; Neves, José Ivanildo; Oliveira, Paulo Sergio Lopes de; Alves, Maria Julia Manso; Devi, Lakshmi A; Schechtman, Deborah

    2016-02-25

    Protein kinase C (PKC) plays a regulatory role in key pathways in cancer. However, since phosphorylation is a step for classical PKC (cPKC) maturation and does not correlate with activation, there is a lack of tools to detect active PKC in tissue samples. Here, a structure-based rational approach was used to select a peptide to generate an antibody that distinguishes active from inactive cPKC. A peptide conserved in all cPKCs, C2Cat, was chosen since modeling studies based on a crystal structure of PKCβ showed that it is localized at the interface between the C2 and catalytic domains of cPKCs in an inactive kinase. Anti-C2Cat recognizes active cPKCs at least two-fold better than inactive kinase in ELISA and immunoprecipitation assays, and detects the temporal dynamics of cPKC activation upon receptor or phorbol stimulation. Furthermore, the antibody is able to detect active PKC in human tissue. Higher levels of active cPKC were observed in the more aggressive triple negative breast cancer tumors as compared to the less aggressive estrogen receptor positive tumors. Thus, this antibody represents a reliable, hitherto unavailable and a valuable tool to study PKC activation in cells and tissues. Similar structure-based rational design strategies can be broadly applied to obtain active-state specific antibodies for other signal transduction molecules.

  7. Acadesine kills chronic myelogenous leukemia (CML cells through PKC-dependent induction of autophagic cell death.

    Directory of Open Access Journals (Sweden)

    Guillaume Robert

    Full Text Available CML is an hematopoietic stem cell disease characterized by the t(9;22 (q34;q11 translocation encoding the oncoprotein p210BCR-ABL. The effect of acadesine (AICAR, 5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside a compound with known antileukemic effect on B cell chronic lymphoblastic leukemia (B-CLL was investigated in different CML cell lines. Acadesine triggered loss of cell metabolism in K562, LAMA-84 and JURL-MK1 and was also effective in killing imatinib-resistant K562 cells and Ba/F3 cells carrying the T315I-BCR-ABL mutation. The anti-leukemic effect of acadesine did not involve apoptosis but required rather induction of autophagic cell death. AMPK knock-down by Sh-RNA failed to prevent the effect of acadesine, indicating an AMPK-independent mechanism. The effect of acadesine was abrogated by GF109203X and Ro-32-0432, both inhibitor of classical and new PKCs and accordingly, acadesine triggered relocation and activation of several PKC isoforms in K562 cells. In addition, this compound exhibited a potent anti-leukemic effect in clonogenic assays of CML cells in methyl cellulose and in a xenograft model of K562 cells in nude mice. In conclusion, our work identifies an original and unexpected mechanism by which acadesine triggers autophagic cell death through PKC activation. Therefore, in addition to its promising effects in B-CLL, acadesine might also be beneficial for Imatinib-resistant CML patients.

  8. Role of protein kinase C in regulation of Na+- and K +-dependent ATPase activity and pump function in corneal endothelial cells.

    Science.gov (United States)

    Hatou, Shin; Yamada, Masakazu; Mochizuki, Hiroshi; Nishida, Teruo

    2009-05-01

    Na+- and K+-dependent ATPase (Na,K-ATPase) plays an important role in the pump function of the corneal endothelium. We investigated the possible role of protein kinase C (PKC) in regulation of Na,K-ATPase activity and pump function in corneal endothelial cells. Confluent monolayers of mouse corneal endothelial cells were exposed to phorbol 12,13-dibutyrate (PDBu) to induce activation of PKC. ATPase activity of the cells was evaluated by using ammonium molybdate in spectrophotometric measurement of phosphate released from ATP, with Na,K-ATPase activity being defined as the portion of total ATPase activity sensitive to ouabain. Pump function of the cells was measured with a Ussing chamber, with the pump function attributable to Na,K-ATPase activity being defined as the portion of the total short-circuit current sensitive to ouabain. PDBu (10(-7) M) increased the Na,K-ATPase activity and pump function of the cultured cells. These effects of PDBu were potentiated by the cyclooxygenase inhibitor indomethacin and the cytochrome P(450) inhibitor resorufin and were blocked by okadaic acid, an inhibitor of protein phosphatases 1 and 2A. Our results suggest that PKC bidirectionally regulates Na,K-ATPase activity in mouse corneal endothelial cells: it inhibits Na,K-ATPase activity in a cyclooxygenase- and cytochrome P(450)-dependent manner, whereas it stimulates such activity by activating protein phosphatases 1 or 2A.

  9. PKC/ROS-Mediated NLRP3 Inflammasome Activation Is Attenuated by Leishmania Zinc-Metalloprotease during Infection

    Science.gov (United States)

    Jung, Jee Yong; Chang, Kwang-Poo; Olivier, Martin

    2015-01-01

    Parasites of the Leishmania genus infect and survive within macrophages by inhibiting several microbicidal molecules, such as nitric oxide and pro-inflammatory cytokines. In this context, various species of Leishmania have been reported to inhibit or reduce the production of IL-1β both in vitro and in vivo. However, the mechanism whereby Leishmania parasites are able to affect IL-1β production and secretion by macrophages is still not fully understood. Dependent on the stimulus at hand, the maturation of IL-1β is facilitated by different inflammasome complexes. The NLRP3 inflammasome has been shown to be of pivotal importance in the detection of danger molecules such as inorganic crystals like asbestos, silica and malarial hemozoin, (HZ) as well as infectious agents. In the present work, we investigated whether Leishmania parasites modulate NLRP3 inflammasome activation. Using PMA-differentiated THP-1 cells, we demonstrate that Leishmania infection effectively inhibits macrophage IL-1β production upon stimulation. In this context, the expression and activity of the metalloprotease GP63 - a critical virulence factor expressed by all infectious Leishmania species - is a prerequisite for a Leishmania-mediated reduction of IL-1β secretion. Accordingly, L. mexicana, purified GP63 and GP63-containing exosomes, caused the inhibition of macrophage IL-1β production. Leishmania-dependent suppression of IL-1β secretion is accompanied by an inhibition of reactive oxygen species (ROS) production that has previously been shown to be associated with NLRP3 inflammasome activation. The observed loss of ROS production was due to an impaired PKC-mediated protein phosphorylation. Furthermore, ROS-independent inflammasome activation was inhibited, possibly due to an observed GP63-dependent cleavage of inflammasome and inflammasome-related proteins. Collectively for the first time, we herein provide evidence that the protozoan parasite Leishmania, through its surface

  10. Atypical protein kinase C activity is required for extracellular matrix degradation and invasion by Src-transformed cells.

    Science.gov (United States)

    Rodriguez, Elena M; Dunham, Elizabeth E; Martin, G Steven

    2009-10-01

    Atypical protein kinase C (aPKC) isoforms have been shown to mediate Src-dependent signaling in response to growth factor stimulation. To determine if aPKC activity contributes to the transformed phenotype of cells expressing oncogenic Src, we have examined the activity and function of aPKCs in 3T3 cells expressing viral Src (v-Src). aPKC activity and tyrosine phosphorylation were found to be elevated in some but not all clones of mouse fibroblasts expressing v-Src. aPKC activity was inhibited either by addition of a membrane-permeable pseudosubstrate, by expression of a dominant-negative aPKC, or by RNAi-mediated knockdown of specific aPKC isoforms. aPKC activity contributes to morphological transformation and stress fiber disruption, and is required for migration of Src-transformed cells and for their ability to polarize at the edge of a monolayer. The lambda isoform of aPKC is specifically required for invasion through extracellular matrix in Boyden chamber assays and for degradation of the extracellular matrix in in situ zymography assays. Tyrosine phosphorylation of aPKClambda is required for its ability to promote cell invasion. The defect in invasion upon aPKC inhibition appears to result from a defect in the assembly and/or function of podosomes, invasive adhesions on the ventral surface of the cell that are sites of protease secretion. aPKC was also found to localize to podosomes of v-Src transformed cells, suggesting a direct role for aPKC in podosome assembly and/or function. We conclude that basal or elevated aPKC activity is required for the ability of Src-transformed cells to degrade and invade the extracellular matrix. Copyright 2009 Wiley-Liss, Inc.

  11. Cellulase Activity in Solid State Fermentation of Palm Kernel Cake with Trichoderma sp.

    Directory of Open Access Journals (Sweden)

    Massaud, M. B. N.

    2012-01-01

    Full Text Available Aims: The effect of different types of fungal inocula to the cellulase activity measured on palm kernel cake (PKC was studied. Methodology and Results: Isolate Pro-A1 which was identified as Trichoderma sp. was selected as a potential producer of cellulase via solid state fermentation technique (SSF. Two types of PKCs were used; raw PKC (containing residual oil and defatted PKC. The PKCs were inoculated with different concentrations of conidia and varying amounts (g of solid mycelia plugs (SMP for SSF. The effect of ultrafiltered crude fungal filtrate (CFF as inocula was also being tested. The highest cellulase activity of 2.454 FPU/mL was detected with 60% (wt/wt SMP applied to the raw PKC. Conversely, 2.059 FPU/mL of cellulase activity was measured when 80% (wt/wt of SMP was applied to the defatted PKC which is 62.3% higher than the untreated defatted PKC; and more than 100% increase in enzymatic activity compared to raw PKC. The cellulase activity in the SSF inoculated with 8 x 106 conidia /mL and 12 x 106 conidia /mL were 1.704 FPU/mL for raw PKC and 1.856 FPU/mL for defatted PKC, an enhancement of about 46% from uninoculated batch. Inoculation with CFF bears corresponding maximum improvement of the cellulase activity on both PKCs of 13.58% (raw and 2.86% (defatted. Conclusion, significance and impact of study: The current study proves that Trichoderma sp. in the form of SMP can enhance the cellulase activity on PKCs effectively with more than 100% increment. Fungal conidia are also a better choice in enhancing cellulase activity of Trichoderma sp. permitted that the PKC used is devoid of oil. From this study, Trichoderma sp. holds the potential of converting lignocellulosic materials into products of commercial and industrial values such as glucose and other biofuels.

  12. Activation of Protein Kinase C and Protein Kinase D in Human Natural Killer Cells: Effects of Tributyltin, Dibutyltin, and Tetrabromobisphenol A

    Science.gov (United States)

    Rana, Krupa; Whalen, Margaret M.

    2015-01-01

    Up to now, the ability of target cells to activate protein kinase C (PKC) and protein kinase D (PKD) (which is often a downstream target of PKC) has not been examined in natural killer (NK) lymphocytes. Here we examined whether exposure of human NK cells to lysis sensitive tumor cells activated PKC and PKD. The results of these studies show for the first time that activation of PKC and PKD occurs in response to target cell binding to NK cells. Exposure of NK cells to K562 tumor cells for 10 and 30 minutes increased phosphorylation/activation of both PKC and PKD by roughly 2 fold. Butyltins (tributyltin (TBT); dibutyltin (DBT)) and brominated compounds (tetrabromobisphenol A (TBBPA)) are environmental contaminants that are found in human blood. Exposures of NK cells to TBT, DBT or TBBPA decrease NK cell lytic function in part by activating the mitogen activated protein kinases (MAPKs) that are part of the NK lytic pathway. We established that PKC and PKD are part of the lytic pathway upstream of MAPKs and thus we investigated whether DBT, TBT, and TBBPA exposures activated PKC and PKD. TBT activated PKC by 2–3 fold at 10 min at concentrations ranging from 50–300 nM while DBT caused a 1.3 fold activation at 2.5 μM at 10 min. Both TBT and DBT caused an approximately 2 fold increase in phosphorylation/activation of PKC. Exposures to TBBPA caused no statistically significant changes in either PKC or PKD activation. PMID:26228090

  13. Role of protein kinase C in TBT-induced inhibition of lytic function and MAPK activation in human natural killer cells.

    Science.gov (United States)

    Abraha, Abraham B; Rana, Krupa; Whalen, Margaret M

    2010-11-01

    Human natural killer (NK) cells are lymphocytes that destroy tumor and virally infected cells. Previous studies have shown that exposure of NK cells to tributyltin (TBT) greatly diminishes their ability to destroy tumor cells (lytic function) while activating mitogen-activated protein kinases (MAPK) (p44/42, p38, and JNK) in NK cells. The signaling pathway that regulates NK lytic function appears to include activation of protein kinase C(PKC) as well as MAPK activity. TBT-induced activation of MAPKs would trigger a portion of the NK lytic signaling pathway, which would then leave the NK cell unable to trigger this pathway in response to a subsequent encounter with a target cell. In the present study we evaluated the involvement of PKC in inhibition of NK lysis of tumor cells and activation of MAPKs caused by TBT exposure. TBT caused a 2–3-fold activation of PKC at concentrations ranging from 50 to 300 nM (16–98 ng/ml),indicating that activation of PKC occurs in response to TBT exposure. This would then leave the NK cell unable to respond to targets. Treatment with the PKC inhibitor, bisindolylmaleimide I, caused an 85% decrease in the ability of NK cells to lyse tumor cells, validating the involvement of PKC in the lytic signaling pathway. The role of PKC in the activation of MAPKs by TBT was also investigated using bisindolylmaleimide I. The results indicated that, in NK cells where PKC activation was blocked, there was no activation of the MAPK, p44/42 in response to TBT.However, TBT-induced activation of the MAPKs, p38 and JNK did not require PKC activation. These results indicate the pivotal role of PKC in the TBT-induced loss of NK lytic function including activation of p44/42 by TBT in NK cells.

  14. Role of protein kinase C in the TBT-induced inhibition of lytic function and MAPK activation in human natural killer cells

    Science.gov (United States)

    Abraha, Abraham B.; Rana, Krupa; Whalen, Margaret M.

    2010-01-01

    Human natural killer (NK) cells are lymphocytes that destroy tumor and virally infected cells. Previous studies have shown that exposures of NK cells to tributyltin (TBT) greatly diminish their ability to destroy tumor cells (lytic function) while activating mitogen-activated protein kinases (MAPK) (p44/42, p38, and JNK) in the NK cells. The signaling pathway that regulates NK lytic function appears to include activation of protein kinase C (PKC) as well as MAPK activity. The TBT-induced activation of MAPKs would trigger a portion of the NK lytic signaling pathway, which would then leave the NK cell unable to trigger this pathway in response to a subsequent encounter with a target cell. In the present study we evaluated the involvement of PKC in the inhibition of NK lysis of tumor cells and activation of MAPKs caused by TBT exposures. TBT caused a 2–3 fold activation of PKC at concentrations ranging from 50–300 nM (16–98 ng/mL), indicating that activation of PKC occurs in response to TBT exposures. This would then leave the NK cell unable to respond to targets. Treatment with the PKC inhibitor, bisindolylmaleimide I, caused an 85% decrease in the ability of NK cells to lyse tumor cells validating the involvement of PKC in the lytic signaling pathway. The role of PKC in the activation of MAPKs by TBT was also investigated using bisindolylmaleimide I. The results indicated that in NK cells where PKC activation was blocked there was no activation of the MAPK, p44/42 in response to TBT. However, TBT-induced activation of the MAPKs, p38 and JNK did not require PKC activation. These results indicate the pivotal role of PKC in the TBT-induced loss of NK lytic function including the activation of p44/42 by TBT in NK cells. PMID:20390410

  15. ARC (NSC 188491 has identical activity to Sangivamycin (NSC 65346 including inhibition of both P-TEFb and PKC

    Directory of Open Access Journals (Sweden)

    Hollingshead Melinda G

    2009-02-01

    Full Text Available Abstract Background The nucleoside analog, ARC (NSC 188491 is a recently characterized transcriptional inhibitor that selectively kills cancer cells and has the ability to perturb angiogenesis in vitro. In this study, the mechanism of action of ARC was further investigated by comparing in vitro and in vivo activity with other anti-neoplastic purines. Methods Structure-based homology searches were used to identify those compounds with similarity to ARC. Comparator compounds were then evaluated alongside ARC in the context of viability, cell cycle and apoptosis assays to establish any similarities. Following this, biological overlap was explored in detail using gene-expression analysis and kinase inhibition assays. Results Results demonstrated that sangivamycin, an extensively characterized pro-apoptotic nucleoside isolated from Streptomyces, had identical activity to ARC in terms of 1 cytotoxicity assays, 2 ability to induce a G2/M block, 3 inhibitory effects on RNA/DNA/protein synthesis, 4 transcriptomic response to treatment, 5 inhibition of protein kinase C, 6 inhibition of positive transcription elongation factor b (P-TEFb, 7 inhibition of VEGF secretion, and 8 activity within hollow fiber assays. Extending ARC activity to PKC inhibition provides a molecular basis for ARC cancer selectivity and anti-angiogenic effects. Furthermore, functional overlap between ARC and sangivamycin suggests that development of ARC may benefit from a retrospective of previous sangivamycin clinical trials. However, ARC was found to be inactive in several xenograft models, likely a consequence of rapid serum clearance. Conclusion Overall, these data expand on the biological properties of ARC but suggest additional studies are required before it can be considered a clinical trials candidate.

  16. Mechanism of phorbol ester-mediated protein kinase C activation in EL4 thymoma cells

    International Nuclear Information System (INIS)

    Huang, F.L.; Arora, P.K.; Hanna, E.E.; Huang, K.P.

    1987-01-01

    Mouse thymoma EL4 cells respond to phorbol 12-myristate 13-acetate (PMA) in interleukin-2 secretion and growth inhibition. A rapid translocation of protein kinase C (PKC) from cytosol to the particulate fraction and followed by proteolytic degradation occur when EL4 cells are incubated with PMA. In the present study the translocated membrane-associated PKC (PP-PKC) was solubilized by buffer containing NP-40 and its behavior on column chromatography, molecular weight, and kinetic properties were compared to the cytosolic PKC (CS-PKC) from untreated cells. From DE-52 cellulose column, CS-PKC could be eluted by buffer containing 0.1 M KCl, whereas PP-PKC was eluted with buffer containing 0.25 M KCl and 0.2% NP-40. On gel filtration the partially purified PP-PKC from DE-52 was separated into two species: a high Mr species, which was a complex of 82KDa PKC, PMA, and lipid as evidenced by immunoblot analysis and labeling with [ 3 H]PMA and [ 3 H]myristic acid, and a 82KDa species, which was free of PMA and lipid. This 82KDa PP-PKC, though similar to the CS-PKC in molecular weight, is distinguishable from the CS-PKC in having lower Ka values for both Ca 2+ and PS and no longer requires diacylglycerol for maximum activation. These results indicate that upon PMA treatment of EL4 cells, the CS-PKC was modified through enhancing the kinase activity and affinity for membrane lipid. The modification results in the translocation and complexing of PKC with membrane lipid and PMA and subsequent degradation

  17. Activation of protein kinase C by elevation of glucose concentration: Proposal for a mechanism in the development of diabetic vascular complications

    International Nuclear Information System (INIS)

    Lee, Tianshing; Saltsman, K.A.; Ohashi, Hiromi; King, G.L.

    1989-01-01

    Hyperglycemia is believed to be the major cause of diabetic vascular complications involving both microvessels and arteries as in the retina, renal glomeruli, and aorta. It is unclear by which mechanism hyperglycemia is altering the metabolism and functions of vascular cells, although changes in nonenzymatic protein glycosylation and increases in cellular sorbitol levels have been postulated to be involved. Previously, the authors have reported that the elevation of extracellular glucose levels with cultured bovine retinal capillary endothelial cells causes an increase in protein kinase C (PKC) activity of the membranous pool with a parallel decrease in the cytosol without alteration of its total activity. Now they demonstrate that the mechanism for the activation of PKC is due to an enhanced de novo synthesis of diacylglycerol as indicated by a 2-fold increase of [ 14 C]diacylglycerol labeling from [ 14 C]glucose. The elevated diacylglycerol de novo synthesis is secondarily due to increased formation of precursors derived from glucose metabolism; this formation is enhanced by hyperglycemia as substantiated by elevated [ 3 H]glucose conversion into water. This effect of hyperglycemia on PKC is also observed in cultured aortic smooth muscle and endothelial cells and the retina and kidney of diabetic rats, but not in the brain. Since PKC in vascular cells has been shown to modulate hormone receptor turnover, neovascularization in vitro, and cell growth, they propose that this mechanism of enhancing the membranous PKC activities by hyperglycemia plays an important role in the development of diabetic vascular complications

  18. Isolamento e caracterização de um mutante de saccharomyces cerevisiae com características fenotípicas opostas à cepa pkc

    OpenAIRE

    Gomes, Katia das Neves

    2004-01-01

    Em leveduras, a proteína quinase C participa da regulação da via bioquímica responsável pela transcrição de uma subunidade da enzima glucano sintase, a qual está envolvida na síntese da parede celular. A via PKC MAP quinase consiste das enzimas Bck1, Mkk1/2 e Mpk1 que são ativadas por fosforilação. Recentemente, nós descobrimos que o mutante pkc1 D, contrariamente aos demais mutantes da cascata Map quinase, exibe dois principais defeitos no controle do metabolismo de carbono. A cepa pkc1 D ap...

  19. 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. PKCactivity 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 PKCactivation. 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

  20. Role of mitochondrial ATP-sensitive potassium channel-mediated PKC-ε in delayed protection against myocardial ischemia/reperfusion injury in isolated hearts of sevoflurane-preconditioned rats

    Energy Technology Data Exchange (ETDEWEB)

    Wang, C. [Department of Anesthesiology and Critical Care, The Second Affiliate Hospital, Soochow University, Suzhou (China); Institute of Neuroscience, Soochow University, Suzhou (China); Hu, S.M. [Institute of Neuroscience, Soochow University, Suzhou (China); Xie, H.; Qiao, S.G. [Department of Anesthesiology and Critical Care, The Second Affiliate Hospital, Soochow University, Suzhou (China); Liu, H. [Department of Anesthesiology and Pain Medicine, University of California Davis Health System, Davis, CA (United States); Liu, C.F. [Institute of Neuroscience, Soochow University, Suzhou (China)

    2015-03-27

    This study aimed to determine the role of mitochondrial adenosine triphosphate-sensitive potassium (mitoK{sub ATP}) channels and protein kinase C (PKC)-ε in the delayed protective effects of sevoflurane preconditioning using Langendorff isolated heart perfusion models. Fifty-four isolated perfused rat hearts were randomly divided into 6 groups (n=9). The rats were exposed for 60 min to 2.5% sevoflurane (the second window of protection group, SWOP group) or 33% oxygen inhalation (I/R group) 24 h before coronary occlusion. The control group (CON) and the sevoflurane group (SEVO) group were exposed to 33% oxygen and 2.5% sevoflurane for 60 min, respectively, without coronary occlusion. The mitoK{sub ATP} channel inhibitor 5-hydroxydecanoate (5-HD) was given 30 min before sevoflurane preconditioning (5-HD+SWOP group). Cardiac function indices, infarct sizes, serum cardiac troponin I (cTnI) concentrations, and the expression levels of phosphorylated PKC-ε (p-PKC-ε) and caspase-8 were measured. Cardiac function was unchanged, p-PKC-ε expression was upregulated, caspase-8 expression was downregulated, cTnI concentrations were decreased, and the infarcts were significantly smaller (P<0.05) in the SWOP group compared with the I/R group. Cardiac function was worse, p-PKC-ε expression was downregulated, caspase-8 expression was upregulated, cTnI concentration was increased and infarcts were larger in the 5-HD+SWOP group (P<0.05) compared with the SWOP group. The results suggest that mitoK{sub ATP} channels are involved in the myocardial protective effects of sevoflurane in preconditioning against I/R injury, by regulating PKC-ε phosphorylation before ischemia, and by downregulating caspase-8 during reperfusion.

  1. Immune responses of mussel hemocyte subpopulations are differentially regulated by enzymes of the PI 3-K, PKC, and ERK kinase families.

    Science.gov (United States)

    García-García, Erick; Prado-Alvarez, Maria; Novoa, Beatriz; Figueras, Antonio; Rosales, Carlos

    2008-01-01

    Various hemocyte cell types have been described in invertebrates, but for most species a functional characterization of different hemocyte cell types is still lacking. In order to characterize some immunological properties of mussel (Mytilus galloprovincialis) hemocytes, cells were separated by flow cytometry and their capacity for phagocytosis, production of reactive oxygen species (ROS), and production of nitric oxide (NO), was examined. Phosphatidylinositol 3-kinase (PI 3-K), protein kinase C (PKC), and extracellular signal-regulated kinase (ERK) inhibitors were also used to biochemically characterize these cell responses. Four morphologically distinct subpopulations, designated R1-R4, were detected. R1, R2, and R3 cells presented different levels of phagocytosis towards zymosan, latex beads, and two bacteria species. Similarly, R1 to R3, but not R4, cells produced ROS, while all subpopulations produced NO, in response to zymosan. Internalization of all phagocytic targets was blocked by PI 3-K inhibition. In addition, internalization of latex particles, but not of bacteria, was partially blocked by PKC or ERK inhibition. Interestingly, phagocytosis of zymosan was impaired by PKC, or ERK inhibitors, only in R2 cells. Zymosan-induced ROS production was blocked by PI 3-K inhibition, but not by PKC, or ERK inhibition. In addition, zymosan-stimulated NO production was affected by PI 3-K inhibition in R1 and R2, but not in R3 or R4 cells. NO production in all cell types was unaffected by PKC inhibition, but ERK inhibition blocked it in R2 cells. These data reveal the existence of profound functional and biochemical differences in mussel hemocytes and indicate that M. galloprovincialis hemocytes are specialized cells fulfilling specific tasks in the context of host defense.

  2. Activation of protein kinase C alters the intracellular distribution and mobility of cardiac Na+ channels.

    Science.gov (United States)

    Hallaq, Haifa; Wang, Dao W; Kunic, Jennifer D; George, Alfred L; Wells, K Sam; Murray, Katherine T

    2012-02-01

    Na(+) current derived from expression of the cardiac isoform SCN5A is reduced by receptor-mediated or direct activation of protein kinase C (PKC). Previous work has suggested a possible role for loss of Na(+) channels at the plasma membrane in this effect, but the results are controversial. In this study, we tested the hypothesis that PKC activation acutely modulates the intracellular distribution of SCN5A channels and that this effect can be visualized in living cells. In human embryonic kidney cells that stably expressed SCN5A with green fluorescent protein (GFP) fused to the channel COOH-terminus (SCN5A-GFP), Na(+) currents were suppressed by an exposure to PKC activation. Using confocal microscopy, colocalization of SCN5A-GFP channels with the plasma membrane under control and stimulated conditions was quantified. A separate population of SCN5A channels containing an extracellular epitope was immunolabeled to permit temporally stable labeling of the plasma membrane. Our results demonstrated that Na(+) channels were preferentially trafficked away from the plasma membrane by PKC activation, with a major contribution by Ca(2+)-sensitive or conventional PKC isoforms, whereas stimulation of protein kinase A (PKA) had the opposite effect. Removal of the conserved PKC site Ser(1503) or exposure to the NADPH oxidase inhibitor apocynin eliminated the PKC-mediated effect to alter channel trafficking, indicating that both channel phosphorylation and ROS were required. Experiments using fluorescence recovery after photobleaching demonstrated that both PKC and PKA also modified channel mobility in a manner consistent with the dynamics of channel distribution. These results demonstrate that the activation of protein kinases can acutely regulate the intracellular distribution and molecular mobility of cardiac Na(+) channels in living cells.

  3. Different associations of CD45 isoforms with STAT3, PKC and ERK regulate IL-6-induced proliferation in myeloma.

    Directory of Open Access Journals (Sweden)

    Xu Zheng

    Full Text Available In response to interleukin 6 (IL-6 stimulation, both CD45RO and CD45RB, but not CD45RA, translocate to lipid rafts. However, the significance of this distinct translocation and the downstream signals in CD45 isoforms-participated IL-6 signal are not well understood. Using sucrose fractionation, we found that phosphorylated signal transducer and activator of transcription (STAT3 and STAT1 were mainly localized in lipid rafts in response to IL-6 stimulation, despite both STAT3 and STAT1 localizing in raft and non-raft fractions in the presence or absence of IL-6. On the other hand, extracellular signal-regulated kinase (ERK, and phosphorylated ERK were localized in non-raft fractions regardless of the existence of IL-6. The rafts inhibitor significantly impeded the phosphorylation of STAT3 and STAT1 and nuclear translocation, but had little effect on (and only postponing the phosphorylation of ERK. This data suggests that lipid raft-dependent STAT3 and STAT1 pathways are dominant pathways of IL-6 signal in myeloma cells. Interestingly, the phosphorylation level of STAT3 but not STAT1 in CD45+ cells was significantly higher compared to that of CD45- cells, while the phosphorylation level of ERK in CD45+ myeloma cells was relatively low. Furthermore, exogenously expressed CD45RO/RB significantly enhanced STAT3, protein kinase C (PKC and downstream NF-κB activation; however, CD45RA/RB inhibited IL-6-induced ERK phosphorylation. CD45 also enhanced the nuclear localization of STAT3 but not that of STAT1. In response to IL-6 stimulation, CD45RO moved into raft compartments and formed a complex with STAT3 and PKC in raft fraction, while CD45RA remained outside of lipid rafts and formed a complex with ERK in non-raft fraction. This data suggests a different role of CD45 isoforms in IL-6-induced signaling, indicating that while CD45RA/RB seems inhibit the rafts-unrelated ERK pathway, CD45RO/RB may actually work to enhance the rafts-related STAT3 and PKC

  4. Role of PKC and CaV1.2 in detrusor overactivity in a model of obesity associated with insulin resistance in mice.

    Directory of Open Access Journals (Sweden)

    Luiz O Leiria

    Full Text Available Obesity/metabolic syndrome are common risk factors for overactive bladder. This study aimed to investigate the functional and molecular changes of detrusor smooth muscle (DSM in high-fat insulin resistant obese mice, focusing on the role of protein kinase C (PKC and Ca(v1.2 in causing bladder dysfunction. Male C57BL/6 mice were fed with high-fat diet for 10 weeks. In vitro functional responses and cystometry, as well as PKC and Ca(v1.2 expression in bladder were evaluated. Obese mice exhibited higher body weight, epididymal fat mass, fasting glucose and insulin resistance. Carbachol (0.001-100 µM, α,β-methylene ATP (1-10 µM, KCl (1-300 mM, extracellular Ca(2+ (0.01-100 mM and phorbol-12,13-dibutyrate (PDBu; 0.001-3 µM all produced greater DSM contractions in obese mice, which were fully reversed by the Ca(v1.2 blocker amlodipine. Cystometry evidenced augmented frequency, non-void contractions and post-void pressure in obese mice that were also prevented by amlodipine. Metformin treatment improved the insulin sensitivity, and normalized the in vitro bladder hypercontractility and cystometric dysfunction in obese mice. The PKC inhibitor GF109203X (1 µM also reduced the carbachol induced contractions. PKC protein expression was markedly higher in bladder tissues from obese mice, which was normalized by metformin treatment. The Ca(v1.2 channel protein expression was not modified in any experimental group. Our findings show that Ca(v1.2 blockade and improvement of insulin sensitization restores the enhanced PKC protein expression in bladder tissues and normalizes the overactive detrusor. It is likely that insulin resistance importantly contributes for the pathophysiology of this urological disorder in obese mice.

  5. Lipopolysaccharide stimulates endogenous β-glucuronidase via PKC/NF-κB/c-myc signaling cascade: a possible factor in hepatolithiasis formation.

    Science.gov (United States)

    Yao, Dianbo; Dong, Qianze; Tian, Yu; Dai, Chaoliu; Wu, Shuodong

    2017-11-29

    Hepatolithiasis is commonly encountered in Southeastern and Eastern Asian countries, but the pathogenesis mechanism of stone formation is still not well understood. Now, the role of endogenous β-glucuronidase in pigment stones formation is being gradually recognized. In this study, the mechanism of increased expression and secretion of endogenous β-glucuronidase during hepatolithiasis formation was investigated. We assessed the endogenous β-glucuronidase, c-myc, p-p65, and p-PKC expression in liver specimens with hepatolithiasis by immunohistochemical staining, and found that compared with that in normal liver samples, the expression of endogenous β-glucuronidase, c-myc, p-p65, and p-PKC in liver specimens with hepatolithiasis significantly increased, and their expressions were positively correlated with each other. Lipopolysaccharide (LPS) induced increased expression of endogenous β-glucuronidase and c-myc in hepatocytes and intrahepatic biliary epithelial cells in a dose- and time-dependent manner, and endogenous β-glucuronidase secretion increased, correspondingly. C-myc siRNA transfection effectively inhibited the LPS-induced expression of endogenous β-glucuronidase. Furthermore, NF-κB inhibitor pyrrolidine dithiocarbamate or PKC inhibitor chelerythrine could effectively inhibit the LPS-induced expression of c-myc and endogenous β-glucuronidase, and the expression of p-p65 was also partly inhibited by chelerythrine. Our clinical observations and experimental data indicate that LPS could induce the increased expression and secretion of endogenous β-glucuronidase via a signaling cascade of PKC/NF-κB/c-myc in hepatocytes and intrahepatic biliary epithelial cells, and endogenous β-glucuronidase might play a possible role in the formation of hepatolithiasis.

  6. In Vitro and In Vivo Antitumor Activity of [Pt(O,O'-acac)(γ-acac)(DMS)] in Malignant Pleural Mesothelioma.

    Science.gov (United States)

    Muscella, Antonella; Vetrugno, Carla; Cossa, Luca Giulio; Antonaci, Giovanna; De Nuccio, Francesco; De Pascali, Sandra Angelica; Fanizzi, Francesco Paolo; Marsigliante, Santo

    2016-01-01

    Malignant pleural mesothelioma (MPM) is an aggressive malignancy highly resistant to chemotherapy. There is an urgent need for effective therapy inasmuch as resistance, intrinsic and acquired, to conventional therapies is common. Among Pt(II) antitumor drugs, [Pt(O,O'-acac)(γ-acac)(DMS)] (Ptac2S) has recently attracted considerable attention due to its strong in vitro and in vivo antiproliferative activity and reduced toxicity. The purpose of this study was to examine the efficacy of Ptac2S treatment in MPM. We employed the ZL55 human mesothelioma cell line in vitro and in a murine xenograft model in vivo, to test the antitumor activity of Ptac2S. Cytotoxicity assays and Western blottings of different apoptosis and survival proteins were thus performed. Ptac2S increases MPM cell death in vitro and in vivo compared with cisplatin. Ptac2S was more efficacious than cisplatin also in inducing apoptosis characterized by: (a) mitochondria depolarization, (b) increase of bax expression and its cytosol-to-mitochondria translocation and decrease of Bcl-2 expression, (c) activation of caspase-7 and -9. Ptac2S activated full-length PKC-δ and generated a PKC-δ fragment. Full-length PKC-δ translocated to the nucleus and membrane, whilst PKC-δ fragment concentrated to mitochondria. Ptac2S was also responsible for the PKCactivation that provoked phosphorylation of p38. Both PKC-δ and PKC-ε inhibition (by PKC-siRNA) reduced the apoptotic death of ZL55 cells. Altogether, our results confirm that Ptac2S is a promising therapeutic agent for malignant mesothelioma, providing a solid starting point for its validation as a suitable candidate for further pharmacological testing.

  7. The phosphatidylinositol 3-kinase inhibitor, wortmannin, inhibits insulin-induced activation of phosphatidylcholine hydrolysis and associated protein kinase C translocation in rat adipocytes.

    OpenAIRE

    Standaert, M L; Avignon, A; Yamada, K; Bandyopadhyay, G; Farese, R V

    1996-01-01

    We questioned whether phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase C (PKC) function as interrelated signalling mechanisms during insulin action in rat adipocytes. Insulin rapidly activated a phospholipase D that hydrolyses phosphatidylcholine (PC), and this activation was accompanied by increases in diacylglycerol and translocative activation of PKC-alpha and PKC-beta in the plasma membrane. Wortmannin, an apparently specific PI 3-kinase inhibitor, inhibited insulin-stimulat...

  8. ARF6 and GASP-1 are post-endocytic sorting proteins selectively involved in the intracellular trafficking of dopamine D2 receptors mediated by GRK and PKC in transfected cells

    Science.gov (United States)

    Cho, DI; Zheng, M; Min, C; Kwon, KJ; Shin, CY; Choi, HK; Kim, KM

    2013-01-01

    Background and Purpose GPCRs undergo both homologous and heterologous regulatory processes in which receptor phosphorylation plays a critical role. The protein kinases responsible for each pathway are well established; however, other molecular details that characterize each pathway remain unclear. In this study, the molecular mechanisms that determine the differences in the functional roles and intracellular trafficking between homologous and PKC-mediated heterologous internalization pathways for the dopamine D2 receptor were investigated. Experimental Approach All of the S/T residues located within the intracellular loops of D2 receptor were mutated, and the residues responsible for GRK- and PKC-mediated internalization were determined in HEK-293 cells and SH-SY5Y cells. The functional role of receptor internalization and the cellular components that determine the post-endocytic fate of internalized D2 receptors were investigated in the transfected cells. Key Results T134, T225/S228/S229 and S325 were involved in PKC-mediated D2 receptor desensitization. S229 and adjacent S/T residues mediated the PKC-dependent internalization of D2 receptors, which induced down-regulation and desensitization. S/T residues within the second intracellular loop and T225 were the major residues involved in GRK-mediated internalization of D2 receptors, which induced receptor resensitization. ARF6 mediated the recycling of D2 receptors internalized in response to agonist stimulation. In contrast, GASP-1 mediated the down-regulation of D2 receptors internalized in a PKC-dependent manner. Conclusions and Implications GRK- and PKC-mediated internalizations of D2 receptors occur through different intracellular trafficking pathways and mediate distinct functional roles. Distinct S/T residues within D2 receptors and different sorting proteins are involved in the dissimilar regulation of D2 receptors by GRK2 and PKC. PMID:23082996

  9. Direct binding and activation of protein kinase C isoforms by steroid hormones.

    LENUS (Irish Health Repository)

    Alzamora, Rodrigo

    2008-10-01

    The non-genomic action of steroid hormones regulates a wide variety of cellular responses including regulation of ion transport, cell proliferation, migration, death and differentiation. In order to achieve such plethora of effects steroid hormones utilize nearly all known signal transduction pathways. One of the key signalling molecules regulating the non-genomic action of steroid hormones is protein kinase C (PKC). It is thought that rapid action of steroids hormones results from the activation of plasma membrane receptors; however, their molecular identity remains elusive. In recent years, an increasing number of studies have pointed at the selective binding and activation of specific PKC isoforms by steroid hormones. This has led to the hypothesis that PKC could act as a receptor as well as a transducer of the non-genomic effects of these hormones. In this review we summarize the current knowledge of the direct binding and activation of PKC by steroid hormones.

  10. L-ARGININE PREVENTS METABOLIC EFFECTS OF HIGH GLUCOSE IN DIABETIC MICE

    OpenAIRE

    West, Matthew B.; Ramana, Kota V.; Kaiserova, Karin; Srivastava, Satish K.; Bhatnagar, Aruni

    2008-01-01

    We tested the hypothesis that activation of the polyol pathway and protein kinase C (PKC) during diabetes is due to loss of NO. Our results show that after 4 weeks of streptozotocin-induced diabetes, treatment with L-arginine restored NO levels and prevented tissue accumulation of sorbitol in mice, which was accompanied by an increase in glutathiolation of aldose reductase. L-arginine treatment decreased superoxide generation in the aorta, total PKC activity and PKC-βII phosphorylation in the...

  11. The roles of MCP-1 and protein kinase C delta activation in human eosinophilic leukemia EoL-1 cells.

    Science.gov (United States)

    Lee, Ji-Sook; Yang, Eun Ju; Kim, In Sik

    2009-12-01

    Idiopathic hypereosinophilc syndrome is a disorder associated with clonally eosinophilic proliferation. The importance of FIP1-like-1-platelet-derived growth factor receptor-alpha (FIP1L1-PDGFRA) in the pathogenesis and classification of HES has been recently reported. In this study, we investigated the contribution of monocyte chemoattractant protein-1 (MCP-1)/CCL2 to chemotactic activity and protein kinase C delta (PKC delta in the human eosinophilic leukemia cell line EoL-1. These cells express CCR2 protein among the CC chemokine receptors (CCR1-5). MCP-1 induces strong migration of EoL-1 cells and the chemotaxis signal in response to MCP-1 involves a G(i)/G(o) protein, phospholipase C (PLC), PKC delta, p38 MAPK and NF-kappaB. MCP-1 activates p38 MAPK via G(i)/G(o) protein, PLC and PKC delta cascade. MCP-1 also induces NF-kappaB translocation and the activation is inhibited by PKC delta activation. The increase in the basal expression and activity of PKC delta in EoL-1 cells, compared to normal eosinophils, inhibits apoptosis in EoL-1 cells. Anti-apoptotic mechanism of PKC delta is related to inhibition of caspase 3 and caspase 9, but not to FIP1L1-PDGFRA. PKC delta functions as an anti-apoptotic molecule, and is involved in EoL-1 cell movement stimulated by MCP-1. This study contributes to an understanding of MCP-1 in eosinophil biology and pathogenic mechanism of eosinophilic disorders.

  12. Influence of cations on activity and distribution of protein kinase C in S49 lymphoma cells

    International Nuclear Information System (INIS)

    Brunton, L.; Watson, M.; Schultz, M.; Trejo, J.; Speizer, L.

    1987-01-01

    In S49 lymphoma cells, the distribution of protein kinase C (PKC) between soluble and membrane fractions can be regulated by the concentration of Ca ++ in the homogenization buffer. When cells are fractionated with 10μM Ca ++ and low Mg ++ (0.3mM), PKC is largely (56%) membrane-bound. Mg ++ inhibits this effect of Ca ++ by 75%; the EC 50 for Mg ++ reducing the translocation induced by 10μM Ca ++ is 1mM, as detected by binding of [ 3 H] phorbol dibutyrate ([ 3 H]PDB). Other divalent cations have different effects. When Cu ++ (1mM) is included in the homogenization buffer, both the enzymic activity of PKC and its capacity to bind [ 3 H]PDB are lost in both the cytosolic and membrane fractions. Cd ++ and Zn ++ (at 1mM) also inhibit the binding of [ 3 H]PDB to PKC in cytosolic fractions. K + , Li + , Co ++ and Mn ++ at 1mM do not mimic these effects. With Ca ++ at 500μM, the EC 50 for inhibition by Cu ++ of [ 3 H]PDB binding and enzymic activity of PKC are 25μM and 75μM, respectively. These effects of Cu ++ are also noticeable when the cation is added to intact S49 cells. The effect of Cu ++ on PKC is only relatively specific: [Cu ++ ] ≥ 100μM inhibits the activity of cyclic AMP-dependent protein kinase in vitro. Knowledge of these effects of heavy metals on PKC may prove helpful in manipulation of the enzyme pharmacologically as well as in determining the role of PKC in the cellular responses to heavy metals

  13. Role of LPAR3, PKC and EGFR in LPA-induced cell migration in oral squamous carcinoma cells

    International Nuclear Information System (INIS)

    Brusevold, Ingvild J; Tveteraas, Ingun H; Aasrum, Monica; Ødegård, John; Sandnes, Dagny L; Christoffersen, Thoralf

    2014-01-01

    Oral squamous cell carcinoma is an aggressive neoplasm with serious morbidity and mortality, which typically spreads through local invasive growth. Lysophosphatidic acid (LPA) is involved in a number of biological processes, and may have a role in cancer cell migration and invasiveness. LPA is present in most tissues and can activate cells through six different LPA receptors (LPAR1-6). Although LPA is predominantly promigratory, some of the receptors may have antimigratory effects in certain cells. The signalling mechanisms of LPA are not fully understood, and in oral carcinoma cells the specific receptors and pathways involved in LPA-stimulated migration are unknown. The oral carcinoma cell lines E10, SCC-9, and D2 were investigated. Cell migration was studied in a scratch wound assay, and invasion was demonstrated in organotypic three dimensional co-cultures. Protein and mRNA expression of LPA receptors was studied with Western blotting and qRT-PCR. Activation of signalling proteins was examined with Western blotting and isoelectric focusing, and signalling mechanisms were further explored using pharmacological agents and siRNA directed at specific receptors and pathways. LPA stimulated cell migration in the two oral carcinoma cell lines E10 and SCC-9, but was slightly inhibitory in D2. The receptor expression profile and the effects of specific pharmacological antagonist and agonists indicated that LPA-stimulated cell migration was mediated through LPAR3 in E10 and SCC-9. Furthermore, in both these cell lines, the stimulation by LPA was dependent on PKC activity. However, while LPA induced transactivation of EGFR and the stimulated migration was blocked by EGFR inhibitors in E10 cells, LPA did not induce EGFR transactivation in SCC-9 cells. In D2 cells, LPA induced EGFR transactivation, but this was associated with slowing of a very high inherent migration rate in these cells. The results demonstrate LPA-stimulated migration in oral carcinoma cells through LPAR3

  14. Atypical PKC-iota Controls Stem Cell Expansion via Regulation of the Notch Pathway

    Directory of Open Access Journals (Sweden)

    In Kyoung Mah

    2015-11-01

    Full Text Available The number of stem/progenitor cells available can profoundly impact tissue homeostasis and the response to injury or disease. Here, we propose that an atypical PKC, Prkci, is a key player in regulating the switch from an expansion to a differentiation/maintenance phase via regulation of Notch, thus linking the polarity pathway with the control of stem cell self-renewal. Prkci is known to influence symmetric cell division in invertebrates; however a definitive role in mammals has not yet emerged. Using a genetic approach, we find that loss of Prkci results in a marked increase in the number of various stem/progenitor cells. The mechanism used likely involves inactivation and symmetric localization of NUMB, leading to the activation of NOTCH1 and its downstream effectors. Inhibition of atypical PKCs may be useful for boosting the production of pluripotent stem cells, multipotent stem cells, or possibly even primordial germ cells by promoting the stem cell/progenitor fate.

  15. Phorbol 12,13-dibutyrate-induced protein kinase C activation triggers sustained contracture in human myometrium in vitro.

    Science.gov (United States)

    Massenavette, Laurence; Paul, Wilène; Corriveau, Stéphanie; Pasquier, Jean-Charles; Rousseau, Éric

    2017-09-01

    Although physiologic transition from rhythmic contractions to uterine retraction postpartum remains a poorly understood process, it has been shown that the latter is essential in the prevention of hemorrhage and its negative consequences. To investigate the transition from oscillatory contractions to tonic contracture in human myometrium after delivery, a mechanism purported to facilitate postpartum hemostasis. Protein kinase C (PKC) plays a key regulatory role in human uterine contractions because it can prevent dephosphorylation of regulatory proteins and sensitize the contractile machinery to low Ca 2+ . Thus, activation of PKC by phorbol 12,13-dibutyrate (PDBu) may act as a strong uterotonic agent. Uterine biopsies were obtained from consenting women undergoing elective caesarian delivery at term without labor (N = 19). Isometric tension measurements were performed on uterine strips (n = 114). The amplitudes and area under the curve of phasic contractions and tonic responses were measured and compared. A total of 1 μM PDBu was added to the isolated organ baths, and maximal tension of the uterine contracture was determined in the absence and presence of either 1 μM of staurosporine, 100 nM nifedipine, or 10 μM cyclopiazonic acid to assess the role of PKC and calcium sensitivity on uterine contractility. On the addition of PDBu on either basal or oxytocin-induced activity, consistent contractures were obtained concomitant with complete inhibition of phasic contractions. After a 30-minute incubation period, the mean amplitude of the PDBu-induced tone represented 65.3% of the amplitude of spontaneous contraction. Staurosporine, a protein kinase inhibitor, induced a 91.9% inhibition of PDBu contractures, a process not affected by nifedipine or cyclopiazonic acid, thus indicating that this mechanism is largely Ca 2+ independent. Pharmacologic activation of PKC leads to a significant contracture of the myometrium. Together, these data suggest that the up

  16. Parathyroid Hormone Activates Phospholipase C (PLC)-Independent Protein Kinase C Signaling Pathway via Protein Kinase A (PKA)-Dependent Mechanism: A New Defined Signaling Route Would Induce Alternative Consideration to Previous Conceptions.

    Science.gov (United States)

    Tong, Guojun; Meng, Yue; Hao, Song; Hu, Shaoyu; He, Youhua; Yan, Wenjuan; Yang, Dehong

    2017-04-20

    BACKGROUND Parathyroid hormone (PTH) is an effective anti-osteoporosis agent, after binding to its receptor PTHR1, several signaling pathways, including cAMP/protein kinase A (PKA) and phospholipase C (PLC)/protein kinase C (PKC), are initiated through G proteins; with the cAMP/PKA pathway as the major pathway. Earlier studies have reported that PTHR1 might also activate PKC via a PLC-independent mechanism, but this pathway remains unclear. MATERIAL AND METHODS In HEK293 cells, cAMP accumulation was measured with ELISA and PKC was measured with fluorescence resonance energy transfer (FRET) analysis using CKAR plasmid. In MC3T3-E1 cells, real-time PCR was performed to examine gene expressions. Then assays for cell apoptosis, cell differentiation, alkaline phosphatase activity, and mineralization were performed. RESULTS The FRET analysis found that PTH(1-34), [G1,R19]PTH(1-34) (GR(1-34), and [G1,R19]PTH(1-28) (GR(1-28) were all activated by PKC. The PKC activation ability of GR(1-28) was blocked by cAMP inhibitor (Rp-cAMP) and rescued with the addition of active PKA-α and PKA-β. The PKC activation ability of GR(1-34) was partially inhibited by Rp-cAMP. In MC3T3-E1 cells, gene expressions of ALP, CITED1, NR4a2, and OSX that was regulated by GR(1-28) were significantly changed by the pan-PKC inhibitor Go6983. After pretreatment with Rp-cAMP, the gene expressions of ALP, CITED1, and OPG were differentially regulated by GR(1-28) or GR(1-34), and the difference was blunted by Go6983. PTH(1-34), GR(1-28), and GR(1-34) significantly decreased early apoptosis and augmented osteoblastic differentiation in accordance with the activities of PKA and PKC. CONCLUSIONS PLC-independent PKC activation induced by PTH could be divided into two potential mechanisms: one was PKA-dependent and associated with PTH(1-28); the other was PKA-independent and associated with PTH(29-34). We also found that PTH could activate PLC-independent PKC via PKA-dependent mechanisms.

  17. Protein kinase C promotes restoration of calcium homeostasis to platelet activating factor-stimulated human neutrophils by inhibition of phospholipase C

    Directory of Open Access Journals (Sweden)

    Anderson Ronald

    2009-10-01

    Full Text Available Abstract Background The role of protein kinase C (PKC in regulating the activity of phospholipase C (PLC in neutrophils activated with the chemoattractant, platelet-activating factor (PAF, 20 and 200 nM, was probed in the current study using the selective PKC inhibitors, GF10903X (0.5 - 1 μM and staurosporine (400 nM. Methods Alterations in cytosolic Ca2+, Ca2+ influx, inositol triphosphate (IP3, and leukotriene B4 production were measured using spectrofluorimetric, radiometric and competitive binding radioreceptor and immunoassay procedures, respectively. Results Activation of the cells with PAF was accompanied by an abrupt increase in cytosolic Ca2+ followed by a gradual decline towards basal levels. Pretreatment of neutrophils with the PKC inhibitors significantly increased IP3 production with associated enhanced Ca2+ release from storage vesicles, prolongation of the peak cytosolic Ca2+ transients, delayed clearance and exaggerated reuptake of the cation, and markedly increased synthesis of LTB4. The alterations in Ca2+ fluxes observed with the PKC inhibitors were significantly attenuated by U73122, a PLC inhibitor, as well as by cyclic AMP-mediated upregulation of the Ca2+-resequestering endomembrane ATPase. Taken together, these observations are compatible with a mechanism whereby PKC negatively modulates the activity of PLC, with consequent suppression of IP3 production and down-regulation of Ca2+ mediated pro-inflammatory responses of PAF-activated neutrophils. Conclusion Although generally considered to initiate and/or amplify intracellular signalling cascades which activate and sustain the pro-inflammatory activities of neutrophils and other cell types, the findings of the current study have identified a potentially important physiological, anti-inflammatory function for PKC, at least in neutrophils.

  18. PKC in motorneurons underlies self-learning, a form of motor learning in Drosophila

    Directory of Open Access Journals (Sweden)

    Julien Colomb

    2016-04-01

    Full Text Available Tethering a fly for stationary flight allows for exquisite control of its sensory input, such as visual or olfactory stimuli or a punishing infrared laser beam. A torque meter measures the turning attempts of the tethered fly around its vertical body axis. By punishing, say, left turning attempts (in a homogeneous environment, one can train a fly to restrict its behaviour to right turning attempts. It was recently discovered that this form of operant conditioning (called operant self-learning, may constitute a form of motor learning in Drosophila. Previous work had shown that Protein Kinase C (PKC and the transcription factor dFoxP were specifically involved in self-learning, but not in other forms of learning. These molecules are specifically involved in various forms of motor learning in other animals, such as compulsive biting in Aplysia, song-learning in birds, procedural learning in mice or language acquisition in humans. Here we describe our efforts to decipher which PKC gene is involved in self-learning in Drosophila. We also provide evidence that motorneurons may be one part of the neuronal network modified during self-learning experiments. The collected evidence is reminiscent of one of the simplest, clinically relevant forms of motor learning in humans, operant reflex conditioning, which also relies on motorneuron plasticity.

  19. Neural cell adhesion molecule-stimulated neurite outgrowth depends on activation of protein kinase C and the Ras-mitogen-activated protein kinase pathway

    DEFF Research Database (Denmark)

    Kolkova, K; Novitskaya, V; Pedersen, N

    2000-01-01

    , inhibitors of the nonreceptor tyrosine kinase p59(fyn), PLC, PKC and MEK and an activator of PKC, phorbol-12-myristate-13-acetate (PMA). MEK2 transfection rescued cells treated with all inhibitors. The same was found for PMA treatment, except when cells concomitantly were treated with the MEK inhibitor....... Arachidonic acid rescued cells treated with antibodies to the FGF receptor or the PLC inhibitor, but not cells in which the activity of PKC, p59(fyn), FAK, Ras, or MEK was inhibited. Interaction of NCAM with a synthetic NCAM peptide ligand, known to induce neurite outgrowth, was shown to stimulate...... phosphorylation of the MAP kinases extracellular signal-regulated kinases ERK1 and ERK2. The MAP kinase activation was sustained, because ERK1 and ERK2 were phosphorylated in PC12-E2 cells and primary hippocampal neurons even after 24 hr of cultivation on NCAM-expressing fibroblasts. Based on these results, we...

  20. Visual Snapshots of Intracellular Kinase Activity At The Onset of Mitosis

    Science.gov (United States)

    Dai, Zhaohua; Dulyaninova, Natalya G.; Kumar, Sanjai; Bresnick, Anne R.; Lawrence, David S.

    2007-01-01

    Summary Visual snapshots of intracellular kinase activity can be acquired with exquisite temporal control using a light-activatable (caged) sensor, thereby providing a means to interrogate enzymatic activity at any point during the cell division cycle. Robust protein kinase activity transpires just prior to, but not immediately following, nuclear envelope breakdown (NEB). Furthermore, kinase activity is required for progression from prophase into metaphase. Finally, the application of selective protein kinase C (PKC) inhibitors, in combination with the caged sensor, correlates the action of the PKC β isoform with subsequent NEB. PMID:18022564

  1. AMP-activated protein kinase downregulates Kv7.1 cell surface expression

    DEFF Research Database (Denmark)

    Andersen, Martin N; Krzystanek, Katarzyna; Jespersen, Thomas

    2012-01-01

    in response to polarization of the epithelial Madin-Darby canine kidney (MDCK) cell line and that this was mediated by activation of protein kinase C (PKC). In this study, the pathway downstream of PKC, which leads to internalization of Kv7.1 upon cell polarization, is elucidated. We show by confocal...... microscopy that Kv7.1 is endocytosed upon initiation of the polarization process and sent for degradation by the lysosomal pathway. The internalization could be mimicked by pharmacological activation of the AMP-activated protein kinase (AMPK) using three different AMPK activators. We demonstrate...

  2. Correlation between protein kinase C alpha activity and membrane phase behavior.

    Science.gov (United States)

    Micol, V; Sánchez-Piñera, P; Villalaín, J; de Godos, A; Gómez-Fernández, J C

    1999-02-01

    Lipid activation of protein kinase C alpha (PKC alpha) was studied by using a model mixture containing 1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1, 2-dimyristoyl-sn-glycero-3-phosphoserine (DMPS), and 1, 2-dimyristoyl-sn-glycerol (1,2-DMG). This lipid mixture was physically characterized by differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR), and 31P-nuclear magnetic resonance (31P-NMR). Based on these techniques, a phase diagram was constructed by keeping a constant DMPC/DMPS molar ratio of 4:1 and changing the concentration of 1,2-DMG. This phase diagram displayed three regions and two compounds: compound 1 (C1), with 45 mol% 1,2-DMG, and compound 2 (C2), with 60 mol% 1,2-DMG. When the phase diagram was elaborated in the presence of Ca2+ and Mg2+, at concentrations similar to those used in the PKC alpha activity assay, the boundaries between the regions changed slightly and C1 had 35 mol% 1,2-DMG. The activity of PKC alpha was studied at several temperatures and at different concentrations of 1,2-DMG, with a maximum of activity reached at 30 mol% 1,2-DMG and lower values at higher concentrations. In the presence of Ca2+ and Mg2+, maximum PKC alpha activity occurred at concentrations of 1,2-DMG that were close to the boundary in the phase diagram between region 1, where compound C1 and the pure phospholipid coexisted in the gel phase, and region 2, where compounds C1 and C2 coexisted. These results suggest that the membrane structure corresponding to a mixture of 1,2-DMG/phospholipid complex and free phospholipid is better able to support the activity of PKC alpha than the 1,2-DMG/phospholipid complex alone.

  3. Domain-specific phosphomimetic mutation allows dissection of different protein kinase C (PKC) isotype-triggered activities of the RNA binding protein HuR.

    Science.gov (United States)

    Schulz, Sebastian; Doller, Anke; Pendini, Nicole R; Wilce, Jacqueline A; Pfeilschifter, Josef; Eberhardt, Wolfgang

    2013-12-01

    The ubiquitous mRNA binding protein human antigen R (HuR) participates in the post-transcriptional regulation of many AU-rich element (ARE)-bearing mRNAs. Previously, by using in vitro kinase assay, we have identified serines (Ser) 158, 221 and 318 as targets of protein kinase C (PKC)-triggered phosphorylation. In this study, we tested whether GFP- or GST-tagged HuR constructs bearing a phosphomimetic Ser (S)-to-Asp (D) substitution at the different PKC target sites, would affect different HuR functions including HuR nucleo-cytoplasmic redistribution and binding to different types of ARE-containing mRNAs. The phosphomimetic GFP-tagged HuR protein bearing a phosphomimetic substitution in the hinge region of HuR (HuR-S221D) showed an increased cytoplasmic abundance when compared to wild-type HuR. Conversely, data from in vitro kinase assay and electrophoretic mobility shift assay (EMSA), implicates that phosphorylation at Ser 221 is not relevant for mRNA binding of HuR. Quantification of in vitro binding affinities of GST-tagged wild-type HuR and corresponding HuR proteins bearing a phosphomimetic substitution in either RRM2 (HuR-S158D) or in RRM3 (HuR-S318D) by microscale thermophoresis (MST) indicates a specific binding of wild-type HuR to type I, II or type III-ARE-oligonucleotides in the high nanomolar range. Interestingly, phosphomimetic mutation at position 158 or 318 had a negative influence on HuR binding to type I- and type II-ARE-mRNAs whereas it significantly enhanced HuR affinity to a type III-ARE substrate. Our data suggest that differential phosphorylation of HuR by PKCs at different HuR domains coordinates subcellular HuR distribution and leads to a preferential binding to U-rich bearing target mRNA. © 2013.

  4. A PKC-dependent recruitment of MMP-2 controls semaphorin-3A growth-promoting effect in cortical dendrites.

    Directory of Open Access Journals (Sweden)

    Bertrand Gonthier

    Full Text Available There is increasing evidence for a crucial role of proteases and metalloproteinases during axon growth and guidance. In this context, we recently described a functional link between the chemoattractive Sema3C and Matrix metalloproteinase 3 (MMP3. Here, we provide data demonstrating the involvement of MMP-2 to trigger the growth-promoting effect of Sema3A in cortical dendrites. The in situ analysis of MMP-2 expression and activity is consistent with a functional growth assay demonstrating in vitro that the pharmacological inhibition of MMP-2 reduces the growth of cortical dendrites in response to Sema3A. Hence, our results suggest that the selective recruitment and activation of MMP-2 in response to Sema3A requires a PKC alpha dependent mechanism. Altogether, we provide a second set of data supporting MMPs as effectors of the growth-promoting effects of semaphorins, and we identify the potential signalling pathway involved.

  5. Mutation of the protein kinase C site in borna disease virus phosphoprotein abrogates viral interference with neuronal signaling and restores normal synaptic activity.

    Directory of Open Access Journals (Sweden)

    Christine M A Prat

    2009-05-01

    Full Text Available Understanding the pathogenesis of infection by neurotropic viruses represents a major challenge and may improve our knowledge of many human neurological diseases for which viruses are thought to play a role. Borna disease virus (BDV represents an attractive model system to analyze the molecular mechanisms whereby a virus can persist in the central nervous system (CNS and lead to altered brain function, in the absence of overt cytolysis or inflammation. Recently, we showed that BDV selectively impairs neuronal plasticity through interfering with protein kinase C (PKC-dependent signaling in neurons. Here, we tested the hypothesis that BDV phosphoprotein (P may serve as a PKC decoy substrate when expressed in neurons, resulting in an interference with PKC-dependent signaling and impaired neuronal activity. By using a recombinant BDV with mutated PKC phosphorylation site on P, we demonstrate the central role of this protein in BDV pathogenesis. We first showed that the kinetics of dissemination of this recombinant virus was strongly delayed, suggesting that phosphorylation of P by PKC is required for optimal viral spread in neurons. Moreover, neurons infected with this mutant virus exhibited a normal pattern of phosphorylation of the PKC endogenous substrates MARCKS and SNAP-25. Finally, activity-dependent modulation of synaptic activity was restored, as assessed by measuring calcium dynamics in response to depolarization and the electrical properties of neuronal networks grown on microelectrode arrays. Therefore, preventing P phosphorylation by PKC abolishes viral interference with neuronal activity in response to stimulation. Our findings illustrate a novel example of viral interference with a differentiated neuronal function, mainly through competition with the PKC signaling pathway. In addition, we provide the first evidence that a viral protein can specifically interfere with stimulus-induced synaptic plasticity in neurons.

  6. PKC/CREB pathway mediates the expressions of GABAA receptor subunits in cultured hippocampal neurons after low-Mg2+ solution treatment.

    Science.gov (United States)

    Wu, Guofeng; Yu, Jinpeng; Wang, Likun; Ren, Siying; Zhang, Yixia

    2018-02-01

    To investigate the potential effects of the PKC/CREB pathway on the expressions of GABA A receptor subunits α1, γ2, and δ in cultured hippocampal neurons using a model of epilepsy that employed conditions of low magnesium (Mg 2+ ). A total of 108 embryonic rats at the age of 18 embryonic days (E18)prepared from adult female SD rats were used as experimental subjects. Primary rat hippocampal cultures were prepared from the embryonic 18 days rats. The cultured hippocampal neurons were then treated with artificial cerebrospinal fluid containing low Mg 2+ solutions to generate a low Mg 2+ model of epilepsy. The low Mg 2+ stimulation lasted for 3 h and then returned to in maintenance medium for 20 h. The changes of the GABA A receptor subunit α1, γ2, δ were observed by blocking or activating the function of the CREB. The quantification of the GABA A receptor subunit α1, γ2, δ and the CREB were determined by a qRT-PCR and a Western blot method. After the neurons were exposed to a low-Mg 2+ solution for 3 h, GABA A receptor mRNA expression markedly increased compared to the control, and then gradually decreased. In contrast, CREB mRNA levels exhibited a dramatic down-regulation 3 h after terminating low-Mg 2+ treatment, and then peaked at 9 h. Western blot analyses verified that staurosporine suppressed CREB phosphorylation (p-CREB). The mRNA expression of GABA A receptor subunit α1 increased only in the presence of staurosporine, whereas the expressions of subunits γ2 and δ significantly increased in the presence of either KG-501 or staurosporine. Furthermore, phorbol 12-myristate 13-acetate (PMA) decreased the expressions of GABA A subunits α1, γ2, and δ when administered alone. However, the administration of either KG-501 or staurosporine reversed the inhibitory effects of PMA. The PKC/CREB pathway may negatively regulate the expressions of GABA A receptor subunits α1, γ2, and δ in cultured hippocampal neurons in low Mg 2+ model of

  7. Insulin stimulates phospholipase D-dependent phosphatidylcholine hydrolysis, Rho translocation, de novo phospholipid synthesis, and diacylglycerol/protein kinase C signaling in L6 myotubes.

    Science.gov (United States)

    Standaert, M L; Bandyopadhyay, G; Zhou, X; Galloway, L; Farese, R V

    1996-07-01

    Previous studies have provided conflicting findings on whether insulin activates certain, potentially important, phospholipid signaling systems in skeletal muscle preparations. In particular, insulin effects on the hydrolysis of phosphatidylcholine (PC) and subsequent activation of protein kinase C (PKC) have not been apparent in some studies. Presently, we examined insulin effects on phospholipid signaling systems, diacylglycerol (DAG) production, and PKC translocation/activation in L6 myotubes. We found that insulin provoked rapid increases in phospholipase D (PLD)-dependent hydrolysis of PC, as evidenced by increases in choline release and phosphatidylethanol production in cells incubated in the presence of ethanol. In association with PC-PLD activation, Rho, a small G protein that is known to activate PC-PLD activation, translocated from the cytosol to the membrane fraction in response to insulin treatment. PC-PLD activation was also accompanied by increases in total DAG production and increases in the translocation of both PKC enzyme activity and DAG-sensitive PKC-alpha, -beta, -delta, and -epsilon from the cytosol to the membrane fraction. A potential role for PKC or a related protein kinase in insulin action was suggested by the finding that RO 31-8220 inhibited both PKC enzyme activity and insulin-stimulated [3H]2-deoxyglucose uptake. Our findings provide the first evidence that insulin stimulates Rho translocation and activates PC-PLD in L6 skeletal muscle cells. Moreover, this signaling system appears to lead to increases in DAG/PKC signaling, which, along with other related signaling factors, may regulate certain metabolic processes, such as glucose transport, in these cells.

  8. Phosphorylation of protein kinase C sites Ser42/44 decreases Ca2+-sensitivity and blunts enhanced length-dependent activation in response to protein kinase A in human cardiomyocytes

    NARCIS (Netherlands)

    Wijnker, P.J.M.; Sequeira Oliveira, V.; Witjas-Paalberends, E.R.; Foster, D.B.; dos Remedios, C.G.; Murphy, A.M.; Stienen, G.J.M.; van der Velden, J.

    2014-01-01

    Protein kinase C (PKC)-mediated phosphorylation of troponin I (cTnI) at Ser42/44 is increased in heart failure. While studies in rodents demonstrated that PKC-mediated Ser42/44 phosphorylation decreases maximal force and ATPase activity, PKC incubation of human cardiomyocytes did not affect maximal

  9. Stabilization and activation of p53 are regulated independently by different phosphorylation events

    Science.gov (United States)

    Chernov, Mikhail V.; Ramana, Chilakamarti V.; Adler, Victor V.; Stark, George R.

    1998-01-01

    Treatment of mouse or human cells with the protein kinase C (PKC) inhibitors H7 or bisindolylmaleimide I induced an increase in the lifetime of p53, leading to its accumulation. In inhibitor-treated cells, p53 translocated to the nuclei and bound to DNA but was not competent to induce transcription. However, transactivation could be induced by subsequent DNA damage. Phorbol ester, a potent activator of PKC, significantly inhibited the accumulation of p53 after DNA damage. Therefore, constitutive PKC-dependent phosphorylation of p53 itself, or of a protein that interacts with p53, is required for the rapid degradation of p53 in untreated cells. Furthermore, an increase in the lifetime of p53 is not accompanied necessarily by its activation. Treatment with the PKC inhibitors decreased the overall level of p53 phosphorylation but led to the appearance of a phosphopeptide not seen in tryptic digests of p53 from untreated cells. Therefore, the lifetime and activities of p53 are likely to be regulated by distinct alterations of the phosphorylation pattern of p53, probably caused by the actions of different kinases. PMID:9482877

  10. Amplification of the uvrA gene product of Escherichia coli to 7% of cellular protein by linkage to the p/sub L/ promoter of pKC30

    International Nuclear Information System (INIS)

    Yoakum, G.H.; Yeung, A.T.; Mattes, W.B.; Grossman, L.

    1982-01-01

    Researchers have constructed a hybrid pKC30-uvrA plasmid (pGHY5003) in which transcription of the uvrA gene can be induced under p/sub L/ control to amplify the uvrA gene product to 7% of cellular protein. To construct pGHY5003, researchers developed a genetic selection using the basal level of expression (30 0 C) from p/sub L/ in thermosensitive cI857 lysogens to isolate appropriately tailored repair genes inserted at the Hpa I site of pKC30 from recombinant DNA mixtures with a variety of products. In addition, a post-uv-irradiation radiolabeling method was adapted to screen inserts for temperature-inducible polypeptide synthesis directed by transcription under p/sub L/ control rapidly. This should prove generally useful for isolating genes inserted at the Hpa I site of plasmid pKC30 with the following characteristics: (1) genetically functional hybrid plasmids selected from a large population of exonucleolytically tailored fragments ligated into Hpa I of pKC30 and (2) production of high-level amplification for the gene product of interest by screening for post-uv-irradiation temperature inducibility of polypeptides synthesized from hybrid plasmids. The level of amplification obtained for the uvrA gene product from pGHY5003 is approximately 10,000-fold higher than estimates of the level of uvrA protein in logarithmic phase Escherichia coli

  11. Staurosporine potentiates platelet activating factor stimulated phospholipase C activity in rabbit platelets but does not block desensitization by platelet activating factor

    International Nuclear Information System (INIS)

    Morrison, W.J.; Dhar, A.; Shukla, S.D.

    1989-01-01

    The possible involvement of protein kinase C activation in regulating PAF-stimulated PLC activity was studied in rabbit platelets. PAF stimulated incorporation of 32 P into proteins and caused [ 3 H]InsP 3 levels to increase about 260% of control. These responses were compared after platelets were pretreated with either PAF, phorbol 12-myristate 13-acetate (PMA) or staurosporine and also after pretreatments with staurosporine followed by PAF or PMA. Pretreating platelets with staurosporine potentiated PAF-stimulated [ 3 H]InsP 3 levels by 54% and blocked protein phosphorylation. Pretreatments with PAF and PMA caused PAF-stimulated [ 3 H]InsP 3 levels to decrease to 115 and 136%, respectively. Staurosporine pretreatment blocked the decrease caused by the PMA pretreatment but not that by PAF. This study demonstrates that PAF-stimulated PLC activity is negatively affected by protein kinase C (PKC) activation and that inhibition of PKC activity did not prevent desensitization of PLC by PAF

  12. Effects of protein kinase C activators on phorbol ester-sensitive and -resistant EL4 thymoma cells.

    Science.gov (United States)

    Sansbury, H M; Wisehart-Johnson, A E; Qi, C; Fulwood, S; Meier, K E

    1997-09-01

    Phorbol ester-sensitive EL4 murine thymoma cells respond to phorbol 12-myristate 13-acetate with activation of ERK mitogen-activated protein kinases, synthesis of interleukin-2, and death, whereas phorbol ester-resistant variants of this cell line do not exhibit these responses. Additional aspects of the resistant phenotype were examined, using a newly-established resistant cell line. Phorbol ester induced morphological changes, ERK activation, calcium-dependent activation of the c-Jun N-terminal kinase (JNK), interleukin-2 synthesis, and growth inhibition in sensitive but not resistant cells. A series of protein kinase C activators caused membrane translocation of protein kinase C's (PKCs) alpha, eta, and theta in both cell lines. While PKC eta was expressed at higher levels in sensitive than in resistant cells, overexpression of PKC eta did not restore phorbol ester-induced ERK activation to resistant cells. In sensitive cells, PKC activators had similar effects on cell viability and ERK activation, but differed in their abilities to induce JNK activation and interleukin-2 synthesis. PD 098059, an inhibitor of the mitogen activated protein (MAP)/ERK kinase kinase MEK, partially inhibited ERK activation and completely blocked phorbol ester-induced cell death in sensitive cells. Thus MEK and/or ERK activation, but not JNK activation or interleukin-2 synthesis, appears to be required for phorbol ester-induced toxicity. Alterations in phorbol ester response pathways, rather than altered expression of PKC isoforms, appear to confer phorbol ester resistance to EL4 cells.

  13. Different protein kinase C isoenzymes mediate inhibition of cardiac rapidly activating delayed rectifier K+ current by different G-protein coupled receptors.

    Science.gov (United States)

    Liu, Xueli; Wang, Yuhong; Zhang, Hua; Shen, Li; Xu, Yanfang

    2017-12-01

    Elevated angiotensin II (Ang II) and sympathetic activity contributes to a high risk of ventricular arrhythmias in heart disease. The rapidly activating delayed rectifier K + current (I Kr ) carried by the hERG channels plays a critical role in cardiac repolarization, and decreased I Kr is involved in increased cardiac arrhythmogenicity. Stimulation of α 1A -adrenoreceptors or angiotensin II AT 1 receptors is known to inhibit I Kr via PKC. Here, we have identified the PKC isoenzymes mediating the inhibition of I Kr by activation of these two different GPCRs. The whole-cell patch-clamp technique was used to record I Kr in guinea pig cardiomyocytes and HEK293 cells co-transfected with hERG and α 1A -adrenoreceptor or AT 1 receptor genes. A broad spectrum PKC inhibitor Gö6983 (not inhibiting PKCε), a selective cPKC inhibitor Gö6976 and a PKCα-specific inhibitor peptide, blocked the inhibition of I Kr by the α 1A -adrenoreceptor agonist A61603. However, these inhibitors did not affect the reduction of I Kr by activation of AT 1 receptors, whereas the PKCε-selective inhibitor peptide did block the effect. The effects of angiotensin II and the PKCε activator peptide were inhibited in mutant hERG channels in which 17 of the 18 PKC phosphorylation sites were deleted, whereas a deletion of the N-terminus of the hERG channels selectively prevented the inhibition elicited by A61603 and the cPKC activator peptide. Our results indicated that inhibition of I Kr by activation of α 1A -adrenoreceptors or AT 1 receptors were mediated by PKCα and PKCε isoforms respectively, through different molecular mechanisms. © 2017 The British Pharmacological Society.

  14. Activation of cAMP-dependent signaling induces oxidative modification of the cardiac Na+-K+ pump and inhibits its activity.

    Science.gov (United States)

    White, Caroline N; Liu, Chia-Chi; Garcia, Alvaro; Hamilton, Elisha J; Chia, Karin K M; Figtree, Gemma A; Rasmussen, Helge H

    2010-04-30

    Cellular signaling can inhibit the membrane Na(+)-K(+) pump via protein kinase C (PKC)-dependent activation of NADPH oxidase and a downstream oxidative modification, glutathionylation, of the beta(1) subunit of the pump alpha/beta heterodimer. It is firmly established that cAMP-dependent signaling also regulates the pump, and we have now examined the hypothesis that such regulation can be mediated by glutathionylation. Exposure of rabbit cardiac myocytes to the adenylyl cyclase activator forskolin increased the co-immunoprecipitation of NADPH oxidase subunits p47(phox) and p22(phox), required for its activation, and increased superoxide-sensitive fluorescence. Forskolin also increased glutathionylation of the Na(+)-K(+) pump beta(1) subunit and decreased its co-immunoprecipitation with the alpha(1) subunit, findings similar to those already established for PKC-dependent signaling. The decrease in co-immunoprecipitation indicates a decrease in the alpha(1)/beta(1) subunit interaction known to be critical for pump function. In agreement with this, forskolin decreased ouabain-sensitive electrogenic Na(+)-K(+) pump current (arising from the 3:2 Na(+):K(+) exchange ratio) of voltage-clamped, internally perfused myocytes. The decrease was abolished by the inclusion of superoxide dismutase, the inhibitory peptide for the epsilon-isoform of PKC or inhibitory peptide for NADPH oxidase in patch pipette solutions that perfuse the intracellular compartment. Pump inhibition was also abolished by inhibitors of protein kinase A and phospholipase C. We conclude that cAMP- and PKC-dependent inhibition of the cardiac Na(+)-K(+) pump occurs via a shared downstream oxidative signaling pathway involving NADPH oxidase activation and glutathionylation of the pump beta(1) subunit.

  15. Protease-activated receptor-2 activation exaggerates TRPV1-mediated cough in guinea pigs.

    Science.gov (United States)

    Gatti, Raffaele; Andre, Eunice; Amadesi, Silvia; Dinh, Thai Q; Fischer, Axel; Bunnett, Nigel W; Harrison, Selena; Geppetti, Pierangelo; Trevisani, Marcello

    2006-08-01

    A lowered threshold to the cough response frequently accompanies chronic airway inflammatory conditions. However, the mechanism(s) that from chronic inflammation results in a lowered cough threshold is poorly understood. Irritant agents, including capsaicin, resiniferatoxin, and citric acid, elicit cough in humans and in experimental animals through the activation of the transient receptor potential vanilloid 1 (TRPV1). Protease-activated receptor-2 (PAR2) activation plays a role in inflammation and sensitizes TRPV1 in cultured sensory neurons by a PKC-dependent pathway. Here, we have investigated whether PAR2 activation exaggerates TRPV1-dependent cough in guinea pigs and whether protein kinases are involved in the PAR2-induced cough modulation. Aerosolized PAR2 agonists (PAR2-activating peptide and trypsin) did not produce any cough per se. However, they potentiated citric acid- and resiniferatoxin-induced cough, an effect that was completely prevented by the TRPV1 receptor antagonist capsazepine. In contrast, cough induced by hypertonic saline, a stimulus that provokes cough in a TRPV1-independent manner, was not modified by aerosolized PAR2 agonists. The PKC inhibitor GF-109203X, the PKA inhibitor H-89, and the cyclooxygenase inhibitor indomethacin did not affect cough induced by TRPV1 agonists, but abated the exaggeration of this response produced by PAR2 agonists. In conclusion, PAR2 stimulation exaggerates TRPV1-dependent cough by activation of diverse mechanism(s), including PKC, PKA, and prostanoid release. PAR2 activation, by sensitizing TRPV1 in primary sensory neurons, may play a role in the exaggerated cough observed in certain airways inflammatory diseases such as asthma and chronic obstructive pulmonary disease.

  16. TOTAL 2003 activities report in brief

    International Nuclear Information System (INIS)

    2004-01-01

    This activities report presents the activities of the petroleum industry Group Total for the year 2003. The following topics are detailed: the corporate social responsibility with the environment stewardship, the energy future management, the safety enhancing, the human resources and the ethics and local development; the shareholder information with the Total share and the share-holding structure; the activities with informations on the Group, the main events, the upstream exploration and production,, the downstream refining, marketing, trading and shipping, the chemicals with overview 2003, base chemical and polymers, performance and specialities. (A.L.B.)

  17. The roles of serine protease, intracellular and extracellular phenoloxidase in activation of prophenoloxidase system, and characterization of phenoloxidase from shrimp haemocytes induced by lipopolysaccharide or dopamine

    Science.gov (United States)

    Xie, Peng; Pan, Luqing; Xu, Wujie; Yue, Feng

    2013-09-01

    We investigated the effects of lipopolysaccharide (LPS) and dopamine (DA) on the activation of the prophenoloxidase (proPO) system of Litopenaeus vannamei. LPS and DA were shown with a negative dose-dependent effect on hyalne cells (HC), semi-granular cells (SGC), large granular cells (LGC), and total haemocyte count (THC). When haemocytes were treated with LPS or DA, serine proteinase activity and intracellular phenoloxidase (PO) activity were significantly reduced, but extracellular PO activity increased significantly. These findings indicated that the reduction in haemocyte counts was mainly because of the degranulation and activation of the proPO system from semi-granule and large granule cells. The PKC inhibitor, chelerythrine, and the TPK inhibitor, genistein, had an inhibitory effect on extracellular PO activity, while serine proteinase and intracellular PO activity increased. This suggests that the LPS and DA induce the activation of proPO in haemocytes via PKC and TPK-related signaling pathways, but serine proteinase may be activated only by PKC, as the genistein effects were not statistically significant. Electrophoresis analysis revealed that POs induced by LPS or DA have the same molecular mass and high diphenolase activity. Two PO bands at 526 kDa and 272 kDa were observed in PAGE, while in the haemocyte lysate supernatant (HLS), only a 272-kDa band was observed. This band was resolved after SDS-PAGE under non-reducing and reducing conditions into two groups of POs, 166 kDa and 126 kDa, and 78.1 kDa and 73.6 kDa, respectively, suggesting that PO in L. vannamei is an oligomer, which may have different compositions intra- and extracellularly.

  18. Involvement of atypical protein kinase C in the regulation of cardiac glucose and long-chain fatty acid uptake

    Directory of Open Access Journals (Sweden)

    Daphna D.J. Habets

    2012-09-01

    Full Text Available Aim: The signaling pathways involved in the regulation of cardiac GLUT4 translocation/glucose uptake and CD36 translocation/ long-chain fatty acid uptake are not fully understood. We compared in heart/muscle-specific PKC-λ knockout mice the roles of atypical PKCs (PKC-ζ and PKC-λ in regulating cardiac glucose and fatty acid uptake. Results: Neither insulin-stimulated nor AMPK-mediated glucose and fatty acid uptake were inhibited upon genetic PKC-λ ablation in cardiomyocytes. In contrast, myristoylated PKC-ζ pseudosubstrate inhibited both insulin-stimulated and AMPK-mediated glucose and fatty acid uptake by >80% in both wild-type and PKC-λ-knockout cardiomyocytes. In PKC-λ knockout cardiomyocytes, PKC-ζ is the sole remaining atypical PKC isoform, and its expression level is not different from wild-type cardiomyocytes, in which it contributes to 29% and 17% of total atypical PKC expression and phosphorylation, respectively. Conclusion: Taken together, atypical PKCs are necessary for insulin-stimulated and AMPK-mediated glucose uptake into the heart, as well as for insulin-stimulated and AMPK-mediated fatty acid uptake. However, the residual PKCactivity in PKC-λ-knockout cardiomyocytes is sufficient to allow optimal stimulation of glucose and fatty acid uptake, indicating that atypical PKCs are necessary but not rate-limiting in the regulation of cardiac substrate uptake and that PKC-λ and PKC-ζ have interchangeable functions in these processes.

  19. ω-3 and ω-6 Fatty Acids Modulate Conventional and Atypical Protein Kinase C Activities in a Brain Fatty Acid Binding Protein Dependent Manner in Glioblastoma Multiforme

    Directory of Open Access Journals (Sweden)

    Marwa E. Elsherbiny

    2018-04-01

    Full Text Available Glioblastoma multiforme (GBM is a highly infiltrative brain cancer with a dismal prognosis. High levels of brain fatty acid binding protein (B-FABP are associated with increased migration/infiltration in GBM cells, with a high ratio of arachidonic acid (AA to docosahexaenoic acid (DHA driving B-FABP-mediated migration. Since several protein kinase Cs (PKCs are overexpressed in GBM and linked to migration, we explored a possible relationship between B-FABP and levels/activity of different PKCs, as a function of AA and DHA supplementation. We report that ectopic expression of B-FABP in U87 cells alters the levels of several PKCs, particularly PKCζ. Upon analysis of PKCζ RNA levels in a panel of GBM cell lines and patient-derived GBM neurospheres, we observed a trend towards moderate positive correlation (r = 0.624, p = 0.054 between B-FABP and PKCζ RNA levels. Analysis of PKC activity in U87 GBM cells revealed decreased typical PKC activity (23.4% in B-FABP-expressing cells compared with nonexpressing cells, with no difference in novel and atypical PKC activities. AA and DHA modulated both conventional and atypical PKC activities in a B-FABP-dependent manner, but had no effect on novel PKC activity. These results suggest that conventional and atypical PKCs are potential downstream effectors of B-FABP/fatty acid-mediated alterations in GBM growth properties.

  20. Physical activity of elderly patients after total hip arthroplasty.

    Science.gov (United States)

    Cukras, Zbigniew; Praczko, Katarzyna; Kostka, Tomasz; Jegier, Anna

    2007-01-01

    Total hip arthroplasty (THA) is the most common method of treatment of severe hip osteoarthritis. There is little data concerning the physical activity of total hip arthroplasty patients in Poland and investigations to explore this area are useful. The aim of the study was to describe the post-operative physical activity of total hip arthroplasty patients. A total of 146 adult people were examined, among which 28 men and 41 women had undergone total hip arthroplasty due to primary osteoarthritis of the hip, while another 32 men and 41 women matched for age who had not undergone hip surgery for osteoarthritis served as controls. The physical activity of study participants was assessed with the 7-Day Physical Activity Recall Questionnaire. All participants were also asked about the type and amount of physical activity they engaged in to maintain good health. Physical activity measured as the total amount of calories expended through physical activity per week was similar in the post-THA patients compared to the controls. The only differences were a smaller amount of calories expended during low-intensity physical activity by men after total hip arthroplasty compared to men who had not undergone surgery for osteoarthritis and a smaller amount of calories expended through high-intensity physical activity by women after total hip arthroplasty compared to female controls. The kinds of recreational physical activity most commonly practised by patients a mean of two years after total hip arthroplasty were marching, bicycling and general body conditioning exercises (usually the continuation of exercises recommended during post-operative rehabilitation). The percentage of post-THA patients undertaking physical activity for the prevention of non-communicable diseases was low. Physical activity should be more effectively encouraged in patients after total hip arthroplasty.

  1. Calcitonin gene-related peptide promotes the wound healing of human bronchial epithelial cells via PKC and MAPK pathways.

    Science.gov (United States)

    Zhou, Yong; Zhang, Min; Sun, Guo-Ying; Liu, Yong-Ping; Ran, Wen-Zhuo; Peng, Li; Guan, Cha-Xiang

    2013-06-10

    Calcitonin gene-related peptide (CGRP) is a 37-amino acid neuropeptide derived from the calcitonin gene. CGRP is widely distributed in the central and peripheral neuronal systems. In the lung, CGRP could modulate dendritic cell function, stimulate proliferation of alveolar epithelial cells and mediate lung injury in mice. In this study, we investigated the effect of CGRP on the wound healing of human bronchial epithelial cells (HBECs) in vitro. The results showed that CGRP accelerated the recovery of wound area of monolayer HBECs in a dose-dependent manner. CGRP inhibited the lipopolysaccharide-induced apoptosis in HBECs. The percentage of S phase and G2/M phase was increased in HBECs after CGRP treatment. CGRP upregulated the expression of Ki67 in a dose-dependent manner. Some pathway inhibitors were used to investigate the signal pathway in which CGRP was involved. We found out that PKC pathway inhibitor (H-7) and MAPK pathway inhibitor (PD98059) could partially attenuate the effect of CGRP, which indicated that CGRP might promote the wound healing of HBECs via PKC and/or MAPK dependent pathway by accelerating migration and proliferation, and inhibiting apoptosis. Copyright © 2013 Elsevier B.V. All rights reserved.

  2. Agonist-mediated activation of Bombyx mori diapause hormone receptor signals to extracellular signal-regulated kinases 1 and 2 through Gq-PLC-PKC-dependent cascade.

    Science.gov (United States)

    Jiang, Xue; Yang, Jingwen; Shen, Zhangfei; Chen, Yajie; Shi, Liangen; Zhou, Naiming

    2016-08-01

    Diapause is a developmental strategy adopted by insects to survive in challenging environments such as the low temperatures of a winter. This unique process is regulated by diapause hormone (DH), which is a neuropeptide hormone that induces egg diapause in Bombyx mori and is involved in terminating pupal diapause in heliothis moths. An G protein-coupled receptor from the silkworm, B. mori, has been identified as a specific cell surface receptor for DH. However, the detailed information on the DH-DHR system and its mechanism(s) involved in the induction of embryonic diapause remains unknown. Here, we combined functional assays with various specific inhibitors to elucidate the DHR-mediated signaling pathways. Upon activation by DH, B. mori DHR is coupled to the Gq protein, leading to a significant increase of intracellular Ca(2+) and cAMP response element-driven luciferase activity in an UBO-QIC, a specific Gq inhibitor, sensitive manner. B. mori DHR elicited ERK1/2 phosphorylation in a dose- and time-dependent manner in response to DH. This effect was almost completely inhibited by co-incubation with UBO-QIC and was also significantly suppressed by PLC inhibitor U73122, PKC inhibitors Gö6983 and the Ca(2+) chelator EGTA. Moreover, DHR-induced activation of ERK1/2 was significantly attenuated by treatment with the Gβγ specific inhibitors gallein and M119K and the PI3K specific inhibitor Wortmannin, but not by the Src specific inhibitor PP2. Our data also demonstrates that the EGFR-transactivation pathway is not involved in the DHR-mediated ERK1/2 phosphorylation. Future efforts are needed to clarify the role of the ERK1/2 signaling pathway in the DH-mediated induction of B. mori embryonic diapause. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Synaptic activity-related classical protein kinase C isoform localization in the adult rat neuromuscular synapse.

    Science.gov (United States)

    Besalduch, Núria; Tomàs, Marta; Santafé, Manel M; Garcia, Neus; Tomàs, Josep; Lanuza, Maria Angel

    2010-01-10

    Protein kinase C (PKC) is essential for signal transduction in a variety of cells, including neurons and myocytes, and is involved in both acetylcholine release and muscle fiber contraction. Here, we demonstrate that the increases in synaptic activity by nerve stimulation couple PKC to transmitter release in the rat neuromuscular junction and increase the level of alpha, betaI, and betaII isoforms in the membrane when muscle contraction follows the stimulation. The phosphorylation activity of these classical PKCs also increases. It seems that the muscle has to contract in order to maintain or increase classical PKCs in the membrane. We use immunohistochemistry to show that PKCalpha and PKCbetaI were located in the nerve terminals, whereas PKCalpha and PKCbetaII were located in the postsynaptic and the Schwann cells. Stimulation and contraction do not change these cellular distributions, but our results show that the localization of classical PKC isoforms in the membrane is affected by synaptic activity.

  4. Role of insulin in regulation of Na+-/K+-dependent ATPase activity and pump function in corneal endothelial cells.

    Science.gov (United States)

    Hatou, Shin; Yamada, Masakazu; Akune, Yoko; Mochizuki, Hiroshi; Shiraishi, Atsushi; Joko, Takeshi; Nishida, Teruo; Tsubota, Kazuo

    2010-08-01

    The Na(+)-/K(+)-dependent ATPase (Na,K-ATPase) expressed in the basolateral membrane of corneal endothelial cells plays an important role in the pump function of the corneal endothelium. The role of insulin in the regulation of Na,K-ATPase activity and pump function in corneal endothelial cells was investigated. Confluent monolayers of mouse corneal endothelial cells were exposed to insulin. ATPase activity was evaluated by spectrophotometric measurement of phosphate released from ATP with the use of ammonium molybdate; Na,K-ATPase activity was defined as the portion of total ATPase activity sensitive to ouabain. Pump function was measured with the use of a Ussing chamber; pump function attributable to Na,K-ATPase activity was defined as the portion of the total short-circuit current sensitive to ouabain. Western blot analysis and immunocytochemistry were performed to measure the expression of the Na,K-ATPase alpha(1)-subunit. Insulin increased the Na,K-ATPase activity and pump function of cultured corneal endothelial cells. These effects were blocked by protein kinase C (PKC) inhibitors and protein phosphatases 1 and 2A inhibitor. Western blot analysis indicated that insulin decreased the ratio of the inactive Na,K-ATPase alpha(1)-subunit. Immunocytochemistry indicated that insulin increased the cell surface expression of the Na,K-ATPase alpha(1)-subunit. These results suggest that insulin increases the Na,K-ATPase activity and pump function of cultured corneal endothelial cells. The effect of insulin is mediated by PKC and presumably results in the activation of PP1, 2A, or both, which are essential for activating Na,K-ATPase by alpha(1)-subunit dephosphorylation.

  5. Bortezomib induces neuropathic pain through protein kinase C-mediated activation of presynaptic NMDA receptors in the spinal cord.

    Science.gov (United States)

    Xie, Jing-Dun; Chen, Shao-Rui; Chen, Hong; Pan, Hui-Lin

    2017-09-01

    Chemotherapeutic drugs, including bortezomib, often cause painful peripheral neuropathy, which is a severe dose-limiting adverse effect experienced by many cancer patients. The glutamate N-methyl-d-aspartate receptors (NMDARs) at the spinal cord level are critically involved in the synaptic plasticity associated with neuropathic pain. In this study, we determined whether treatment with bortezomib, a proteasome inhibitor, affects the NMDAR activity of spinal dorsal horn neurons. Systemic treatment with bortezomib in rats did not significantly affect postsynaptic NMDAR currents elicited by puff application of NMDA directly to dorsal horn neurons. Bortezomib treatment markedly increased the baseline frequency of miniature excitatory postsynaptic currents (EPSCs), which was completely normalized by the NMDAR antagonist 2-amino-5-phosphonopentanoic acid (AP5). AP5 also reduced the amplitude of monosynaptic EPSCs evoked by dorsal root stimulation in bortezomib-treated, but not vehicle-treated, rats. Furthermore, inhibition of protein kinase C (PKC) with chelerythrine fully reversed the increased frequency of miniature EPSCs and the amplitude of evoked EPSCs in bortezomib-treated rats. Intrathecal injection of AP5 and chelerythrine both profoundly attenuated mechanical allodynia and hyperalgesia induced by systemic treatment with bortezomib. In addition, treatment with bortezomib induced striking membrane translocation of PKC-βII, PKC-δ, and PKC-ε in the dorsal root ganglion. Our findings indicate that bortezomib treatment potentiates nociceptive input from primary afferent nerves via PKC-mediated tonic activation of presynaptic NMDARs. Targeting presynaptic NMDARs and PKC at the spinal cord level may be an effective strategy for treating chemotherapy-induced neuropathic pain. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Mururins A-C, three new lignoids from Brosimum acutifolium and their protein kinase inhibitory activity.

    Science.gov (United States)

    Takashima, Junko; Asano, Shoichi; Ohsaki, Ayumi

    2002-07-01

    Two new flavonolignans, mururins A and B ( 1 and 2), and a new lignan, mururin C ( 3), were isolated from the bark of Brosimum acutifolium Huber together with three known lignans. Their structures were elucidated by spectroscopic means and chemical modifications. They were tested for protein kinase A (PKA) and protein kinase C (PKC) inhibitory activity. Mururin A showed 3 % and 63 % inhibition to PKA and PKC, respectively, at 20 microM. Mururin B showed 58 % and 38 % inhibition, respectively. Mururin C did not have significant activity.

  7. Evidence against roles for phorbol binding protein Munc13-1, ADAM adaptor Eve-1, or vesicle trafficking phosphoproteins Munc18 or NSF as phospho-state-sensitive modulators of phorbol/PKC-activated Alzheimer APP ectodomain shedding

    Directory of Open Access Journals (Sweden)

    Lovestone Simon

    2007-12-01

    Full Text Available Abstract Background Shedding of the Alzheimer amyloid precursor protein (APP ectodomain can be accelerated by phorbol esters, compounds that act via protein kinase C (PKC or through unconventional phorbol-binding proteins such as Munc13-1. We have previously demonstrated that application of phorbol esters or purified PKC potentiates budding of APP-bearing secretory vesicles at the trans-Golgi network (TGN and toward the plasma membrane where APP becomes a substrate for enzymes responsible for shedding, known collectively as α-secretase(s. However, molecular identification of the presumptive "phospho-state-sensitive modulators of ectodomain shedding" (PMES responsible for regulated shedding has been challenging. Here, we examined the effects on APP ectodomain shedding of four phorbol-sensitive proteins involved in regulation of vesicular membrane trafficking of APP: Munc13-1, Munc18, NSF, and Eve-1. Results Overexpression of either phorbol-sensitive wildtype Munc13-1 or phorbol-insensitive Munc13-1 H567K resulted in increased basal APP ectodomain shedding. However, in contrast to the report of Roßner et al (2004, phorbol ester-dependent APP ectodomain shedding from cells overexpressing APP and Munc13-1 wildtype was indistinguishable from that observed following application of phorbol to cells overexpressing APP and Munc13-1 H567K mutant. This pattern of similar effects on basal and stimulated APP shedding was also observed for Munc18 and NSF. Eve-1, an ADAM adaptor protein reported to be essential for PKC-regulated shedding of pro-EGF, was found to play no obvious role in regulated shedding of sAPPα. Conclusion Our results indicate that, in the HEK293 system, Munc13-1, Munc18, NSF, and EVE-1 fail to meet essential criteria for identity as PMES for APP.

  8. Evidence against roles for phorbol binding protein Munc13-1, ADAM adaptor Eve-1, or vesicle trafficking phosphoproteins Munc18 or NSF as phospho-state-sensitive modulators of phorbol/PKC-activated Alzheimer APP ectodomain shedding.

    Science.gov (United States)

    Ikin, Annat F; Causevic, Mirsada; Pedrini, Steve; Benson, Lyndsey S; Buxbaum, Joseph D; Suzuki, Toshiharu; Lovestone, Simon; Higashiyama, Shigeki; Mustelin, Tomas; Burgoyne, Robert D; Gandy, Sam

    2007-12-09

    Shedding of the Alzheimer amyloid precursor protein (APP) ectodomain can be accelerated by phorbol esters, compounds that act via protein kinase C (PKC) or through unconventional phorbol-binding proteins such as Munc13-1. We have previously demonstrated that application of phorbol esters or purified PKC potentiates budding of APP-bearing secretory vesicles at the trans-Golgi network (TGN) and toward the plasma membrane where APP becomes a substrate for enzymes responsible for shedding, known collectively as alpha-secretase(s). However, molecular identification of the presumptive "phospho-state-sensitive modulators of ectodomain shedding" (PMES) responsible for regulated shedding has been challenging. Here, we examined the effects on APP ectodomain shedding of four phorbol-sensitive proteins involved in regulation of vesicular membrane trafficking of APP: Munc13-1, Munc18, NSF, and Eve-1. Overexpression of either phorbol-sensitive wildtype Munc13-1 or phorbol-insensitive Munc13-1 H567K resulted in increased basal APP ectodomain shedding. However, in contrast to the report of Rossner et al (2004), phorbol ester-dependent APP ectodomain shedding from cells overexpressing APP and Munc13-1 wildtype was indistinguishable from that observed following application of phorbol to cells overexpressing APP and Munc13-1 H567K mutant. This pattern of similar effects on basal and stimulated APP shedding was also observed for Munc18 and NSF. Eve-1, an ADAM adaptor protein reported to be essential for PKC-regulated shedding of pro-EGF, was found to play no obvious role in regulated shedding of sAPPalpha. Our results indicate that, in the HEK293 system, Munc13-1, Munc18, NSF, and EVE-1 fail to meet essential criteria for identity as PMES for APP.

  9. Mechanism of the protective effects of the combined treatment with rhynchophylla total alkaloids and sinapine thiocyanate against a prothrombotic state caused by vascular endothelial cell inflammatory damage.

    Science.gov (United States)

    Li, Yunlun; Zhang, Xinya; Yang, Wenqing; Li, Chao; Chu, Yanjun; Jiang, Haiqiang; Shen, Zhenzhen

    2017-06-01

    The aim of the present study was to investigate the effect and the underlying mechanism of the combined treatment of rhynchophylla total alkaloids (RTA) and sinapine thiocyanate for protection against a prothrombotic state (PTS) associated with the tumor necrosis factor-alpha (TNF-α)-induced inflammatory injury of vascular endothelial cells (VECs). A TNF-α-induced VEC inflammatory injury model was established, and cell morphology of VECs was evaluated using scanning electron microscopy. In addition, reverse transcription-quantitative polymerase chain reaction and western blot analysis were performed to examine the mRNA and protein expression of coagulation-related factors, including nuclear factor-κB (NF-κB), transforming growth factor-β1 (TGF-β1), tissue factor (TF), plasminogen activator inhibitor (PAI-1), protease-activation receptors (PAR-1) and protein kinase C (PKC-α) in VECs. Combined treatment with RTA and sinapine thiocyanate was demonstrated to reduce, to a varying extent, the mRNA and protein expression of NF-κB, TGF-β1, TF, PAR-1, PKC-α and PAI-1. Furthermore, combined treatment with RTA and sinapine thiocyanate was able to downregulate the expression of coagulation-related factors in injured VECs, thereby inhibiting the PTS induced by vascular endothelial injury. The underlying mechanism is partially associated with the TF-mediated activation of the thrombin-receptor signaling pathway that suppresses coagulation during inflammation and balances fibrinolysis in order to inhibit fibrin generation and deposition.

  10. Multimerization of the cytoplasmic domain of syndecan-4 is required for its ability to activate protein kinase C

    DEFF Research Database (Denmark)

    Oh, E S; Woods, A; Couchman, J R

    1997-01-01

    of syndecan-4 (4L) containing a membrane-proximal basic sequence did not form higher order oligomers and could not regulate the activity of PKCalphabetagamma unless induced to aggregate by phosphatidylinositol 4,5-bisphosphate. Oligomerization and PKC regulatory activity of the 4V peptide were both increased...... by addition of N-terminal cysteine and reduced by phosphorylation of the cysteine thiol group. Concentration of syndecan-4 at sites of focal adhesion formation may enhance multimerization and both localize PKC and potentiate its activity to induce stable complex formation....

  11. Protein kinase C epsilon mediates the inhibition of angiotensin II on the slowly activating delayed-rectifier potassium current through channel phosphorylation.

    Science.gov (United States)

    Gou, Xiangbo; Wang, Wenying; Zou, Sihao; Qi, Yajuan; Xu, Yanfang

    2018-03-01

    The slowly activating delayed rectifier K + current (I Ks ) is one of the main repolarizing currents in the human heart. Evidence has shown that angiotensin II (Ang II) regulates I Ks through the protein kinase C (PKC) pathway, but the related results are controversial. This study was designed to identify PKC isoenzymes involved in the regulation of I Ks by Ang II and the underlying molecular mechanism. The whole-cell patch-clamp technique was used to record I Ks in isolated guinea pig ventricular cardiomyocytes and in human embryonic kidney (HEK) 293 cells co-transfected with human KCNQ1/KCNE1 genes and Ang II type 1 receptor genes. Ang II inhibited I Ks in a concentration-dependent manner in native cardiomyocytes. A broad PKC inhibitor Gö6983 (not inhibiting PKCε) and a selective cPKC inhibitor Gö6976 did not affect the inhibitory action of Ang II. In contrast, the inhibition was significantly attenuated by PKCε-selective peptide inhibitor εV1-2. However, direct activation of PKC by phorbol 12-myristate 13-acetate (PMA) increased the cloned human I Ks in HEK293 cells. Similarly, the cPKC peptide activator significantly enhanced the current. In contrast, the PKCε peptide activator inhibited the current. Further evidence showed that PKCε knockdown by siRNA antagonized the Ang II-induced inhibition on KCNQ1/KCNE1 current, whereas knockdown of cPKCs (PKCα and PKCβ) attenuated the potentiation of the current by PMA. Moreover, deletion of four putative phosphorylation sites in the C-terminus of KCNQ1 abolished the action of PMA. Mutation of two putative phosphorylation sites in the N-terminus of KCNQ1 and one site in KCNE1 (S102) blocked the inhibition of Ang II. Our results demonstrate that PKCε isoenzyme mediates the inhibitory action of Ang II on I Ks and by phosphorylating distinct sites in KCNQ1/KCNE1, cPKC and PKCε isoenzymes produce the contrary regulatory effects on the channel. These findings have provided new insight into the molecular mechanism

  12. Structural insight with mutational impact on tyrosinase and PKC-β interaction from Homo sapiens: Molecular modeling and docking studies for melanogenesis, albinism and increased risk for melanoma.

    Science.gov (United States)

    Banerjee, Arundhati; Ray, Sujay

    2016-10-30

    Human tyrosinase, is an important protein for biosynthetic pathway of melanin. It was studied to be phosphorylated and activated by protein kinase-C, β-subunit (PKC-β) through earlier experimentations with in vivo evidences. Documentation documents that mutation in two essentially vital serine residues in C-terminal end of tyrosinase leads to albinism. Due to the deficiency of protective shield like enzyme; melanin, albinos are at an increased peril for melanoma and other skin cancers. So, computational and residue-level insight including a mutational exploration with evolutionary importance into this mechanism lies obligatory for future pathological and therapeutic developments. Therefore, functional tertiary models of the relevant proteins were analyzed after satisfying their stereo-chemical features. Evolutionarily paramount residues for the activation of tyrosinase were perceived via multiple sequence alignment phenomena. Mutant-type tyrosinase protein (S98A and S102A) was thereby modeled, maintaining the wild-type proteins' functionality. Furthermore, this present comparative study discloses the variation in the stable residual participation (for mutant-type and wild-type tyrosinase-PKCβ complex). Mainly, an increased number of polar negatively charged residues from the wild-type tyrosinase participated with PKC-β, predominantly. Fascinatingly supported by evaluation of statistical significances, mutation even led to a destabilizing impact in tyrosinase accompanied by conformational switches with a helix-to-coil transition in the mutated protein. Even the allosteric sites in the protein got poorly hampered upon mutation leading to weaker tendency for binding partners to interact. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Up-regulation of interleukin-4 production via NF-AT/AP-1 activation in T cells by biochanin A, a phytoestrogen and its metabolites

    International Nuclear Information System (INIS)

    Park, Jin; Chung, Su Wol; Kim, Seung Hyun; Kim, Tae Sung

    2006-01-01

    Phytoestrogens are naturally occurring compounds derived from plants. Although phytoestrogens exhibit many biological functions including estrogen agonist/antagonist properties, the effect on allergic responses remains unclear. In this study, we investigated whether biochanin A, a phytoestrogen and its metabolites, genistein, p-ethylphenol and phenolic acid, affect production of IL-4, a pro-inflammatory cytokine closely associated with allergic immune responses, in primary CD4 + T cells and EL4 T lymphoma cells. Biochanin A, genistein and p-ethylphenol significantly enhanced IL-4 production from both CD4 + T cells and EL4 cells in a dose-dependent manner, while phenolic acid did not. Biochanin A, genistein and p-ethylphenol also enhanced IL-4 gene promoter activity in EL4 cells transiently transfected with IL-4 promoter constructs, but this effect was impaired in EL4 cells transfected with an IL-4 promoter construct deleted of a P4 site carrying NF-AT and AP-1 binding sites. In addition, biochanin A, genistein and p-ethylphenol increased both NF-AT and AP-1 DNA binding activities, indicating that they might enhance IL-4 production via NF-AT/AP-1 activation. Furthermore, biochanin A, genistein and p-ethylphenol increased p38 MAPK phosphorylation and PKC activity, while they did not affect ERK phosphorylation. The enhanced NF-AT DNA binding activities were suppressed by inhibitors for PI3-K and PKC, but not by p38 MAPK inhibitors. In contrast, the enhanced AP-1 DNA binding activities and p38 MAPK phosphorylation were significantly suppressed by specific inhibitors for PKC and p38 MAPK, but not by PI3-K inhibitors. These results demonstrate, for the first time, that biochanin A, genistein and p-ethylphenol enhance IL-4 production in activated T cells by two independent pathways, PI3-K/PKC/NF-AT and PKC/p38 MAPK/AP-1

  14. Screening of protein kinase inhibitors identifies PKC inhibitors as inhibitors of osteoclastic acid secretion and bone resorption

    Directory of Open Access Journals (Sweden)

    Boutin Jean A

    2010-10-01

    Full Text Available Abstract Background Bone resorption is initiated by osteoclastic acidification of the resorption lacunae. This process is mediated by secretion of protons through the V-ATPase and chloride through the chloride antiporter ClC-7. To shed light on the intracellular signalling controlling extracellular acidification, we screened a protein kinase inhibitor library in human osteoclasts. Methods Human osteoclasts were generated from CD14+ monocytes. The effect of different kinase inhibitors on lysosomal acidification in human osteoclasts was investigated using acridine orange for different incubation times (45 minutes, 4 and 24 hours. The inhibitors were tested in an acid influx assay using microsomes isolated from human osteoclasts. Bone resorption by human osteoclasts on bone slices was measured by calcium release. Cell viability was measured using AlamarBlue. Results Of the 51 compounds investigated only few inhibitors were positive in both acidification and resorption assays. Rottlerin, GF109203X, Hypericin and Ro31-8220 inhibited acid influx in microsomes and bone resorption, while Sphingosine and Palmitoyl-DL-carnitine-Cl showed low levels of inhibition. Rottlerin inhibited lysosomal acidification in human osteoclasts potently. Conclusions In conclusion, a group of inhibitors all indicated to inhibit PKC reduced acidification in human osteoclasts, and thereby bone resorption, indicating that acid secretion by osteoclasts may be specifically regulated by PKC in osteoclasts.

  15. PASSIVE-AVOIDANCE TRAINING INDUCES ENHANCED LEVELS OF IMMUNOREACTIVITY FOR MUSCARINIC ACETYLCHOLINE-RECEPTOR AND COEXPRESSED PKC-GAMMA AND MAP-2 IN RAT CORTICAL-NEURONS

    NARCIS (Netherlands)

    VANDERZEE, EA; DOUMA, BRK; BOHUS, B; LUITEN, PGM

    1994-01-01

    Changes in neocortical immunoreactivity (ir) for muscarinic acetylcholine receptors (mAChRs), protein kinase C gamma (PKC gamma), microtubule-associated protein 2 (MAP-2), and the calcium-binding protein parvalbumin (PARV) induced by the performance of a one-trial passive shock avoidance (PSA) task

  16. Stabilization and activation of p53 are regulated independently by different phosphorylation events

    OpenAIRE

    Chernov, Mikhail V.; Ramana, Chilakamarti V.; Adler, Victor V.; Stark, George R.

    1998-01-01

    Treatment of mouse or human cells with the protein kinase C (PKC) inhibitors H7 or bisindolylmaleimide I induced an increase in the lifetime of p53, leading to its accumulation. In inhibitor-treated cells, p53 translocated to the nuclei and bound to DNA but was not competent to induce transcription. However, transactivation could be induced by subsequent DNA damage. Phorbol ester, a potent activator of PKC, significantly inhibited the accumulation of p53 after DNA damage. Therefore, constitut...

  17. D-Saccharic acid 1,4-lactone protects diabetic rat kidney by ameliorating hyperglycemia-mediated oxidative stress and renal inflammatory cytokines via NF-κB and PKC signaling

    Energy Technology Data Exchange (ETDEWEB)

    Bhattacharya, Semantee [Department of Life Sciences and Biotechnology, Jadavpur University, 188, Raja S C Mullick Road, Kolkata 700 032 (India); Manna, Prasenjit [Division of Molecular Medicine, Bose Institute, P-1/12, CIT Scheme VII M, Kolkata-700054 (India); Gachhui, Ratan [Department of Life Sciences and Biotechnology, Jadavpur University, 188, Raja S C Mullick Road, Kolkata 700 032 (India); Sil, Parames C., E-mail: parames@bosemain.boseinst.ac.in [Division of Molecular Medicine, Bose Institute, P-1/12, CIT Scheme VII M, Kolkata-700054 (India)

    2013-02-15

    Increasing evidence suggests that oxidative stress is involved in the pathogenesis of diabetic nephropathy (DN) and this can be attenuated by antioxidants. D-Saccharic acid 1,4-lactone (DSL) is known for its detoxifying and antioxidant properties. Our early investigation showed that DSL can ameliorate alloxan (ALX) induced diabetes mellitus and oxidative stress in rats by inhibiting pancreatic β-cell apoptosis. In the present study we, therefore, investigated the protective role of DSL against renal injury in ALX induced diabetic rats. ALX exposure (at a dose of 120 mg/kg body weight, i. p., once) elevated the blood glucose level, serum markers related to renal injury, the production of reactive oxygen species (ROS), and disturbed the intra-cellular antioxidant machineries. Oral administration of DSL (80 mg/kg body weight) restored all these alterations close to normal. In addition, DSL could also normalize the aldose reductase activity which was found to increase in the diabetic rats. Investigating the mechanism of its protective activity, we observed the activation of different isoforms of PKC along with the accumulation of matrix proteins like collagen and fibronectin. The diabetic rats also showed nuclear translocation of NF-κB and increase in the concentration of inflammatory cytokines in the renal tissue. The activation of mitochondria dependent apoptotic pathway was observed in the diabetic rat kidneys. However, treatment of diabetic rats with DSL counteracted all these changes. These findings, for the first time, demonstrated that DSL could ameliorate renal dysfunction in diabetic rats by suppressing the oxidative stress related signalling pathways. - Highlights: ► Sustained hyperglycemia and oxidative stress lead to diabetic renal injury. ► D-saccharic acid 1,4-lactone prevents renal damage in alloxan-induced diabetes. ► It restores intra-cellular antioxidant machineries and kidney apoptosis. ► DSL reduces hyperglycemia-mediated oxidative stress

  18. Changes in total carbohydrate and total antioxidant activity induced by gamma irradiation of wheat flour

    International Nuclear Information System (INIS)

    Manupriya, B.R.; Shenoy, K. Bhasker; Patil, Shrikant L.; Somashekarappa, H.M.

    2015-01-01

    Wheat is a staple food grain in India after rice and occupies number one position in the world. The wheat crop not only gives food grains but also gives fodder for animals. Among many preservation methods irradiation is a current technique used to overcome infestation, contamination and spoilage of stored grains. The present study is aimed to check the changes in composition of irradiated wheat flour. Wheat flour was exposed to five different irradiation doses (0.25 KGy, 0.5KGy, 1KGy, 5KGy and 10 KGy) by using 60 Co gamma-irradiation chamber. Irradiated flour was stored in air sealed polyethylene pouch and plastic container at room temperature for different time intervals (0 th day, 1 month and 3 months). The stored flour was checked for total antioxidant activity by phosphomolybdate method and total carbohydrates concentration by phenol-sulphuric acid method. On 0 th day total antioxidant activity and total carbohydrate concentration was found to be increased at 0.5KGy (0.113 mg/ml and 0.045 mg/ml respectively) when compared to control (0.79 mg/ml and 39.5 mg/ml). Similarly for 1 month stored samples of air sealed polyethylene pouch total antioxidant activity and total carbohydrate concentration was observed to be increased at 0.5KGy (0.117 mg/ml and 0.045mg/ml respectively) when compared to control (0.096 mg/ml and 0.035 mg/ml). But in case of stored samples of plastic container total antioxidant activity increased at 0.25KGy (0.060 mg/ml) and total carbohydrate increased at 5KGy (0.051 mg/ml). Increased and decreased values were found in both factors for 3 months stored samples of air sealed polyethylene pouch and plastic container. Total antioxidant activity increased at 5KGy (0.072 mg/ml) for polyethylene bag samples and at 0.5KGy (0.137 mg/ml) for plastic container sample. Same way total carbohydrate concentration increased at 0.25KGy (0.046 mg/ml) and at 1KGy (0.045 mg/ml) respectively. This increase is due to affects of γ-irradiation on biomolecules by

  19. Modulation of Immune Function in Rats Using Oligosaccharides Extracted from Palm Kernel Cake.

    Science.gov (United States)

    Faseleh Jahromi, Mohammd; Shokryazdan, Parisa; Idrus, Zulkifli; Ebrahimi, Rohollah; Bashokouh, Fatemeh; Liang, Juan Boo

    2017-01-01

    To investigate the prebiotic and immunomodulatory effects of PKC extract (OligoPKC) a total of 24 male rats were randomly assigned to three treatment groups receiving basal diet (control), basal diet containing 0.5% OligoPKC, or basal diet containing 1% OligoPKC for four weeks. We found that OligoPKC had no significant effect on the tested growth parameters. However, it increased the size of the total and beneficial bacterial populations while reducing pathogen populations. OligoPKC increased the concentration of immunoglobulins in the serum and cecal contents of rats. It also enhanced the antioxidant capacity of the liver while reducing lipid peroxidation in liver tissue. OligoPKC affected the expression of genes involved in immune system function in the intestine. Therefore, OligoPKC could be considered a potential mannan-based prebiotic for humans and animals due to its beneficial effects on the health and well-being of the model rats.

  20. Duodenal mucosal protein kinase C-δ regulates glucose production in rats.

    Science.gov (United States)

    Kokorovic, Andrea; Cheung, Grace W C; Breen, Danna M; Chari, Madhu; Lam, Carol K L; Lam, Tony K T

    2011-11-01

    Activation of protein kinase C (PKC) enzymes in liver and brain alters hepatic glucose metabolism, but little is known about their role in glucose regulation in the gastrointestinal tract. We investigated whether activation of PKC-δ in the duodenum is sufficient and necessary for duodenal nutrient sensing and regulates hepatic glucose production through a neuronal network in rats. In rats, we inhibited duodenal PKC and evaluated whether nutrient-sensing mechanisms, activated by refeeding, have disruptions in glucose regulation. We then performed gain- and loss-of-function pharmacologic and molecular experiments to target duodenal PKC-δ; we evaluated the impact on glucose production regulation during the pancreatic clamping, while basal levels of insulin were maintained. PKC-δ was detected in the mucosal layer of the duodenum; intraduodenal infusion of PKC inhibitors disrupted glucose homeostasis during refeeding, indicating that duodenal activation of PKC-δ is necessary and sufficient to regulate glucose homeostasis. Intraduodenal infusion of the PKC activator 1-oleoyl-2-acetyl-sn-glycerol (OAG) specifically activated duodenal mucosal PKC-δ and a gut-brain-liver neuronal pathway to reduce glucose production. Molecular and pharmacologic inhibition of duodenal mucosal PKC-δ negated the ability of duodenal OAG and lipids to reduce glucose production. In the duodenal mucosa, PKC-δ regulates glucose homeostasis. Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.

  1. Programming of neurotoxic cofactor CXCL-10 in HIV-1-associated dementia: abrogation of CXCL-10-induced neuro-glial toxicity in vitro by PKC activator

    Directory of Open Access Journals (Sweden)

    Mehla Rajeev

    2012-10-01

    Full Text Available Abstract Background More than 50% of patients undergoing lifelong suppressive antiviral treatment for HIV-1 infection develop minor HIV-1-associated neurocognitive disorders. Neurological complications during HIV-1 infection are the result of direct neuronal damage by proinflammatory products released from HIV-1-infected or -uninfected activated lymphocytes, monocytes, macrophages, microglia and astrocytes. The specific pro-inflammatory products and their roles in neurotoxicity are far from clear. We investigated proinflammatory cytokines and chemokines in the cerebrospinal fluid (CSF of HIV-demented (HIV-D and HIV-nondemented (HIV-ND patients and studied their affect on neuroglial toxicity. Methods and results Bioplex array showed elevated levels of signatory chemokines or cytokines (IL-6, IFN-γ, CXCL10, MCP-1 and PDGF in the CSF of HIV-D patients (n = 7 but not in that of HIV-ND patients (n = 7. Among the signatory cytokines and chemokines, CXCL10 was distinctly upregulated in-vitro in HIV-1 (NLENG1-activated human fetal astrocytes, HIV-1 (Ba-L-infected macrophages, and HIV-1 (NLENG1-infected lymphocytes. Virus-infected macrophages also had increased levels of TNF-α. Consistently, human fetal astrocytes treated with HIV-1 and TNF-α induced the signatory molecules. CXCL10 in combination with HIV-1 synergistically enhanced neuronal toxicity and showed chemotactic activity (~ 40 fold for activated peripheral blood mononuclear cells (PBMC, suggesting the intersection of signaling events imparted by HIV-1 and CXCL10 after binding to their respective surface receptors, CXCR4 and CXCR3, on neurons. Blocking CXCR3 and its downstream MAP kinase (MAPK signaling pathway suppressed combined CXCL10 and HIV-1-induced neurotoxicity. Bryostatin, a PKC modulator and suppressor of CXCR4, conferred neuroprotection against combined insult with HIV-1 and CXCL10. Bryostatin also suppressed HIV-1 and CXCL10-induced PBMC chemotaxis. Although, therapeutic targeting

  2. Mechanisms of activation of NHE by cell shrinkage and by calyculin A in Ehrlich ascites tumor cells

    DEFF Research Database (Denmark)

    Pedersen, Stine Helene Falsig; Varming, Camilla; Hoffmann, E K

    2002-01-01

    The Na+/H+ exchanger isoforms NHE1, NHE2, and NHE3 were all found to be expressed in Ehrlich ascites tumor cells, as evaluated by Western blotting and confocal microscopy. Under unstimulated conditions, NHE1 was found predominantly in the plasma membrane, NHE3 intracellularly, and NHE2 in both co....... It is concluded that shrinkage-induced NHE activation is dependent on PKC and p38 MAPK, but not on MLCK or ERK1/2. NHE activity under both iso- and hypertonic conditions is increased by inhibition of serine/threonine phosphatases, and this effect appears to be PKC-dependent....

  3. Effect of Antrodia camphorata on Inflammatory Arterial Thrombosis-Mediated Platelet Activation: The Pivotal Role of Protein Kinase C

    Directory of Open Access Journals (Sweden)

    Wan-Jung Lu

    2014-01-01

    Full Text Available Antrodia camphorata is a rare Taiwanese medicinal mushroom. Antrodia camphorata extract has been reported to exhibit antioxidant, anti-inflammation, antimetastasis, and anticancer activities and plays a role in liver fibrosis, vasorelaxation, and immunomodulation. Critical vascular inflammation leads to vascular dysfunction and cardiovascular diseases, including abdominal aortic aneurysms, hypertension, and atherosclerosis. Platelet activation plays a crucial role in intravascular thrombosis, which is involved in a wide variety of cardiovascular diseases. However, the effect of Antrodia camphorata on platelet activation remains unclear. We examined the effects of Antrodia camphorata on platelet activation. In the present study, Antrodia camphorata treatment (56–224 μg/mL inhibited platelet aggregation induced by collagen, but not U46619, an analogue of thromboxane A2, thrombin, and arachidonic acid. Antrodia camphorata inhibited collagen-induced calcium (Ca2+ mobilization and phosphorylation of protein kinase C (PKC and Akt. In addition, Antrodia camphorata significantly reduced the aggregation and phosphorylation of PKC in phorbol-12, 13-dibutyrate (PDBu activated platelets. In conclusion, Antrodia camphorata may inhibit platelet activation by inhibiting of Ca2+ and PKC cascade and the Akt pathway. Our study suggests that Antrodia camphorata may be a potential therapeutic agent for preventing or treating thromboembolic disorders.

  4. The C1 domain-targeted isophthalate derivative HMI-1b11 promotes neurite outgrowth and GAP-43 expression through PKCα activation in SH-SY5Y cells.

    Science.gov (United States)

    Talman, Virpi; Amadio, Marialaura; Osera, Cecilia; Sorvari, Salla; Boije Af Gennäs, Gustav; Yli-Kauhaluoma, Jari; Rossi, Daniela; Govoni, Stefano; Collina, Simona; Ekokoski, Elina; Tuominen, Raimo K; Pascale, Alessia

    2013-07-01

    Protein kinase C (PKC) is a family of serine/threonine phosphotransferases ubiquitously expressed and involved in multiple cellular functions, such as proliferation, apoptosis and differentiation. The C1 domain of PKC represents an attractive drug target, especially for developing PKC activators. Dialkyl 5-(hydroxymethyl)isophthalates are a novel group of synthetic C1 domain ligands that exhibit antiproliferative effect in HeLa cervical carcinoma cells. Here we selected two isophthalates, HMI-1a3 and HMI-1b11, and characterized their effects in the human neuroblastoma cell line SH-SY5Y. Both of the active isophthalates exhibited significant antiproliferative and differentiation-inducing effects. Since HMI-1b11 did not impair cell survival even at the highest concentration tested (20μM), and supported neurite growth and differentiation of SH-SY5Y cells, we focused on studying its downstream signaling cascades and effects on gene expression. Consistently, genome-wide gene expression microarray and gene set enrichment analysis indicated that HMI-1b11 (10μM) induced changes in genes mainly related to cell differentiation. In particular, further studies revealed that HMI-1b11 exposure induced up-regulation of GAP-43, a marker for neurite sprouting and neuronal differentiation. These effects were induced by a 7-min HMI-1b11 treatment and specifically depended on PKCα activation, since pretreatment with the selective inhibitor Gö6976 abolished the up-regulation of GAP-43 protein observed at 12h. In parallel, we found that a 7-min exposure to HMI-1b11 induced PKCα accumulation to the cytoskeleton, an effect that was again prevented by pretreatment with Gö6976. Despite similar binding affinities to PKC, the isophthalates had different effects on PKC-dependent ERK1/2 signaling: HMI-1a3-induced ERK1/2 phosphorylation was transient, while HMI-1b11 induced a rapid but prolonged ERK1/2 phosphorylation. Overall our data are in accordance with previous studies showing that

  5. Extraction process of palm kernel cake as a source of mannan for feed additive on poultry diet

    Science.gov (United States)

    Tafsin, M.; Hanafi, N. D.; Yusraini, E.

    2017-05-01

    Palm Kernel Cake (PKC) is a by-product of palm kernel oil extraction and found in large quantity in Indonesia. The inclusion of PKC on poultry diet are limited due to some nutritional problems such as anti-nutritional properties (mannan). On the other hand, mannan containing polysaccharides play in various biological functions particularly in enhancing the immune response and to control pathogen in poultry. The research objective to find out the extraction process of PKC and conducted at animal nutrition and Feed Science Laboratory, Agricultural Faculty, University of Sumatera Utara. Various extraction methode were used in this experiment, including fraction analysis used 7 number sieves, and followed by water and acetic acid extraction. The result indicated that PKC had different particle size according to sieve size and dominated by particle size 850 um. The analysis of sugar content indicated that each particle size had different characteristic on treatment by hot water extraction. The particle size 180—850 um had higher sugar content than coarse PKC (2000—3000 um). The total sugar content were recovered vary between 0.9—3,2% from PKC were extracted. Treatment grinding method followed by hot water extraction (100—120 °C, 1 h) increased total sugar content than previous treatments and reach 8% from PKC were extracted. Utilisation acetic acid decreased the total amount of total sugar from PKC were extracted. It is concluded that treatment by hot temperature (110—120 °C) for 1 h show highest yield to extract sugar from PKC.

  6. Acute stress enhances learning and memory by activating acid-sensing ion channels in rats.

    Science.gov (United States)

    Ye, Shunjie; Yang, Rong; Xiong, Qiuju; Yang, Youhua; Zhou, Lianying; Gong, Yeli; Li, Changlei; Ding, Zhenhan; Ye, Guohai; Xiong, Zhe

    2018-04-15

    Acute stress has been shown to enhance learning and memory ability, predominantly through the action of corticosteroid stress hormones. However, the valuable targets for promoting learning and memory induced by acute stress and the underlying molecular mechanisms remain unclear. Acid-sensing ion channels (ASICs) play an important role in central neuronal systems and involves in depression, synaptic plasticity and learning and memory. In the current study, we used a combination of electrophysiological and behavioral approaches in an effort to explore the effects of acute stress on ASICs. We found that corticosterone (CORT) induced by acute stress caused a potentiation of ASICs current via glucocorticoid receptors (GRs) not mineralocorticoid receptors (MRs). Meanwhile, CORT did not produce an increase of ASICs current by pretreated with GF109203X, an antagonist of protein kinase C (PKC), whereas CORT did result in a markedly enhancement of ASICs current by bryostatin 1, an agonist of PKC, suggesting that potentiation of ASICs function may be depended on PKC activating. More importantly, an antagonist of ASICs, amiloride (10 μM) reduced the performance of learning and memory induced by acute stress, which is further suggesting that ASICs as the key components involves in cognitive processes induced by acute stress. These results indicate that acute stress causes the enhancement of ASICs function by activating PKC signaling pathway, which leads to potentiated learning and memory. Copyright © 2018 Elsevier Inc. All rights reserved.

  7. The PreS2 activator MHBst of hepatitis B virus activates c-raf-1/Erk2 signaling in transgenic mice

    Science.gov (United States)

    Hildt, Eberhard; Munz, Barbara; Saher, Gesine; Reifenberg, Kurt; Hofschneider, Peter Hans

    2002-01-01

    The large hepatitis B virus (HBV) surface protein (LHBs) and C-terminally truncated middle size surface proteins (MHBst) form the family of the PreS2 activator proteins of HBV. Their transcriptional activator function is based on the cytoplasmic orientation of the PreS2 domain. MHBst activators are paradigmatic for this class of activators. Here we report that MHBst is protein kinase C (PKC)-dependently phosphorylated at Ser28. The integrity of the phosphorylation site is essential for the activator function. MHBst triggers PKC-dependent activation of c-Raf-1/Erk2 signaling that is a prerequisite for MHBst-dependent activation of AP-1 and NF-κB. To analyze the pathophysiological relevance of these data in vivo, transgenic mice were established that produce the PreS2 activator MHBst specifically in the liver. In these mice, a permanent PreS2-dependent specific activation of c-Raf-1/Erk2 signaling was observed, resulting in an increased hepatocyte proliferation rate. In transgenics older than 15 months, an increased incidence of liver tumors occurs. These data suggest that PreS2 activators LHBs and MHBst exert a tumor promoter-like function by activation of key enzymes of proliferation control. PMID:11847101

  8. Cardiomyopathy-Associated Gene 1-Sensitive PKC-Dependent Connexin 43 Expression and Phosphorylation in Left Ventricular Noncompaction Cardiomyopathy

    Directory of Open Access Journals (Sweden)

    Yuanyuan Xie

    2017-11-01

    Full Text Available Background/Aims: Cardiomyopathy-associated gene 1 (CMYA1 plays an important role in embryonic cardiac development, postnatal cardiac remodeling and myocardial injury repair. Abnormal CMYA1 expression may be involved in cardiac dysplasia and primary cardiomyopathy. Our study aims to establish the relationship between CMYA1 and Left ventricular noncompaction cardiomyopathy (LVNC pathogenesis. Methods: We explored the effects of CMYA1 on connexins (Cx, which contribute to gap junction intercellular communication (GJIC, and the underlying signaling pathway in human normal tissues, LVNC myocardial tissues and HL1 cells by means of western blotting, RT-qPCR, immunohistochemistry, immunofluorescence, co-immunoprecipitation and scrape loading-dye transfer. Results: CMYA1 expression was inversely associated with Cx43 and Cx40 expression, as determined by gap junction PCR array analysis. An increased expression and disordered distribution of CMYA1 at the intercalated discs in LVNC myocardial tissue was also observed. CMYA1 and Cx43 are co-expressed and interact in myocardial cells. CMYA1 expression was positively correlated with p-Cx43 (S368 via the Protein kinase C (PKC signaling pathway in myocardial tissue and HL1 cells. The diffusion distance of Lucifer Yellow in the HL1 cells in which CMYA1 was over-expressed or knocked down was significantly less or more than that of the control group, respectively. Conclusion: Abnormal CMYA1 expression affects the expression and phosphorylation of Cx43 through the PKC signaling pathway, which is involved in the regulation of GJIC. CMYA1 participates in the molecular mechanism of LVNC pathogenesis.

  9. PKCη confers protection against apoptosis by inhibiting the pro-apoptotic JNK activity in MCF-7 cells

    International Nuclear Information System (INIS)

    Rotem-Dai, Noa; Oberkovitz, Galia; Abu-Ghanem, Sara; Livneh, Etta

    2009-01-01

    Apoptosis is frequently regulated by different protein kinases including protein kinase C family enzymes. Both inhibitory and stimulatory effects were demonstrated for several of the different PKC isoforms. Here we show that the novel PKC isoform, PKCη, confers protection against apoptosis induced by the DNA damaging agents, UVC irradiation and the anti-cancer drug - Camptothecin, of the breast epithelial adenocarcinoma MCF-7 cells. The induced expression of PKCη in MCF-7 cells, under the control of the tetracycline-responsive promoter, resulted in increased cell survival and inhibition of cleavage of the apoptotic marker PARP-1. Activation of caspase-7 and 9 and the release of cytochrome c were also inhibited by the inducible expression of PKCη. Furthermore, JNK activity, required for apoptosis in MCF-7, as indicated by the inhibition of both caspase-7 cleavage and cytochrome c release from the mitochondria in the presence of the JNK inhibitor SP600125, was also suppressed by PKCη expression. Hence, in contrast to most PKC isoforms enhancing JNK activation, our studies show that PKCη is an anti-apoptotic protein, acting as a negative regulator of JNK activity. Thus, PKCη could represent a target for intervention aimed to reduce resistance to anti-cancer treatments.

  10. NMDA-induced potentiation of mGluR5 is mediated by activation of protein phosphatase 2B/calcineurin

    Science.gov (United States)

    Alagarsamy, Sudar; Saugstad, Julie; Warren, Lee; Mansuy, Isabelle M.; Gereau, Robert W.; Conn, P. Jeffrey

    2010-01-01

    Previous reports have shown that activation of N-methyl-D-aspartate (NMDA) receptors potentiates responses to activation of the group I metabotropic glutamate receptor mGluR5 by reversing PKC-mediated desensitization of this receptor. NMDA-induced reversal of mGluR5 desensitization is dependent on activation of protein phosphatases. However, the specific protein phosphatase involved and the precise mechanism by which NMDA receptor activation reduces mGluR desensitization are not known. We have performed a series of molecular, biochemical, and genetic studies to show that NMDA-induced regulation of mGluR5 is dependent on activation of calcium-dependent protein phosphatase 2B/calcineurin (PP2B/CaN). Furthermore, we report that purified calcineurin directly dephosphorylates the C-terminal tail of mGluR5 at sites that are phosphorylated by PKC. Finally, immunoprecipitation and GST fusion protein pull-down experiments reveal that calcineurin interacts with mGluR5, suggesting that these proteins could be colocalized in a signaling complex. Taken together with previous studies, these data suggest that activation of NMDA receptors leads to activation of calcineurin and that calcineurin modulates mGluR5 function by directly dephosphorylating mGluR5 at PKC sites that are involved in desensitization of this receptor. 2005 Elsevier Ltd. All rights reserved. PMID:16005030

  11. Protein kinase Cη activates NF-κB in response to camptothecin-induced DNA damage

    International Nuclear Information System (INIS)

    Raveh-Amit, Hadas; Hai, Naama; Rotem-Dai, Noa; Shahaf, Galit; Gopas, Jacob; Livneh, Etta

    2011-01-01

    Highlights: → Protein kinase C-eta (PKCη) is an upstream regulator of the NF-κB signaling pathway. → PKCη activates NF-κB in non-stressed conditions and in response to DNA damage. → PKCη regulates NF-κB by activating IκB kinase (IKK) and inducing IκB degradation. -- Abstract: The nuclear factor κB (NF-κB) family of transcription factors participates in the regulation of genes involved in innate- and adaptive-immune responses, cell death and inflammation. The involvement of the Protein kinase C (PKC) family in the regulation of NF-κB in inflammation and immune-related signaling has been extensively studied. However, not much is known on the role of PKC in NF-κB regulation in response to DNA damage. Here we demonstrate for the first time that PKC-eta (PKCη) regulates NF-κB upstream signaling by activating the IκB kinase (IKK) and the degradation of IκB. Furthermore, PKCη enhances the nuclear translocation and transactivation of NF-κB under non-stressed conditions and in response to the anticancer drug camptothecin. We and others have previously shown that PKCη confers protection against DNA damage-induced apoptosis. Our present study suggests that PKCη is involved in NF-κB signaling leading to drug resistance.

  12. The PreS2 activator MHBs(t) of hepatitis B virus activates c-raf-1/Erk2 signaling in transgenic mice.

    Science.gov (United States)

    Hildt, Eberhard; Munz, Barbara; Saher, Gesine; Reifenberg, Kurt; Hofschneider, Peter Hans

    2002-02-15

    The large hepatitis B virus (HBV) surface protein (LHBs) and C-terminally truncated middle size surface proteins (MHBs(t)) form the family of the PreS2 activator proteins of HBV. Their transcriptional activator function is based on the cytoplasmic orientation of the PreS2 domain. MHBs(t) activators are paradigmatic for this class of activators. Here we report that MHBs(t) is protein kinase C (PKC)-dependently phosphorylated at Ser28. The integrity of the phosphorylation site is essential for the activator function. MHBs(t) triggers PKC-dependent activation of c-Raf-1/Erk2 signaling that is a prerequisite for MHBs(t)-dependent activation of AP-1 and NF-kappaB. To analyze the pathophysiological relevance of these data in vivo, transgenic mice were established that produce the PreS2 activator MHBs(t) specifically in the liver. In these mice, a permanent PreS2-dependent specific activation of c-Raf-1/Erk2 signaling was observed, resulting in an increased hepatocyte proliferation rate. In transgenics older than 15 months, an increased incidence of liver tumors occurs. These data suggest that PreS2 activators LHBs and MHBs(t) exert a tumor promoter-like function by activation of key enzymes of proliferation control.

  13. Phrenic long-term facilitation requires PKCθ activity within phrenic motor neurons.

    Science.gov (United States)

    Devinney, Michael J; Fields, Daryl P; Huxtable, Adrianne G; Peterson, Timothy J; Dale, Erica A; Mitchell, Gordon S

    2015-05-27

    Acute intermittent hypoxia (AIH) induces a form of spinal motor plasticity known as phrenic long-term facilitation (pLTF); pLTF is a prolonged increase in phrenic motor output after AIH has ended. In anesthetized rats, we demonstrate that pLTF requires activity of the novel PKC isoform, PKCθ, and that the relevant PKCθ is within phrenic motor neurons. Whereas spinal PKCθ inhibitors block pLTF, inhibitors targeting other PKC isoforms do not. PKCθ is highly expressed in phrenic motor neurons, and PKCθ knockdown with intrapleural siRNAs abolishes pLTF. Intrapleural siRNAs targeting PKCζ, an atypical PKC isoform expressed in phrenic motor neurons that underlies a distinct form of phrenic motor plasticity, does not affect pLTF. Thus, PKCθ plays a critical role in spinal AIH-induced respiratory motor plasticity, and the relevant PKCθ is localized within phrenic motor neurons. Intrapleural siRNA delivery has considerable potential as a therapeutic tool to selectively manipulate plasticity in vital respiratory motor neurons. Copyright © 2015 the authors 0270-6474/15/358107-11$15.00/0.

  14. Reciprocal regulation of platelet responses to P2Y and thromboxane receptor activation.

    Science.gov (United States)

    Barton, J F; Hardy, A R; Poole, A W; Mundell, S J

    2008-03-01

    Thromboxane A(2) and ADP are two major platelet agonists that stimulate two sets of G protein-coupled receptors to activate platelets. Although aggregation responses to ADP and thromboxane desensitize, there are no reports currently addressing whether activation by one agonist may heterologously desensitize responses to the other. To demonstrate whether responses to ADP or U46619 may be modulated by prior treatment of platelets with the alternate agonist, revealing a level of cross-desensitization between receptor systems. Here we show that pretreatment of platelets with either agonist substantially desensitizes aggregation responses to the other agonist. Calcium responses to thromboxane receptor activation are desensitized by preactivation of P2Y(1) but not P2Y(12) receptors. This heterologous desensitization is mediated by a protein kinase C (PKC)-independent mechanism. Reciprocally, calcium responses to ADP are desensitized by pretreatment of platelets with the thromboxane analogue, U46619, and P2Y(12)-mediated inhibition of adenylate cyclase is also desensitized by pretreatment with U46619. In this direction, desensitization is comprised of two components, a true heterologous component that is PKC-independent, and a homologous component that is mediated through stimulated release of dense granule ADP. This study reveals cross-desensitization between ADP and thromboxane receptor signaling in human platelets. Cross-desensitization is mediated by protein kinases, involving PKC-dependent and independent pathways, and indicates that alterations in the activation state of one receptor may have effects upon the sensitivity of the other receptor system.

  15. PKC signaling is involved in the regulation of progranulin (acrogranin/PC-cell-derived growth factor/granulin-epithelin precursor) protein expression in human ovarian cancer cell lines.

    Science.gov (United States)

    Diaz-Cueto, Laura; Arechavaleta-Velasco, Fabian; Diaz-Arizaga, Adriana; Dominguez-Lopez, Pablo; Robles-Flores, Martha

    2012-07-01

    Overexpression of progranulin (also named acrogranin, PC-cell-derived growth factor, or granulin-epithelin precursor) is associated with ovarian cancer, specifically with cell proliferation, malignancy, chemoresistance, and shortened overall survival. The objective of the current study is to identify the signaling pathways involved in the regulation of progranulin expression in ovarian cancer cell lines. We studied the relation of protein kinase C (PKC), phosphatidylinositol 3-kinase, protein kinase A, P38, extracellular signal-regulated kinase, and Akt pathways on the modulation of progranulin expression levels in NIH-OVCAR-3 and SK-OV-3 ovarian cancer cell lines. The different pathways were examined using pharmacological inhibitors (calphostin C, LY294002, H89, SB203580, PD98059, and Akt Inhibitor), and mRNA and protein progranulin expression were analyzed by reverse transcriptase polymerase chain reaction and Western blot techniques, respectively. Inhibition of PKC signal transduction pathway by calphostin C decreased in a dose-dependent manner protein but not mRNA levels of progranulin in both ovarian cancer cell lines. LY294002 but not wortmannin, which are phosphatidylinositol 3-kinase inhibitors, also diminished the expression of progranulin in both cell lines. In addition, LY294002 treatment produced a significant reduction in cell viability. Inhibition of protein kinase A, P38, extracellular signal-regulated kinase, and Akt did not affect progranulin protein expression. These results suggest that the PKC signaling is involved in the regulation of progranulin protein expression in 2 different ovarian cancer cell lines. Inhibiting these intracellular signal transduction pathways may provide a future therapeutic target for hindering the cellular proliferation and invasion in ovarian cancer produced by progranulin.

  16. Deregulation of the actin cytoskeleton and macropinocytosis in response to phorbol ester by the mutant protein kinase C gamma that causes spinocerebellar ataxia type 14

    Directory of Open Access Journals (Sweden)

    Kazuhiro eYamamoto

    2014-04-01

    Full Text Available Several missense mutations in the protein kinase Cγ (γPKC gene have been found to cause spinocerebellar ataxia type 14 (SCA14, an autosomal dominant neurodegenerative disease. γPKC is a neuron-specific member of the classical PKCs and is activated and translocated to subcellular regions as a result of various stimuli, including diacylglycerol synthesis, increased intracellular Ca2+ and phorbol esters. We investigated whether SCA14 mutations affect the γPKC-related functions by stimulating HeLa cells with TPA (12-O-tetradecanoylpholbol 13-acetate, a type of phorbol ester. Wild-type (WT γPKC-GFP was translocated to the plasma membrane within 10 min of TPA stimulation, followed by its perinuclear translocation and cell shrinkage, in a PKC kinase activity- and microtubule-dependent manner. On the other hand, although SCA14 mutant γPKC-GFP exhibited a similar translocation to the plasma membrane, the subsequent perinuclear translocation and cell shrinkage were significantly impaired in response to TPA. Translocated WT γPKC colocalized with F-actin and formed large vesicular structures in the perinuclear region. The uptake of FITC-dextran, a marker of macropinocytosis, was promoted by TPA stimulation in cells expressing WT γPKC, and FITC-dextran was surrounded by γPKC-positive vesicles. Moreover, TPA induced the phosphorylation of MARCKS, which is a membrane-substrate of PKC, resulting in the translocation of phosphorylated MARCKS to the perinuclear region, suggesting that TPA induces macropinocytosis via γPKC activation. However, TPA failed to activate macropinocytosis and trigger the translocation of phosphorylated MARCKS in cells expressing the SCA14 mutant γPKC. These findings suggest that γPKC is involved in the regulation of the actin cytoskeleton and macropinocytosis in HeLa cells, while SCA14 mutant γPKC fails to regulate these processes due to its reduced kinase activity at the plasma membrane. This property might be involved in

  17. cAMP-dependent kinase does not modulate the Slack sodium-activated potassium channel.

    Science.gov (United States)

    Nuwer, Megan O; Picchione, Kelly E; Bhattacharjee, Arin

    2009-09-01

    The Slack gene encodes a Na(+)-activated K(+) channel and is expressed in many different types of neurons. Like the prokaryotic Ca(2+)-gated K(+) channel MthK, Slack contains two 'regulator of K(+) conductance' (RCK) domains within its carboxy terminal, domains likely involved in Na(+) binding and channel gating. It also contains multiple consensus protein kinase C (PKC) and protein kinase A (PKA) phosphorylation sites and although regulated by protein kinase C (PKC) phosphorylation, modulation by PKA has not been determined. To test if PKA directly regulates Slack, nystatin-perforated patch whole-cell currents were recorded from a human embryonic kidney (HEK-293) cell line stably expressing Slack. Bath application of forskolin, an adenylate cyclase activator, caused a rapid and complete inhibition of Slack currents however, the inactive homolog of forskolin, 1,9-dideoxyforskolin caused a similar effect. In contrast, bath application of 8-bromo-cAMP did not affect the amplitude nor the activation kinetics of Slack currents. In excised inside-out patch recordings, direct application of the PKA catalytic subunit to patches did not affect the open probability of Slack channels nor was open probability affected by direct application of protein phosphatase 2B. Preincubation of cells with the protein kinase A inhibitor KT5720 also did not change current density. Finally, mutating the consensus phosphorylation site located between RCK domain 1 and domain 2 from serine to glutamate did not affect current activation kinetics. We conclude that unlike PKC, phosphorylation by PKA does not acutely modulate the function and gating activation kinetics of Slack channels.

  18. Membrane Receptor-Induced Changes of the Protein Kinases A and C Activity May Play a Leading Role in Promoting Developmental Synapse Elimination at the Neuromuscular Junction.

    Science.gov (United States)

    Tomàs, Josep M; Garcia, Neus; Lanuza, Maria A; Nadal, Laura; Tomàs, Marta; Hurtado, Erica; Simó, Anna; Cilleros, Víctor

    2017-01-01

    Synapses that are overproduced during histogenesis in the nervous system are eventually lost and connectivity is refined. Membrane receptor signaling leads to activity-dependent mutual influence and competition between axons directly or with the involvement of the postsynaptic cell and the associated glial cell/s. Presynaptic muscarinic acetylcholine (ACh) receptors (subtypes mAChR; M 1 , M 2 and M 4 ), adenosine receptors (AR; A 1 and A 2A ) and the tropomyosin-related kinase B receptor (TrkB), among others, all cooperate in synapse elimination. Between these receptors there are several synergistic, antagonic and modulatory relations that clearly affect synapse elimination. Metabotropic receptors converge in a limited repertoire of intracellular effector kinases, particularly serine protein kinases A and C (PKA and PKC), to phosphorylate protein targets and bring about structural and functional changes leading to axon loss. In most cells A 1 , M 1 and TrkB operate mainly by stimulating PKC whereas A 2A , M 2 and M 4 inhibit PKA. We hypothesize that a membrane receptor-induced shifting in the protein kinases A and C activity (inhibition of PKA and/or stimulation of PKC) in some nerve endings may play an important role in promoting developmental synapse elimination at the neuromuscular junction (NMJ). This hypothesis is supported by: (i) the tonic effect (shown by using selective inhibitors) of several membrane receptors that accelerates axon loss between postnatal days P5-P9; (ii) the synergistic, antagonic and modulatory effects (shown by paired inhibition) of the receptors on axonal loss; (iii) the fact that the coupling of these receptors activates/inhibits the intracellular serine kinases; and (iv) the increase of the PKA activity, the reduction of the PKC activity or, in most cases, both situations simultaneously that presumably occurs in all the situations of singly and paired inhibition of the mAChR, AR and TrkB receptors. The use of transgenic animals and

  19. Membrane Receptor-Induced Changes of the Protein Kinases A and C Activity May Play a Leading Role in Promoting Developmental Synapse Elimination at the Neuromuscular Junction

    Directory of Open Access Journals (Sweden)

    Josep M. Tomàs

    2017-08-01

    Full Text Available Synapses that are overproduced during histogenesis in the nervous system are eventually lost and connectivity is refined. Membrane receptor signaling leads to activity-dependent mutual influence and competition between axons directly or with the involvement of the postsynaptic cell and the associated glial cell/s. Presynaptic muscarinic acetylcholine (ACh receptors (subtypes mAChR; M1, M2 and M4, adenosine receptors (AR; A1 and A2A and the tropomyosin-related kinase B receptor (TrkB, among others, all cooperate in synapse elimination. Between these receptors there are several synergistic, antagonic and modulatory relations that clearly affect synapse elimination. Metabotropic receptors converge in a limited repertoire of intracellular effector kinases, particularly serine protein kinases A and C (PKA and PKC, to phosphorylate protein targets and bring about structural and functional changes leading to axon loss. In most cells A1, M1 and TrkB operate mainly by stimulating PKC whereas A2A, M2 and M4 inhibit PKA. We hypothesize that a membrane receptor-induced shifting in the protein kinases A and C activity (inhibition of PKA and/or stimulation of PKC in some nerve endings may play an important role in promoting developmental synapse elimination at the neuromuscular junction (NMJ. This hypothesis is supported by: (i the tonic effect (shown by using selective inhibitors of several membrane receptors that accelerates axon loss between postnatal days P5–P9; (ii the synergistic, antagonic and modulatory effects (shown by paired inhibition of the receptors on axonal loss; (iii the fact that the coupling of these receptors activates/inhibits the intracellular serine kinases; and (iv the increase of the PKA activity, the reduction of the PKC activity or, in most cases, both situations simultaneously that presumably occurs in all the situations of singly and paired inhibition of the mAChR, AR and TrkB receptors. The use of transgenic animals and various

  20. PKCδ-dependent p47phox activation mediates methamphetamine-induced dopaminergic neurotoxicity.

    Science.gov (United States)

    Dang, Duy-Khanh; Shin, Eun-Joo; Kim, Dae-Joong; Tran, Hai-Quyen; Jeong, Ji Hoon; Jang, Choon-Gon; Ottersen, Ole Petter; Nah, Seung-Yeol; Hong, Jau-Shyong; Nabeshima, Toshitaka; Kim, Hyoung-Chun

    2018-02-01

    Protein kinase C (PKC) has been recognized to activate NADPH oxidase (PHOX). However, the interaction between PKC and PHOX in vivo remains elusive. Treatment with methamphetamine (MA) resulted in a selective increase in PKCδ expression out of PKC isoforms. PKCδ co-immunoprecipitated with p47phox, and facilitated phosphorylation and membrane translocation of p47phox. MA-induced increases in PHOX activity and reactive oxygen species were attenuated by knockout of p47phox or PKCδ. In addition, MA-induced impairments in the Nrf-2-related glutathione synthetic system were also mitigated by knockout of p47phox or PKCδ. Glutathione-immunoreactivity was co-localized in Iba-1-labeled microglial cells and in NeuN-labeled neurons, but not in GFAP-labeled astrocytes, reflecting the necessity for self-protection against oxidative stress by mainly microglia. Buthionine-sulfoximine, an inhibitor of glutathione biosynthesis, potentiated microglial activation and pro-apoptotic changes, leading to dopaminergic losses. These neurotoxic processes were attenuated by rottlerin, a pharmacological inhibitor of PKCδ, genetic inhibitions of PKCδ [i.e., PKCδ knockout mice (KO) and PKCδ antisense oligonucleotide (ASO)], or genetic inhibition of p47phox (i.e., p47phox KO or p47phox ASO). Rottlerin did not exhibit any additive effects against the protective activity offered by genetic inhibition of p47phox. Therefore, we suggest that PKCδ is a critical regulator for p47phox activation induced by MA, and that Nrf-2-dependent GSH induction via inhibition of PKCδ or p47phox, is important for dopaminergic protection against MA insult. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Doxazosin stimulates galectin-3 expression and collagen synthesis in HL-1 cardiomyocytes independent of protein kinase C pathway

    Directory of Open Access Journals (Sweden)

    Xiaoqian Qian

    2016-12-01

    Full Text Available Doxazosin, a drug commonly prescribed for hypertension and prostate disease, increases heart failure risk. However, the underlying mechanism remains unclear. Galectin-3 is an important mediator that plays a pathogenic role in cardiac hypertrophy and heart failure. In the present study, we investigated whether doxazosin could stimulate galectin-3 expression and collagen synthesis in cultured HL-1 cardiomyocytes. We found that doxazosin dose-dependently induced galectin-3 protein expression, with a statistically significant increase in expression with a dose as low as 0.01 μM. Doxazosin upregulated collagen I and α-smooth muscle actin (α-SMA protein levels and also induced apoptotic protein caspase-3 in HL-1 cardiomyocytes. Although we previously reported that activation of protein kinase C (PKC stimulates galectin-3 expression, blocking the PKC pathway with the PKC inhibitor chelerythrine did not prevent doxazosin-induced galectin-3 and collagen expression. Consistently, doxazosin treatment did not alter total and phosphorylated PKC. These results suggest that doxazosin-stimulated galectin-3 is independent of PKC pathway. To determine if the α1-adrenergic pathway is involved, we pretreated the cells with the irreversible α-adrenergic receptor blocker phenoxybenzamine and found that doxazosin-stimulated galectin-3 and collagen expression was similar to controls, suggesting that doxazosin acts independently of α1-adrenergic receptor blockade. Collectively, we show a novel effect of doxazosin on cardiomycytes by stimulating heart fibrosis factor galectin-3 expression. The mechanism of action of doxazosin is not mediated through either activation of the PKC pathway or antagonism of α1-adrenergic receptors.

  2. VHH Activators and Inhibitors for Protein Kinase C Epsilon

    NARCIS (Netherlands)

    Summanen, M.M.I.

    2012-01-01

    Protein kinase C epsilon (PKCε), which is one of the novel PKC isozymes, is widely expressed throughout the body and has important roles in the function of the nervous, cardiovascular and immune systems. In order to better understand PKCε regulated pathways, isozyme specific activity modulators are

  3. Interaction between protein kinase C and protein kinase A can modulate transmitter release at the rat neuromuscular synapse.

    Science.gov (United States)

    Santafé, M M; Garcia, N; Lanuza, M A; Tomàs, M; Tomàs, J

    2009-02-15

    We used intracellular recording to investigate the functional interaction between protein kinase C (PKC) and protein kinase A (PKA) signal transduction cascades in the control of transmitter release in the neuromuscular synapses from adult rats. Our results indicate that: 1) PKA and PKC are independently involved in asynchronous release. 2) Evoked acetylcholine (ACh) release is enhanced with the PKA agonist Sp-8-BrcAMP and the PKC agonist phorbol ester (PMA). 3) PKA has a constitutive role in promoting a component of normal evoked transmitter release because, when the kinase is inhibited with H-89, the release diminishes. However, the PKC inhibitor calphostin C (CaC) does not affect ACh release. 4) PKA regulates neurotransmission without PKC involvement because, after PMA or CaC modulation of the PKC activity, coupling to the ACh release of PKA can normally be stimulated with Sp-8-BrcAMP or inhibited with H-89. 5) After PKA inhibition with H-89, PKC stimulation with PMA (or inhibition with CaC) does not lead to any change in evoked ACh release. However, in PKA-stimulated preparations with Sp-8-BrcAMP, PKC becomes tonically active, thus potentiating a component of release that can now be blocked with CaC. In normal conditions, therefore, PKA was able to modulate ACh release independently of PKC activity, whereas PKA stimulation caused the PKC coupling to evoked release. In contrast, PKA inhibition prevent PKC stimulation (with the phorbol ester) and coupling to ACh output. There was therefore some dependence of PKC on PKA activity in the fine control of the neuromuscular synaptic functionalism and ACh release.

  4. Total phenolics and antioxidant activity of five medicinal plant

    International Nuclear Information System (INIS)

    Sousa, Cleyton Marcos de M.; Silva, Hilris Rocha e; Vieira-Junior, Gerardo Magela; Ayres, Mariane Cruz C.; Costa, Charllyton Luis S. da; Araajo, Delton Servulo; Cavalcante, Luis Carlos D.; Barros, Elcio Daniel S.; Araujo, Paulo Breitner de M.; Brandao, Marcela S.; Chaves, Mariana H.

    2007-01-01

    This paper describes total phenolics content and antioxidant activity in the ethanolic extract of leaves, bark and roots of five medicinal plants: Terminalia brasiliensis Camb., Terminalia fagifolia Mart. and Zucc., Copernicia cerifera (Miller) H.E. Moore, Cenostigma macrophyllum Tul. var. acuminata Teles Freire and Qualea grandiflora Mart. The total phenolics content of the plant extracts, determined by the Folin-Ciocalteu method, varied from 250.0 ±8,2 to 763,63 ±13.03 mg of gallic acid equivalent/g dry EtOH extract. The antioxidant activity of extracts was evaluated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay system. Extract of bark from T. brasiliensis, the most active, with an EC 50 value of 27.59 ± 0.82 μg/mL, was comparable to rutin (EC 50 = 27.80 ± 1.38) and gallic acid (EC 50 = 24.27 ± 0.31), used as positive controls. The relationship between total phenolic content and antioxidant activity was positive and significant for T. brasiliensis, C. macrophyllum and C. cerifera. (author)

  5. Phytochemical screening, total phenolic, total flavonoids contents and antioxidant activity of cinchona ledgeriana leaves ethanol extract

    Science.gov (United States)

    Sundowo, Andini; Artanti, Nina; Hanafi, M.; Minarti, Primahana, Gian

    2017-11-01

    C ledgeriana is a medicinal plant that contains alkaloids, especially on the barks for commercial production of quinine as antimalarial. The main alkaloids in this plant are cinchonine, cinchonidine, quinine and quinidine. Besides for antiamalarial this plant is also commonly used to treat whooping cough, influenza and dysentery. Compare to other medicinal plants, nowadays only very few studies were conducted in Cinchona species. Our current study aims to determine the content of phytochemical, total phenol and total flavonoids from C. ledgeriana leaves 70% ethanol extract. The extraction was performed by maceration method using 70% ethanol solvent and then fractionated into hexane, ethylacetate and butanol. Phytochemical screening was performed to determine the content of alkaloids, flavonoids, terpenoids, tannins and saponins. Total phenol and flavonoid contents of the extract were determined by Folin-Ciocalteu and alumunium chloride colorimetric methods using gallic acid and quercetin as standards. The antioxidant activity was determined by using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity. The results of phytochemical screening showed that the 70% ethanol extract of C. ledgeriana leaves contained alkaloids, flavonoids, terpenoids, tannins and saponins. The total phenol and total flavonoids analysis showed that ethyl acetate fraction had the highest total phenol (40.23%) and total flavonoids (65.34%).

  6. Effect of feeding palm oil by-products based diets on total bacteria, cellulolytic bacteria and methanogenic archaea in the rumen of goats.

    Science.gov (United States)

    Abubakr, Abdelrahim; Alimon, Abdul Razak; Yaakub, Halimatun; Abdullah, Norhani; Ivan, Michael

    2014-01-01

    Rumen microorganisms are responsible for digestion and utilization of dietary feeds by host ruminants. Unconventional feed resources could be used as alternatives in tropical areas where feed resources are insufficient in terms of quality and quantity. The objective of the present experiment was to evaluate the effect of diets based on palm oil (PO), decanter cake (DC) or palm kernel cake (PKC) on rumen total bacteria, selected cellulolytic bacteria, and methanogenic archaea. Four diets: control diet (CD), decanter cake diet (DCD), palm kernel cake diet (PKCD) and CD plus 5% PO diet (CPOD) were fed to rumen cannulated goats and rumen samples were collected at the start of the experimental diets (day 0) and on days 4, 6, 8, 12, 18, 24 and 30 post dietary treatments. Feeding DCD and PKCD resulted in significantly higher (Pgoats fed PKCD and CPOD and the trend showed a severe reduction on days 4 and 6 post experimental diets. In conclusion, results indicated that feeding DCD and PKC increased the populations of cellulolytic bacteria and decreased the density of methanogenic archaea in the rumen of goats.

  7. Protein kinase C prevents oligodendrocyte differentiation : Modulation of actin cytoskeleton and cognate polarized membrane traffic

    NARCIS (Netherlands)

    Baron, W; de Vries, EJ; de Vries, H; Hoekstra, D

    1999-01-01

    In a previous study, we showed that activation of protein kinase C (PKC) prevents oligodendrocyte differentiation at the pro-oligodendrocyte stage. The present study was undertaken to identify downstream targets of PKC action in oligodendrocyte progenitor cells. Activation of PKC induced the

  8. The antitumor natural product tanshinone IIA inhibits protein kinase C and acts synergistically with 17-AAG.

    Science.gov (United States)

    Lv, Chao; Zeng, Hua-Wu; Wang, Jin-Xin; Yuan, Xing; Zhang, Chuang; Fang, Ting; Yang, Pei-Ming; Wu, Tong; Zhou, Yu-Dong; Nagle, Dale G; Zhang, Wei-Dong

    2018-02-07

    Tanshinone IIA (Tan IIA), the primary bioactive compound derived from the traditional Chinese medicine (TCM) Salvia miltiorrhiza Bunge, has been reported to possess antitumor activity. However, its antitumor mechanisms are not fully understood. To resolve the potential antitumor mechanism(s) of Tan IIA, its gene expression profiles from our database was analyzed by connectivity map (CMAP) and the CMAP-based mechanistic predictions were confirmed/validated in further studies. Specifically, Tan IIA inhibited total protein kinase C (PKC) activity and selectively suppressed the expression of cytosolic and plasma membrane PKC isoforms ζ and ε. The Ras/MAPK pathway that is closely regulated by the PKC signaling is also inhibited by Tan IIA. While Tan IIA did not inhibit heat shock protein 90 (Hsp90), it synergistically enhanced the antitumor efficacy of the Hsp90 inhibitors 17-AAG and ganetespib in human breast cancer MCF-7 cells. In addition, Tan IIA significantly inhibited PI3K/Akt/mTOR signaling, and induced both cell cycle arrest and autophagy. Collectively, these studies provide new insights into the molecular mechanisms responsible for antitumor activity of Tan IIA.

  9. Antioxidant Activities of Total Pigment Extract from Blackberries

    Directory of Open Access Journals (Sweden)

    Jiechao Liu

    2005-01-01

    Full Text Available Total pigment has been extracted from blackberries and its antioxidant activity against lipid peroxidation and scavenging capacities towards superoxide anion radicals, hydroxyl radicals and nitrite in different in vitro systems have been investigated. The total pigment extract from blackberries (TPEB exhibited strong antioxidant activity against lipid peroxidation in a linoleic acid model system and scavenging capacities towards superoxide anion radicals, generated by a pyrogallol autoxidation system or by an illuminating riboflavin system, hydroxyl radicals generated by Fenton reaction, and nitrite. Furthermore, the antioxidant activities were correlated with the concentrations of the TPEB. In the test concentration range, the maximum inhibition percentage against linoleic acid peroxidation was 98.32 % after one week’s incubation, and the maximum scavenging percentages for the free radicals and nitrite inhibition in the above reactive systems reached 90.48, 96.48, 93.58 and 98.94 %, respectively. The TPEB is a natural, edible colorant with excellent antioxidant activities and health benefits and it seems to be applicable in both healthy food and medicine.

  10. Brief pressure overload of the left ventricle reduces myocardial infarct size via activation of protein kinase C.

    Science.gov (United States)

    Tang, Chia-Yu; Lai, Chang-Chi; Chiang, Shu-Chiung; Tseng, Kuo-Wei; Huang, Cheng-Hsiung

    2015-09-01

    We have previously reported that brief pressure overload of the left ventricle reduced myocardial infarct (MI) size. However, the role of protein kinase C (PKC) remains uncertain. In this study, we investigated whether pressure overload reduces MI size by activating PKC. MI was induced by a 40-minute occlusion of the left anterior descending coronary artery and a 3-hour reperfusion in anesthetized Sprague-Dawley rats. MI size was determined using triphenyl tetrazolium chloride staining. Brief pressure overload was achieved by two 10-minute partial snarings of the ascending aorta, raising the systolic left ventricular pressure 50% above the baseline value. Ischemic preconditioning was elicited by two 10-minute coronary artery occlusions and 10-minute reperfusions. Dimethyl sulfoxide (vehicle) or calphostin C (0.1 mg/kg, a specific inhibitor of PKC) was administered intravenously as pretreatment. The MI size, expressed as the percentage of the area at risk, was significantly reduced in the pressure overload group and the ischemic preconditioning group (19.0 ± 2.9% and 18.7 ± 3.0% vs. 26.1 ± 2.6% in the control group, where p overload and ischemic preconditioning (25.2 ± 2.4% and 25.0 ± 2.3%, where p overload of the left ventricle reduced MI size. Since calphostin C significantly limited the decrease of MI size, our results suggested that brief pressure overload reduces MI size via activation of PKC. Copyright © 2015. Published by Elsevier Taiwan.

  11. Corydalis edulis Maxim. Promotes Insulin Secretion via the Activation of Protein Kinase Cs (PKCs) in Mice and Pancreatic β Cells.

    Science.gov (United States)

    Zheng, Jiao; Zhao, Yunfang; Lun, Qixing; Song, Yuelin; Shi, Shepo; Gu, Xiaopan; Pan, Bo; Qu, Changhai; Li, Jun; Tu, Pengfei

    2017-01-16

    Corydalis edulis Maxim., a widely grown plant in China, had been proposed for the treatment for type 2 diabetes mellitus. In this study, we found that C. edulis extract (CE) is protective against diabetes in mice. The treatment of hyperglycemic and hyperlipidemic apolipoprotein E (ApoE)-/- mice with a high dose of CE reduced serum glucose by 28.84% and serum total cholesterol by 17.34% and increased insulin release. We also found that CE significantly enhanced insulin secretion in a glucose-independent manner in hamster pancreatic β cell (HIT-T15). Further investigation revealed that CE stimulated insulin exocytosis by a protein kinase C (PKC)-dependent signaling pathway and that CE selectively activated novel protein kinase Cs (nPKCs) and atypical PKCs (aPKCs) but not conventional PKCs (cPKCs) in HIT-T15 cells. To the best of our knowledge, our study is the first to identify the PKC pathway as a direct target and one of the major mechanisms underlying the antidiabetic effect of CE. Given the good insulinotropic effect of this herbal medicine, CE is a promising agent for the development of new drugs for treating diabetes.

  12. Topical application of a protein kinase C inhibitor reduces skin and hair pigmentation

    NARCIS (Netherlands)

    Park, Hee-Young; Lee, Jin; González, Salvador; Middelkamp-Hup, Maritza A.; Kapasi, Sameer; Peterson, Shaun; Gilchrest, Barbara A.

    2004-01-01

    To determine whether inhibition of PKC-beta activity decreases pigmentation, paired cultures of primary human melanocytes were first pretreated with bisindolylmaleimide (Bis), a selective PKC inhibitor, or vehicle alone for 30 min, and then treated with TPA for an additional 90 min to activate PKC

  13. The hepatitis B virus large surface protein (LHBs) is a transcriptional activator.

    Science.gov (United States)

    Hildt, E; Saher, G; Bruss, V; Hofschneider, P H

    1996-11-01

    It has been shown that a C-terminally truncated form of the middle-sized hepatitis B virus (HBV) surface protein (MHBst) functions as a transcriptional activator. This function is dependent on the cytosolic orientation of the N-terminal PreS2 domain of MHBst, but in the case of wild-type MHBs, the PreS2 domain is contranslationally translocated into the ER lumen. Recent reports demonstrated that the PreS2 domain of the large HBV surface protein (LHBs) initially remains on the cytosolic side of the ER membrane after translation. Therefore, the question arose as to whether the LHBs protein exhibits the same transcriptional activator function as MHBst. We show that LHBs, like MHBst, is indeed able to activate a variety of promoter elements. There is evidence for a PKC-dependent activation of AP-1 and NF-kappa B by LHBs. Downstream of the PKC the functionality of c-Raf-1 kinase is a prerequisite for LHBs-dependent activation of AP-1 and NF-kappa B since inhibition of c-Raf-1 kinase abolishes LHBs-dependent transcriptional activation of AP-1 and NF-kappa B.

  14. Protein kinase C activation decreases cell surface expression of the GLT-1 subtype of glutamate transporter. Requirement of a carboxyl-terminal domain and partial dependence on serine 486.

    Science.gov (United States)

    Kalandadze, Avtandil; Wu, Ying; Robinson, Michael B

    2002-11-29

    Na(+)-dependent glutamate transporters are required for the clearance of extracellular glutamate and influence both physiological and pathological effects of this excitatory amino acid. In the present study, the effects of a protein kinase C (PKC) activator on the cell surface expression and activity of the GLT-1 subtype of glutamate transporter were examined in two model systems, primary co-cultures of neurons and astrocytes that endogenously express GLT-1 and C6 glioma cells transfected with GLT-1. In both systems, activation of PKC with phorbol ester caused a decrease in GLT-1 cell surface expression. This effect is opposite to the one observed for the EAAC1 subtype of glutamate transporter (Davis, K. E., Straff, D. J., Weinstein, E. A., Bannerman, P. G., Correale, D. M., Rothstein, J. D., and Robinson, M. B. (1998) J. Neurosci. 18, 2475-2485). Several recombinant chimeric proteins between GLT-1 and EAAC1 transporter subtypes were generated to identify domains required for the subtype-specific redistribution of GLT-1. We identified a carboxyl-terminal domain consisting of 43 amino acids (amino acids 475-517) that is required for PKC-induced GLT-1 redistribution. Mutation of a non-conserved serine residue at position 486 partially attenuated but did not completely abolish the PKC-dependent redistribution of GLT-1. Although we observed a phorbol ester-dependent incorporation of (32)P into immunoprecipitable GLT-1, mutation of serine 486 did not reduce this signal. We also found that chimeras containing the first 446 amino acids of GLT-1 were not functional unless amino acids 475-517 of GLT-1 were also present. These non-functional transporters were not as efficiently expressed on the cell surface and migrated to a smaller molecular weight, suggesting that a subtype-specific interaction is required for the formation of functional transporters. These studies demonstrate a novel effect of PKC on GLT-1 activity and define a unique carboxyl-terminal domain as an

  15. Splenectomy after partial hepatectomy accelerates liver regeneration in mice by promoting tight junction formation via polarity protein Par 3-aPKC.

    Science.gov (United States)

    Liu, Guoxing; Xie, Chengzhi; Fang, Yu; Qian, Ke; Liu, Qiang; Liu, Gao; Cao, Zhenyu; Du, Huihui; Fu, Jie; Xu, Xundi

    2018-01-01

    Several experimental studies have demonstrated that removal of the spleen accelerates liver regeneration after partial hepatectomy. While the mechanism of splenectomy promotes liver regeneration by the improvement of the formation of tight junction and the establishment of hepatocyte polarity is still unknown. We analyzed the cytokines, genes and proteins expression between 70% partial hepatectomy mice (PHx) and simultaneous 70% partial hepatectomy and splenectomy mice (PHs) at predetermined timed points. Compared with the PHx group mice, splenectomy accelerated hepatocyte proliferation in PHs group. The expression of Zonula occludens-1 (ZO-1) indicated that splenectomy promotes the formation of tight junction during liver regeneration. TNF-α, IL-6, HGF, TSP-1 and TGF-β1 were essential factors for the formation of tight junction and the establishment of hepatocytes polarity in liver regeneration. After splenectomy, Partitioning defective 3 homolog (Par 3) and atypical protein kinase C (aPKC) regulate hepatocyte localization and junctional structures in regeneration liver. Our data suggest that the time course expression of TNF-α, IL-6, HGF, TSP-1, and TGF-β1 and the change of platelets take part in liver regeneration. Combination with splenectomy accelerates liver regeneration by improvement of the tight junction formation which may help to establish hepatocyte polarity via Par 3-aPKC. This may provide a clue for us that splenectomy could accelerate liver regeneration after partial hepatectomy of hepatocellular carcinoma and living donor liver transplantation. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Protein kinase C isoforms at the neuromuscular junction: localization and specific roles in neurotransmission and development.

    Science.gov (United States)

    Lanuza, Maria A; Santafe, Manel M; Garcia, Neus; Besalduch, Núria; Tomàs, Marta; Obis, Teresa; Priego, Mercedes; Nelson, Phillip G; Tomàs, Josep

    2014-01-01

    The protein kinase C family (PKC) regulates a variety of neural functions including neurotransmitter release. The selective activation of a wide range of PKC isoforms in different cells and domains is likely to contribute to the functional diversity of PKC phosphorylating activity. In this review, we describe the isoform localization, phosphorylation function, regulation and signalling of the PKC family at the neuromuscular junction. Data show the involvement of the PKC family in several important functions at the neuromuscular junction and in particular in the maturation of the synapse and the modulation of neurotransmission in the adult. © 2013 Anatomical Society.

  17. Cyclic nucleotides and mitogen-activated protein kinases: regulation of simvastatin in platelet activation

    Directory of Open Access Journals (Sweden)

    Hou Ssu-Yu

    2010-06-01

    Full Text Available Abstract Background 3-Hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA reductase inhibitors (statins have been widely used to reduce cardiovascular risk. These statins (i.e., simvastatin may exert other effects besides from their cholesterol-lowering actions, including inhibition of platelet activation. Platelet activation is relevant to a variety of coronary heart diseases. Although the inhibitory effect of simvastatin in platelet activation has been studied; the detailed signal transductions by which simvastatin inhibit platelet activation has not yet been completely resolved. Methods The aim of this study was to systematically examine the detailed mechanisms of simvastatin in preventing platelet activation. Platelet aggregation, flow cytometric analysis, immunoblotting, and electron spin resonance studies were used to assess the antiplatelet activity of simvastatin. Results Simvastatin (20-50 μM exhibited more-potent activity of inhibiting platelet aggregation stimulated by collagen than other agonists (i.e., thrombin. Simvastatin inhibited collagen-stimulated platelet activation accompanied by [Ca2+]i mobilization, thromboxane A2 (TxA2 formation, and phospholipase C (PLCγ2, protein kinase C (PKC, and mitogen-activated protein kinases (i.e., p38 MAPK, JNKs phosphorylation in washed platelets. Simvastatin obviously increased both cyclic AMP and cyclic GMP levels. Simvastatin markedly increased NO release, vasodilator-stimulated phosphoprotein (VASP phosphorylation, and endothelial nitric oxide synthase (eNOS expression. SQ22536, an inhibitor of adenylate cyclase, markedly reversed the simvastatin-mediated inhibitory effects on platelet aggregation, PLCγ2 and p38 MAPK phosphorylation, and simvastatin-mediated stimulatory effects on VASP and eNOS phosphorylation. Conclusion The most important findings of this study demonstrate for the first time that inhibitory effect of simvastatin in platelet activation may involve activation of the cyclic AMP

  18. Bisphenol A differentially activates protein kinase C isoforms in murine placental tissue

    Energy Technology Data Exchange (ETDEWEB)

    Tan, Wenjuan; Huang, Hui; Wang, Yanfei [Biochemistry Programme, School of Life Sciences, Faculty of Science, The Chinese University of Hong Kong, Shatin, N.T. (Hong Kong); Wong, Tsz Yan [Food and Nutritional Sciences Programme, School of Life Sciences, Faculty of Science, The Chinese University of Hong Kong, Shatin, N.T. (Hong Kong); Wang, C.C. [Department of Obstetrics and Gynecology, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, N.T. (Hong Kong); Leung, Lai K., E-mail: laikleung@cuhk.edu.hk [Biochemistry Programme, School of Life Sciences, Faculty of Science, The Chinese University of Hong Kong, Shatin, N.T. (Hong Kong); Food and Nutritional Sciences Programme, School of Life Sciences, Faculty of Science, The Chinese University of Hong Kong, Shatin, N.T. (Hong Kong)

    2013-06-01

    Bisphenol A is utilized to make polycarbonate plastics and is an environmental pollutant. Recent research has indicated that it is an endocrine disruptor and may interfere with reproductive processes. Our lab has previously shown that bisphenol A could regulate corticotrophin releasing hormone and aromatase in cultured placental cells. In the present study, the effect of bisphenol A on these two genes in the placenta was investigated in mice. Pregnant ICR mice were gavaged with bisphenol A at 2, 20 and 200 mg/kg body weight/day from E13 to E16 and were euthanized at E17. Compared to the control mice, increased plasma estrogen and corticotrophin releasing hormone were observed in bisphenol A-treated mice. Messenger RNA quantification indicated that placental crh but not cyp19 was induced in mice treated with bisphenol A. Tracking the related signaling pathway, we found that protein kinase C ζ/λ and δ were activated in the placentas of bisphenol A-treated mice. As the gene promoter of crh contains CRE and the half site of ERE, either phospho-PKC or estrogen could stimulate the gene transactivation. These results indicate that bisphenol A might increase plasma concentrations of estradiol, testosterone, corticotrophin releasing hormone and placental phospho-PKC ζ/λ and δ in mice. Ultimately, the incidence of premature birth in these mice could increase. - Highlights: • The pollutant bisphenol A differentially activated PKC isoforms in the placenta. • CRE-binding activity in the nuclear protein of placenta was increased. • Bisphenol A induces CRH mRNA expression in mice.

  19. Bisphenol A differentially activates protein kinase C isoforms in murine placental tissue

    International Nuclear Information System (INIS)

    Tan, Wenjuan; Huang, Hui; Wang, Yanfei; Wong, Tsz Yan; Wang, C.C.; Leung, Lai K.

    2013-01-01

    Bisphenol A is utilized to make polycarbonate plastics and is an environmental pollutant. Recent research has indicated that it is an endocrine disruptor and may interfere with reproductive processes. Our lab has previously shown that bisphenol A could regulate corticotrophin releasing hormone and aromatase in cultured placental cells. In the present study, the effect of bisphenol A on these two genes in the placenta was investigated in mice. Pregnant ICR mice were gavaged with bisphenol A at 2, 20 and 200 mg/kg body weight/day from E13 to E16 and were euthanized at E17. Compared to the control mice, increased plasma estrogen and corticotrophin releasing hormone were observed in bisphenol A-treated mice. Messenger RNA quantification indicated that placental crh but not cyp19 was induced in mice treated with bisphenol A. Tracking the related signaling pathway, we found that protein kinase C ζ/λ and δ were activated in the placentas of bisphenol A-treated mice. As the gene promoter of crh contains CRE and the half site of ERE, either phospho-PKC or estrogen could stimulate the gene transactivation. These results indicate that bisphenol A might increase plasma concentrations of estradiol, testosterone, corticotrophin releasing hormone and placental phospho-PKC ζ/λ and δ in mice. Ultimately, the incidence of premature birth in these mice could increase. - Highlights: • The pollutant bisphenol A differentially activated PKC isoforms in the placenta. • CRE-binding activity in the nuclear protein of placenta was increased. • Bisphenol A induces CRH mRNA expression in mice

  20. Effect of extraction solvent on total phenol content, total flavonoid content, and antioxidant activity of Limnophila aromatica

    Directory of Open Access Journals (Sweden)

    Quy Diem Do

    2014-09-01

    Full Text Available Limnophila aromatica is commonly used as a spice and a medicinal herb in Southeast Asia. In this study, water and various concentrations (50%, 75%, and 100% of methanol, ethanol, and acetone in water were used as solvent in the extraction of L. aromatica. The antioxidant activity, total phenolic content, and total flavonoid content of the freeze-dried L. aromatica extracts were investigated using various in vitro assays. The extract obtained by 100% ethanol showed the highest total antioxidant activity, reducing power and DPPH (2,2-diphenyl-1-picrylhydrazyl radical scavenging activity. The same extract also exhibited the highest phenolic content (40.5 mg gallic acid equivalent/g of defatted L. aromatica and the highest flavonoid content (31.11 mg quercetin equivalent/g of defatted L. aromatica. The highest extraction yield was obtained by using 50% aqueous acetone. These results indicate that L. aromatica can be used in dietary applications with a potential to reduce oxidative stress.

  1. Endo- and exocytic rate constants for spontaneous and protein kinase C-activated T cell receptor cycling

    DEFF Research Database (Denmark)

    Menné, Charlotte; Møller Sørensen, Tine; Siersma, Volkert

    2002-01-01

    To determine the rate constants of spontaneous and activated TCR cycling, we examined TCR endo- and exocytosis in the human T cell line Jurkat by three different methods. Using a simple kinetic model for TCR cycling and non-linear regression analyses, we found that the spontaneous endocytic rate...... constant of the TCR was low (approximately 0.012 min(-1)) whereas the spontaneous exocytic rate constant was similar to that of other cycling receptors (approximately 0.055 min(-1)). Following protein kinase C activation (PKC) the endocytic rate constant was increased tenfold (to approximately 0.128 min(-1......)) whereas the exocytic rate constant was unaffected. Thus, the TCR becomes a rapidly cycling receptor with kinetics similar to classical cycling receptors subsequent to PKC activation. This results in a reduction of the half-life of cell surface expressed TCR from approximately 58 to 6 min and allows rapid...

  2. Association of atypical protein kinase C isotypes with the docker protein FRS2 in fibroblast growth factor signaling.

    Science.gov (United States)

    Lim, Y P; Low, B C; Lim, J; Wong, E S; Guy, G R

    1999-07-02

    FRS2 is a docker protein that recruits signaling proteins to the plasma membrane in fibroblast growth factor signal transduction. We report here that FRS2 was associated with PKC lambda when Swiss 3T3 cells were stimulated with basic fibroblast growth factor. PKC zeta, the other member of the atypical PKC subfamily, could also bind FRS2. The association between FRS2 and PKC lambda is likely to be direct as shown by yeast two-hybrid analysis. The C-terminal fragments of FRS2 (amino acid residues 300-508) and SNT2 (amino acids 281-492), an isoform bearing 50% identity to FRS2, interacted with PKC lambda at a region (amino acids 240-562) that encompasses the catalytic domain. In vitro kinase assays revealed neither FRS2 nor SNT2 was a substrate of PKC lambda or zeta. Mutation of the alanine residue (Ala-120) to glutamate in the pseudo-substrate region of PKC lambda results in a constitutively active kinase that exhibited more than 2-fold greater binding to FRS2 in vitro than its "closed" wild-type counterpart. Tyrosine phosphorylation of FRS2 did not affect its binding to the constitutively active PKC lambda mutant, suggesting that the activation of PKC lambda is necessary and sufficient for its association with FRS2. It is likely that FRS2 serves as an anchoring protein for targeting activated atypical PKCs to the cell plasma membrane in signaling pathways.

  3. PKC-alpha modulation by miR-483-3p in platinum-resistant ovarian carcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Arrighetti, Noemi, E-mail: Noemi.Arrighetti@istitutotumori.mi.it [Molecular Pharmacology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, via Amadeo 42, Milan 20133 (Italy); Cossa, Giacomo, E-mail: Gia.Cossa@gmail.com [Molecular Pharmacology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, via Amadeo 42, Milan 20133 (Italy); De Cecco, Loris, E-mail: Loris.Dececco@istitutotumori.mi.it [Functional Genomics and Bioinformatics, Fondazione IRCCS Istituto Nazionale dei Tumori, via Amadeo 42, Milan 20133 (Italy); Stucchi, Simone, E-mail: Simone.Stucchi@istitutotumori.mi.it [Molecular Pharmacology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, via Amadeo 42, Milan 20133 (Italy); Carenini, Nives, E-mail: Nives.Carenini@istitutotumori.mi.it [Molecular Pharmacology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, via Amadeo 42, Milan 20133 (Italy); Corna, Elisabetta, E-mail: Elisabetta.Corna@istitutotumori.mi.it [Molecular Pharmacology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, via Amadeo 42, Milan 20133 (Italy); Gandellini, Paolo, E-mail: Paolo.Gandellini@istitutotumori.mi.it [Molecular Pharmacology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, via Amadeo 42, Milan 20133 (Italy); Zaffaroni, Nadia, E-mail: Nadia.Zaffaroni@istitutotumori.mi.it [Molecular Pharmacology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, via Amadeo 42, Milan 20133 (Italy); Perego, Paola, E-mail: paola.perego@istitutotumori.mi.it [Molecular Pharmacology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, via Amadeo 42, Milan 20133 (Italy); Gatti, Laura, E-mail: Laura.Gatti@istitutotumori.mi.it [Molecular Pharmacology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, via Amadeo 42, Milan 20133 (Italy)

    2016-11-01

    The occurrence of drug resistance limits the efficacy of platinum compounds in the cure of ovarian carcinoma. Since microRNAs (miRNAs) may contribute to this phenomenon by regulating different aspects of tumor cell response, the aim of this study was to exploit the analysis of expression of miRNAs in platinum sensitive/resistant cells in an attempt to identify potential regulators of drug response. MiR-483-3p, which may participate in apoptosis and cell proliferation regulation, was found up-regulated in 4 platinum resistant variants, particularly in the IGROV-1/Pt1 subline, versus parental cells. Transfection of a synthetic precursor of miR-483-3p in IGROV-1 parental cells elicited a marked up-regulation of the miRNA levels. Growth-inhibition and colony-forming assays indicated that miR-483-3p over-expression reduced cell growth and conferred mild levels of cisplatin resistance in IGROV-1 cells, by interference with their proliferative potential. Predicted targets of miR-483-3p included PRKCA (encoding PKC-alpha), previously reported to be associated to platinum-resistance in ovarian carcinoma. We found that miR-483-3p directly targeted PRKCA in IGROV-1 cells. In keeping with this finding, cisplatin sensitivity of IGROV-1 cells decreased upon molecular/pharmacological inhibition of PKC-alpha. Overall, our results suggest that overexpression of miR-483-3p by ovarian carcinoma platinum-resistant cells may interfere with their proliferation, thus protecting them from DNA damage induced by platinum compounds and ultimately representing a drug-resistance mechanism. The impairment of cell growth may account for low levels of drug resistance that could be relevant in the clinical setting. - Highlights: • miR-483-3p is up-regulated in ovarian carcinoma cells resistant to platinum drugs. • Ectopic expression of miR-483-3p in IGROV-1 confers mild levels of Pt-resistance. • Overexpression of miR-483-3p down-regulates PRKCA levels in ovarian carcinoma cells. • miR 483

  4. Differential effects of phorbol 12-myristate 13-acetate and diacylglycerols on thromboxane A2-independent phospholipase A2 activation in collage-stimulated human platelets.

    Science.gov (United States)

    Reddy, S; Rao, G H; Murthy, M

    1994-04-01

    We investigated the priming effects of protein kinase C (PKC) activators such as phorbol 12-myristate 13-acetate (PMA), 1,2-DiC8 and OAG, and 1,3-DiC8 (a poor activator of PKC) on thromboxane A2 (TxA2)-independent phospholipase A2 (PLA2) activation in human platelets using collagen and A23187 as agonists. We measured PLA2 activation in collagen-stimulated platelets in the presence of BW755C, which abolished TxA2 synthesis, rise in cytosolic Ca2+, and aggregation. In the presence of PMA (50 nM), the amount of arachidonic acid (AA) released in platelets stimulated with collagen and A23187 represented 300% (13.85 nmol versus 4.5 nmol) and 400% (28 nmol versus 7 nmol) of controls (without PMA), respectively, while 1,2-DiC8, OAG, and 1,3-DiC8 increased TxA2-independent AA release by 50% in A23187-stimulated platelets and had no effect on the release of AA in collagen-stimulated platelets. Interestingly, 1,3-DiC8, which is a poor activator of PKC, was as effective as the other two DAGs (OAG and 1,2-DiC8) in priming TxA2-independent PLA2 activation, but was less effective than PMA in platelets stimulated with A23187. These results suggest that the TXA2-dependent IP3-mediated rise in cytosolic Ca2+ may not be obligatory for priming PLA2 activation in the presence of PMA in collagen-stimulated platelets. In contrast, 1,2-DiC8, OAG, and 1,3-DiC8 likely enhanced PLA2 activation via intracellular Ca2+ as they selectively affect this enzyme only in A23187-stimulated platelets. We also observed a significant increase in both saturated (palmitic and stearic acids) and unsaturated fatty acids (oleic and linoleic acids) in platelets stimulated by collagen or A23187 in the presence of PMA (50 nM), but not in the presence of DAGs. These findings imply that PMA may also affect the activation of DAG/MAG lipases, PLA1, or nonspecific PLA2. Since both 1,2-DiC8 and OAG exert no significant effect on the release of these fatty acids, the effects observed with PMA on DAG lipase/PLA1 may not

  5. Physical education in schools, sport activity and total physical activity in adolescents

    Directory of Open Access Journals (Sweden)

    Priscila Missaki Nakamura

    2013-07-01

    Full Text Available Less than half of adolescents reach the recommended300 minutes per week of physical activity (PA. Physical educationclasses and sports participation provideopportunities for adolescents to accumulate moretime for PA practice; however, littleis known about the influence of these variables onthe level of total physical activity ofadolescents. The aim of this study was toinvestigate the association between the practiceof physical education (PE in schools and sportsactivities (SA with the practice oftotal PA of adolescents. The study wascross-sectional and involved 467 adolescents ofhigh school (15.8 ± 0.9 years-old from the city ofRio Claro, in the State of São Paulo. Participants completed the Physical ActivityQuestionnaire to Older Children (PAQ-Cand questions related to the practice of PE and SAin schools. We performed a logisticregression with p<0.05 using SPSS. Girls hadlower prevalence of PA than boys, 9.4% and26.8%, respectively. Boys who did not participateof PE classes (OR=0.25, CI95%=0.09-0.66 and SA in schools (OR=0.34, CI95%=0.12-0.95were less likely to be active in PAthan boys who practiced these activities. Theparticipation in PE classes or engagementin some SA were positively associated with thepractice of total PA in boys.

  6. A cell-death-defying factor, anamorsin mediates cell growth through inactivation of PKC and p38MAPK

    International Nuclear Information System (INIS)

    Saito, Yuri; Shibayama, Hirohiko; Tanaka, Hirokazu; Tanimura, Akira; Kanakura, Yuzuru

    2011-01-01

    Research highlights: → Anamorsin (AM) (also called CIAPIN-1) is a cell-death-defying factor. → Biological mechanisms of AM functions have not been elucidated yet. → PKCθ , PKCδ and p38MAPK were more phosphorylated in AM deficient MEF cells. → AM may negatively regulates PKCs and p38MAPK in MEF cells. -- Abstract: Anamorsin (AM) plays crucial roles in hematopoiesis and embryogenesis. AM deficient (AM KO) mice die during late gestation; AM KO embryos are anemic and very small compared to wild type (WT) embryos. To determine which signaling pathways AM utilizes for these functions, we used murine embryonic fibroblast (MEF) cells generated from E-14.5 AM KO or WT embryos. Proliferation of AM KO MEF cells was markedly retarded, and PKCθ, PKCδ, and p38MAPK were more highly phosphorylated in AM KO MEF cells. Expression of cyclinD1, the target molecule of p38MAPK, was down-regulated in AM KO MEF cells. p38MAPK inhibitor as well as PKC inhibitor restored expression of cyclinD1 and cell growth in AM KO MEF cells. These data suggest that PKCθ, PKCδ, and p38MAPK activation lead to cell cycle retardation in AM KO MEF cells, and that AM may negatively regulate novel PKCs and p38MAPK in MEF cells.

  7. Suppression of the lipopolysaccharide-induced expression of MARCKS-related protein (MRP) affects transmigration in activated RAW264.7 cells.

    Science.gov (United States)

    Chun, Kwang-Rok; Bae, Eun Mi; Kim, Jae-Kwan; Suk, Kyoungho; Lee, Won-Ha

    2009-01-01

    The molecular action mechanism of MRP, one of the protein kinase C (PKC) substrates, has been under intense investigation, but reports on its role in macrophage function remain controversial. The treatment of macrophage cell lines with bacterial lipopolysaccharide (LPS) induced a high level of MRP expression suggesting that MRP plays a role in the function of activated macrophages. In order to investigate the role of MRP in activated RAW264.7 cells, we stably transfected MRP-specific shRNA expression constructs and tested for alterations in macrophage-related functions. The down-regulation of MRP expression resulted in a marked reduction in chemotaxis toward MCP-1 or extracellular matrix proteins. Furthermore, pharmacological inhibitors of PKC significantly inhibited the chemotaxis in RAW264.7 cells. These data reveals the pivotal role of MRP in the transmigration of activated RAW264.7 cells.

  8. BDNF-TrkB Signaling Coupled to nPKCε and cPKCβI Modulate the Phosphorylation of the Exocytotic Protein Munc18-1 During Synaptic Activity at the Neuromuscular Junction.

    Science.gov (United States)

    Simó, Anna; Just-Borràs, Laia; Cilleros-Mañé, Víctor; Hurtado, Erica; Nadal, Laura; Tomàs, Marta; Garcia, Neus; Lanuza, Maria A; Tomàs, Josep

    2018-01-01

    Munc18-1, a neuron-specific member of the Sec1/Munc18 family, is involved in neurotransmitter release by binding tightly to syntaxin. Munc18-1 is phosphorylated by PKC on Ser-306 and Ser-313 in vitro which reduces the amount of Munc18-1 able to bind syntaxin. We have previously identified that PKC is involved in neurotransmitter release when continuous electrical stimulation imposes a moderate activity on the NMJ and that muscle contraction through TrkB has an important impact on presynaptic PKC isoforms levels, specifically cPKCβI and nPKCε. Therefore, the present study was designed to understand how Munc18-1 phosphorylation is affected by (1) synaptic activity at the neuromuscular junction, (2) nPKCε and cPKCβI isoforms activity, (3) muscle contraction per se , and (4) the BDNF/TrkB signaling in a neuromuscular activity-dependent manner. We performed immunohistochemistry and confocal techniques to evidence the presynaptic location of Munc18-1 in the rat diaphragm muscle. To study synaptic activity, we stimulated the phrenic nerve (1 Hz, 30 min) with or without contraction (abolished by μ-conotoxin GIIIB). Specific inhibitory reagents were used to block nPKCε and cPKCβI activity and to modulate the tropomyosin receptor kinase B (TrkB). Main results obtained from Western blot experiments showed that phosphorylation of Munc18-1 at Ser-313 increases in response to a signaling mechanism initiated by synaptic activity and directly mediated by nPKCε. Otherwise, cPKCβI and TrkB activities work together to prevent this synaptic activity-induced Munc18-1 phosphorylation by a negative regulation of cPKCβI over nPKCε. Therefore, a balance between the activities of these PKC isoforms could be a relevant cue in the regulation of the exocytotic apparatus. The results also demonstrate that muscle contraction prevents the synaptic activity-induced Munc18-1 phosphorylation through a mechanism that opposes the TrkB/cPKCβI/nPKCε signaling.

  9. Endothelin receptors and activity differ in human, dog, and rabbit lung.

    Science.gov (United States)

    McKay, K O; Armour, C L; Black, J L

    1996-01-01

    In this study, we have examined dog and rabbit airways as potential models for human airways in regard to the activity of endothelin. The receptors involved in the response to endothelin-1 (ET-1) in airway tissue from human, rabbit, and dog lung were investigated, as was the mechanism responsible for the contraction to ET-1 in tissue from the three species. By using specific endothelin receptor agonists and antagonists, we have demonstrated that ETB receptors predominate in rabbit and human airways and ETA receptors in dog airways. The contraction to ET-1 is not dependent on cyclooxygenase products of arachidonic acid, as indomethacin had no effect on the response to ET-1. Extracellular calcium influx via voltage-dependent channels is necessary for contraction to ET-1 in rabbit and dog airways. These results are in contrast to our previously reported results in human airways, in which neither removal of extracellular calcium nor verapamil affected the ET-1 response. The sustained phase of the contraction to ET-1 in all three species may be mediated in part by activation of protein kinase C (PKC), as the inhibitor staurosporine significantly altered the time course of the response to endothelin. We therefore conclude that in rabbit airways ET-1 activates ETB receptors, triggers the influx of extracellular calcium through voltage-dependent channels, and induces a contractile response that is, in part, dependent upon stimulation of PKC. The same mechanism is triggered in dog bronchus; however, the receptors involved in this species are of the ETA type. Finally, in human airways, the contractile response to ET-1, while independent of extracellular calcium influx, is dependent upon PKC activation after binding of the peptide to ETB receptors.

  10. Method to determine the activity concentration and total activity of radioactive waste

    International Nuclear Information System (INIS)

    Angeles C, A.

    2001-02-01

    A characteristic system of radioactive waste is described to determine the concentration of radionuclides activity and the total activity of bundles of radioactive waste. The system this integrated by three subsystems: - Elevator of drums. - Electromechanics. - Gamma spectroscopy. In the system it is analyzed waste of issuing gamma specifically, and this designed for materials of relative low density and it analyzes materials of cylindrical recipients

  11. Inhibition of total oxygen uptake by silica nanoparticles in activated sludge

    Energy Technology Data Exchange (ETDEWEB)

    Sibag, Mark [Department of Environment and Energy, Sejong University, 98 Gunja-Dong, Gwangjin-Gu, Seoul 143-747 (Korea, Republic of); Choi, Byeong-Gyu [School of Civil, Environmental and Architectural Engineering, Korea University, 145, Anam-ro, Sungbuk-ku, Seoul 136-701 (Korea, Republic of); Suh, Changwon [Energy Lab, Environment Group, Samsung Advanced Institute of Technology, 130 Samsung-ro, Yeongtong-gu, Suwon-si, Gyeonggi-do 443-803 (Korea, Republic of); Lee, Kwan Hyung; Lee, Jae Woo [Department of Environmental Engineering and Program in Environmental Technology and Policy, Korea University, Sejong 339-700 (Korea, Republic of); Maeng, Sung Kyu [Department of Civil and Environmental Engineering, Sejong University, 98 Gunja-Dong, Gwangjin-Gu, Seoul 143-747 (Korea, Republic of); Cho, Jinwoo, E-mail: jinwoocho@sejong.edu [Department of Environment and Energy, Sejong University, 98 Gunja-Dong, Gwangjin-Gu, Seoul 143-747 (Korea, Republic of)

    2015-02-11

    Highlights: • Silica nanoparticles (SNP) inhibit total oxygen uptake in activated sludge. • Relatively smaller SNP are inhibitorier than larger SNP. • SNP alters C15:0, C16:0 and C18:0 in activated sludge fatty acid methyl ester profile. - Abstract: Nanoparticle toxicity to biological activities in activated sludge is largely unknown. Among the widely used nanoparticles, silica nanoparticles (SNP) have a limited number of studies associated with inhibition to the activated sludge process (ASP). We demonstrated SNP inhibition of activated sludge respiration through oxygen uptake rate (OUR) measurement. Based on the percentage inhibition of total oxygen consumption (I{sub T}), we observed that smaller SNPs (12 nm, I{sub T} = 33 ± 3%; 151 nm, I{sub T} = 23 ± 2%) were stronger inhibitors than larger SNPs (442 and 683 nm, I{sub T} = 5 ± 1%). Transmission electron micrographs showed that some of the SNPs were adsorbed on and/or apparently embedded somewhere in the microbial cell membrane. Whether SNPs are directly associated with the inhibition of total oxygen uptake warrants further studies. However, it is clear that SNPs statistically significantly altered the composition of microbial membrane lipids, which was more clearly described by principal component analysis and weighted Euclidian distance (PCA-ED) of the fatty acid methyl ester (FAME) data. This study suggests that SNPs potentially affect the biological activity in activated sludge through the inhibition of total oxygen uptake.

  12. Time-lapse cinematography study of the germinal vesicle behaviour in mouse primary oocytes treated with activators of protein kinases A and C.

    Science.gov (United States)

    Alexandre, H; Mulnard, J

    1988-12-01

    A passive erratic movement of the germinal vesicle (GV), already visible in small incompetent oocytes, is followed by an active scalloping of the nuclear membrane soon before GV breakdown (GVBD) in cultured competent oocytes. Maturation can be inhibited by activators of protein kinase A (PK-A) and protein kinase C (PK-C). Our time-lapse cinematography analysis allowed us to describe an unexpected behaviour of the GV when PK-C, but not PK-A, is activated: GV undergoes a displacement toward the cortex according to the same biological clock which triggers the programmed translocation of the spindle in control oocytes. It is concluded that, when oocytes become committed to undergo maturation, the cytoplasm acquires a PK-A-controlled "centrifugal displacement property" which is not restricted to the spindle.

  13. Go-6976 Reverses Hyperglycemia-Induced Insulin Resistance Independently of cPKC Inhibition in Adipocytes

    Science.gov (United States)

    Robinson, Katherine A.; Hegyi, Krisztina; Hannun, Yusuf A.; Buse, Maria G.; Sethi, Jaswinder K.

    2014-01-01

    Chronic hyperglycemia induces insulin resistance by mechanisms that are incompletely understood. One model of hyperglycemia-induced insulin resistance involves chronic preincubation of adipocytes in the presence of high glucose and low insulin concentrations. We have previously shown that the mTOR complex 1 (mTORC1) plays a partial role in the development of insulin resistance in this model. Here, we demonstrate that treatment with Go-6976, a widely used “specific” inhibitor of cPKCs, alleviates hyperglycemia-induced insulin resistance. However, the effects of mTOR inhibitor, rapamycin and Go-6976 were not additive and only rapamycin restored impaired insulin-stimulated AKT activation. Although, PKCα, (but not –β) was abundantly expressed in these adipocytes, our studies indicate cPKCs do not play a major role in causing insulin-resistance in this model. There was no evidence of changes in the expression or phosphorylation of PKCα, and PKCα knock-down did not prevent the reduction of insulin-stimulated glucose transport. This was also consistent with lack of IRS-1 phosphorylation on Ser-24 in hyperglycemia-induced insulin-resistant adipocytes. Treatment with Go-6976 did inhibit a component of the mTORC1 pathway, as evidenced by decreased phosphorylation of S6 ribosomal protein. Raptor knock-down enhanced the effect of insulin on glucose transport in insulin resistant adipocytes. Go-6976 had the same effect in control cells, but was ineffective in cells with Raptor knock-down. Taken together these findings suggest that Go-6976 exerts its effect in alleviating hyperglycemia-induced insulin-resistance independently of cPKC inhibition and may target components of the mTORC1 signaling pathway. PMID:25330241

  14. Physical education in schools, sport activity and total physical activity in adolescents

    Directory of Open Access Journals (Sweden)

    Priscila Missaki Nakamura

    2013-06-01

    Full Text Available DOI: http://dx.doi.org/10.5007/1980-0037.2013v15n5p517 Less than half of adolescents reach the recommended300 minutes per week of physical activity (PA. Physical educationclasses and sports participation provideopportunities for adolescents to accumulate moretime for PA practice; however, littleis known about the influence of these variables onthe level of total physical activity ofadolescents. The aim of this study was toinvestigate the association between the practiceof physical education (PE in schools and sportsactivities (SA with the practice oftotal PA of adolescents. The study wascross-sectional and involved 467 adolescents ofhigh school (15.8 ± 0.9 years-old from the city ofRio Claro, in the State of São Paulo. Participants completed the Physical ActivityQuestionnaire to Older Children (PAQ-Cand questions related to the practice of PE and SAin schools. We performed a logisticregression with p<0.05 using SPSS. Girls hadlower prevalence of PA than boys, 9.4% and26.8%, respectively. Boys who did not participateof PE classes (OR=0.25, CI95%=0.09-0.66 and SA in schools (OR=0.34, CI95%=0.12-0.95were less likely to be active in PAthan boys who practiced these activities. Theparticipation in PE classes or engagementin some SA were positively associated with thepractice of total PA in boys.

  15. The CD3 gamma leucine-based receptor-sorting motif is required for efficient ligand-mediated TCR down-regulation

    DEFF Research Database (Denmark)

    von Essen, Marina; Menné, Charlotte; Nielsen, Bodil L

    2002-01-01

    . The other pathway is dependent on protein kinase C (PKC)-mediated activation of the CD3 gamma di-leucine-based receptor-sorting motif. Previous studies have failed to demonstrate a connection between ligand- and PKC-induced TCR down-regulation. Thus, although an apparent paradox, the dogma has been...... that ligand- and PKC-induced TCR down-regulations are not interrelated. By analyses of a newly developed CD3 gamma-negative T cell variant, freshly isolated and PHA-activated PBMC, and a mouse T cell line, we challenged this dogma and demonstrate in this work that PKC activation and the CD3 gamma di...

  16. Shigella IpaH0722 E3 Ubiquitin Ligase Effector Targets TRAF2 to Inhibit PKC–NF-κB Activity in Invaded Epithelial Cells

    Science.gov (United States)

    Ashida, Hiroshi; Nakano, Hiroyasu; Sasakawa, Chihiro

    2013-01-01

    NF-κB plays a central role in modulating innate immune responses to bacterial infections. Therefore, many bacterial pathogens deploy multiple mechanisms to counteract NF-κB activation. The invasion of and subsequent replication of Shigella within epithelial cells is recognized by various pathogen recognition receptors as pathogen-associated molecular patterns. These receptors trigger innate defense mechanisms via the activation of the NF-κB signaling pathway. Here, we show the inhibition of the NF-κB activation by the delivery of the IpaH E3 ubiquitin ligase family member IpaH0722 using Shigella's type III secretion system. IpaH0722 dampens the acute inflammatory response by preferentially inhibiting the PKC-mediated activation of NF-κB by ubiquitinating TRAF2, a molecule downstream of PKC, and by promoting its proteasome-dependent degradation. PMID:23754945

  17. Chapter 4: Measurements of total beta-activity in the fallout

    International Nuclear Information System (INIS)

    Duggleby, J.C.; Johannessen, J.C.; Kotler, L.H.; Stewart, F.M.

    1974-01-01

    In order to provide information on fresh fission products in fallout reaching Australia from nuclear tests being conducted by France in Polynesia, measurements were made of total beta activity in daily fallout deposition of 25 Australian sampling stations covering a three month period from 25 July to 23 October 1973. The methods employed to measure the radioactivity of the samples are described and the data on total beta-activity, and the calculated external gamma radiation doses from fresh fallout are presented. (R.L.)

  18. Spinal NMDA receptor activation constrains inactivity-induced phrenic motor facilitation in Charles River Sprague-Dawley rats.

    Science.gov (United States)

    Streeter, K A; Baker-Herman, T L

    2014-10-01

    Reduced spinal synaptic inputs to phrenic motor neurons elicit a unique form of spinal plasticity known as inactivity-induced phrenic motor facilitation (iPMF). iPMF requires tumor necrosis factor-α (TNF-α) and atypical protein kinase C (aPKC) activity within spinal segments containing the phrenic motor nucleus to stabilize early, transient increases in phrenic burst amplitude into long-lasting iPMF. Here we tested the hypothesis that spinal N-methyl-d-aspartate receptor (NMDAR) activation constrains long-lasting iPMF in some rat substrains. Phrenic motor output was recorded in anesthetized, ventilated Harlan (HSD) and Charles River (CRSD) Sprague-Dawley rats exposed to a 30-min central neural apnea. HSD rats expressed a robust, long-lasting (>60 min) increase in phrenic burst amplitude (i.e., long-lasting iPMF) when respiratory neural activity was restored. By contrast, CRSD rats expressed an attenuated, transient (∼15 min) iPMF. Spinal NMDAR inhibition with DL-2-amino-5-phosphonopentanoic acid (APV) before neural apnea or shortly (4 min) prior to the resumption of respiratory neural activity revealed long-lasting iPMF in CRSD rats that was phenotypically similar to that in HSD rats. By contrast, APV did not alter iPMF expression in HSD rats. Spinal TNF-α or aPKC inhibition impaired long-lasting iPMF enabled by NMDAR inhibition in CRSD rats, suggesting that similar mechanisms give rise to long-lasting iPMF in CRSD rats with NMDAR inhibition as those giving rise to long-lasting iPMF in HSD rats. These results suggest that NMDAR activation can impose constraints on TNF-α-induced aPKC activation after neural apnea, impairing stabilization of transient iPMF into long-lasting iPMF. These data may have important implications for understanding differential responses to reduced respiratory neural activity in a heterogeneous human population. Copyright © 2014 the American Physiological Society.

  19. PI3 kinase is important for Ras, MEK and Erk activation of Epo-stimulated human erythroid progenitors

    Directory of Open Access Journals (Sweden)

    Schmidt Enrico K

    2004-05-01

    Full Text Available Abstract Background Erythropoietin is a multifunctional cytokine which regulates the number of erythrocytes circulating in mammalian blood. This is crucial in order to maintain an appropriate oxygen supply throughout the body. Stimulation of primary human erythroid progenitors (PEPs with erythropoietin (Epo leads to the activation of the mitogenic kinases (MEKs and Erks. How this is accomplished mechanistically remained unclear. Results Biochemical studies with human cord blood-derived PEPs now show that Ras and the class Ib enzyme of the phosphatidylinositol-3 kinase (PI3K family, PI3K gamma, are activated in response to minimal Epo concentrations. Surprisingly, three structurally different PI3K inhibitors block Ras, MEK and Erk activation in PEPs by Epo. Furthermore, Erk activation in PEPs is insensitive to the inhibition of Raf kinases but suppressed upon PKC inhibition. In contrast, Erk activation induced by stem cell factor, which activates c-Kit in the same cells, is sensitive to Raf inhibition and insensitive to PI3K and PKC inhibitors. Conclusions These unexpected findings contrast with previous results in human primary cells using Epo at supraphysiological concentrations and open new doors to eventually understanding how low Epo concentrations mediate the moderate proliferation of erythroid progenitors under homeostatic blood oxygen levels. They indicate that the basal activation of MEKs and Erks in PEPs by minimal concentrations of Epo does not occur through the classical cascade Shc/Grb2/Sos/Ras/Raf/MEK/Erk. Instead, MEKs and Erks are signal mediators of PI3K, probably the recently described PI3K gamma, through a Raf-independent signaling pathway which requires PKC activity. It is likely that higher concentrations of Epo that are induced by hypoxia, for example, following blood loss, lead to additional mitogenic signals which greatly accelerate erythroid progenitor proliferation.

  20. BDNF-TrkB Signaling Coupled to nPKCε and cPKCβI Modulate the Phosphorylation of the Exocytotic Protein Munc18-1 During Synaptic Activity at the Neuromuscular Junction

    Directory of Open Access Journals (Sweden)

    Anna Simó

    2018-06-01

    Full Text Available Munc18-1, a neuron-specific member of the Sec1/Munc18 family, is involved in neurotransmitter release by binding tightly to syntaxin. Munc18-1 is phosphorylated by PKC on Ser-306 and Ser-313 in vitro which reduces the amount of Munc18-1 able to bind syntaxin. We have previously identified that PKC is involved in neurotransmitter release when continuous electrical stimulation imposes a moderate activity on the NMJ and that muscle contraction through TrkB has an important impact on presynaptic PKC isoforms levels, specifically cPKCβI and nPKCε. Therefore, the present study was designed to understand how Munc18-1 phosphorylation is affected by (1 synaptic activity at the neuromuscular junction, (2 nPKCε and cPKCβI isoforms activity, (3 muscle contraction per se, and (4 the BDNF/TrkB signaling in a neuromuscular activity-dependent manner. We performed immunohistochemistry and confocal techniques to evidence the presynaptic location of Munc18-1 in the rat diaphragm muscle. To study synaptic activity, we stimulated the phrenic nerve (1 Hz, 30 min with or without contraction (abolished by μ-conotoxin GIIIB. Specific inhibitory reagents were used to block nPKCε and cPKCβI activity and to modulate the tropomyosin receptor kinase B (TrkB. Main results obtained from Western blot experiments showed that phosphorylation of Munc18-1 at Ser-313 increases in response to a signaling mechanism initiated by synaptic activity and directly mediated by nPKCε. Otherwise, cPKCβI and TrkB activities work together to prevent this synaptic activity–induced Munc18-1 phosphorylation by a negative regulation of cPKCβI over nPKCε. Therefore, a balance between the activities of these PKC isoforms could be a relevant cue in the regulation of the exocytotic apparatus. The results also demonstrate that muscle contraction prevents the synaptic activity–induced Munc18-1 phosphorylation through a mechanism that opposes the TrkB/cPKCβI/nPKCε signaling.

  1. Lead acetate induces EGFR activation upstream of SFK and PKCα linkage to the Ras/Raf-1/ERK signaling

    International Nuclear Information System (INIS)

    Wang, C.-Y.; Wang, Y.-T.; Tzeng, D.-W.; Yang, J.-L.

    2009-01-01

    Lead acetate (Pb), a probable human carcinogen, can activate protein kinase C (PKC) upstream of extracellular signal-regulated kinase 1 and 2 (ERK1/2). Yet, it remains unclear whether Pb activation of PKC → ERK1/2 involves receptor/non-receptor tyrosine kinases and the Ras signaling transducer. Here we demonstrate a novel mechanism elicited by Pb for transmitting ERK1/2 signaling in CL3 human non-small-cell lung adenocarcinoma cells. Pb induction of higher steady-state levels of Ras-GTP was essential for increasing phospho-Raf-1 S338 and phospho-ERK1/2. Pre-treatment of the cells with a conventional PKC inhibitor Goe6976 or depleting PKCα using specific small interfering RNA blocked Pb induction of Ras-GTP. Pb also activated cellular tyrosine kinases. Specific pharmacological inhibitors, PD153035 for epidermal growth factor receptor (EGFR) and SU6656 for Src family tyrosine kinases (SFK), but not AG1296 for platelet-derived growth factor receptor, could suppress the Pb-induced tyrosine kinases, PKCα, Ras-GTP, phospho-Raf-1 S338 and phospho-ERK1/2. Furthermore, phosphorylation of tyrosines on the EGFR multiple autophosphorylation sites and the conserved SFK autophosphorylation site occurred during exposure of cells to Pb for 1-5 min and 5-30 min, respectively. Intriguingly, Pb activation of EGFR required the intrinsic kinase activity but not dimerization of the receptor. Inhibition of SFK or PKCα activities did not affect EGFR phosphorylation, while knockdown of EGFR blocked SFK phosphorylation and PKCα activation following Pb. Together, these results indicate that immediate activation of EGFR in response to Pb is obligatory for activation of SFK and PKCα and subsequent the Ras-Raf-1-MKK1/2-ERK1/2 signaling cascade

  2. Protein kinase C isoforms in bovine aortic endothelial cells: role in regulation of P2Y- and P2U-purinoceptor-stimulated prostacyclin release.

    Science.gov (United States)

    Patel, V; Brown, C; Boarder, M R

    1996-05-01

    1. Enhanced synthesis of prostacyclin (PGI2) and inositol polyphosphates in bovine aortic endothelial cells in response to ATP and ADP is mediated by co-existing P2Y- and P2U-purinoceptors. Here we examine the regulation of these responses by isoforms of protein kinase C (PKC). 2. Immunoblots with antisera specific for 8 different PKC isoforms revealed the presence of alpha, epsilon and zeta, while no immunoreactivity was found for beta, gamma, delta, eta and theta isoforms. PKC-alpha was largely cytosolic in unstimulated cells and almost all translocated to the membrane (Triton X-100 soluble) after a 1 min treatment with the PKC activating phorbol myristate acetate (PMA); PKC-epsilon was always in a Triton X-100 insoluble membrane fraction, while PKC-zeta was found in both soluble and membrane bound (Triton X-100 soluble) forms in the unstimulated cells and was unaffected by PMA. 3. Treatment with PMA for 6 h led to a 90% downregulation of PKC-alpha, while the immunoreactivity to the epsilon and zeta isoforms remained largely unchanged. 4. After either 10 min or 6 h exposure to PMA the PGI2 response to activation of both receptors was enhanced, while the inositol 1,4,5-trisphosphate response to P2Y-purinoceptor activation was substantially attenuated and the P2U-purinoceptor response was unchanged. Thus the PGI2 response to PMA under conditions when 90% of the PKC-alpha was lost resembles that seen on acute stimulation of PKC by PMA, and the PGI2 response does not correlate with phospholipase C response. 5. Inhibition of PKC with the isoform non-selective inhibitors, Ro 31-8220 and Go 6850 abolished the PGI2 response to both P2U- and P2Y-purinoceptor stimulation. However, Go 6976, which preferentially inhibits Ca2+ sensitive isoforms (such as PKC-alpha) and not Ca2+ insensitive isoforms (such as PKC-epsilon), had no effect on the PGI2 response. 6. The results show that there is a requirement for PKC in the stimulation of PGI2 production by endothelial P2Y- and P2U

  3. Protein Kinase C Inhibitors as Modulators of Vascular Function and Their Application in Vascular Disease

    Directory of Open Access Journals (Sweden)

    Raouf A. Khalil

    2013-03-01

    Full Text Available Blood pressure (BP is regulated by multiple neuronal, hormonal, renal and vascular control mechanisms. Changes in signaling mechanisms in the endothelium, vascular smooth muscle (VSM and extracellular matrix cause alterations in vascular tone and blood vessel remodeling and may lead to persistent increases in vascular resistance and hypertension (HTN. In VSM, activation of surface receptors by vasoconstrictor stimuli causes an increase in intracellular free Ca2+ concentration ([Ca2+]i, which forms a complex with calmodulin, activates myosin light chain (MLC kinase and leads to MLC phosphorylation, actin-myosin interaction and VSM contraction. Vasoconstrictor agonists could also increase the production of diacylglycerol which activates protein kinase C (PKC. PKC is a family of Ca2+-dependent and Ca2+-independent isozymes that have different distributions in various blood vessels, and undergo translocation from the cytosol to the plasma membrane, cytoskeleton or the nucleus during cell activation. In VSM, PKC translocation to the cell surface may trigger a cascade of biochemical events leading to activation of mitogen-activated protein kinase (MAPK and MAPK kinase (MEK, a pathway that ultimately increases the myofilament force sensitivity to [Ca2+]i, and enhances actin-myosin interaction and VSM contraction. PKC translocation to the nucleus may induce transactivation of various genes and promote VSM growth and proliferation. PKC could also affect endothelium-derived relaxing and contracting factors as well as matrix metalloproteinases (MMPs in the extracellular matrix further affecting vascular reactivity and remodeling. In addition to vasoactive factors, reactive oxygen species, inflammatory cytokines and other metabolic factors could affect PKC activity. Increased PKC expression and activity have been observed in vascular disease and in certain forms of experimental and human HTN. Targeting of vascular PKC using PKC inhibitors may function in

  4. Further characterization of protein kinase C in mouse mast cells

    International Nuclear Information System (INIS)

    White, J.R.; Ishizaka, T.

    1986-01-01

    Bridging of cell-bound IgE antibody molecules on colony stimulating factor dependent mouse mast cell line (PT-18) cells by multivalent antigen induces the mobilization and uptake of Ca 2+ monitored by Quin-2 and the production of diacylglycerol. Exposure of the sensitized cells to antigen also induces a substantial increase in protein kinase C (PKC) activity in the plasma membrane (340 units to 1375 units: 1 unit = 1 pmol of 32 P incorporated into Histone H-1/min/10 7 cells), within 30 seconds. There is also an increase in 3 H phorbol-12, 13-dibutyrate ( 3 H-PDB) binding which parallels the increase in PKC activity both in kinetics and antigen dose dependency. Determination of K/sub m/ and V/sub max/ for PKC revealed no difference between the cytosolic and membranous forms of PKC. Partial purification of PKC from the membrane of sensitized mast cells which had been labeled with 32 P and stimulated with DNP-HSA revealed a protein of 80-84,000 molecular weight, which migrated on polyacrylamide gel electrophoresis just above an authentic standard of PKC purified from rat brain. Treatment of the PKC from mouse mast cell membrane with alkaline phosphatase resulted in a reduction of phosphorylating activity and bindability of 3 H-PDB. In conclusion, the authors speculate that activation of mouse mast cells by cross-linking IgE results in the phosphorylation of a silent-pool of PKC converting it from an inactive state to an activated form

  5. In vitro antioxidant, antibacterial and anti-tumor activities of total ...

    African Journals Online (AJOL)

    Purpose: To investigate the in vitro antioxidant, antibacterial and anti-tumor activities of total flavonoids from Elsholtzia densa Benth of Sichuan Province, China. Methods: The total flavonoids of Elsholtzia densa Bent were extracted utilizing the ultrasonic extraction method, and purified by D101 macroporous adsorption resin ...

  6. Modulation of Na+/K+ ATPase Activity by Hydrogen Peroxide Generated through Heme in L. amazonensis.

    Directory of Open Access Journals (Sweden)

    Nathália Rocco-Machado

    Full Text Available Leishmania amazonensis is a protozoan parasite that occurs in many areas of Brazil and causes skin lesions. Using this parasite, our group showed the activation of Na+/K+ ATPase through a signaling cascade that involves the presence of heme and protein kinase C (PKC activity. Heme is an important biomolecule that has pro-oxidant activity and signaling capacity. Reactive oxygen species (ROS can act as second messengers, which are required in various signaling cascades. Our goal in this work is to investigate the role of hydrogen peroxide (H2O2 generated in the presence of heme in the Na+/K+ ATPase activity of L. amazonensis. Our results show that increasing concentrations of heme stimulates the production of H2O2 in a dose-dependent manner until a concentration of 2.5 μM heme. To confirm that the effect of heme on the Na+/K+ ATPase is through the generation of H2O2, we measured enzyme activity using increasing concentrations of H2O2 and, as expected, the activity increased in a dose-dependent manner until a concentration of 0.1 μM H2O2. To investigate the role of PKC in this signaling pathway, we observed the production of H2O2 in the presence of its activator phorbol 12-myristate 13-acetate (PMA and its inhibitor calphostin C. Both showed no effect on the generation of H2O2. Furthermore, we found that PKC activity is increased in the presence of H2O2, and that in the presence of calphostin C, H2O2 is unable to activate the Na+/K+ ATPase. 100 μM of Mito-TEMPO was capable of abolishing the stimulatory effect of heme on Na+/K+ ATPase activity, indicating that mitochondria might be the source of the hydrogen peroxide production induced by heme. The modulation of L. amazonensis Na+/K+ ATPase by H2O2 opens new possibilities for understanding the signaling pathways of this parasite.

  7. Microgravity modifies protein kinase C isoform translocation in the human monocytic cell line U937 and human peripheral blood T-cells

    Science.gov (United States)

    Hatton, Jason P.; Gaubert, Francois; Cazenave, Jean-Pierre; Schmitt, Didier; Hashemi, B. B. (Principal Investigator); Hughes-Fulford, M. (Principal Investigator)

    2002-01-01

    Individual protein kinase C (PKC) isoforms fulfill distinct roles in the regulation of the commitment to differentiation, cell cycle arrest, and apoptosis in both monocytes and T-cells. The human monocyte like cell line U937 and T-cells were exposed to microgravity, during spaceflight and the translocation (a critical step in PKC signaling) of individual isoforms to cell particulate fraction examined. PKC activating phorbol esters induced a rapid translocation of several PKC isoforms to the particulate fraction of U937 monocytes under terrestrial gravity (1 g) conditions in the laboratory. In microgravity, the translocation of PKC beta II, delta, and epsilon in response to phorbol esters was reduced in microgravity compared to 1 g, but was enhanced in weak hypergravity (1.4 g). All isoforms showed a net increase in particulate PKC following phorbol ester stimulation, except PKC delta which showed a net decrease in microgravity. In T-cells, phorbol ester induced translocation of PKC delta was reduced in microgravity, compared to 1 g, while PKC beta II translocation was not significantly different at the two g-levels. These data show that microgravity differentially alters the translocation of individual PKC isoforms in monocytes and T-cells, thus providing a partial explanation for the modifications previously observed in the activation of these cell types under microgravity.

  8. Involvement of protein kinase C in the modulation of morphine-induced analgesia and the inhibitory effects of exposure to 60-hz magnetic fields in the land snail, Cepaea nemoralis

    Energy Technology Data Exchange (ETDEWEB)

    Kavaliers, M.; Ossenkopp, K.P. (Univ. of Western Ontario, London (Canada))

    1990-02-26

    One of the more consistent and dramatic effects of exposure to magnetic fields is the attenuation of morphine-induced analgesia. Results of previous studies have implicated alterations in calcium channel functioning and Ca{sup ++} flux in the mediation of these effects. It is generally accepted that Ca{sup ++}-activated-phospholipid-dependent protein kinase (Protein kinase C; PKC) plays an important role in relaying trans-membrane signaling in diverse Ca{sup ++} dependent cellular processes. In experiment 1 we observed that morphine-induced analgesia in the land snail, Cepaea nemoralis, as measured by the latency of an avoidance behavior to a warmed surface, was reduced by the PKC activator, SC-9, and was enhanced by the PKC inhibitors, H-7 and H-9. In contrast, HA-10004, a potent inhibitor of other protein kinases, but only a very weak inhibitor of PKC, had no effect on morphine-induced analgesia. In experiment 2 exposure of snails for 30 minutes to a 1.0 gauss (rms) 60-Hz magnetic field reduced morphine-induced analgesia. This inhibitory effect of the magnetic field was reduced by the PKC inhibitors, H-7 and H-9, and was augmented by the PKC activator SC-9. These results suggest that: (i) PKC is involved in the modulation of morphine-induced analgesia and, (ii) the inhibitory effects of magnetic fields involve PKC.

  9. Valorization of By-Products from Palm Oil Mills for the Production of Generic Fermentation Media for Microbial Oil Synthesis.

    Science.gov (United States)

    Tsouko, Erminda; Kachrimanidou, Vasiliki; Dos Santos, Anderson Fragoso; do Nascimento Vitorino Lima, Maria Eduarda; Papanikolaou, Seraphim; de Castro, Aline Machado; Freire, Denise Maria Guimarães; Koutinas, Apostolis A

    2017-04-01

    This study demonstrates the production of a generic nutrient-rich feedstock using by-product streams from palm oil production that could be used as a substitute for commercial fermentation supplements. Solid-state fermentations of palm kernel cake (PKC) and palm-pressed fiber (PPF) were conducted in tray bioreactors and a rotating drum bioreactor by the fungal strain Aspergillus oryzae for the production of crude enzymes. The production of protease was optimized (319.3 U/g) at an initial moisture content of 55 %, when PKC was used as the sole substrate. The highest free amino nitrogen (FAN) production (5.6 mg/g) obtained via PKC hydrolysis using the crude enzymes produced via solid-state fermentation was achieved at 50 °C. Three initial PKC concentrations (48.7, 73.7, and 98.7 g/L) were tested in hydrolysis experiments, leading to total Kjeldahl nitrogen to FAN conversion yields up to 27.9 %. Sequential solid-state fermentation followed by hydrolysis was carried out in the same rotating drum bioreactor, leading to the production of 136.7 U/g of protease activity during fermentation and 196.5 mg/L of FAN during hydrolysis. Microbial oil production was successfully achieved with the oleaginous yeast strain Lipomyces starkeyi DSM 70296 cultivated on the produced PKC hydrolysate mixed with commercial carbon sources, including glucose, xylose, mannose, galactose, and arabinose.

  10. Development of diacyltetrol lipids as activators for the C1 domain of protein kinase C.

    Science.gov (United States)

    Mamidi, Narsimha; Gorai, Sukhamoy; Mukherjee, Rakesh; Manna, Debasis

    2012-04-01

    The protein kinase C (PKC) family of serine/threonine kinases is an attractive drug target for the treatment of cancer and other diseases. Diacylglycerol (DAG), phorbol esters and others act as ligands for the C1 domain of PKC isoforms. Inspection of the crystal structure of the PKCδ C1b subdomain in complex with phorbol-13-O-acetate shows that one carbonyl group and two hydroxyl groups play pivotal roles in recognition of the C1 domain. To understand the importance of two hydroxyl groups of phorbol esters in PKC binding and to develop effective PKC activators, we synthesized DAG like diacyltetrols (DATs) and studied binding affinities with C1b subdomains of PKCδ and PKCθ. DATs, with the stereochemistry of natural DAGs at the sn-2 position, were synthesized from (+)-diethyl L-tartrate in four to seven steps as single isomers. The calculated EC(50) values for the short and long chain DATs varied in the range of 3-6 μM. Furthermore, the fluorescence anisotropy values of the proteins were increased in the presence of DATs in a similar manner to that of DAGs. Molecular docking of DATs (1b-4b) with PKCδ C1b showed that the DATs form hydrogen bonds with the polar residues and backbone of the protein, at the same binding site, as that of DAG and phorbol esters. Our findings reveal that DATs represent an attractive group of C1 domain ligands that can be used as research tools or further structurally modified for potential drug development.

  11. Rosiglitazone attenuates NF-κB-dependent ICAM-1 and TNF-α production caused by homocysteine via inhibiting ERK1/2/p38MAPK activation

    International Nuclear Information System (INIS)

    Bai, Yong-Ping; Liu, Yu-Hui; Chen, Jia; Song, Tao; You, Yu; Tang, Zhen-Yan; Li, Yuan-Jian; Zhang, Guo-Gang

    2007-01-01

    Previous studies demonstrated an important interaction between nuclear factor-kappaB (NF-κB) activation and homocysteine (Hcy)-induced cytokines expression in endothelial cells and vascular smooth muscle cells. However, the underlying mechanism remains illusive. In this study, we investigated the effects of Hcy on NF-κB-mediated sICAM-1, TNF-α production and the possible involvement of ERK 1/2 /p38MAPK pathway. The effects of rosiglitazone intervention were also examined. Our results show that Hcy increased the levels of sICAM-1 and TNF-α in cultured human umbilical vein endothelial cells (HUVECs) in a time- and concentration-dependent manner. This effect was significantly depressed by rosiglitazone and different inhibitors (PDTC, NF-κB inhibitor; PD98059, MEK inhibitor; SB203580, p38MAPK specific inhibitor; and staurosporine, PKC inhibitor). Next, we investigated the effect of Hcy on ERK 1/2 /p38MAPK pathway and NF-κB activity in HUVECs. The results show that Hcy activated both ERK 1/2 /p38MAPK pathway and NF-κB-DNA-binding activity. These effects were markedly inhibited by rosiglitazone as well as other inhibitors (SB203580, PD98059, and PDTC). Further, the pretreatment of staurosporine abrogated ERK 1/2 /p38MAPK phosphorylation, suggesting that Hcy-induced ERK 1/2 /p38MAPK activation is associated with PKC activity. Our results provide evidence that Hcy-induced NF-κB activation was mediated by activation of ERK 1/2 /p38MAPK pathway involving PKC activity. Rosiglitazone reduces the NF-κB-mediated sICAM-1 and TNF-α production induced by Hcy via inhibition of ERK 1/2 /p38MAPK pathway

  12. Control of ADAM17 activity by regulation of its cellular localisation

    Science.gov (United States)

    Lorenzen, Inken; Lokau, Juliane; Korpys, Yvonne; Oldefest, Mirja; Flynn, Charlotte M.; Künzel, Ulrike; Garbers, Christoph; Freeman, Matthew; Grötzinger, Joachim; Düsterhöft, Stefan

    2016-01-01

    An important, irreversible step in many signalling pathways is the shedding of membrane-anchored proteins. A Disintegrin And Metalloproteinase (ADAM) 17 is one of the major sheddases involved in a variety of physiological and pathophysiological processes including regeneration, differentiation, and cancer progression. This central role in signalling implies that ADAM17 activity has to be tightly regulated, including at the level of localisation. Most mature ADAM17 is localised intracellularly, with only a small amount at the cell surface. We found that ADAM17 is constitutively internalised by clathrin-coated pits and that physiological stimulators such as GPCR ligands induce ADAM17-mediated shedding, but do not alter the cell-surface abundance of the protease. In contrast, the PKC-activating phorbol ester PMA, often used as a strong inducer of ADAM17, causes not only proteolysis by ADAM17 but also a rapid increase of the mature protease at the cell surface. This is followed by internalisation and subsequent degradation of the protease. Eventually, this leads to a substantial downregulation of mature ADAM17. Our results therefore imply that physiological activation of ADAM17 does not rely on its relocalisation, but that PMA-induced PKC activity drastically dysregulates the localisation of ADAM17. PMID:27731361

  13. PDK1 Is a Regulator of Epidermal Differentiation that Activates and Organizes Asymmetric Cell Division

    Directory of Open Access Journals (Sweden)

    Teruki Dainichi

    2016-05-01

    Full Text Available Asymmetric cell division (ACD in a perpendicular orientation promotes cell differentiation and organizes the stratified epithelium. However, the upstream cues regulating ACD have not been identified. Here, we report that phosphoinositide-dependent kinase 1 (PDK1 plays a critical role in establishing ACD in the epithelium. Production of phosphatidyl inositol triphosphate (PIP3 is localized to the apical side of basal cells. Asymmetric recruitment of atypical protein kinase C (aPKC and partitioning defective (PAR 3 is impaired in PDK1 conditional knockout (CKO epidermis. PDK1CKO keratinocytes do not undergo calcium-induced activation of aPKC or IGF1-induced activation of AKT and fail to differentiate. PDK1CKO epidermis shows decreased expression of Notch, a downstream effector of ACD, and restoration of Notch rescues defective expression of differentiation-induced Notch targets in vitro. We therefore propose that PDK1 signaling regulates the basal-to-suprabasal switch in developing epidermis by acting as both an activator and organizer of ACD and the Notch-dependent differentiation program.

  14. A leukocyte activation test identifies food items which induce release of DNA by innate immune peripheral blood leucocytes.

    Science.gov (United States)

    Garcia-Martinez, Irma; Weiss, Theresa R; Yousaf, Muhammad N; Ali, Ather; Mehal, Wajahat Z

    2018-01-01

    Leukocyte activation (LA) testing identifies food items that induce a patient specific cellular response in the immune system, and has recently been shown in a randomized double blinded prospective study to reduce symptoms in patients with irritable bowel syndrome (IBS). We hypothesized that test reactivity to particular food items, and the systemic immune response initiated by these food items, is due to the release of cellular DNA from blood immune cells. We tested this by quantifying total DNA concentration in the cellular supernatant of immune cells exposed to positive and negative foods from 20 healthy volunteers. To establish if the DNA release by positive samples is a specific phenomenon, we quantified myeloperoxidase (MPO) in cellular supernatants. We further assessed if a particular immune cell population (neutrophils, eosinophils, and basophils) was activated by the positive food items by flow cytometry analysis. To identify the signaling pathways that are required for DNA release we tested if specific inhibitors of key signaling pathways could block DNA release. Foods with a positive LA test result gave a higher supernatant DNA content when compared to foods with a negative result. This was specific as MPO levels were not increased by foods with a positive LA test. Protein kinase C (PKC) inhibitors resulted in inhibition of positive food stimulated DNA release. Positive foods resulted in CD63 levels greater than negative foods in eosinophils in 76.5% of tests. LA test identifies food items that result in release of DNA and activation of peripheral blood innate immune cells in a PKC dependent manner, suggesting that this LA test identifies food items that result in release of inflammatory markers and activation of innate immune cells. This may be the basis for the improvement in symptoms in IBS patients who followed an LA test guided diet.

  15. Chemical Composition of the Essential Oil, Total Phenolics, Total Flavonoids and Antioxidant Activity of Methanolic Extracts of Satureja montana L.

    Directory of Open Access Journals (Sweden)

    Avni Hajdari

    2016-05-01

    Full Text Available Aerial parts of Satureja montana L. (Lamiaceae were collected from seven growing wild populations (four populations in Kosovo, two in Albania and one in Montenegro in 2013 with the aim of assessing the natural variation in the chemical composition of the essential oils, total flavonoids, total phenolics and the antioxidant activity of their methanolic extracts. Essential oils were obtained by steam distillation and analysed using GC-FID and GC-MS, whereas total flavonoids, total phenolics and antioxidant activities were determined using spectrophotometric methods. Sixty-one volatile constituents were identified. The main constituents were myrcene, p-cymene, γ-terpinene, linalool, thymol, carvacrol and viridiflorol. Total phenolics ranged from 68.1 to 102.6 mg/g dry mass, the total flavonoid content ranged from 38.3 to 67.0 mg/g dm, and the antioxidant activity according to the DPPH assay ranged from 253.3 to 342.9 mg TE/g dm and according to the FRAP assay ranged from 8.9 to 11.4 mg TE/g dm. Hierarchical cluster analysis and principal component analyses were used to assess the geographical variations in the essential oil composition. Statistical analysis revealed that the analysed populations are grouped into four main clusters that appear to reflect the environmental impact on the chemical composition, which is influenced by differences in habitat composition, altitude and microclimatic conditions.

  16. Activity and Quality of Life after Total Hip Arthroplasty.

    Science.gov (United States)

    Świtoń, Anna; Wodka-Natkaniec, Ewa; Niedźwiedzki, Łukasz; Gaździk, Tadeusz; Niedźwiedzki, Tadeusz

    2017-10-31

    Coxarthrosis is a chronic musculoskeletal condition that causes severe pain and considerable limi-tation of the patient's motor performance. Total hip arthroplasty is one of the most common and effective methods used in the treatment of advanced degenerative changes. The aim of the present study was to evaluate the activity and quality of life of patients after unilateral total hip arthroplasty. The study was conducted in a group of 189 patients who had undergone unilateral total hip arthroplasty. Goniometry was used to determine the range of motion of both hip joints. Patients' physical ability and pain severity were assessed based on the Harris Hip Score (HHS) questionnaire. The examination of the range of motion in the lower extremities revealed statistically significant diffe-rences in flexion (p<0.01), abduction (p=<0.01), adduction (p<0.01) and external rotation (p<0.01) between the operated and the healthy extremity. The greatest limitation of motion was demonstrated for external rotation (<14°). Approximately 14% of the patients were not able to perform this motion in their healthy hip joint, while 17.5% of them could not do so in the affected hip joint. Analysis of HHS results (mean = 79 pts) revealed that more than 50% of the patients described their functional ability and quality of life as good and excellent. It was demonstrated that 54% of patients did not suffer from pain, whereas minor or mild pain was noted in 35%. 1. A subjective clinical assessment of patients after total hip arthroplasty showed that their quality of life had improved. 2. It is necessary to perform physiotherapy after total hip arthroplasty, on both the operated and healthy side. 3. Exacerbation of pain and impaired activity in patients after total hip arthroplasty were associated with the female sex to a considerable extent.

  17. Antioxidant activity, total phenols and flavonoids of lichens from Venezuelan Andes.

    Directory of Open Access Journals (Sweden)

    Claudia M. Plaza

    2014-10-01

    Full Text Available Context:The biological potential of lichens has been documented through their use in traditional medicine. Secondary lichen metabolites exert a wide variety of biological actions, including their use as antioxidants. Aims:To evaluate the antioxidant activity, total phenol content, and flavonoids of four lichen fungal taxa collected in Mérida (Venezuela, and statistically evaluate the correlation between the antioxidant activity and the amount of phenols and flavonoids in the samples. Methods: Extracts were prepared with water, ethanol and dichloromethane from Cladonia aff. rappii, Cora aff. glabrata, Peltigera laciniata and Thamnolia vermicularis. The antioxidant capacity assessment was determined using DPPH• radical method and reducing power, with ascorbic acid as control. Total phenols were determined by means of the Folin-Ciocalteu method with gallic acid. Total flavonoids were estimated according to the modified Dowd method, using quercetin as standard. Results:The ethanolic extracts of the tested lichens showed the highest scavenging activity and reducing power compared to water and dichloromethane extracts at 4 mg/mL. The highest antiradical power value was found in ethanolic extract of Peltigera laciniata (2.28 mL/mgand the lowest in dichloromethane extract of Cora aff. glabrata (0.30 mL/mg. The correlation between antioxidant activity and total phenolic content was moderate. The flavonoids content of ethanolic extracts was highly significant but negative (p < 0.05. There was good correlation in dichloromethane extracts. The ethanolic extract of P. laciniata exhibited the highest antiradical activity despite showing the lowest flavonoid content. Conclusions: The ethanolic extracts of lichens tested showed to have the higher antioxidant activity and may be used as natural sources of new antioxidants.

  18. Response of total phenolic content and antioxidant activities of bush ...

    African Journals Online (AJOL)

    The positive health benefits associated with tea are made possible by the antioxidant activity of phenolic compounds present in tea. The total phenolic content and antioxidant activity of bush tea (Athrixia phylicoides DC.) and special tea (Monsonia burkeana) were studied. The extractions were done in triplicate using cold ...

  19. Radio frequency radiation effects on protein kinase C activity in rats' brain

    International Nuclear Information System (INIS)

    Paulraj, R.; Behari, J.

    2004-01-01

    The present work describes the effect of amplitude modulated radio frequency (rf) radiation (112 MHz amplitude-modulated at 16 Hz) on calcium-dependent protein kinase C (PKC) activity on developing rat brain. Thirty-five days old Wistar rats were used for this study. The rats were exposed 2 h per day for 35 days at a power density of 1.0 mW/cm 2 (SAR=1.48 W/kg). After exposure, rats were sacrificed and PKC was determined in whole brain, hippocampus and whole brain minus hippocampus separately. A significant decrease in the enzyme level was observed in the exposed group as compared to the sham exposed group. These results indicate that this type of radiation could affect membrane bound enzymes associated with cell signaling, proliferation and differentiation. This may also suggest an affect on the behavior of chronically exposed rats

  20. The Effect of Structured Exercise Intervention on Intensity and Volume of Total Physical Activity

    Directory of Open Access Journals (Sweden)

    Niko Wasenius

    2014-12-01

    Full Text Available This study aimed to investigate the effects of a 12-week structured exercise intervention on total physical activity and its subcategories. Twenty-three overweight or obese middle aged men with impaired glucose regulation were randomized into a 12-week Nordic walking group, a power-type resistance training group, and a non-exercise control group. Physical activity was measured with questionnaires before the intervention (1–4 weeks and during the intervention (1–12 weeks and was expressed in metabolic equivalents of task. No significant change in the volume of total physical activity between or within the groups was observed (p > 0.050. The volume of total leisure-time physical activity (structured exercises + non-structured leisure-time physical activity increased significantly in the Nordic walking group (p 0.050 compared to the control group. In both exercise groups increase in the weekly volume of total leisure-time physical activity was inversely associated with the volume of non-leisure-time physical activities. In conclusion, structured exercise intervention did not increase the volume of total physical activity. Albeit, endurance training can increase the volume of high intensity physical activities, however it is associated with compensatory decrease in lower intensity physical activities. To achieve effective personalized exercise program, individuality in compensatory behavior should be recognised.

  1. Assessment of total flavonoid content and antioxidant activity of Mullein (Verbascum songaricum ecotypes

    Directory of Open Access Journals (Sweden)

    2017-11-01

    Full Text Available Background and objectives: The Mullein genus is the largest genus of Scrophulariaceae family which has extensive natural habitat in southwest of Iran. Mullein contains compounds such as phenolic compounds, mucilage, saponins and anthocyanin. The aim of this study was to evaluate the total flavonoid content and antioxidant activity of mullein ecotypes in Iran. Methods: Six ecotypes of the Verbascum songaricum were evaluated. Determination of total flavonoid content was performed bythealuminium chloride colourimetric method. The antioxidant activity of the flower extracts was measured using the DPPH method. Results: The results showed that total flavonoid content and antioxidant activity were different among ecotypes.  The highest and lowest amounts of total flavonoidwas obtained  from Shermard ecotype (13.42 mg rutin /g DW and Klar ecotypes(10.10 mg rutin /g DW, respectively. The highest amounts of antioxidant activity were obtained from the Shermard ecotype (IC50 246.35 μg/mL. The correlation analysis showed that a significant relation between flavonoid, antioxidant activity and habitat elevation. Conclusion: Total flavonoid content and antioxidant activity of the samples were affected by habitat climatic.  The present data indicated that the highest antioxidant activity may be due to higher flavonoid content and the habitat elevation was effective on the flavonoid content. Due to the high amounts of flavonoid and antioxidant activity of mullein extract, it seems to be a good herbal option as an antioxidant in complementary therapies.

  2. In Ovo and dietary administration of oligosaccharides extracted from palm kernel cake influence general health of pre- and neonatal broiler chicks.

    Science.gov (United States)

    Faseleh Jahromi, Mohammad; Shokryazdan, Parisa; Idrus, Zulkifli; Ebrahimi, Rohollah; Liang, Juan Boo

    2017-01-01

    Palm kernel cake (PKC) is the main byproduct from the palm oil industry in several tropical countries that contains considerable amounts of oligosaccharide. We earlier demonstrated beneficial prebiotic effects of oligosaccharides extract of PKC (OligoPKC) in starter and finisher broiler birds. This study was envisaged to elucidate the effects of in ovo and/or oral administration of the OligoPKC on prenatal and post-hatched broiler chicks. A total of 140 broiler (Cobb500) eggs were randomly divided into two groups (n = 70 each), and on day 12 of incubation, eggs in one group received in ovo injection of 0.1 mL (containing 20 mg) of OligoPKC, while those in the other group received 0.1 mL of saline (placebo) solution. Of these in ovo placebo or OligoPKC injected eggs, after hatching, six chicks from each group were sampled for day-one analysis, while 48 chicks from each group were randomly allocated to two dietary regimes involving either no feeding or feeding of OligoPKC through basal diet for a 14 days experiment forming the experimental groups as: (i) saline-injected (Control, C), (ii) OligoPKC-injected (PREBovo), (iii) saline-injected, but fed 1% OligoPKC (PREBd), and (iv) OligoPKC-injected and also 1% OligoPKC (PREBovo+d). In ovo injection of prebiotic OligoPKC had no effect on body weight and serum immunoglobulins concentrations of day old chicks, except for IgG, which was increased significantly (P C and PREBovo), but lesser influenced by in ovo OligoPKC injection. Irrespective of its prior in ovo exposure, chicks fed OligoPKC diets had lower population of pathogenic bacteria. Overall serum immunoglobulin status of birds was improved by feeding of OligoPKC but in ovo OligoPKC injection had minor effect on that. In most cases, in ovo OligoPKC injection and feeding of OligoPKC reduced the expression of nutrient transporters in the intestine and improved antioxidant capacity of liver and serum. It is concluded that in ovo injection of OligoPKC increased Ig

  3. Interação entre células do cumulus e atividade da proteína quinase C em diferentes fases da maturação nuclear de oócitos bovinos Interaction between cumulus cells and the activity of protein kinase C at different stages of bovine oocyte nuclear maturation

    Directory of Open Access Journals (Sweden)

    A.C. Bertagnolli

    2004-08-01

    Full Text Available Verificou-se a influência da proteína quinase C (PK-C no reinício e na progressão da meiose em oócitos bovinos, determinando se as células do cumulus são mediadoras da PK-C na regulação da maturação dos oócitos. Complexos cumulus-oócitos (CCO e oócitos desnudos (OD, distribuídos aleatoriamente em seis tratamentos (T com base na presença de um ativador da PK-C (PMA (T1 e T2, de um forbol éster incapaz de ativar a PK-C (4alfa-PDD-controle (T3 e T4 ou de apenas o meio básico (TCM-199-controle (T5 e T6, foram cultivados por 7, 9, 12, 18 e 22 horas. A percentagem de rompimento da vesícula germinativa no grupo cultivado com PMA foi maior do que nos dois grupos controle, com e sem células do cumulus. O cultivo de CCO e OD por 12 e 18 horas demonstrou que a PK-C influencia a progressão para os estádios de metáfase I (MI e metáfase II (MII de maneira dependente das células do cumulus. Nos períodos de 9 e 22 horas, não foi possível observar diferença entre os grupos quanto aos diferentes estádios de maturação. A ativação da PK-C acelera o reinício da meiose independentemente das células somáticas e acelera a progressão até os estádios de MI e MII na dependência das células do cumulus.The aim of this study was to evaluate the effect of protein kinase C (PK-C on the meiotic resumption and progression in bovine oocyte, and to determine if the cumulus cells mediate the PK-C action in the regulation of bovine oocyte nuclear maturation. Cumulus-oocyte complexes (COC and denuded oocytes (DO, randomly allotted to 6 treatments (T based on the presence of an activator of PK-C (PMA (T1 and T2, or a phorbol ester unable to activate PK-C (4alphaPDD-control (T3 and T4 or a basic culture medium (T5 and T6, were cultivated for 7, 9, 12, 18 and 22 hours. The percentage of germinal vesicle breakdown (GVBD was higher when the oocytes were cultured with PMA than in the control groups with and without cumulus cells. However, PK-C was

  4. Total phenol content and antioxidant activity of water solutions of plant extracts

    Directory of Open Access Journals (Sweden)

    Mirela Kopjar

    2009-01-01

    Full Text Available Water solutions of extracts were investigated for total phenol content, flavonoid content and antioxidant activity. Susceptibility to degradation of water solutions of plant extracts, under light and in the dark, during storage at room temperature was investigated in order to determine their stability prior to their application for fortification of food products. Large dispersion of total phenol (TP content in the investigated model solutions of selected extracts (olive leaves, green tea, red grape, red wine, pine bark PE 5:1, pine bark PE 95 %, resveratrol, ranging from 11.10 mg GAE/100 mL to 92.19 mg GAE/100 mL was observed. Consequently, large dispersion of total flavonoids (TF content (8.89 mg to 61.75 mg CTE/100 mL was also observed. Since phenols have been mostly responsible for antioxidant activity of extracts, in most cases, antioxidant activity followed the TP content. That was proven by estimation of correlation coefficient between the total phenol content and antioxidant activity. Correlation coefficients between investigated parameters ranged from 0.5749 to 0.9604. During storage of 5 weeks at room temperature loss of phenols and flavonoids occurred. Antioxidant activity decreased with the decrease of TP and TF content. Degradations of phenols and flavonoids were more pronounced in samples stored at light.

  5. Protein kinase C signaling and cell cycle regulation

    Directory of Open Access Journals (Sweden)

    Adrian R Black

    2013-01-01

    Full Text Available A link between T cell proliferation and the protein kinase C (PKC family of serine/threonine kinases has been recognized for about thirty years. However, despite the wealth of information on PKC-mediated control of T cell activation, understanding of the effects of PKCs on the cell cycle machinery in this cell type remains limited. Studies in other systems have revealed important cell cycle-specific effects of PKC signaling that can either positively or negatively impact proliferation. The outcome of PKC activation is highly context-dependent, with the precise cell cycle target(s and overall effects determined by the specific isozyme involved, the timing of PKC activation, the cell type, and the signaling environment. Although PKCs can regulate all stages of the cell cycle, they appear to predominantly affect G0/G1 and G2. PKCs can modulate multiple cell cycle regulatory molecules, including cyclins, cyclin-dependent kinases (cdks, cdk inhibitors and cdc25 phosphatases; however, evidence points to Cip/Kip cdk inhibitors and D-type cyclins as key mediators of PKC-regulated cell cycle-specific effects. Several PKC isozymes can target Cip/Kip proteins to control G0/G1→S and/or G2→M transit, while effects on D-type cyclins regulate entry into and progression through G1. Analysis of PKC signaling in T cells has largely focused on its roles in T cell activation; thus, observed cell cycle effects are mainly positive. A prominent role is emerging for PKCθ, with non-redundant functions of other isozymes also described. Additional evidence points to PKCδ as a negative regulator of the cell cycle in these cells. As in other cell types, context-dependent effects of individual isozymes have been noted in T cells, and Cip/Kip cdk inhibitors and D-type cyclins appear to be major PKC targets. Future studies are anticipated to take advantage of the similarities between these various systems to enhance understanding of PKC-mediated cell cycle regulation in

  6. Protein kinase C isozymes, novel phorbol ester receptors and cancer chemotherapy.

    LENUS (Irish Health Repository)

    Barry, O P

    2012-02-03

    Recent years have seen extensive growth in the understanding of the role(s) of the various PKC isozymes and novel receptors for the phorbol ester tumor promoters. The PKC family of serine-threonine kinases is an important regulator of signaling cascades that control cell proliferation and death, and therefore represent targets for cancer therapy. While past interests have focused on PKC-selective inhibitors, more recently, intensive research has been underway for selective activators and inhibitors for each individual PKC isozyme. In the past few years a large number of PKC activators and inhibitors with potential as anticancer agents have been developed. A number of these compounds are already in Phase II clinical testing. As a new generation of cancer chemotherapeutic agents are designed, developed and put through a series of rigorous clinical trials, we can anticipate achieving exquisite control over PKC-mediated regulatory pathways, leading ultimately to a greater understanding of different cancers.

  7. Total. Group dynamics and activities. Competitive environment and strategic perspectives. Release - July 2017

    International Nuclear Information System (INIS)

    2017-07-01

    After a synthesis which notably proposes a SWOT analysis of the Total group, this report proposes a presentation of the Total Group (general overview, presentation of activities, human resources, shareholder structure and stock market data, competitive environment). It gives an overview of the Total group dynamics and of its activities through a presentation of an environment analysis (world oil demand, refining-chemistry activity, hydrocarbon prices), a presentation of the group activity (turnover, turnover per segment, operational income and financial results of competitors). It comments important events and development axes: four strategic orientations, strengthening of the upstream pole, restructuring of refining and chemical activities, widening of the energy provision, consolidation of positions in the marketing and services sector. Financial data are presented along with the main economic and financial indicators. Important statistical data are provided

  8. Possible involvement of integrin-mediated signalling in oocyte activation: evidence that a cyclic RGD-containing peptide can stimulate protein kinase C and cortical granule exocytosis in mouse oocytes

    Directory of Open Access Journals (Sweden)

    Carbone Maria

    2006-09-01

    Full Text Available Abstract Background Mammalian sperm-oocyte interaction at fertilization involves several combined interactions between integrins on the oocyte and integrin ligands (disintegrins on the sperm. Recent research has indicated the ability of peptides containing the RGD sequence that characterized several sperm disintegrins, to induce intracellular Ca2+ transients and to initiate parthenogenetic development in amphibian and bovine oocytes. In the present study, we investigate the hypothesis that an integrin-associated signalling may participate in oocyte activation signalling by determining the ability of a cyclic RGD-containing peptide to stimulate the activation of protein kinase C (PKC and the exocytosis of cortical granules in mouse oocytes. Methods An In-Vitro-Fertilization assay (IVF was carried in order to test the condition under which a peptide containing the RGD sequence, cyclo(Arg-Gly-Asp-D-Phe-Val, was able to inhibit sperm fusion with zona-free mouse oocytes at metaphase II stage. PKC activity was determined by means of an assay based on the ability of cell lysates to phosphorylate MARKS peptide, a specific PKC substrate. Loss of cortical granules was evaluated by measuring density in the oocyte cortex of cortical granules stained with LCA-biotin/Texas red-streptavidin. In all the experiments, effects of a control peptide containing a non RGD sequence, cyclo(Arg-Ala-Asp-D-Phe-Val, were evaluated. Results The IVF assay revealed that the fusion rate declined significantly when insemination was carried out in the presence of cyclic RGD peptide at concentrations > or = 250 microM (P Conclusion The presents results provide evidence that a cyclic RGD peptide highly effective in inhibiting sperm-oocyte interaction stimulates in mouse oocytes the activation of PKC and the exocytosis of cortical granules. These data support the view that RGD-binding receptors may function as signalling receptors giving rise integrated signalling not sufficient for

  9. Protein kinase C and extracellular signal-regulated kinase regulate movement, attachment, pairing and egg release in Schistosoma mansoni.

    Directory of Open Access Journals (Sweden)

    Margarida Ressurreição

    2014-06-01

    Full Text Available Protein kinases C (PKCs and extracellular signal-regulated kinases (ERKs are evolutionary conserved cell signalling enzymes that coordinate cell function. Here we have employed biochemical approaches using 'smart' antibodies and functional screening to unravel the importance of these enzymes to Schistosoma mansoni physiology. Various PKC and ERK isotypes were detected, and were differentially phosphorylated (activated throughout the various S. mansoni life stages, suggesting isotype-specific roles and differences in signalling complexity during parasite development. Functional kinase mapping in adult worms revealed that activated PKC and ERK were particularly associated with the adult male tegument, musculature and oesophagus and occasionally with the oesophageal gland; other structures possessing detectable activated PKC and/or ERK included the Mehlis' gland, ootype, lumen of the vitellaria, seminal receptacle and excretory ducts. Pharmacological modulation of PKC and ERK activity in adult worms using GF109203X, U0126, or PMA, resulted in significant physiological disturbance commensurate with these proteins occupying a central position in signalling pathways associated with schistosome muscular activity, neuromuscular coordination, reproductive function, attachment and pairing. Increased activation of ERK and PKC was also detected in worms following praziquantel treatment, with increased signalling associated with the tegument and excretory system and activated ERK localizing to previously unseen structures, including the cephalic ganglia. These findings support roles for PKC and ERK in S. mansoni homeostasis, and identify these kinase groups as potential targets for chemotherapeutic treatments against human schistosomiasis, a neglected tropical disease of enormous public health significance.

  10. Lead (Pb+2) impairs long-term memory and blocks learning-induced increases in hippocampal protein kinase C activity

    International Nuclear Information System (INIS)

    Vazquez, Adrinel; Pena de Ortiz, Sandra

    2004-01-01

    The long-term storage of information in the brain known as long-term memory (LTM) depends on a variety of intracellular signaling cascades utilizing calcium (Ca 2+ ) and cyclic adenosine monophosphate as second messengers. In particular, Ca +2 /phospholipid-dependent protein kinase C (PKC) activity has been proposed to be necessary for the transition from short-term memory to LTM. Because the neurobehavioral toxicity of lead (Pb +2 ) has been associated to its interference with normal Ca +2 signaling in neurons, we studied its effects on spatial learning and memory using a hippocampal-dependent discrimination task. Adult rats received microinfusions of either Na + or Pb +2 acetate in the CA1 hippocampal subregion before each one of four training sessions. A retention test was given 7 days later to examine LTM. Results suggest that intrahippocampal Pb +2 did not affect learning of the task, but significantly impaired retention. The effects of Pb +2 selectively impaired reference memory measured in the retention test, but had no effect on the general performance because it did not affect the latency to complete the task during the test. Finally, we examined the effects of Pb +2 on the induction of hippocampal Ca +2 /phospholipid-dependent PKC activity during acquisition training. The results showed that Pb +2 interfered with the learning-induced activation of Ca +2 /phospholipid-dependent PKC on day 3 of acquisition. Overall, our results indicate that Pb +2 causes cognitive impairments in adult rats and that such effects might be subserved by interference with Ca +2 -related signaling mechanisms required for normal LTM

  11. How much locomotive activity is needed for an active physical activity level: analysis of total step counts

    Directory of Open Access Journals (Sweden)

    Ohkawara Kazunori

    2011-11-01

    Full Text Available Abstract Background Although physical activity recommendations for public health have focused on locomotive activity such as walking and running, it is uncertain how much these activities contribute to overall physical activity level (PAL. The purpose of the present study was to determine the contribution of locomotive activity to PAL using total step counts measured in a calorimeter study. Methods PAL, calculated as total energy expenditure divided by basal metabolic rate, was evaluated in 11 adult men using three different conditions for 24-hour human calorimeter measurements: a low-activity day (L-day targeted at a low active level of PAL (1.45, and a high-frequency moderate activity day (M-day or a high-frequency vigorous activity day (V-day targeted at an active level of PAL (1.75. These subjects were permitted only light activities except prescribed activities. In a separate group of 41 adults, free-living PAL was evaluated using doubly-labeled water (DLW. In both experiments, step counts per day were also measured using an accelerometer. Results In the human calorimeter study, PAL and step counts were 1.42 ± 0.10 and 8,973 ± 543 steps/d (L-day, 1.82 ± 0.14 and 29,588 ± 1,126 steps/d (M-day, and 1.74 ± 0.15 and 23,755 ± 1,038 steps/d (V-day, respectively. In the DLW study, PAL and step counts were 1.73 ± 0.15 and 10,022 ± 2,605 steps/d, and there was no significant relationship between PAL and daily step counts. Conclusions These results indicate that an enormous number of steps are needed for an active level of PAL if individuals extend physical activity-induced energy expenditure by only locomotive activity. Therefore, non-locomotive activity such as household activity should also play a significant role in increasing PAL under free-living conditions.

  12. Involvement of protein kinase C in the mechanism of action of Escherichia coli heat-stable enterotoxin (STa) in a human colonic carcinoma cell line, COLO-205

    International Nuclear Information System (INIS)

    Gupta, Dyuti Datta; Saha, Subhrajit; Chakrabarti, Manoj K.

    2005-01-01

    The present study was undertaken to determine the involvement of calcium-protein kinase C pathway in the mechanism of action of Escherichia coli heat stable enterotoxin (STa) apart from STa-induced activation of guanylate cyclase in human colonic carcinoma cell line COLO-205, which was used as a model cultured cell line to study the mechanism of action of E. coli STa. In response to E. coli STa, protein kinase C (PKC) activity was increased in a time-dependent manner with its physical translocation from cytosol to membrane. Inhibition of the PKC activity in membrane fraction and inhibition of its physical translocation in response to IP 3 -mediated calcium release inhibitor dantrolene suggested the involvement of intracellular store depletion in the regulation of PKC activity. Among different PKC isoforms, predominant involvement of calcium-dependent protein kinase C (PKCα) was specified using isotype-specific pseudosubstrate, which showed pronounce enzyme activity. Inhibition of enzyme activity by PKCα-specific inhibitor Goe6976 and immunoblott study employing isotype-specific antibody further demonstrated the involvement of calcium-dependent isoform of PKC in the mechanism of action of E. coli STa. Moreover, inhibition of guanylate cyclase activity by PKCα-specific inhibitor Goe6976 suggested the involvement of PKCα in the regulation of guanylate cyclase activity

  13. Study on Total Quality Management Activities in Taiwan Tourism Factories

    Directory of Open Access Journals (Sweden)

    Yi-Chan Chung

    2016-08-01

    Full Text Available As competition becomes increasingly fierce, how to integrate the concept of information technology involvement into total quality management activities and better serve the customers, thus increasing the effectiveness of their services, has become an important focus for many tourism factories in their selection of an appropriate operational strategy. With the tourism factories in Taiwan as the subjects, this study was therefore set out to investigate the impact of operational strategies, information technology involvement and motivation on the executive degree of total quality management (TQM activities, as well as the correlation between the executive degree of TQM activities and operational performance. This research was conducted from March to April 2016 using 56 effective questionnaires through a questionnaire survey method. According to the research findings, the higher the executive degree of TQM activities was, the more significant a positive impact it would have on operational performance; the execution of differentiation strategies would have a positive impact on the execution of TQM activities; the more a company was involved in information technology, the more significant a positive impact it would have on the execution of TQM activities; the higher the intrinsic motivation was, the more significant a positive impact it would have on the executive degrees of TQM activities. It is therefore advisable for tourism factories to implement TQM activities, adopt differentiation strategies, seek greater information technology involvement and become more strongly motivated toward TQM implementation in order to improve operational performance.

  14. Correction of metabolic abnormalities in a rodent model of obesity, metabolic syndrome, and type 2 diabetes mellitus by inhibitors of hepatic protein kinase C-ι

    Science.gov (United States)

    Sajan, Mini P.; Nimal, Sonali; Mastorides, Stephen; Acevedo-Duncan, Mildred; Kahn, C. Ronald; Fields, Alan P.; Braun, Ursula; Leitges, Michael; Farese, Robert V.

    2013-01-01

    Excessive activity of hepatic atypical protein kinase (aPKC) is proposed to play a critical role in mediating lipid and carbohydrate abnormalities in obesity, the metabolic syndrome, and type 2 diabetes mellitus. In previous studies of rodent models of obesity and type 2 diabetes mellitus, adenoviral-mediated expression of kinase-inactive aPKC rapidly reversed or markedly improved most if not all metabolic abnormalities. Here, we examined effects of 2 newly developed small-molecule PKC-ι/λ inhibitors. We used the mouse model of heterozygous muscle-specific knockout of PKC-λ, in which partial deficiency of muscle PKC-λ impairs glucose transport in muscle and thereby causes glucose intolerance and hyperinsulinemia, which, via hepatic aPKC activation, leads to abdominal obesity, hepatosteatosis, hypertriglyceridemia, and hypercholesterolemia. One inhibitor, 1H-imidazole-4-carboxamide, 5-amino-1-[2,3-dihydroxy-4-[(phosphonooxy)methyl]cyclopentyl-[1R-(1a,2b,3b,4a)], binds to the substrate-binding site of PKC-λ/ι, but not other PKCs. The other inhibitor, aurothiomalate, binds to cysteine residues in the PBl-binding domains of aPKC-λ/ι/ζ and inhibits scaffolding. Treatment with either inhibitor for 7 days inhibited aPKC, but not Akt, in liver and concomitantly improved insulin signaling to Akt and aPKC in muscle and adipocytes. Moreover, both inhibitors diminished excessive expression of hepatic, aPKC-dependent lipogenic, proinflammatory, and gluconeogenic factors; and this was accompanied by reversal or marked improvements in hyperglycemia, hyperinsulinemia, abdominal obesity, hepatosteatosis, hypertriglyceridemia, and hypercholesterolemia. Our findings highlight the pathogenetic importance of insulin signaling to hepatic PKC-ι in obesity, the metabolic syndrome, and type 2 diabetes mellitus and suggest that 1H-imidazole-4-carboxamide, 5-amino-1-[2,3-dihydroxy-4-[(phosphonooxy)methyl]cyclopentyl-[1R-(1a,2b,3b,4a)] and aurothiomalate or similar agents that

  15. Ethanol activation of protein kinase A regulates GABA-A receptor subunit expression in the cerebral cortex and contributes to ethanol-induced hypnosis

    Directory of Open Access Journals (Sweden)

    Sandeep eKumar

    2012-04-01

    Full Text Available Protein kinases are implicated in neuronal cell functions such as modulation of ion channel function, trafficking and synaptic excitability. Both protein kinase C (PKC and A (PKA are involved in regulation of γ-aminobutyric acid type A (GABA-A receptors through phosphorylation. However, the role of PKA in regulating GABA-A receptors following acute ethanol exposure is not known. The present study investigated the role of PKA in ethanol effects on GABA-A receptor α1 subunit expression in the P2 synaptosomal fraction of the rat cerebral cortex. Additionally, GABA-related behaviors were also examined. Rats were administered ethanol (2.0 – 3.5 g/kg or saline and PKC, PKA and GABA-A receptor α1 subunit levels were measured by Western blot analysis. Ethanol (3.5 g/kg transiently increased GABA-A receptor α1 subunit expression and PKA RIIβ subunit expression at similar time points whereas PKA RIIα was increased at later time points. In contrast, PKC isoform expression remained unchanged. Notably, the moderate ethanol dose (2.0g/kg had no effect on GABA-A α1 subunit levels although PKA RIIα and RIIβ were increased at 10 and 60 minutes, when PKC isozymes are also known to be elevated. To determine if PKA activation was responsible for the ethanol-induced elevation of GABA-A α1 subunits, the PKA antagonist H89 was administered to rats prior to ethanol exposure. H89 administration prevented ethanol-induced increases in GABA-A receptor α1 subunit expression. Moreover, increasing PKA activity intracerebroventricularly with Sp-cAMP prior to a hypnotic dose of ethanol increased ethanol-induced loss of righting reflex duration. This effect appears to be mediated in part by GABA-A receptors as increasing PKA activity also increased the duration of muscimol-induced loss of righting reflex. Overall these data suggest that PKA mediates ethanol-induced GABA-A receptor expression and contributes to ethanol behavioral effects involving GABA-A receptors.

  16. Differential regulation of histamine- and bradykinin-stimulated phospholipase C in adrenal chromaffin cells: evidence for involvement of different protein kinase C isoforms.

    Science.gov (United States)

    Sena, C M; Rosário, L M; Parker, P J; Patel, V; Boarder, M R

    1996-03-01

    In this report we investigate the isoforms of protein kinase C (PKC) present in cultured adrenal chromaffin cells with respect to their modulation by treatment with phorbol ester and their possible differential involvement in the regulation of responses to histamine and bradykinin. The presence of individual isoforms of PKC was investigated by using eight isoform specific antisera, as a result of which PKC-alpha, epsilon, and zeta were identified. To characterize down-regulation of these enzymes, cells were incubated for 6-48 h with 1 microM phorbol myristate acetate (PMA). PKC-epsilon down-regulated more rapidly than PKC-alpha. At 12 h, PMA pretreatment, for example, PKC-epsilon was maximally down-regulated (23 +/- 4% of controls), whereas PKC-alpha was unchanged. PKC-alpha showed partial down-regulation by 24 h of PMA pretreatment. PKC-zeta did not down-regulate at any of the times tested. Translocation from cytosol to membrane in response to PMA was also more rapid for PKC-epsilon than for PKC-alpha. The accumulation of total 3H-inositol (poly) phosphates in response to bradykinin or histamine was essentially abolished by prior treatment with 10-min PMA treatment (1 microM). However, with 12-h exposure to PMA, the bradykinin response was restored to the level seen with no prior PMA exposure. The histamine response showed no recovery by 12 h of PMA, but showed partial recovery by 24 h of PMA pretreatment. These observations showed that the restoration of the response to bradykinin corresponds to the loss of PKC-epsilon, whereas the restoration of the histamine response corresponds to the loss of PKC-alpha. This picture was confirmed with further studies on cytosolic Ca2+. The results show that chromaffin cells exhibit an unusual pattern of down-regulation of PKC isoforms on prolonged exposure to PMA, and that there is a differential effect of exposure to PMA on the histamine and bradykinin responses, suggesting that different PLC-linked receptors in chromafin

  17. Change in active travel and changes in recreational and total physical activity in adults: longitudinal findings from the iConnect study

    Science.gov (United States)

    2013-01-01

    Background To better understand the health benefits of promoting active travel, it is important to understand the relationship between a change in active travel and changes in recreational and total physical activity. Methods These analyses, carried out in April 2012, use longitudinal data from 1628 adult respondents (mean age 54 years; 47% male) in the UK-based iConnect study. Travel and recreational physical activity were measured using detailed seven-day recall instruments. Adjusted linear regression models were fitted with change in active travel defined as ‘decreased’ (15 min/week) as the primary exposure variable and changes in (a) recreational and (b) total physical activity (min/week) as the primary outcome variables. Results Active travel increased in 32% (n=529), was maintained in 33% (n=534) and decreased in 35% (n=565) of respondents. Recreational physical activity decreased in all groups but this decrease was not greater in those whose active travel increased. Conversely, changes in active travel were associated with commensurate changes in total physical activity. Compared with those whose active travel remained unchanged, total physical activity decreased by 176.9 min/week in those whose active travel had decreased (adjusted regression coefficient −154.9, 95% CI −195.3 to −114.5) and was 112.2 min/week greater among those whose active travel had increased (adjusted regression coefficient 135.1, 95% CI 94.3 to 175.9). Conclusion An increase in active travel was associated with a commensurate increase in total physical activity and not a decrease in recreational physical activity. PMID:23445724

  18. Immunomodulatory role of interleukin-10 in visceral leishmaniasis: defective activation of protein kinase C-mediated signal transduction events.

    Science.gov (United States)

    Bhattacharyya, S; Ghosh, S; Jhonson, P L; Bhattacharya, S K; Majumdar, S

    2001-03-01

    Leishmania donovani, an intracellular protozoan parasite, challenges host defense mechanisms by impairing the signal transduction of macrophages. In this study we investigated whether interleukin-10 (IL-10)-mediated alteration of signaling events in a murine model of visceral leishmaniasis is associated with macrophage deactivation. Primary in vitro cultures of macrophages infected with leishmanial parasites markedly elevated the endogenous release of IL-10. Treatment with either L. donovani or recombinant IL-10 (rIL-10) inhibited both the activity and expression of the Ca2+-dependent protein kinase C (PKC) isoform. However, preincubation with neutralizing anti-IL-10 monoclonal antibody (MAb) restored the PKC activity in the parasitized macrophage. Furthermore, we observed that coincubation of macrophages with rIL-10 and L. donovani increased the intracellular parasite burden, which was abrogated by anti-IL-10 MAb. Consistent with these observations, generation of superoxide (O2-) and nitric oxide and the release of murine tumor necrosis factor-alpha were attenuated in response to L. donovani or rIL-10 treatment. On the other hand, preincubation of the infected macrophages with neutralizing anti-IL-10 MAb significantly blocked the inhibition of nitric oxide and murine tumor necrosis factor-alpha release by the infected macrophages. These findings imply that infection with L. donovani induces endogenous secretion of murine IL-10, which in turn facilitates the intracellular survival of the protozoan and orchestrates several immunomodulatory roles via selective impairment of PKC-mediated signal transduction.

  19. Activation of muscarinic M-1 cholinoceptors by curcumin to increase contractility in urinary bladder isolated from Wistar rats.

    Science.gov (United States)

    Cheng, Tse-Chou; Lu, Chih-Cheng; Chung, Hsien-Hui; Hsu, Chih-Chieh; Kakizawa, Nozomi; Yamada, Shizuo; Cheng, Juei-Tang

    2010-04-05

    Curcumin is an active principle contained in rhizome of Curcuma longa, and it has been recently mentioned to show affinity to muscarinic M-1 cholinoceptors (M(1)-mAChR). In the present study, we found that curcumin caused a concentration-dependent increase of muscle tone in urinary bladder isolated from Wistar rats. This action was inhibited by pirenzepine at concentration enough to block M(1)-mAChR. In radioligand-binding assay, specific binding of [(3)H]-oxotremorine (OXO-M) in the rat bladder homogenates was also displaced by curcumin in a concentration-dependent manner. In the presence of inhibitors for PLC-PKC pathway, either U73122 (phospholipase C inhibitor) or chelerythrine (protein kinase C inhibitor), curcumin-stimulated contraction in urinary bladder was markedly reduced. In conclusion, the obtained results suggest that curcumin can activate M(1)-mAChR at concentrations lower than to scavenge free radicals to increase of muscle tone in urinary bladder through PLC-PKC pathway.

  20. Cytotoxicity, Total Phenolic Contents and Antioxidant Activity of the ...

    African Journals Online (AJOL)

    The leaves of Annona muricata were extracted using ethanol and the extracts were evaluated for cytotoxicity using Brine Shrimp Lethality Assay, total phenolic content (TPC) and antioxidant activity using DPPH radical scavenging assay. The crude extract showed 73.33 % mortality at 1000 μg/mL concentration and its ...

  1. A novel Leishmania infantum nuclear phosphoprotein Lepp12 which stimulates IL1-beta synthesis in THP-1 transfectants

    Directory of Open Access Journals (Sweden)

    Mograbi Baharia

    2003-04-01

    Full Text Available Abstract Background We report cloning and characterization of a novel Leishmania infantum protein which we termed Lepp12, and we examine its possible implication in the interference with intramacrophage signaling pathways. Results The protein Lepp12 contains 87 amino acid sequence and exhibits 5 potential phosphorylation sites by protein kinase C (PKC. Recombinant GST-Lepp12 is phosphorylated in vitro by exogenous PKC and by PKC-like activities present in promastigote and in the myelomonocytic THP-1 cell line, indicating that at least one phosphorylation site is functional on the recombinant Lepp12. The natural Lepp12 protein is present in L. infantum promastigotes, as evidenced using specific anti-Lepp12 antibodies produced by immunopurification from acute phase VL patient sera. Interestingly, human patient sera are strongly reactive with GST-Lepp12, demonstrating immunogenic properties of Lepp12 in man, but no immune response to Lepp12 is detectable in experimentally infected animals. When isolated from promastigotes, Lepp12 migrates as two species of apparent MW of 18.3 kDa (major and 14 kDa (minor, localizes in the nuclear fraction and appears constitutively phosphorylated. Natural Lepp12 is phosphorylable in vitro by both exogenous PKC and PKC-like activity present in THP-1 extracts. The intracellular Lepp12 transfected into THP-1 cells activates these cells to produce IL-1beta and induces an enhancing effect on PMA stimulated IL-1beta synthesis, as demonstrated using GST-Lepp12 transfectants. Conclusions Together these results indicate that Lepp12 represents a substrate for PKC or other PKC-like activities present in the promastigote form and the host cell and therefore may interfere with signal transduction pathways involving PKC.

  2. Total phenolic content, antioxidant and antimicrobial activities of Blepharis edulis extracts

    Directory of Open Access Journals (Sweden)

    Mohaddese Mahboubi

    2013-02-01

    Full Text Available Blepharis edulis is traditionally used as an antiseptic, purgative, aphrodisiac and anti-inflammatory agent. The extractsof plant aerial parts were screened for total phenolic content (TPC gallic acid equivalents (GAE, total flavonoid compound(TFC quercetin equivalents (QE, antioxidant capacity and its antimicrobial activity by micro broth dilution assay. The 50%-inhibition values of BHT and 70% (v/v aqueous ethanol, 70% (v/v aqueous methanol, methanol, and water extracts of B.edulis according to the DPPH method were found to be 19.6, 71.2, 73.7, 81.4, and 218.4 g/ml, respectively. TPC ranged from38.9 to 102.7 mg GAE/g dry extracts. The antimicrobial activity showed that yeast and fungi were sensitive and resistantmicroorganisms to the extracts. The 70%-methanol extract showed more drastic antimicrobial activity than the others. Theantimicrobial activity of ethanolic extract is the same as of the methanolic extract; water extract had the weakest antimicrobialactivity.

  3. Radiation-induced adaptive response and intracellular signal transduction pathways

    International Nuclear Information System (INIS)

    Tachibana, Akira

    2009-01-01

    As an essential biological function, cells can sense the radiation even at low dose and respond to it, and which is one of bases of the radiation-induced adaptive response (AR) where effects caused by high dose radiation are reduced by prior exposure to low dose radiation (LDR). Here described are studies of AR in well established m5S cells on the intracellular signal transduction that involves sensing of LDR and transmitting of its signal within the cell network. The first signal for AR yielded by LDR on the cell membrane is exactly unknown though hydrogen peroxide and phorbol ester (PMA) can reportedly cause AR. As PMA activates protein kinase C (PKC) and its inhibitors suppress AR, participation of PKC in AR has been suggested and supported by studies showing PKCα activation by LDR. In addition, p38 mitogen-activated protein kinase (MAPK) is shown to participate in AR by those facts that the enzyme is activated by LDR, a p38 MAPK inhibitor suppresses AR, and PKC inhibitors suppress the enzyme activation, which also suggesting that the signaling from PKC to p38 MAPK can become operative by LDR. However, the possible reverse signaling is also suggested, and thus the activation of positive feedback mechanism is postulated in PKC/p38 MAPK/phospholipase δ1/ PKC pathway. Cells introduced with siRNA against Prkca gene (coding PKCs) produce reduced amount of the enzyme, particularly, of PKCα. In those cells, AR by 5 Gy X-ray is not observed and thereby PKCα is involved in AR. The signaling in AR is only partly elucidated at present as above, and more detailed studies including identification of more PKC subtypes and signaling to DNA repair system are considered necessary. (K.T.)

  4. Down-Regulation of Desmosomes in Cultured Cells: The Roles of PKC, Microtubules and Lysosomal/Proteasomal Degradation

    Science.gov (United States)

    McHarg, Selina; Hopkins, Gemma; Lim, Lusiana; Garrod, David

    2014-01-01

    Desmosomes are intercellular adhesive junctions of major importance for tissue integrity. To allow cell motility and migration they are down-regulated in epidermal wound healing. Electron microscopy indicates that whole desmosomes are internalised by cells in tissues, but the mechanism of down-regulation is unclear. In this paper we provide an overview of the internalisation of half-desmosomes by cultured cells induced by calcium chelation. Our results show that: (i) half desmosome internalisation is dependent on conventional PKC isoforms; (ii) microtubules transport internalised half desmosomes to the region of the centrosome by a kinesin-dependent mechanism; (iii) desmosomal proteins remain colocalised after internalisation and are not recycled to the cell surface; (iv) internalised desmosomes are degraded by the combined action of lysosomes and proteasomes. We also confirm that half desmosome internalisation is dependent upon the actin cytoskeleton. These results suggest that half desmosomes are not disassembled and recycled during or after internalisation but instead are transported to the centrosomal region where they are degraded. These findings may have significance for the down-regulation of desmosomes in wounds. PMID:25291180

  5. Spinal TNFα is necessary for inactivity-induced phrenic motor facilitation

    Science.gov (United States)

    Broytman, Oleg; Baertsch, Nathan A; Baker-Herman, Tracy L

    2013-01-01

    A prolonged reduction in central neural respiratory activity elicits a form of plasticity known as inactivity-induced phrenic motor facilitation (iPMF), a ‘rebound’ increase in phrenic burst amplitude apparent once respiratory neural activity is restored. iPMF requires atypical protein kinase C (aPKC) activity within spinal segments containing the phrenic motor nucleus to stabilize an early transient increase in phrenic burst amplitude and to form long-lasting iPMF following reduced respiratory neural activity. Upstream signal(s) leading to spinal aPKC activation are unknown. We tested the hypothesis that spinal tumour necrosis factor-α (TNFα) is necessary for iPMF via an aPKC-dependent mechanism. Anaesthetized, ventilated rats were exposed to a 30 min neural apnoea; upon resumption of respiratory neural activity, a prolonged increase in phrenic burst amplitude (42 ± 9% baseline; P phrenic motor nucleus prior to neural apnoea blocked long-lasting iPMF (2 ± 8% baseline; P > 0.05). Intrathecal TNFα without neural apnoea was sufficient to elicit long-lasting phrenic motor facilitation (pMF; 62 ± 7% baseline; P < 0.05). Similar to iPMF, TNFα-induced pMF required spinal aPKC activity, as intrathecal delivery of a ζ-pseudosubstrate inhibitory peptide (PKCζ-PS) 35 min following intrathecal TNFα arrested TNFα-induced pMF (28 ± 8% baseline; P < 0.05). These data demonstrate that: (1) spinal TNFα is necessary for iPMF; and (2) spinal TNFα is sufficient to elicit pMF via a similar aPKC-dependent mechanism. These data are consistent with the hypothesis that reduced respiratory neural activity elicits iPMF via a TNFα-dependent increase in spinal aPKC activity. PMID:23878370

  6. A role for protein kinase C in the regulation of membrane fluidity and Ca²(+) flux at the endoplasmic reticulum and plasma membranes of HEK293 and Jurkat cells.

    Science.gov (United States)

    Chen, Lihong; Meng, Qingli; Jing, Xian; Xu, Pingxiang; Luo, Dali

    2011-02-01

    Protein kinase C (PKC) plays a prominent role in the regulation of a variety of cellular functions, including Ca²(+) signalling. In HEK293 and Jurkat cells, the Ca²(+) release and Ca²(+) uptake stimulated by several different activators were attenuated by activation of PKC with phorbol myristate acetate (PMA) or 1-oleoyl-2-acetyl-sn-glycerol (OAG) and potentiated by PKC inhibition with Gö6983 or knockdown of PKCα or PKCβ using shRNA. Immunostaining and Western blotting analyses revealed that PKCα and PKCβII accumulated at the plasma membrane (PM) and that these isoforms, along with PKCβI, also translocated to the endoplasmic reticulum (ER) upon activation with PMA. Measurements of membrane fluidity showed that, like the cell membrane stabilizers bovine serum albumin (BSA) and ursodeoxycholate (UDCA), PMA and OAG significantly reduced the fluidity of both the PM and ER membranes; these effects were blocked in PKC-knockdown cells. Interestingly, both BSA and UDCA inhibited the Ca²(+) responses to agonists to the same extent as PMA, whereas Tween 20, which increases membrane fluidity, raised the internal Ca²(+) concentration. Thus, activation of PKC induces both translocation of PKC to the PM and ER membranes and downregulation of membrane fluidity, thereby negatively modulating Ca²(+) flux. Copyright © 2010 Elsevier Inc. All rights reserved.

  7. Method to determine the activity concentration and total activity of radioactive waste; Metodo para determinar la concentracion de actividad y actividad total de desechos radiactivos

    Energy Technology Data Exchange (ETDEWEB)

    Angeles C, A

    2001-02-15

    A characteristic system of radioactive waste is described to determine the concentration of radionuclides activity and the total activity of bundles of radioactive waste. The system this integrated by three subsystems: - Elevator of drums. - Electromechanics. - Gamma spectroscopy. In the system it is analyzed waste of issuing gamma specifically, and this designed for materials of relative low density and it analyzes materials of cylindrical recipients.

  8. Activation of Telomerase by Ionizing Radiation: Differential Response to the Inhibition of DNA Double-Strand Break Repair by Abrogation of Poly(ADP-ribosyl)ation, by LY294002, or by Wortmannin

    International Nuclear Information System (INIS)

    Neuhof, Dirk; Zwicker, Felix; Kuepper, Jan-Heiner; Debus, Juergen; Weber, Klaus-Josef

    2007-01-01

    Purpose: Telomerase activity represents a radiation-inducible function, which may be targeted by a double-strand break (DSB)-activated signal transduction pathway. Therefore, the effects of DNA-PK inhibitors (Wortmannin and LY294002) on telomerase upregulation after irradiation were studied. In addition, the role of trans-dominant inhibition of poly(ADP-ribosyl)ation, which strongly reduces DSB rejoining, was assessed in comparison with 3-aminobenzamide. Methods and Materials: COM3 rodent cells carry a construct for the dexamethasone-inducible overexpression of the DNA-binding domain of PARP1 and exhibit greatly impaired DSB rejoining after irradiation. Telomerase activity was measured using polymerase chain reaction ELISA 1 h after irradiation with doses up to 10 Gy. Phosphorylation status of PKB/Akt and of PKCα/β II was assessed by western blotting. Results: No telomerase upregulation was detectable for irradiated cells with undisturbed DSB rejoining. In contrast, incubation with LY294002 or dexamethasone yielded pronounced radiation induction of telomerase activity that could be suppressed by Wortmannin. 3-Aminobenzamide not only was unable to induce telomerase activity but also suppressed telomerase upregulation upon incubation with LY294002 or dexamethasone. Phospho-PKB was detectable independent of irradiation or dexamethasone pretreatment, but was undetectable upon incubations with LY294002 or Wortmannin, whereas phospho-PKC rested detectable. Conclusions: Telomerase activation postirradiation was triggered by different treatments that interfere with DNA DSB processing. This telomerase upregulation, however, was not reflected by the phosporylation status of the putative mediators of TERT activation, PKB and PKC. Although an involvement of PKB in TERT activation is not supported by the present findings, a respective role of PKC isoforms other than α/β II cannot be ruled out

  9. PKA, PKC, and AKAP localization in and around the neuromuscular junction

    Directory of Open Access Journals (Sweden)

    Newton Alexandra

    2001-10-01

    Full Text Available Abstract Background One mechanism that directs the action of the second messengers, cAMP and diacylglycerol, is the compartmentalization of protein kinase A (PKA and protein kinase C (PKC. A-kinase anchoring proteins (AKAPs can recruit both enzymes to specific subcellular locations via interactions with the various isoforms of each family of kinases. We found previously that a new class of AKAPs, dual-specific AKAPs, denoted D-AKAP1 and D-AKAP2, bind to RIα in addition to the RII subunits. Results Immunohistochemistry and confocal microscopy were used here to determine that D-AKAP1 colocalizes with RIα at the postsynaptic membrane of the vertebrate neuromuscular junction (NMJ and the adjacent muscle, but not in the presynaptic region. The labeling pattern for RIα and D-AKAP1 overlapped with mitochondrial staining in the muscle fibers, consistent with our previous work showing D-AKAP1 association with mitochondria in cultured cells. The immunoreactivity of D-AKAP2 was distinct from that of D-AKAP1. We also report here that even though the PKA type II subunits (RIIα and RIIβ are localized at the NMJ, their patterns are distinctive and differ from the other R and D-AKAP patterns examined. PKCβ appeared to colocalize with the AKAP, gravin, at the postsynaptic membrane. Conclusions The kinases and AKAPs investigated have distinct patterns of colocalization, which suggest a complex arrangement of signaling micro-environments. Because the labeling patterns for RIα and D-AKAP 1 are similar in the muscle fibers and at the postsynaptic membrane, it may be that this AKAP anchors RIα in these regions. Likewise, gravin may be an anchor of PKCβ at the NMJ.

  10. Anti-oxidant and hepatoprotective activities of total flavonoids of Indocalamus latifolius

    Directory of Open Access Journals (Sweden)

    Juan Tan

    2015-12-01

    Full Text Available The total flavonoids of Indocalamus latifolius were evaluated in term of their anti-oxidant and hepatoprotective activities. The results showed that in vitro hepatoprotective and anti-oxidant activities of total flavonoids at doses of 200 and 400 mg/kg and 100 mg/kg, respectively, were comparable to those of the known hepatoprotective drug silymarin at 100 mg/kg. These data were supplemented with histopathological studies of rat liver sections. Seven of the main flavonoid compounds purified by column chromatography using silica gel, sephadex LH-20 and develosil ODS, and determined to be vitexin, orientin, isovitexin, homoorientin, tricin, tricin-7-O-β-D-glucopyranoside and quercetin-3-O-glucopyranoside.

  11. Growth Factor Inhibiting PKC Sensor in E-coli Environment Using Classification Technique and ANN Method

    Directory of Open Access Journals (Sweden)

    T. K. BASAK

    2011-03-01

    Full Text Available Protein kinease C plays an important role in angiogenesis and apoptosis in cancer. During the phase of angiogenesis the growth factor is up regulated where as during apoptosis the growth factor is down regulated. For down regulation of growth factor the pH environment of intra-cellular fluid has a specific range in the alkaline medium. Protein kinease C along with E-coli through interaction of Selenometabolite is able to maintain that alkaline environment for the apoptosis of the cancer cell with inhibition of the growth factor related to antioxidant/oxidant ratio. The present paper through implementation of Artificial Neural Network and Decision Tree has focused on metastasis linked with Capacitance Relaxation phenomena and down regulation of growth factor (VGEF. In this paper a distributed neural network has been applied to a data mining problem for classification of cancer stages inorder to have proper diagnosis of patient with PKC sensor. The Network was trained off line using 270 patterns each of 6 inputs. Using the weight obtained during training, fresh patterns were tested for accuracy in diagnosis linked with the stages of cancer.

  12. Xanthohumol, a Prenylated Flavonoid from Hops (Humulus lupulus, Prevents Platelet Activation in Human Platelets

    Directory of Open Access Journals (Sweden)

    Ye-Ming Lee

    2012-01-01

    Full Text Available Xanthohumol is the principal prenylated flavonoid in the hop plant (Humulus lupulus L.. Xanthohumol was found to be a very potent cancer chemopreventive agent through regulation of diverse mechanisms. However, no data are available concerning the effects of xanthohumol on platelet activation. The aim of this paper was to examine the antiplatelet effect of xanthohumol in washed human platelets. In the present paper, xanthohumol exhibited more-potent activity in inhibiting platelet aggregation stimulated by collagen. Xanthohumol inhibited platelet activation accompanied by relative [Ca2+]i mobilization, thromboxane A2 formation, hydroxyl radical (OH● formation, and phospholipase C (PLCγ2, protein kinase C (PKC, mitogen-activated protein kinase (MAPK, and Akt phosphorylation. Neither SQ22536, an inhibitor of adenylate cyclase, nor ODQ, an inhibitor of guanylate cyclase, reversed the xanthohumol-mediated inhibitory effect on platelet aggregation. Furthermore, xanthohumol did not significantly increase nitrate formation in platelets. This study demonstrates for the first time that xanthohumol possesses potent antiplatelet activity which may initially inhibit the PI3-kinase/Akt, p38 MAPK, and PLCγ2-PKC cascades, followed by inhibition of the thromboxane A2 formation, thereby leading to inhibition of [Ca2+]i and finally inhibition of platelet aggregation. Therefore, this novel role of xanthohumol may represent a high therapeutic potential for treatment or prevention of cardiovascular diseases.

  13. Ultrasound-Assisted Extraction of Total Flavonoids from Corn Silk and Their Antioxidant Activity

    Directory of Open Access Journals (Sweden)

    Ling-Li Zheng

    2016-01-01

    Full Text Available Object. Ultrasound-assisted extraction of total flavonoids from corn silk and their antioxidant activities were studied. Methods. Response surface methodology was adopted to optimize the extraction conditions and antioxidant activities of the extracted total flavonoids were detected through ferric reducing antioxidant power (FRAP assay. Results. Through a three-level, three-variable Box-Behnken design of response surface methodology (RSM adopting yield as response, the optimal conditions were determined as follows: ultrasonic power 500 W, extraction time 20 min, material solvent ratio 1 : 20, and ethanol concentration 30%. Under the optimum conditions, the extraction yield of total flavonoids was 1.13%. FRAP value of total flavonoids extracted from corn silk was 467.59 μmol/L. Conclusion. The total flavonoids of corn silk could be developed as food natural antioxidant reagents.

  14. Ultrasound-Assisted Extraction of Total Flavonoids from Corn Silk and Their Antioxidant Activity

    OpenAIRE

    Zheng, Ling-Li; Wen, Guan; Yuan, Min-Yong; Gao, Feng

    2016-01-01

    Object. Ultrasound-assisted extraction of total flavonoids from corn silk and their antioxidant activities were studied. Methods. Response surface methodology was adopted to optimize the extraction conditions and antioxidant activities of the extracted total flavonoids were detected through ferric reducing antioxidant power (FRAP) assay. Results. Through a three-level, three-variable Box-Behnken design of response surface methodology (RSM) adopting yield as response, the optimal conditions we...

  15. [Spectrum-effect Relationship Between Total Antioxidant Activity and HPLC Fingerprint of Arctium lappa Root].

    Science.gov (United States)

    Wang, Xiao-juan; Jiang, Lin

    2014-12-01

    To explore the spectrum-effect relationship between the HPLC fingerprint of Arctium lappa root methanol extract and the total antioxidant activity. The experiment was carried out with Gemini C18 110A (250 mm x 4.6 mm, 5 µm) column using methanol-0.04% phosphoric acid as gradient mobile phase at the flow rate of 1.0 mL/min, detection wavelength of 320 nm. The total antioxidant activity was determined by measuring the absorbance of each sample after being reacted with ammonium molybdate reagent. The spectrum-effect relationship was investigated using canonical correlation analysis (CCA). The spectrum-effect relationship between the HPLC fingerprint of Arctium lappa root methanol extract and the total antioxidant activity were established, the similarity of fingerprint of all samples was above 0.9. Peaks 1, 6, 9, 12 and 14 were principle components of Arctium lappa root for the total antioxidant activity. This method contributes to the fast comprehensive evaluation of quality of Arctium lappa root.

  16. Beyond Gap Junction Channel Function: the Expression of Cx43 Contributes to Aldosterone-Induced Mesangial Cell Proliferation via the ERK1/2 and PKC Pathways

    Directory of Open Access Journals (Sweden)

    Aiqing Zhang

    2015-06-01

    Full Text Available Aims: This study aimed to explore the precise mechanism and signaling pathways of mesangial cell (MC proliferation from a new point of view considering Connexin 43 (Cx43. Methods: MC proliferation was measured by the incorporation of 3H-thymidine (3H-TdR. Cx43 was over-expressed in MC cells using lipofectamine 2000, and the expression level was tested with reverse transcription-polymerase chain reaction (RT-PCR and Western blot analyses. The gap junction channel function was explored by Lucifer Yellow scrape loading and dye transfer (SLDT, and the intracellular calcium concentrations ([Ca2+]i were characterized by confocal microscopy on cells loaded with Fura-3/AM. Results: There was an inverse correlation between Cx43 expression and MC proliferation (P0.05. Our data also showed that the mineralcorticoid receptor (MR antagonist spironolactone, ERK1/2 inhibitor PD98059 and PKC inhibitor GF109203X could attenuate the down-regulation of Cx43 expression in Aldo-induced MC proliferation; however, the PI3K inhibitor LY294002 could block MC proliferation without affecting Cx43 expression at either the mRNA or protein level. In addition, Aldo promoted MC proliferation in parallel with increasing [Ca2+]i (PConclusions: Our study provides preliminary evidence that Cx43 is an important regulator of Aldo-promoted MC proliferation. Furthermore, reduced Cx43 expression promoted MC proliferation independent of the gap junction channel function, and this process might be mediated through the ERK1/2- and PKC-dependent pathways.

  17. GnRH signalling pathways and GnRH-induced homologous desensitization in a gonadotrope cell line (alphaT3-1).

    Science.gov (United States)

    Poulin, B; Rich, N; Mas, J L; Kordon, C; Enjalbert, A; Drouva, S V

    1998-07-25

    Exposure of the gonadotrope cells to gonadotropin-releasing hormone (GnRH) reduces their responsiveness to a new GnRH stimulation (homologous desensitization). The time frame as well as the mechanisms underlying this phenomenon are yet unclear. We studied in a gonadotrope cell line (alphaT3-1) the effects of short as well as long term GnRH pretreatments on the GnRH-induced phospholipases-C (PLC), -A2 (PLA2) and -D (PLD) activities, by measuring the production of IP3, total inositol phosphates (IPs), arachidonic acid (AA) and phosphatidylethanol (PEt) respectively. We demonstrated that although rapid desensitization of GnRH-induced IP3 formation did not occur in these cells, persistent stimulation of cells with GnRH or its analogue resulted in a time-dependent attenuation of GnRH-elicited IPs formation. GnRH-induced IPs desensitization was potentiated after direct activation of PKC by the phorbol ester TPA, suggesting the involvement of distinct mechanisms in the uncoupling exerted by either GnRH or TPA on GnRH-stimulated PI hydrolysis. The levels of individual phosphoinositides remained unchanged under any desensitization condition applied. Interestingly, while the GnRH-induced PLA2 activity was rapidly desensitized (2.5 min) after GnRH pretreatments, the neuropeptide-evoked PLD activation was affected at later times, indicating an important time-dependent contribution of these enzymatic activities in the sequential events underlying the GnRH-induced homologous desensitization processes in the gonadotropes. Under GnRH desensitization conditions, TPA was still able to induce PLD activation and to further potentiate the GnRH-evoked PLD activity. AlphaT3-1 cells possess several PKC isoforms which, except PKCzeta, were differentially down-regulated by TPA (PKCalpha, betaII, delta, epsilon, eta) or GnRH (PKCbetaII, delta, epsilon, eta). In spite of the presence of PKC inhibitors or down-regulation of PKC isoforms by TPA, the desensitizing effect of the neuropeptide on

  18. Obligatory Role of Intraluminal O2− in Acute Endothelin-1 and Angiotensin II Signaling to Mediate Endothelial Dysfunction and MAPK Activation in Guinea-Pig Hearts

    Directory of Open Access Journals (Sweden)

    Emilia Wojtera

    2014-10-01

    Full Text Available We hypothesized that, due to a cross-talk between cytoplasmic O2−-sources and intraluminally expressed xanthine oxidase (XO, intraluminal O2− is instrumental in mediating intraluminal (endothelial dysfunction and cytosolic (p38 and ERK1/2 MAPKs phosphorylation manifestations of vascular oxidative stress induced by endothelin-1 (ET-1 and angiotensin II (AT-II. Isolated guinea-pig hearts were subjected to 10-min agonist perfusion causing a burst of an intraluminal O2−. ET-1 antagonist, tezosentan, attenuated AT-II-mediated O2−, indicating its partial ET-1 mediation. ET-1 and Ang-T (AT-II + tezosentan triggered intraluminal O2−, endothelial dysfunction, MAPKs and p47phox phosphorylation, and NADPH oxidase (Nox and XO activation. These effects were: (i prevented by blocking PKC (chelerythrine, Nox (apocynin, mitochondrial ATP-dependent K+ channel (5-HD, complex II (TTFA, and XO (allopurinol; (ii mimicked by the activation of Nox (NADH; and mitochondria (diazoxide, 3-NPA and (iii the effects by NADH were prevented by 5-HD, TTFA and chelerythrine, and those by diazoxide and 3-NPA by apocynin and chelerythrine, suggesting that the agonists coactivate Nox and mitochondria, which further amplify their activity via PKC. The effects by ET-1, Ang-T, NADH, diazoxide, and 3-NPA were opposed by blocking intraluminal O2− (SOD and XO, and were mimicked by XO activation (hypoxanthine. Apocynin, TTFA, chelerythrine, and SOD opposed the effects by hypoxanthine. In conclusion, oxidative stress by agonists involves cellular inside-out and outside-in signaling in which Nox-mitochondria-PKC system and XO mutually maintain their activities via the intraluminal O2−.

  19. The phosphorylation state of CD3gamma influences T cell responsiveness and controls T cell receptor cycling

    DEFF Research Database (Denmark)

    Dietrich, J; Bäckström, T; Lauritsen, J P

    1998-01-01

    The T cell receptor (TCR) is internalized following activation of protein kinase C (PKC) via a leucine (Leu)-based motif in CD3gamma. Some studies have indicated that the TCR is recycled back to the cell surface following PKC-mediated internalization. The functional state of recycled TCR and the ......The T cell receptor (TCR) is internalized following activation of protein kinase C (PKC) via a leucine (Leu)-based motif in CD3gamma. Some studies have indicated that the TCR is recycled back to the cell surface following PKC-mediated internalization. The functional state of recycled TCR...... the phosphorylation state of CD3gamma and T cell responsiveness. Based on these observations a physiological role of CD3gamma and TCR cycling is proposed....

  20. Protein kinase C and α 2-adrenoceptor-mediated inhibition of noradrenaline release from the rat tail artery

    International Nuclear Information System (INIS)

    Bucher, B.; Neuburger, J.; Illes, P.

    1991-01-01

    In isolated rat tail arteries preincubated with [3H]noradrenaline, electrical field stimulation evoked the overflow of tritium. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activating phorbol ester, time-dependently increased the overflow at 1 mumol/L but not at 0.1 mumol/L. In contrast, the overflow was not altered by phorbol 13-acetate (PA, 1 mumol/L), which does not influence the activity of PKC. Polymyxin B (70 mumol/L), an inhibitor of PKC, depressed the overflow when given alone and, in addition, attenuated the effect of PMA, 1 mumol/L. The selective alpha 2-adrenoceptor agonist B-HT 933 depressed the overflow; PMA, 1 mumol/L, did not interfere with the effect of B-HT 933, 10 mumol/L. The results provide evidence for the participation of prejunctionally located PKC in the release of noradrenaline. However, PKC does not seem to be involved in the alpha 2-adrenoceptor-agonist-mediated inhibition of noradrenaline release

  1. Unique in vitro and in vivo thrombopoietic activities of ingenol 3,20 dibenzoate, a Ca(++-independent protein kinase C isoform agonist.

    Directory of Open Access Journals (Sweden)

    Frederick K Racke

    Full Text Available Thrombopoiesis following severe bone marrow injury frequently is delayed, thereby resulting in life-threatening thrombocytopenia for which there are limited treatment options. The reasons for these delays in recovery are not well understood. Protein kinase C (PKC agonists promote megakaryocyte differentiation in leukemia cell lines and primary cells. However, little is known about the megakaryopoietic effects of PKC agonists on primary CD34+ cells grown in culture or in vivo. Here we present evidence that the novel PKC isoform-selective agonist 3,20 ingenol dibenzoate (IDB potently stimulates early megakaryopoiesis of human CD34+ cells. In contrast, broad spectrum PKC agonists failed to do so. In vivo, a single intraperitoneal injection of IDB selectively increased platelets in mice without affecting hemoglobin or white counts. Finally, IDB strongly mitigated radiation-induced thrombocytopenia, even when administered 24 hours after irradiation. Our data demonstrate that novel PKC isoform agonists such as IDB may represent a unique therapeutic strategy for accelerating the recovery of platelet counts following severe marrow injury.

  2. Activities Contributing to Total Energy Expenditure in the United States: Results from the NHAPS Study

    Directory of Open Access Journals (Sweden)

    Block Gladys

    2004-02-01

    Full Text Available Abstract Background Physical activity is increasingly recognized as an important factor influencing health and disease status. Total energy expenditure, both low-intensity and high-intensity, contributes to maintenance of healthy body weight. This paper presents the results of a quantitative approach to determining the activities that contribute to total energy expenditure in the United States. Methods Data from the National Human Activity Pattern Survey (NHAPS were used. In 1992–1994 the NHAPS sampled 4,185 females and 3,330 males, aged 18 years and over, weighted to be representative of the 48 contiguous United States. A detailed report of each activity performed in the previous 24 hours was obtained. A score was created for each activity, by multiplying duration and intensity for each individual and summing across individuals. This score was then used to rank each activity according to its contribution to total population energy expenditure, for the total sample and separately for each gender, race, age, region, and season. Results This analysis reveals our society to be primarily sedentary; leisure time physical activity contributed only approximately 5% of the population's total energy expenditure. Not counting sleeping, the largest contributor to energy expenditure was "Driving a car", followed by "Office work" and "Watching TV". Household activities accounted for 20.1% and 33.3% of energy expenditure for males and females respectively. Conclusion The information presented in this paper may be useful in identifying common activities that could be appropriate targets for behavioral interventions to increase physical activity.

  3. High mobility group box-1 is phosphorylated by protein kinase C zeta and secreted in colon cancer cells

    International Nuclear Information System (INIS)

    Lee, Hanna; Park, Minhee; Shin, Nara; Kim, Gamin; Kim, Yun Gi; Shin, Jeon-Soo; Kim, Hoguen

    2012-01-01

    Highlights: ► Specific enzyme for HMGB1 phosphorylation and its secretion is proposed. ► Inhibition of PKC-ζ leads to significant reduction of the secreted HMGB1. ► Phosphorylation of specific site of HMGB1 redirects its secretion in cancer cells. ► Activation of PKC-ζ in cancers explains the enhanced HMGB1 secretion. -- Abstract: High mobility group box-1 (HMGB1), a nuclear protein, is overexpressed and secreted in cancer cells. Phosphorylation on two different nuclear localization signal regions are known to be important for the nuclear-to-cytoplasmic transport and secretion of HMGB1. However, little is known about the biochemical mechanism of HMGB1 modifications and its subsequent secretion from cancer cells. To identify the specific enzyme and important sites for HMGB1 phosphorylation, we screened the protein kinase C (PKC) family in a colon cancer cell line (HCT116) for HMGB1 binding by pull-down experiments using a 3XFLAG-HMGB1 construct. Strong interactions between atypical PKCs (PKC-ζ, λ, and ι) and cytoplasmic HMGB1 were observed in HCT116 cells. We further identified the most critical PKC isotype that regulates HMGB1 secretion is PKC-ζ by using PKC inhibitors and siRNA experiments. The serine residues at S39, S53 and S181 of HMGB1 were related to enhancing HMGB1 secretion. We also demonstrated overexpression and activation of PKC-ζ in colon cancer tissues. Our findings suggest that PKC-ζ is involved in the phosphorylation of HMGB1, and the phosphorylation of specific serine residues in the nuclear localization signal regions is related to enhanced HMGB1 secretion in colon cancer cells.

  4. Inhibition of JAK3 and PKC via Immunosuppressive Drugs Tofacitinib and Sotrastaurin Inhibits Proliferation of Human B Lymphocytes In Vitro.

    Science.gov (United States)

    Martina, M N; Ramirez Bajo, M J; Bañon-Maneus, E; Moya Rull, D; Hierro-Garcia, N; Revuelta, I; Campistol, J M; Rovira, J; Diekmann, F

    2016-11-01

    Antibody-mediated response in solid organ transplantation is critical for graft dysfunction and loss. The use of immunosuppressive agents partially inhibits the B-lymphocyte response leading to a risk of acute and chronic antibody-mediated rejection. This study evaluated the impact of JAK3 and PKC inhibitors tofacitinib (Tofa) and sotrastaurin (STN), respectively, on B-cell proliferation, apoptosis, and activation in vitro. Human B cells isolated from peripheral blood of healthy volunteers were cocultured with CD40 ligand-transfected fibroblasts as feeder cells in the presence of interleukin (IL) 2, IL-10, and IL-21. The cocultures were treated with immunosuppressants Tofa, STN, and rapamycin (as a control), to analyze the proliferation and apoptosis of B cells by means of Cyquant and flow cytometry, respectively. CD27 and IgG staining were applied to evaluate whether treatments modified the activation of B cells. Tofa and STN were able to inhibit B-cell proliferation to the same extent as rapamycin, without inducing cell apoptosis. After 6 days in coculture with feeder cells, all B cells showed CD27 memory B-cell phenotype. None of the immunosuppressive treatments modified the proportion between class-switched and non-class-switched memory B cells observed in nontreated cultures. The high predominance of CD27 + CD24 + phenotype was not modified by any immunosuppressive treatment. Our results show that Tofa and STN can suppress B-cell antibody responses to an extent similar to rapamycin, in vitro; therefore these compounds may be a useful therapy against antibody-mediated rejection in transplantation. Copyright © 2016. Published by Elsevier Inc.

  5. Antioxidant activity, total phenolic, and total flavonoid of extracts from stems of Jasminum nervosum Lour

    Directory of Open Access Journals (Sweden)

    Wei, Xiangyong

    2011-06-01

    Full Text Available Guangxi traditional Chinese Medical University Universidad de Medicina Tradicional China de Guangxi This study evaluated the antioxidant activities of the extracts of Jasminum nervosum Lour. stems along with the effects of different extract solvents on total phenolics (TP, total flavonoids (TF, and antioxidant potential. The antioxidant activity of the extracts was assessed using the following methods: DPPH, ABTS+ both free radicals scavenging assays, and reducing assays. TP and TF were detected by spectrophotometric and HPLC methods. In former methods, the highest amount of TP content was ethy lacetate extract (EAE, expressed as gallic acid equivalents. The greatest TF content was in the n-butanol extract (BE, expressed as lutin equivalents. No significant difference was observed in the TP/TF content between these two extracts. The antioxidant activity and TP/TF content of three extracts seemed to follow the same trend. This implied that there is a good correlation between antioxidant activities and TP/TF content. But in HPLC methods, EAE contained the highest content of lutin and gallic acid, which decreased in the same order of EAE > BE > PE, the rank order of TP/TF content of EAE and BE were different according to antioxidant ability. The overall results showed that the EAE and BE were richer in phenolics and flavonoids than petroleum ether extract (PE, and may represent a good source of antioxidants.Este estudio evaluó las actividades antioxidantes de extractos de tallos de Jasminum nervosum Lour., y el efecto de diferentes disolventes de extracción en los fenoles totales (TP y flavonoides totales (TF, y su potencial antioxidante. La actividad antioxidante de los extractos fue evaluada usando los siguientes métodos: DPPH, ABTS+ y ensayos reductores. TP y TF fueron detectados por métodos espectroscópicos y por HPLC. Con el primer método, el contenido más alto de TP se obtuvo en el extracto con acetato de etilo (EAE, expresado como

  6. Activation of bradykinin B2 receptor induced the inflammatory responses of cytosolic phospholipase A2 after the early traumatic brain injury.

    Science.gov (United States)

    Chao, Honglu; Liu, Yinlong; Lin, Chao; Xu, Xiupeng; Li, Zheng; Bao, Zhongyuan; Fan, Liang; Tao, Chao; Zhao, Lin; Liu, Yan; Wang, Xiaoming; You, Yongping; Liu, Ning; Ji, Jing

    2018-06-09

    Phospholipase A 2 is a known aggravator of inflammation and deteriorates neurological outcomes after traumatic brain injury (TBI), however the exact inflammatory mechanisms remain unknown. This study investigated the role of bradykinin and its receptor, which are known initial mediators within inflammation activation, as well as the mechanisms of the cytosolic phospholipase A 2 (cPLA 2 )-related inflammatory responses after TBI. We found that cPLA 2 and bradykinin B2 receptor were upregulated after a TBI. Rats treated with the bradykinin B2 receptor inhibitor LF 16-0687 exhibited significantly less cPLA 2 expression and related inflammatory responses in the brain cortex after sustaining a controlled cortical impact (CCI) injury. Both the cPLA 2 inhibitor and the LF16-0687 improved CCI rat outcomes by decreasing neuron death and reducing brain edema. The following TBI model utilized both primary astrocytes and primary neurons in order to gain further understanding of the inflammation mechanisms of the B2 bradykinin receptor and the cPLA 2 in the central nervous system. There was a stronger reaction from the astrocytes as well as a protective effect of LF16-0687 after the stretch injury and bradykinin treatment. The protein kinase C pathway was thought to be involved in the B2 bradykinin receptor as well as the cPLA 2 -related inflammatory responses. Rottlerin, a Protein Kinase C (PKC) δ inhibitor, decreased the activity of the cPLA 2 activity post-injury, and LF16-0687 suppressed both the PKC pathway and the cPLA 2 activity within the astrocytes. These results indicated that the bradykinin B2 receptor-mediated pathway is involved in the cPLA 2 -related inflammatory response from the PKC pathway. Copyright © 2018. Published by Elsevier B.V.

  7. Characterization of Cellulolytic Bacterial Cultures Grown in Different Substrates

    Directory of Open Access Journals (Sweden)

    Mohamed Idris Alshelmani

    2013-01-01

    Full Text Available Nine aerobic cellulolytic bacterial cultures were obtained from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Culture (DSMZ and the American Type Culture Collection (ATCC. The objectives of this study were to characterize the cellulolytic bacteria and to determine the optimum moisture ratio required for solid state fermentation (SSF of palm kernel cake (PKC. The bacteria cultures were grown on reconstituted nutrient broth, incubated at 30∘C and agitated at 200 rpm. Carboxymethyl cellulase, xylanase, and mannanase activities were determined using different substrates and after SSF of PKC. The SSF was conducted for 4 and 7 days with inoculum size of 10% (v/w on different PKC concentration-to-moisture ratios: 1 : 0.2, 1 : 0.3, 1 : 0.4, and 1 : 0.5. Results showed that Bacillus amyloliquefaciens 1067 DSMZ, Bacillus megaterium 9885 ATCC, Paenibacillus curdlanolyticus 10248 DSMZ, and Paenibacillus polymyxa 842 ATCC produced higher enzyme activities as compared to other bacterial cultures grown on different substrates. The cultures mentioned above also produced higher enzyme activities when they were incubated under SSF using PKC as a substrate in different PKC-to-moisture ratios after 4 days of incubation, indicating that these cellulolytic bacteria can be used to degrade and improve the nutrient quality of PKC.

  8. Direct activation of Transient Receptor Potential Vanilloid 1(TRPV1 by Diacylglycerol (DAG

    Directory of Open Access Journals (Sweden)

    Oh Seog

    2008-10-01

    Full Text Available Abstract The capsaicin receptor, known as transient receptor potential channel vanilloid subtype 1 (TRPV1, is activated by a wide range of noxious stimulants and putative ligands such as capsaicin, heat, pH, anandamide, and phosphorylation by protein kinase C (PKC. However, the identity of endogenous activators for TRPV1 under physiological condition is still debated. Here, we report that diacylglycerol (DAG directly activates TRPV1 channel in a membrane-delimited manner in rat dorsal root ganglion (DRG neurons. 1-oleoyl-2-acetyl-sn-glycerol (OAG, a membrane-permeable DAG analog, elicited intracellular Ca2+ transients, cationic currents and cobalt uptake that were blocked by TRPV1-selective antagonists, but not by inhibitors of PKC and DAG lipase in rat DRG neurons or HEK 293 cells heterologously expressing TRPV1. OAG induced responses were about one fifth of capsaicin induced signals, suggesting that OAG displays partial agonism. We also found that endogenously produced DAG can activate rat TRPV1 channels. Mutagenesis of rat TRPV1 revealed that DAG-binding site is at Y511, the same site for capsaicin binding, and PtdIns(4,5P2binding site may not be critical for the activation of rat TRPV1 by DAG in heterologous system. We propose that DAG serves as an endogenous ligand for rat TRPV1, acting as an integrator of Gq/11-coupled receptors and receptor tyrosine kinases that are linked to phospholipase C.

  9. Hippocampal Protein Kinase C Signaling Mediates the Short-Term Memory Impairment Induced by Delta9-Tetrahydrocannabinol.

    Science.gov (United States)

    Busquets-Garcia, Arnau; Gomis-González, Maria; Salgado-Mendialdúa, Victòria; Galera-López, Lorena; Puighermanal, Emma; Martín-García, Elena; Maldonado, Rafael; Ozaita, Andrés

    2018-04-01

    Cannabis affects cognitive performance through the activation of the endocannabinoid system, and the molecular mechanisms involved in this process are poorly understood. Using the novel object-recognition memory test in mice, we found that the main psychoactive component of cannabis, delta9-tetrahydrocannabinol (THC), alters short-term object-recognition memory specifically involving protein kinase C (PKC)-dependent signaling. Indeed, the systemic or intra-hippocampal pre-treatment with the PKC inhibitors prevented the short-term, but not the long-term, memory impairment induced by THC. In contrast, systemic pre-treatment with mammalian target of rapamycin complex 1 inhibitors, known to block the amnesic-like effects of THC on long-term memory, did not modify such a short-term cognitive deficit. Immunoblot analysis revealed a transient increase in PKC signaling activity in the hippocampus after THC treatment. Thus, THC administration induced the phosphorylation of a specific Ser residue in the hydrophobic-motif at the C-terminal tail of several PKC isoforms. This significant immunoreactive band that paralleled cognitive performance did not match in size with the major PKC isoforms expressed in the hippocampus except for PKCθ. Moreover, THC transiently enhanced the phosphorylation of the postsynaptic calmodulin-binding protein neurogranin in a PKC dependent manner. These data demonstrate that THC alters short-term object-recognition memory through hippocampal PKC/neurogranin signaling.

  10. Protein kinase C mediates platelet secretion and thrombus formation through protein kinase D2.

    Science.gov (United States)

    Konopatskaya, Olga; Matthews, Sharon A; Harper, Matthew T; Gilio, Karen; Cosemans, Judith M E M; Williams, Christopher M; Navarro, Maria N; Carter, Deborah A; Heemskerk, Johan W M; Leitges, Michael; Cantrell, Doreen; Poole, Alastair W

    2011-07-14

    Platelets are highly specialized blood cells critically involved in hemostasis and thrombosis. Members of the protein kinase C (PKC) family have established roles in regulating platelet function and thrombosis, but the molecular mechanisms are not clearly understood. In particular, the conventional PKC isoform, PKCα, is a major regulator of platelet granule secretion, but the molecular pathway from PKCα to secretion is not defined. Protein kinase D (PKD) is a family of 3 kinases activated by PKC, which may represent a step in the PKC signaling pathway to secretion. In the present study, we show that PKD2 is the sole PKD member regulated downstream of PKC in platelets, and that the conventional, but not novel, PKC isoforms provide the upstream signal. Platelets from a gene knock-in mouse in which 2 key phosphorylation sites in PKD2 have been mutated (Ser707Ala/Ser711Ala) show a significant reduction in agonist-induced dense granule secretion, but not in α-granule secretion. This deficiency in dense granule release was responsible for a reduced platelet aggregation and a marked reduction in thrombus formation. Our results show that in the molecular pathway to secretion, PKD2 is a key component of the PKC-mediated pathway to platelet activation and thrombus formation through its selective regulation of dense granule secretion.

  11. Transient Receptor Potential Vanilloid 4 Activation-Induced Increase in Glycine-Activated Current in Mouse Hippocampal Pyramidal Neurons

    Directory of Open Access Journals (Sweden)

    Mengwen Qi

    2018-02-01

    Full Text Available Background/Aims: Glycine plays an important role in regulating hippocampal inhibitory/ excitatory neurotransmission through activating glycine receptors (GlyRs and acting as a co-agonist of N-methyl-d-aspartate-type glutamate receptors. Activation of transient receptor potential vanilloid 4 (TRPV4 is reported to inhibit hippocampal A-type γ-aminobutyric acid receptor, a ligand-gated chloride ion channel. GlyRs are also ligand-gated chloride ion channels and this paper aimed to explore whether activation of TRPV4 could modulate GlyRs. Methods: Whole-cell patch clamp recording was employed to record glycine-activated current (IGly and Western blot was conducted to assess GlyRs subunits protein expression. Results: Application of TRPV4 agonist (GSK1016790A or 5,6-EET increased IGly in mouse hippocampal CA1 pyramidal neurons. This action was blocked by specific antagonists of TRPV4 (RN-1734 or HC-067047 and GlyR (strychnine, indicating that activation of TRPV4 increases strychnine-sensitive GlyR function in mouse hippocampal pyramidal neurons. GSK1016790A-induced increase in IGly was significantly attenuated by protein kinase C (PKC (BIM II or D-sphingosine or calcium/calmodulin-dependent protein kinase II (CaMKII (KN-62 or KN-93 antagonists but was unaffected by protein kinase A or protein tyrosine kinase antagonists. Finally, hippocampal protein levels of GlyR α1 α2, α3 and β subunits were not changed by treatment with GSK1016790A for 30 min or 1 h, but GlyR α2, α3 and β subunits protein levels increased in mice that were intracerebroventricularly (icv. injected with GSK1016790A for 5 d. Conclusion: Activation of TRPV4 increases GlyR function and expression, and PKC and CaMKII signaling pathways are involved in TRPV4 activation-induced increase in IGly. This study indicates that GlyRs may be effective targets for TRPV4-induced modulation of hippocampal inhibitory neurotransmission.

  12. Association of total daily physical activity with disability in community-dwelling older persons: a prospective cohort study

    Directory of Open Access Journals (Sweden)

    Shah Raj C

    2012-10-01

    Full Text Available Abstract Background Based on findings primarily using self-report measures, physical activity has been recommended to reduce disability in old age. Collecting objective measures of total daily physical activity in community-dwelling older adults is uncommon, but might enhance the understanding of the relationship of physical activity and disability. We examined whether greater total daily physical activity was associated with less report of disability in the elderly. Methods Data were from the Rush Memory and Aging Project, a longitudinal prospective cohort study of common, age-related, chronic conditions. Total daily physical activity was measured in community-dwelling participants with an average age of 82 using actigraphy for approximately 9 days. Disability was measured via self-reported basic activities of daily living (ADL. The odds ratio and 95% Confidence Interval (CI were determined for the baseline association of total daily physical activity and ADL disability using a logistic regression model adjusted for age, education level, gender and self-report physical activity. In participants without initial report of ADL disability, the hazard ratio and 95% CI were determined for the relationship of baseline total daily physical activity and the development of ADL disability using a discrete time Cox proportional hazard model adjusted for demographics and self-report physical activity. Results In 870 participants, the mean total daily physical activity was 2. 9 × 105 counts/day (range in 105 counts/day = 0.16, 13. 6 and the mean hours/week of self-reported physical activity was 3.2 (SD = 3.6. At baseline, 718 (82.5% participants reported being independent in all ADLs. At baseline, total daily physical activity was protective against disability (OR per 105 counts/day difference = 0.55; 95% CI = 0.47, 0.65. Of the participants without baseline disability, 584 were followed for 3.4 years on average. Each 105 counts/day additional total

  13. pPKCδ activates SC35 splicing factor during H9c2 myoblastic differentiation.

    Science.gov (United States)

    Zara, Susi; Falconi, Mirella; Rapino, Monica; Zago, Michela; Orsini, Giovanna; Mazzotti, Giovanni; Cataldi, Amelia; Teti, Gabriella

    2011-01-01

    Although Protein Kinase C (PKC) isoforms' role in the neonatal and adult cardiac tissue development and ageing has been widely described "in vivo", the interaction of such enzymes with specific nuclear substrates needs to be investigated. The aim of our research has been the study of the expression, localization and interaction with the splicing factor SC35 of PKC isoforms (α, δ, ε, ζ) and their potential role in modulating the transcription machinery. H9c2 cells induced to myoblast differentiation in the presence of 1% Horse Serum (HS) have represented our experimental model. The expression of PKC isoforms, their distribution and interaction with SC35 have been evaluated by western blotting, co-immunoprecipitation and double gold immunolabeling for transmission and scanning electron microscopy. Our results show PKCδ as the most expressed isoform in differentiated cells. Surprisingly, the distribution of PKCδ and SC35 does not show any significant modification between 10%FBS and 1%HS treated samples and no co-localization is observed. Moreover the interaction between the phosphorylated form of PKCδ (pPKCδ) and SC35 increases, is distributed and co-localizes within the nucleus of differentiated H9c2. These data represent reasonable evidence of pPKCδ mediated SC35 splicing factor activation, suggesting its direct effect on transcription via interaction with the transcription machinery. Furthermore, this co-localization represents a crucial event resulting in downstream changes in transcription of components which determine the morphological modifications related to cardiomyoblast differentiated phenotype.

  14. [Smoked sausages and food additives: evaluation of total mutagenic activity].

    Science.gov (United States)

    Dugan, A M; Tkacheva, D L

    2011-01-01

    The paper deals with the evaluation of the total mutagenic activity of samples of the inorganic and organic fractions of three technology smoked sausages (boiled-smoked, semi-smoked, and raw-smoked) and some food additives used to manufacture the above sausages. Their mild and moderate mutagenic effects were recorded in a Salmonella typhimurium bacterial test system with a metabolic activation system. Physicochemical analysis of the fractions of the smoked sausages has shown that their study samples are substantially contaminated with heavy metals and representatives of polycyclic aromatic hydrocarbons, partially causing the mutagenic effects observed.

  15. Sports activity after total joint arthroplasty: recommendations for the counseling physician.

    Science.gov (United States)

    Buza, John A; Fink, Leslie A; Levine, William N

    2013-02-01

    Sports activity after total joint arthroplasty (TJA) has become an increasingly important topic, as many younger patients seeking TJA have higher postoperative expectations with regard to return to athletic activity. Our current knowledge of this area is largely based on retrospective clinical studies and surveys of surgeon recommendations. The decision to participate in sports after TJA depends on the patient's general health, prior athletic experience, type of TJA, and desired sporting activity. Ultimately, patients should discuss these factors with their physician in order to make an educated decision regarding sports activity after TJA. This article summarizes the best available evidence to help guide physicians in their conversation with patients regarding safe and appropriate sports activity after TJA.

  16. High mobility group box-1 is phosphorylated by protein kinase C zeta and secreted in colon cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Hanna; Park, Minhee; Shin, Nara; Kim, Gamin [Department of Pathology, Yonsei University College of Medicine, 134 Shinchon-Dong, Seodaemoon-Ku, Seoul (Korea, Republic of); Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, 134 Shinchon-Dong, Seodaemoon-Ku, Seoul (Korea, Republic of); Kim, Yun Gi [Department of Internal Medicine, Seoul National University College of Medicine, 28 Yongon-dong, Chongno-gu, Seoul 110-744 (Korea, Republic of); Shin, Jeon-Soo [Department of Microbiology, Yonsei University College of Medicine, 134 Shinchon-Dong, Seodaemoon-Ku, Seoul (Korea, Republic of); Kim, Hoguen, E-mail: hkyonsei@yuhs.ac [Department of Pathology, Yonsei University College of Medicine, 134 Shinchon-Dong, Seodaemoon-Ku, Seoul (Korea, Republic of); Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, 134 Shinchon-Dong, Seodaemoon-Ku, Seoul (Korea, Republic of)

    2012-07-27

    Highlights: Black-Right-Pointing-Pointer Specific enzyme for HMGB1 phosphorylation and its secretion is proposed. Black-Right-Pointing-Pointer Inhibition of PKC-{zeta} leads to significant reduction of the secreted HMGB1. Black-Right-Pointing-Pointer Phosphorylation of specific site of HMGB1 redirects its secretion in cancer cells. Black-Right-Pointing-Pointer Activation of PKC-{zeta} in cancers explains the enhanced HMGB1 secretion. -- Abstract: High mobility group box-1 (HMGB1), a nuclear protein, is overexpressed and secreted in cancer cells. Phosphorylation on two different nuclear localization signal regions are known to be important for the nuclear-to-cytoplasmic transport and secretion of HMGB1. However, little is known about the biochemical mechanism of HMGB1 modifications and its subsequent secretion from cancer cells. To identify the specific enzyme and important sites for HMGB1 phosphorylation, we screened the protein kinase C (PKC) family in a colon cancer cell line (HCT116) for HMGB1 binding by pull-down experiments using a 3XFLAG-HMGB1 construct. Strong interactions between atypical PKCs (PKC-{zeta}, {lambda}, and {iota}) and cytoplasmic HMGB1 were observed in HCT116 cells. We further identified the most critical PKC isotype that regulates HMGB1 secretion is PKC-{zeta} by using PKC inhibitors and siRNA experiments. The serine residues at S39, S53 and S181 of HMGB1 were related to enhancing HMGB1 secretion. We also demonstrated overexpression and activation of PKC-{zeta} in colon cancer tissues. Our findings suggest that PKC-{zeta} is involved in the phosphorylation of HMGB1, and the phosphorylation of specific serine residues in the nuclear localization signal regions is related to enhanced HMGB1 secretion in colon cancer cells.

  17. Evaluation of antibacterial and anthelmintic activities with total phenolic contents of Piper betel leaves

    Directory of Open Access Journals (Sweden)

    Kazi Akter

    2014-09-01

    Full Text Available Objective: The study was conducted to investigate the antibacterial and anthelmintic activities and to determine total phenolic contents of methanolic extract of Piper betel leaves. Materials and Methods: The extract was subjected to assay for antibacterial activity using both gram positive and gram negative bacterial strains through disc diffusion method; anthelmintic activity with the determination of paralysis and death time using earthworm (Pheritima posthuma at five different concentrations and the determination of total phenolic contents using the Folin-ciocalteau method. Results: The extract showed significant (p

  18. Differential sensitivity of total and active soil microbial communities to drought and forest management.

    Science.gov (United States)

    Bastida, Felipe; Torres, Irene F; Andrés-Abellán, Manuela; Baldrian, Petr; López-Mondéjar, Rubén; Větrovský, Tomáš; Richnow, Hans H; Starke, Robert; Ondoño, Sara; García, Carlos; López-Serrano, Francisco R; Jehmlich, Nico

    2017-10-01

    Climate change will affect semiarid ecosystems through severe droughts that increase the competition for resources in plant and microbial communities. In these habitats, adaptations to climate change may consist of thinning-that reduces competition for resources through a decrease in tree density and the promotion of plant survival. We deciphered the functional and phylogenetic responses of the microbial community to 6 years of drought induced by rainfall exclusion and how forest management affects its resistance to drought, in a semiarid forest ecosystem dominated by Pinus halepensis Mill. A multiOMIC approach was applied to reveal novel, community-based strategies in the face of climate change. The diversity and the composition of the total and active soil microbiome were evaluated by 16S rRNA gene (bacteria) and ITS (fungal) sequencing, and by metaproteomics. The microbial biomass was analyzed by phospholipid fatty acids (PLFAs), and the microbially mediated ecosystem multifunctionality was studied by the integration of soil enzyme activities related to the cycles of C, N, and P. The microbial biomass and ecosystem multifunctionality decreased in drought-plots, as a consequence of the lower soil moisture and poorer plant development, but this decrease was more notable in unthinned plots. The structure and diversity of the total bacterial community was unaffected by drought at phylum and order level, but did so at genus level, and was influenced by seasonality. However, the total fungal community and the active microbial community were more sensitive to drought and were related to ecosystem multifunctionality. Thinning in plots without drought increased the active diversity while the total diversity was not affected. Thinning promoted the resistance of ecosystem multifunctionality to drought through changes in the active microbial community. The integration of total and active microbiome analyses avoids misinterpretations of the links between the soil microbial

  19. Staurosporine, but not Ro 31-8220, induces interleukin 2 production and synergizes with interleukin 1alpha in EL4 thymoma cells.

    Science.gov (United States)

    Mahon, T M; Matthews, J S; O'Neill, L A

    1997-07-01

    Protein kinase C (PKC) has been implicated in interleukin 1 (IL1) signal transduction in a number of cellular systems, either as a key event in IL1 action or as a negative regulator. Here we have examined the effects of two PKC inhibitors, staurosporine and the more selective agent Ro 31-8220, on IL1 responses in the murine thymoma line EL4.NOB-1. A 1 h pulse of staurosporine was found to strongly potentiate the induction of IL2 by IL1alpha in these cells. In contrast, neither a pulse nor prolonged incubation with Ro 31-8220 affected the response to IL1alpha. Both agents blocked the response to PMA, however. A 1 h pulse of staurosporine was also found to induce IL2 production on its own, activate the transcription factor nuclear factor kappaB (NFkappaB) and increase the expression of a NFkappaB-linked reporter gene. It synergized with IL1alpha in all of these responses. Ro 31-8220 was again without effect, although both staurosporine and Ro 31-8220 blocked the activation of NFkappaB by PMA. Finally, staurosporine caused the translocation of PKC-alpha and -epsilon, and to a lesser extent PKC-beta, but not PKC-θ or -zeta, from the cytosol to the membrane, although a similar effect was observed with Ro 31-8220. The results suggest that PKC is not involved in IL1alpha signalling in EL4 cells. Furthermore, the potentiating effect of staurosporine on IL1alpha action does not involve PKC inhibition, and is likely to be at the level of NFkappaB activation.

  20. Effects of dietary inclusion of palm kernel cake on nutrient utilization, rumen fermentation characteristics and microbial populations of goats fed Paspalum plicatulum hay-based diet

    Directory of Open Access Journals (Sweden)

    Sahutaya Pongprayoon

    2010-12-01

    Full Text Available To investigate the effects of inclusion of palm kernel cake (PKC in the diets on intake, digestibility, rumen fermentationcharacteristics, nitrogen balance and microbial N supply, five goats (initial BW = 20±1 kg were randomly assigned to a55 Latin square design to receive five diets, T1 = concentrate with 15% PKC, T2 = 25% PKC, T3 = 35% PKC, T4 = 45% PKCand T5 = 55% PKC, of dietary dry matter, respectively. Plicatulum hay was offered ad libitum as the roughage. A metabolismtrial lasted for 21 days during which live weight changes and feed intakes were measured. Based on this experiment, therewere no significant differences (p>0.05 among treatment groups regarding dry matter (DM intake and digestion coefficientsof DM, organic matter, crude protein, neutral detergent fiber and acid detergent fiber, except in T4 and T5 (45 and 55% PKCwhich had lower (p0.05, however the concentration of total volatile fatty acids and protozoal populations were slightly lower forgoats fed inclusion of 45-55% PKC as compared with other treatments. Based on this experiment, it could be concluded thatthe optimal level of PKC in concentrate should be 15-35% for goats fed with plicatulum hay and that it may be an effectivemeans of exploiting the use of local feed resources for goat production.

  1. Evaluation of antibacterial and anthelmintic activities with total phenolic contents of Piper betel leaves

    Science.gov (United States)

    Akter, Kazi Nahid; Karmakar, Palash; Das, Abhijit; Anonna, Shamima Nasrin; Shoma, Sharmin Akter; Sattar, Mohammad Mafruhi

    2014-01-01

    Objective: The study was conducted to investigate the antibacterial and anthelmintic activities and to determine total phenolic contents of methanolic extract of Piper betel leaves. Materials and Methods: The extract was subjected to assay for antibacterial activity using both gram positive and gram negative bacterial strains through disc diffusion method; anthelmintic activity with the determination of paralysis and death time using earthworm (Pheritima posthuma) at five different concentrations and the determination of total phenolic contents using the Folin-ciocalteau method. Results: The extract showed significant (pbetel leaves extract, therefore it may be processed for further drug research. PMID:25386394

  2. Protein Kinase C-{delta} mediates down-regulation of heterogeneous nuclear ribonucleoprotein K protein: involvement in apoptosis induction

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Feng-Hou [NO.3 People' s Hospital affiliated to Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 201900 (China); The Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China); Wu, Ying-Li [The Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China); Zhao, Meng [Institute of Health Science, SJTU-SM/Shanghai Institutes for Biological Science, Chinese Academy of Sciences, Shanghai (China); Liu, Chuan-Xu; Wang, Li-Shun [The Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China); Chen, Guo-Qiang, E-mail: chengq@shsmu.edu.cn [The Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China); Institute of Health Science, SJTU-SM/Shanghai Institutes for Biological Science, Chinese Academy of Sciences, Shanghai (China)

    2009-11-15

    We reported previously that NSC606985, a camptothecin analogue, induces apoptosis of acute myeloid leukemia (AML) cells through proteolytic activation of protein kinase C delta ({Delta}PKC-{delta}). By subcellular proteome analysis, heterogeneous nuclear ribonucleoprotein K (hnRNP K) was identified as being significantly down-regulated in NSC606985-treated leukemic NB4 cells. HnRNP K, a docking protein for DNA, RNA, and transcriptional or translational molecules, is implicated in a host of processes involving the regulation of gene expression. However, the molecular mechanisms of hnRNP K reduction and its roles during apoptosis are still not understood. In the present study, we found that, following the appearance of the {Delta}PKC-{delta}, hnRNP K protein was significantly down-regulated in NSC606985, doxorubicin, arsenic trioxide and ultraviolet-induced apoptosis. We further provided evidence that {Delta}PKC-{delta} mediated the down-regulation of hnRNP K protein during apoptosis: PKC-{delta} inhibitor could rescue the reduction of hnRNP K; hnRNP K failed to be decreased in PKC-{delta}-deficient apoptotic KG1a cells; conditional induction of {Delta}PKC-{delta} in U937T cells directly down-regulated hnRNP K protein. Moreover, the proteasome inhibitor also inhibited the down-regulation of hnRNP K protein by apoptosis inducer and the conditional expression of {Delta}PKC-{delta}. More intriguingly, the suppression of hnRNP K with siRNA transfection significantly induced apoptosis. To our knowledge, this is the first demonstration that proteolytically activated PKC-{delta} down-regulates hnRNP K protein in a proteasome-dependent manner, which plays an important role in apoptosis induction.

  3. AKTIVITAS ANTIOKSIDAN DAN KADAR FENOLIK TOTAL DARI GANGGANG MERAH (Gracilaria verrucosa L. [Antioxidant Activity and Total Phenolic Content of Red Sea Weed (Gracilaria verrucosa L.

    Directory of Open Access Journals (Sweden)

    Lydia Ninan Lestario*

    2008-12-01

    Full Text Available The aims of this study were to compare the antioxidant activity and the total phenolic content of red sea weed (Gracilaria verrucosa L. from extract of methanol, ethanol, acetone, chloroform and hexane; and the correlation between total phenolic content and the antioxidant activity of each extract; then to determine the chlorophyll a, chlorophyll b, and carotene content of each extract and their correlation with the free radical scavenging activity as well. The antioxidant activity were measured by free radical scavenging method with DPPH and by reducing power method with K4Fe(CN6 as standard, whereas the total phenolic content was measured by Folin Ciocalteu method with gallic acid as standard. Chlorophyll a, chlorophyll b, and carotene were determined by spectrophotometric method based on Lambert-Beer law. The data of antioxidant activity and total phenolic content were statistically analyzed by Randomized Completely Block Design (RCBD with five kinds of solvents as treatments and five replications. Honestly Significant Difference Test (HSDT was used to compare the difference of treatments; whereas the chlorophyll a, chlorophyll b, and carotene content were not statistically analyzed since they were only supplement data. The results showed that the highest of the antioxidant activity by free radical scavenging method was found in acetone extract : 43.43% (BHT: 84.15%; whereas by reducing power method was found in chloroform extract : 0.1756 meq K4Fe(CN6/g extract (BHT : 6.1767 meq K4Fe(CN6/g extract; and the highest of the total phenolic content was also found in acetone extract : 45.29 mg /g extract. There were close correlation between phenolic content and antioxidant activity both by free radical scavenging method and by reducing power method with r (coefficient correlation respectivelly 0.89 and 0.91.Chlorophyll a and carotene had also close correlation with the free radical scaveging activity, but not for chlorophyll b.

  4. Protein kinase C-mediated ATP stimulation of Na(+)-ATPase activity in LLC-PK1 cells involves a P2Y2 and/or P2Y4 receptor.

    Science.gov (United States)

    Wengert, M; Ribeiro, M C; Abreu, T P; Coutinho-Silva, R; Leão-Ferreira, L R; Pinheiro, A A S; Caruso-Neves, C

    2013-07-15

    ATP-activated P2Y receptors play an important role in renal sodium excretion. The aim of this study was to evaluate the modulation of ATPase-driven sodium reabsorption in the proximal tubule by ATP or adenosine (Ado). LLC-PK1 cells, a model of porcine proximal tubule cells, were used. ATP (10(-6)M) or Ado (10(-6)M) specifically stimulated Na(+)-ATPase activity without any changes in (Na(+)+K(+))-ATPase activity. Our results show that the Ado effect is mediated by its conversion to ATP. Furthermore, it was observed that the effect of ATP was mimicked by UTP, ATPγS and 2-thio-UTP, an agonist of P2Y2 and P2Y4 receptors. In addition, ATP-stimulated Na(+)-ATPase activity involves protein kinase C (PKC). Our results indicate that ATP-induced stimulation of proximal tubule Na(+)-ATPase activity is mediated by a PKC-dependent P2Y2 and/or P2Y4 pathway. These findings provide new perspectives on the role of the effect of P2Y-mediated extracellular ATP on renal sodium handling. Copyright © 2013 Elsevier Inc. All rights reserved.

  5. ANTIOXIDANT ACTIVITY, TOTAL PHENOLIC AND FLAVONOID CONTENT OF MORINDA CITRIFOLIA FRUIT EXTRACTS FROM VARIOUS EXTRACTION PROCESSES

    Directory of Open Access Journals (Sweden)

    PRAVEEN K. RAMAMOORTHY

    2007-04-01

    Full Text Available Soxhlet, Ultrasonic extract of Morinda citrifolia L. fruit and four extracts from high pressure extraction at 10 MPa using ethanol, ethyl acetate as solvent and dried by vacuum oven and spray dryer were analyzed for their antioxidant activity by peroxide value method and diphenylpicrylhydrazyl radical scavenging method. The five extracts along with the reference samples, butylated hydroxyl toluene and tannic acid were further analyzed to determine their total phenolic content by Folin-Ciocalteau method and total flavonoid content by Dowd method. The M. citrifolia extract by high pressure extraction with ethyl acetate as solvent and spray dried was found to exhibit highest antioxidant activity and total flavonoid content. High total phenolic content was determined in the high pressure extract using ethyl acetate as solvent and vacuum dried. It was interesting to note that ultrasonic extract exhibited significant antioxidant activity, total phenolic and flavonoid content. High pressure extracted M. citrifolia in ethanol was found to express lesser values comparatively. The significant difference in activity among the high pressure extracts was found to be due to the polarity of the solvents used for extraction as M. citrifolia fruit contains relatively larger quantity of non-polar antioxidant compounds. It was also found that the drying methods had significant impact on the antioxidant activity, total phenolic and flavonoid content of the extracts.

  6. Metabolic Profiling of Total Physical Activity and Sedentary Behavior in Community-Dwelling Men.

    Directory of Open Access Journals (Sweden)

    Kota Fukai

    Full Text Available Physical activity is known to be preventive against various non-communicable diseases. We investigated the relationship between daily physical activity level and plasma metabolites using a targeted metabolomics approach in a population-based study.A total of 1,193 participants (male, aged 35 to 74 years with fasting blood samples were selected from the baseline survey of a cohort study. Information on daily total physical activity, classified into four levels by quartile of metabolic equivalent scores, and sedentary behavior, defined as hours of sitting per day, was collected through a self-administered questionnaire. Plasma metabolite concentrations were quantified by capillary electrophoresis mass spectrometry method. We performed linear regression analysis models with multivariable adjustment and corrected p-values for multiple testing in the original population (n = 808. The robustness of the results was confirmed by replication analysis in a separate population (n = 385 created by random allocation.Higher levels of total physical activity were associated with various metabolite concentrations, including lower concentrations of amino acids and their derivatives, and higher concentrations of pipecolate (FDR p <0.05 in original population. The findings persisted after adjustment for age, body mass index, smoking, alcohol intake, and energy intake. Isoleucine, leucine, valine, 4-methyl-2-oxoisopentanoate, 2-oxoisopentanoate, alanine, and proline concentrations were lower with a shorter sitting time.Physical activity is related to various plasma metabolites, including known biomarkers for future insulin resistance or type 2 diabetes. These metabolites might potentially play a key role in the protective effects of higher physical activity and/or less sedentary behavior on non-communicable diseases.

  7. Triptolide, a diterpenoid triepoxide, induces antitumor proliferation via activation of c-Jun NH2-terminal kinase 1 by decreasing phosphatidylinositol 3-kinase activity in human tumor cells

    International Nuclear Information System (INIS)

    Miyata, Yoshiki; Sato, Takashi; Ito, Akira

    2005-01-01

    Triptolide, a diterpenoid triepoxide extracted from the Chinese herb Tripterygium wilfordii Hook f., exerts antitumorigenic actions against several tumor cells, but the intracellular target signal molecule(s) for this antitumorigenesis activity of triptolide remains to be identified. In the present study, we demonstrated that triptolide, in a dose-dependent manner, inhibited the proliferation of human fibrosarcoma HT-1080, human squamous carcinoma SAS, and human uterine cervical carcinoma SKG-II cells. In addition, triptolide was found to decrease phosphatidylinositol 3-kinase (PI3K) activity. A PI3K inhibitor, LY-294002, mimicked the triptolide-induced antiproliferative activity in HT-1080, SAS, and SKG-II cells. There was no change in the activity of Akt or protein kinase C (PKC), both of which are downstream effectors in the PI3K pathway. Furthermore, the phosphorylation of Ras, Raf, and mitogen-activated protein/extracellular signal-regulated kinase 1/2 was not modified in HT-1080 cells treated with triptolide. However, the phosphorylation of c-Jun NH 2 -terminal kinase 1 (JNK1) was found to increase in both triptolide- and LY-294002-treated cells. Furthermore, the triptolide-induced inhibition of HT-1080 cell proliferation was not observed by JNK1 siRNA-treatment. These results provide novel evidence that PI3K is a crucial target molecule in the antitumorigenic action of triptolide. They further suggest a possible triptolide-induced inhibitory signal for tumor cell proliferation that is initiated by the decrease in PI3K activity, which in turn leads to the augmentation of JNK1 phosphorylation via the Akt and/or PKC-independent pathway(s). Moreover, it is likely that the activation of JNK1 is required for the triptolide-induced inhibition of tumor proliferation

  8. lgl Regulates the Hippo Pathway Independently of Fat/Dachs, Kibra/Expanded/Merlin and dRASSF/dSTRIPAK

    Energy Technology Data Exchange (ETDEWEB)

    Parsons, Linda M., E-mail: parsonsl@unimelb.edu.au [Cell Cycle and Development Laboratory, Peter MacCallum Cancer Centre, Melbourne, Victoria 3002 (Australia); Department of Genetics, University of Melbourne, Melbourne, Victoria 3010 (Australia); Grzeschik, Nicola A. [Cell Cycle and Development Laboratory, Peter MacCallum Cancer Centre, Melbourne, Victoria 3002 (Australia); Richardson, Helena E., E-mail: n.a.grzeschik@umcg.nl [Cell Cycle and Development Laboratory, Peter MacCallum Cancer Centre, Melbourne, Victoria 3002 (Australia); Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, Victoria 3010 (Australia); Department of Anatomy and Neuroscience, University of Melbourne, Melbourne, Victoria 3010 (Australia); Department of Biochemistry and Molecular Biology, University of Melbourne, Melbourne, Victoria 3010 (Australia); Present address: Department of Cell Biology, University Medical Centre Groningen, Groningen (Netherlands)

    2014-04-16

    In both Drosophila and mammalian systems, the Hippo (Hpo) signalling pathway controls tissue growth by inhibiting cell proliferation and promoting apoptosis. The core pathway consists of a protein kinase Hpo (MST1/2 in mammals) that is regulated by a number of upstream inputs including Drosophila Ras Association Factor, dRASSF. We have previously shown in the developing Drosophila eye epithelium that loss of the apico-basal cell polarity regulator lethal-(2)-giant-larvae (lgl), and the concomitant increase in aPKC activity, results in ectopic proliferation and suppression of developmental cell death by blocking Hpo pathway signalling. Here, we further explore how Lgl/aPKC interacts with the Hpo pathway. Deregulation of the Hpo pathway by Lgl depletion is associated with the mislocalization of Hpo and dRASSF. We demonstrate that Lgl/aPKC regulate the Hpo pathway independently of upstream inputs from Fat/Dachs and the Kibra/Expanded/Merlin complex. We show depletion of Lgl also results in accumulation and mislocalization of components of the dSTRIPAK complex, a major phosphatase complex that directly binds to dRASSF and represses Hpo activity. However, depleting dSTRIPAK components, or removal of dRASSF did not rescue the lgl{sup −/−} or aPKC overexpression phenotypes. Thus, Lgl/aPKC regulate Hpo activity by a novel mechanism, independently of dRASSF and dSTRIPAK. Surprisingly, removal of dRASSF in tissue with increased aPKC activity results in mild tissue overgrowth, indicating that in this context dRASSF acts as a tumor suppressor. This effect was independent of the Hpo and Ras Mitogen Activated Protein Kinase (MAPK) pathways, suggesting that dRASSF regulates a novel pathway to control tissue growth.

  9. Protein kinase C signaling and cell cycle regulation

    OpenAIRE

    Black, Adrian R.; Black, Jennifer D.

    2013-01-01

    A link between T cell proliferation and the protein kinase C (PKC) family of serine/threonine kinases has been recognized for about thirty years. However, despite the wealth of information on PKC-mediated control of T cell activation, understanding of the effects of PKCs on the cell cycle machinery in this cell type remains limited. Studies in other systems have revealed important cell cycle-specific effects of PKC signaling that can either positively or negatively impact proliferation. Th...

  10. Essential oils chemical composition, antioxidant activities and total phenols of Astrodaucus persicus

    Directory of Open Access Journals (Sweden)

    Saeid Goodarzi

    2016-02-01

    Full Text Available Objective(s:Astrodaucus persicus, Apiaceae, is used as vegetable or food additive in some parts of Iran. The essential oils of different parts of Astrodaucus persicus from Kordestan province were analyzed for the first time and compared with other regions. In this study, antioxidant activities and total phenols determination of aerial parts essential oils and root fractions of A. persicus were investigated. Materials and Methods: The essential oils were obtained by hydro-distillation from flowers/fruits, leaves/stems, ripe fruits and roots of plant and analyzed by GC-MS. Crude root extract was fractionated with hexane, chloroform, ethyl acetate and methanol. Antioxidant activities by DPPH and FRAP methods and total phenols by Folin-ciocalteu assay were measured. Results: The abundant compounds of flowers/fruits blue essential oil were α-thujene, β-pinene and α-pinene. The predominant components of blue leaves/stems essential oil were α-thujene, α-pinene and α-fenchene. The major volatiles of ripe fruits blue essential oil were β-pinene, α-thujene and α-pinene. The chief compounds of root yellow essential oil were trans-caryophyllene, bicycogermacrene and germacrene-D. Total root extract and ethyl acetate fraction showed potent antioxidant activities and high amount of total phenols in comparison to other samples. Among volatile oils, the flowers/fruits essential oil showed potent reducing capacity. Conclusion: The major compounds of aerial parts essential oils were hydrocarbon monoterpenes while the chief percentage of roots essential oil constituents were hydrocarbon sesquiterpenes. α-Eudesmol and β-eudesmol were identified as responsible for creation of blue color in aerial parts essential oils. A. persicus was known as a potent antioxidant among Apiaceae.

  11. Essential oils chemical composition, antioxidant activities and total phenols of Astrodaucus persicus.

    Science.gov (United States)

    Goodarzi, Saeid; Hadjiakhoondi, Abbas; Yassa, Narguess; Khanavi, Mahnaz; Tofighi, Zahra

    2016-02-01

    Astrodaucus persicus, Apiaceae, is used as vegetable or food additive in some parts of Iran. The essential oils of different parts of Astrodaucus persicus from Kordestan province were analyzed for the first time and compared with other regions. In this study, antioxidant activities and total phenols determination of aerial parts essential oils and root fractions of A. persicus were investigated. The essential oils were obtained by hydro-distillation from flowers/fruits, leaves/stems, ripe fruits and roots of plant and analyzed by GC-MS. Crude root extract was fractionated with hexane, chloroform, ethyl acetate and methanol. Antioxidant activities by DPPH and FRAP methods and total phenols by Folin-ciocalteu assay were measured. The abundant compounds of flowers/fruits blue essential oil were α-thujene, β-pinene and α-pinene. The predominant components of blue leaves/stems essential oil were α-thujene, α-pinene and α-fenchene. The major volatiles of ripe fruits blue essential oil were β-pinene, α-thujene and α-pinene. The chief compounds of root yellow essential oil were trans-caryophyllene, bicycogermacrene and germacrene-D. Total root extract and ethyl acetate fraction showed potent antioxidant activities and high amount of total phenols in comparison to other samples. Among volatile oils, the flowers/fruits essential oil showed potent reducing capacity. The major compounds of aerial parts essential oils were hydrocarbon monoterpenes while the chief percentage of roots essential oil constituents were hydrocarbon sesquiterpenes. α-Eudesmol and β-eudesmol were identified as responsible for creation of blue color in aerial parts essential oils. A. persicus was known as a potent antioxidant among Apiaceae.

  12. SF-1 (NR5A1) expression is stimulated by the PKA pathway and is essential for the PKA-induced activation of LIPE expression in Y-1 cells.

    Science.gov (United States)

    Kulcenty, K; Holysz, M; Trzeciak, W H

    2015-10-01

    In the adrenal cortex, corticotropin induces the expression of several genes encoding proteins involved in the synthesis and intracellular transport of steroid hormones via the protein kinase A (PKA) signalling pathway, and this process is mediated by steroidogenic factor-1 (SF-1). This study was designed to elucidate the influence of the PKA and PKC pathways on the expression of the SF-1 gene in mouse adrenocortical cells, line Y-1. It has also been attempted to answer the question whether or not SF-1 plays a role in the PKA-induced expression of LIPE gene encoding hormone-sensitive lipase/cholesteryl esterase, which supplies cholesterol for steroid hormone synthesis. In this study, we found that stimulation of the PKA pathway caused a significant increase in SF-1 expression, and that this effect was abolished by the PKA inhibitor, H89. Decreased SF-1 gene transcript levels were seen with the simultaneous activation of PKA and PKC, suggesting a possible interaction between the PKA and PKC pathways. It was also observed that SF-1 increased the transcriptional activity of the LIPE gene by interacting with the SF-1 response element located in promoter A. Moreover, transient silencing of SF-1 expression with specific siRNAs abolished PKA-stimulated transcription of the LIPE gene, indicating that SF-1 is an important regulator of LIPE expression in Y-1 cells and thus could play a role in the regulation of the cholesterol supply for adrenal steroidogenesis.

  13. Comparative utilization of shea butter cake and palm kernel cake by ...

    African Journals Online (AJOL)

    A 2 x 3 factorial experiment combining two factors, SBC and PKC at three levels of inclusion (5%, 10% and 15%) was designed. The six dietary treatments were fed to a total of 180 day-old chicks up to 56 days or age. Broilers fed SBC diets consumed more feed (P < 0.05) than those fed PKC diets between 0-28 days or age.

  14. Drivers shaping the diversity and biogeography of total and active bacterial communities in the South China Sea

    Science.gov (United States)

    Zhang, Yao; Zhao, Zihao; Dai, Minhan; Jiao, Nianzhi; Herndl, Gerhard J

    2014-01-01

    To test the hypothesis that different drivers shape the diversity and biogeography of the total and active bacterial community, we examined the bacterial community composition along two transects, one from the inner Pearl River estuary to the open waters of the South China Sea (SCS) and the other from the Luzon Strait to the SCS basin, using 454 pyrosequencing of the 16S rRNA and 16S rRNA gene (V1-3 regions) and thereby characterizing the active and total bacterial community, respectively. The diversity and biogeographic patterns differed substantially between the active and total bacterial communities. Although the composition of both the total and active bacterial community was strongly correlated with environmental factors and weakly correlated with geographic distance, the active bacterial community displayed higher environmental sensitivity than the total community and particularly a greater distance effect largely caused by the active assemblage from deep waters. The 16S rRNA vs. rDNA relationships indicated that the active bacteria were low in relative abundance in the SCS. This might be due to a high competition between active bacterial taxa as indicated by our community network models. Based on these analyses, we speculate that high competition could cause some dispersal limitation of the active bacterial community resulting in a distinct distance-decay relationship. Altogether, our results indicated that the biogeographic distribution of bacteria in the SCS is the result of both environmental control and distance decay. PMID:24684298

  15. Contractions activate hormone-sensitive lipase in rat muscle by protein kinase C and mitogen-activated protein kinase

    DEFF Research Database (Denmark)

    Donsmark, Morten; Langfort, Jozef; Holm, Cecilia

    2003-01-01

    and contractions. Adrenaline acts via cAMP-dependent protein kinase (PKA). The signalling mediating the effect of contractions is unknown and was explored in this study. Incubated soleus muscles from 70 g male rats were electrically stimulated to perform repeated tetanic contractions for 5 min. The contraction......Intramuscular triacylglycerol is an important energy store and is also related to insulin resistance. The 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 both adrenaline......-induced activation of HSL was abolished by the protein kinase C (PKC) inhibitors bisindolylmaleimide I and calphostin C and reduced 50% by the mitogen-activated protein kinase kinase (MEK) inhibitor U0126, which also completely blocked extracellular signal-regulated kinase (ERK) 1 and 2 phosphorylation. None...

  16. Identification of stable cytotoxic factors in the gas phase extract of cigarette smoke and pharmacological characterization of their cytotoxicity.

    Science.gov (United States)

    Noya, Yoichi; Seki, Koh-Ichi; Asano, Hiroshi; Mai, Yosuke; Horinouchi, Takahiro; Higashi, Tsunehito; Terada, Koji; Hatate, Chizuru; Hoshi, Akimasa; Nepal, Prabha; Horiguchi, Mika; Kuge, Yuji; Miwa, Soichi

    2013-12-06

    Smoking is a major risk factor for atherosclerotic vascular diseases, but the mechanism for its genesis is unknown. We have recently shown that the gas phase of cigarette smoke (nicotine- and tar-free cigarette smoke extract; CSE) likely to reach the systemic circulation contains stable substances which cause cytotoxicity like plasma membrane damage and cell death in cultured cells, and also that the plasma membrane damage is caused through sequential activation of protein kinase C (PKC) and NADPH oxidase (NOX) and the resulting generation of reactive oxygen species (PKC/NOX-dependent mechanism), whereas cell death is caused through PKC/NOX-dependent and -independent mechanisms. To identify these stable substances, the CSE was prepared by passing the main-stream smoke of 10 cigarettes through a Cambridge glass fiber filter, trapping of the smoke in a vessel cooled at -80°C, and subsequent dissolution in 10ml of water. The CSE was fractionated into nine fractions using reversed-phase HPLC, and each fraction was screened for cytotoxicity in cultured cells, using propidium iodide uptake assay for cell membrane damage and MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] reduction assay for cell viability. The cytotoxicity was positive in two of the nine fractions (Fr2 and Fr5). After extraction of the active fractions into dichloromethane, GC/MS analysis identified 2-cyclopenten-1-one (CPO) in Fr5 but none in Fr2. After derivatization of the active fractions with O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine hydrochloride, GC/MS analysis identified acrolein, acetone and propionaldehyde in Fr2, and methyl vinyl ketone (MVK) in Fr5. After 4-h incubation, authentic acrolein and MVK induced concentration-dependent cytotoxicity with EC50 values of 75.9±8.2 and 47.0±8.0μM (mean±SEM; n=3), respectively, whereas acetone, propionaldehyde and CPO were without effect. However, after 24-h incubation, CPO induced concentration

  17. Normal levels of total body sodium and chlorine by neutron activation analysis

    International Nuclear Information System (INIS)

    Kennedy, N.S.J.; Eastell, R.; Smith, M.A.; Tothill, P.

    1983-01-01

    In vivo neutron activation analysis was used to measure total body sodium and chlorine in 18 male and 18 female normal adults. Corrections for body size were developed. Normalisation factors were derived which enable the prediction of the normal levels of sodium and chlorine in a subject. The coefficient of variation of normalised sodium was 5.9% in men and 6.9% in women, and of normalised chlorine 9.3% in men and 5.5% in women. In the range examined (40-70 years) no significant age dependence was observed for either element. Total body sodium was correlated with total body chlorine and total body calcium. Sodium excess, defined as the amount of body sodium in excess of that associated with chlorine, also correlated well with total body calcium. In females there was a mean annual loss of sodium excess of 1.2% after the menopause, similar to the loss of calcium. (author)

  18. The anti-ALS drug riluzole attenuates pericyte loss in the diabetic retinopathy of streptozotocin-treated mice

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Jeong A. [Neural Injury Research Center, Asan Institute for Life Sciences, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Chung, Yoo-Ri [Department of Ophthalmology, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Byun, Hyae-Ran [Neural Injury Research Center, Asan Institute for Life Sciences, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Park, Hwangseo [Department of Bioscience and Biotechnology, Sejong University, Seoul (Korea, Republic of); Koh, Jae-Young, E-mail: jkko@amc.seoul.kr [Neural Injury Research Center, Asan Institute for Life Sciences, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Department of Neurology, Asan Medical Center, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Yoon, Young Hee, E-mail: yhyoon@amc.seoul.kr [Department of Ophthalmology, University of Ulsan College of Medicine, Seoul (Korea, Republic of)

    2017-01-15

    Loss of pericytes, considered an early hallmark of diabetic retinopathy, is thought to involve abnormal activation of protein kinase C (PKC). We previously showed that the anti-amyotrophic lateral sclerosis (ALS) drug riluzole functions as a PKC inhibitor. Here, we examined the effects of riluzole on pathological changes in diabetic retinopathy. Pathological endpoints examined in vivo included the number of pericytes and integrity of retinal vessels in streptozotocin (STZ)-induced diabetic mice. In addition, PKC activation and the induction of monocyte chemotactic protein (MCP1) were assessed in diabetic mice and in human retinal pericytes exposed to advanced glycation end product (AGE) or modified low-density lipoprotein (mLDL). The diameter of retinal vessels and the number of pericytes were severely reduced, and the levels of MCP1 and PKC were increased in STZ-induced diabetic mice. Administration of riluzole reversed all of these changes. Furthermore, the increased expression of MCP1 in AGE- or mLDL-treated cultured retinal pericytes was inhibited by treatment with riluzole or the PKC inhibitor GF109203X. In silico modeling showed that riluzole fits well within the catalytic pocket of PKC. Taken together, our results demonstrate that riluzole attenuates both MCP1 induction and pericyte loss in diabetic retinopathy, likely through its direct inhibitory effect on PKC. - Highlights: • The effects of riluzole were examined in streptozotocin-induced diabetic mice. • The diameter of retinal vessels and the number of pericytes were severely reduced. • The levels of MCP1 and PKC were increased, while riluzole reversed all changes. • Riluzole attenuated the level of MCP1 in AGE- or mLDL-treated retinal pericytes. • Riluzole attenuated both MCP1 induction and pericyte loss in diabetic retinopathy.

  19. The anti-ALS drug riluzole attenuates pericyte loss in the diabetic retinopathy of streptozotocin-treated mice

    International Nuclear Information System (INIS)

    Choi, Jeong A.; Chung, Yoo-Ri; Byun, Hyae-Ran; Park, Hwangseo; Koh, Jae-Young; Yoon, Young Hee

    2017-01-01

    Loss of pericytes, considered an early hallmark of diabetic retinopathy, is thought to involve abnormal activation of protein kinase C (PKC). We previously showed that the anti-amyotrophic lateral sclerosis (ALS) drug riluzole functions as a PKC inhibitor. Here, we examined the effects of riluzole on pathological changes in diabetic retinopathy. Pathological endpoints examined in vivo included the number of pericytes and integrity of retinal vessels in streptozotocin (STZ)-induced diabetic mice. In addition, PKC activation and the induction of monocyte chemotactic protein (MCP1) were assessed in diabetic mice and in human retinal pericytes exposed to advanced glycation end product (AGE) or modified low-density lipoprotein (mLDL). The diameter of retinal vessels and the number of pericytes were severely reduced, and the levels of MCP1 and PKC were increased in STZ-induced diabetic mice. Administration of riluzole reversed all of these changes. Furthermore, the increased expression of MCP1 in AGE- or mLDL-treated cultured retinal pericytes was inhibited by treatment with riluzole or the PKC inhibitor GF109203X. In silico modeling showed that riluzole fits well within the catalytic pocket of PKC. Taken together, our results demonstrate that riluzole attenuates both MCP1 induction and pericyte loss in diabetic retinopathy, likely through its direct inhibitory effect on PKC. - Highlights: • The effects of riluzole were examined in streptozotocin-induced diabetic mice. • The diameter of retinal vessels and the number of pericytes were severely reduced. • The levels of MCP1 and PKC were increased, while riluzole reversed all changes. • Riluzole attenuated the level of MCP1 in AGE- or mLDL-treated retinal pericytes. • Riluzole attenuated both MCP1 induction and pericyte loss in diabetic retinopathy.

  20. Protein kinase C alpha controls erythropoietin receptor signaling

    NARCIS (Netherlands)

    von Lindern, M.; Parren-van Amelsvoort, M.; van Dijk, T.; Deiner, E.; van den Akker, E.; van Emst-de Vries, S.; Willems, P.; Beug, H.; Löwenberg, B.

    2000-01-01

    Protein kinase C (PKC) is implied in the activation of multiple targets of erythropoietin (Epo) signaling, but its exact role in Epo receptor (EpoR) signal transduction and in the regulation of erythroid proliferation and differentiation remained elusive. We analyzed the effect of PKC inhibitors

  1. The determinants of total entrepreneurial activity : a spatial approach / by Albertus Jacobus Meintjes

    OpenAIRE

    Meintjes, Albertus Jakobus

    2006-01-01

    Various bodies and governments from around the world have recognised the importance of entrepreneurship in economic growth and job creation. The rate of start-up businesses and entrepreneurial activity is crucial in every country. The Global Entrepreneurship Monitor (GEM) recognises the contribution of entrepreneurial activity to economic growth and measures it by means of the total entrepreneurial activity (TEA) index. Most recent evidence suggests that regional (or spatial...

  2. Total sitting time, leisure time physical activity and risk of hospitalization due to low back pain

    DEFF Research Database (Denmark)

    Balling, Mie; Holmberg, Teresa; Petersen, Christina B

    2018-01-01

    AIMS: This study aimed to test the hypotheses that a high total sitting time and vigorous physical activity in leisure time increase the risk of low back pain and herniated lumbar disc disease. METHODS: A total of 76,438 adults answered questions regarding their total sitting time and physical...... activity during leisure time in the Danish Health Examination Survey 2007-2008. Information on low back pain diagnoses up to 10 September 2015 was obtained from The National Patient Register. The mean follow-up time was 7.4 years. Data were analysed using Cox regression analysis with adjustment...... disc disease. However, moderate or vigorous physical activity, as compared to light physical activity, was associated with increased risk of low back pain (HR = 1.16, 95% CI: 1.03-1.30 and HR = 1.45, 95% CI: 1.15-1.83). Moderate, but not vigorous physical activity was associated with increased risk...

  3. Trichinella spiralis infection enhances protein kinase C phosphorylation in guinea pig alveolar macrophages.

    Science.gov (United States)

    Dzik, J M; Zieliński, Z; Cieśla, J; Wałajtys-Rode, E

    2010-03-01

    To learn more about the signalling pathways involved in superoxide anion production in guinea pig alveolar macrophages, triggered by Trichinella spiralis infection, protein level and phosphorylation of mitogen activated protein (MAP) kinases and protein kinase C (PKC) were investigated. Infection with T. spiralis, the nematode having 'lung phase' during colonization of the host, enhances PKC phosphorylation in guinea pig alveolar macrophages. Isoenzymes beta and delta of PKC have been found significantly phosphorylated, although their location was not changed as a consequence of T. spiralis infection. Neither in macrophages from T. spiralis-infected guinea pig nor in platelet-activating factor (PAF)-stimulated macrophages from uninfected animals, participation of MAP kinases in respiratory burst activation was statistically significant. The parasite antigens seem to act through macrophage PAF receptors, transducing a signal for enhanced NADPH oxidase activity, as stimulating effect of newborn larvae homogenate on respiratory burst was abolished by specific PAF receptor antagonist CV 6209. A suppressive action of T. spiralis larvae on host alveolar macrophage innate immunological response was reflected by diminished protein level of ERK2 kinase and suppressed superoxide anion production, in spite of high level of PKC phosphorylation.

  4. PKM-ζ is not required for hippocampal synaptic plasticity, learning and memory.

    Science.gov (United States)

    Volk, Lenora J; Bachman, Julia L; Johnson, Richard; Yu, Yilin; Huganir, Richard L

    2013-01-17

    Long-term potentiation (LTP), a well-characterized form of synaptic plasticity, has long been postulated as a cellular correlate of learning and memory. Although LTP can persist for long periods of time, the mechanisms underlying LTP maintenance, in the midst of ongoing protein turnover and synaptic activity, remain elusive. Sustained activation of the brain-specific protein kinase C (PKC) isoform protein kinase M-ζ (PKM-ζ) has been reported to be necessary for both LTP maintenance and long-term memory. Inhibiting PKM-ζ activity using a synthetic zeta inhibitory peptide (ZIP) based on the PKC-ζ pseudosubstrate sequence reverses established LTP in vitro and in vivo. More notably, infusion of ZIP eliminates memories for a growing list of experience-dependent behaviours, including active place avoidance, conditioned taste aversion, fear conditioning and spatial learning. However, most of the evidence supporting a role for PKM-ζ in LTP and memory relies heavily on pharmacological inhibition of PKM-ζ by ZIP. To further investigate the involvement of PKM-ζ in the maintenance of LTP and memory, we generated transgenic mice lacking PKC-ζ and PKM-ζ. We find that both conventional and conditional PKC-ζ/PKM-ζ knockout mice show normal synaptic transmission and LTP at Schaffer collateral-CA1 synapses, and have no deficits in several hippocampal-dependent learning and memory tasks. Notably, ZIP still reverses LTP in PKC-ζ/PKM-ζ knockout mice, indicating that the effects of ZIP are independent of PKM-ζ.

  5. Spinal atypical protein kinase C activity is necessary to stabilize inactivity-induced phrenic motor facilitation

    Science.gov (United States)

    Strey, K.A.; Nichols, N.L.; Baertsch, N.A.; Broytman, O.; Baker-Herman, T.L.

    2012-01-01

    The neural network controlling breathing must establish rhythmic motor output at a level adequate to sustain life. Reduced respiratory neural activity elicits a novel form of plasticity in circuits driving the diaphragm known as inactivity-induced phrenic motor facilitation (iPMF), a rebound increase in phrenic inspiratory output observed once respiratory neural drive is restored. The mechanisms underlying iPMF are unknown. Here, we demonstrate in anesthetized rats that spinal mechanisms give rise to iPMF, and that iPMF consists of at least two mechanistically distinct phases: 1) an early, labile phase that requires atypical PKC (PKCζ and/or PKCΙ/λ) activity to transition to a 2) late, stable phase. Early (but not late) iPMF is associated with increased interactions between PKCζ/Ι and the scaffolding protein ZIP/p62 in spinal regions associated with the phrenic motor pool. Although PKCζ/Ι activity is necessary for iPMF, spinal aPKC activity is not necessary for phrenic long-term facilitation (pLTF) following acute intermittent hypoxia, an activity-independent form of spinal respiratory plasticity. Thus, while iPMF and pLTF both manifest as prolonged increases in phrenic burst amplitude, they arise from distinct spinal cellular pathways. Our data are consistent with the hypotheses that: 1) local mechanisms sense and respond to reduced respiratory-related activity in the phrenic motor pool, and 2) inactivity-induced increases in phrenic inspiratory output require local PKCζ/Ι activity to stabilize into a long-lasting iPMF. Although the physiological role of iPMF is unknown, we suspect that iPMF represents a compensatory mechanism, assuring adequate motor output in a physiological system where prolonged inactivity ends life. PMID:23152633

  6. Physical Activity and Adherence to Mediterranean Diet Increase Total Antioxidant Capacity: The ATTICA Study

    Directory of Open Access Journals (Sweden)

    Stavros A. Kavouras

    2011-01-01

    Full Text Available We studied the association of physical activity and adherence to the Mediterranean diet, in total antioxidant capacity (TAC. A random sample of 1514 men and 1528 women was selected from Attica region. Physical activity was assessed with a translated version of the validated “International Physical Activity Questionnaire” (iPAQ, and dietary intake through a validated Food Frequency Questionnaire (FFQ. Adherence to the Mediterranean diet was assessed by the MedDietScore that incorporated the inherent characteristics of this diet. TAC was positively correlated with the degree of physical activity (P<.05. TAC was also positively correlated with MedDietScore (r=0.24, P<.001. Stratified analysis by diet status revealed that the most beneficial results were observed to highly active people as compared to inactive, who also followed the Mediterranean diet (288  ±  70 μmol/L, 230  ±  50 μmol/L, resp., after adjusting for various confounders. Increased physical activity and greater adherence to the Mediterranean diet were associated with increased total antioxidant capacity.

  7. Measurement of total body chlorine by prompt gamma in vivo neutron activation analysis

    International Nuclear Information System (INIS)

    Beddoe, A.H.; Streat, S.J.; Hill, G.L.

    1987-01-01

    A method of measuring total body chlorine (TBCl) by prompt gamma in vivo neutron activation analysis is described depending on the same NaI(Tl) spectra used for determinations of total body nitrogen. Ratios of chlorine to hydrogen are derived and TBCl determined using a model of body composition depending on measured body weight, total body water (by tritium dilution) and protein (6.25 x nitrogen) as well as estimated body minerals and glycogen. The precision of the method based on scanning an anthropomorphic phantom is approximately 9% (SD), for a patient dose equivalent of less than 0.30 mSv. Spectra collected from 67 normal volunteers (32 male, 35 female) yielded mean values of TBCl of 72 +- 19 (SD) g in males and 53.6 +- 15 g in females, in broad agreement with values reported by workers using delayed gamma methods. Results are presented for two human cadavers analysed by neutron activation and conventional chemical analysis; the ratios of TBCl (neutron activation) to TBCl (chemical) were 0.980 +- 0.028 (SEM) and 0.91 +- 0.09. It is suggested that an improvement in precision will be achieved by increasing the scanning time (thereby increasing the radiation dose equivalent) and by adding two more detectors. (author)

  8. Daily total physical activity level and total cancer risk in men and women: results from a large-scale population-based cohort study in Japan.

    Science.gov (United States)

    Inoue, Manami; Yamamoto, Seiichiro; Kurahashi, Norie; Iwasaki, Motoki; Sasazuki, Shizuka; Tsugane, Shoichiro

    2008-08-15

    The impact of total physical activity level on cancer risk has not been fully clarified, particularly in non-Western, relatively lean populations. The authors prospectively examined the association between daily total physical activity (using a metabolic equivalents/day score) and subsequent cancer risk in the Japan Public Health Center-based Prospective Study. A total of 79,771 general-population Japanese men and women aged 45-74 years who responded to a questionnaire in 1995-1999 were followed for total cancer incidence (4,334 cases) through 2004. Compared with subjects in the lowest quartile, increased daily physical activity was associated with a significantly decreased risk of cancer in both sexes. In men, hazard ratios for the second, third, and highest quartiles were 1.00 (95% confidence interval (CI): 0.90, 1.11), 0.96 (95% CI: 0.86, 1.07), and 0.87 (95% CI: 0.78, 0.96), respectively (p for trend = 0.005); in women, hazard ratios were 0.93 (95% CI: 0.82, 1.05), 0.84 (95% CI: 0.73, 0.96), and 0.84 (95% CI: 0.73, 0.97), respectively (p for trend = 0.007). The decreased risk was more clearly observed in women than in men, especially among the elderly and those who regularly engaged in leisure-time sports or physical exercise. By site, decreased risks were observed for cancers of the colon, liver, and pancreas in men and for cancer of the stomach in women. Increased daily physical activity may be beneficial in preventing cancer in a relatively lean population.

  9. Effect of different cooking methods on total phenolic contents and antioxidant activities of four Boletus mushrooms.

    Science.gov (United States)

    Sun, Liping; Bai, Xue; Zhuang, Yongliang

    2014-11-01

    The influences of cooking methods (steaming, pressure-cooking, microwaving, frying and boiling) on total phenolic contents and antioxidant activities of fruit body of Boletus mushrooms (B. aereus, B. badius, B. pinophilus and B. edulis) have been evaluated. The results showed that microwaving was better in retention of total phenolics than other cooking methods, while boiling significantly decreased the contents of total phenolics in samples under study. Effects of different cooking methods on phenolic acids profiles of Boletus mushrooms showed varieties with both the species of mushroom and the cooking method. Effects of cooking treatments on antioxidant activities of Boletus mushrooms were evaluated by in vitro assays of hydroxyl radical (OH·) -scavenging activity, reducing power and 1, 1-diphenyl-2-picrylhydrazyl radicals (DPPH·) -scavenging activity. Results indicated the changes of antioxidant activities of four Boletus mushrooms were different in five cooking methods. This study could provide some information to encourage food industry to recommend particular cooking methods.

  10. TOTAL PHENOLIC CONTENT, ANTIOXIDANT AND ANTIBACTERIAL ACTIVITIES OF THE EXTRACT OF EPHEDRA PROCERA FISCH. ET MEY.

    Science.gov (United States)

    Dehkordi, Naser Vahed; Kachouie, Mehrdad Ataie; Pirbalouti, Abdollah Ghasemi; Malekpoor, Fatemeh; Rabei, Mohammad

    2015-01-01

    Ephedra prcera belonging to the family Ephedraceae is a poison and medicinal plant. The main aim of present study was to determine total phenolic content and antioxidant and antibacterial activities of ethanolic extract from the aerial parts of E. procera collected from a natural habitat in Chaharmahal va Bakhtiari province, Southwestern Iran. The total phenolic content of the extract by Folin-Ciocalteu method and the antioxidant activity using DPPH assay were determined. The antibacterial activity, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) of the extract were evaluated against five bacteria, including Proteus vulgaris, Pseudomonas aeruginosa, Enteobacter aeogenes, Bacillus ceirus and Staphylococcus aureus. Total phenolic content in the extract of E. procera was 0.718 mg tannic acid/g dry weight extract. The results indicated that the ethanolic extract of E. piocera exhibited radical scavenging activity. In addition, the results of this study confirmed that the ethanolic extract of E. procera exhibited antibacterial activity. In conclusion, the extract of E. piocera could be an important source of phenolic components with antioxidant capacity and antibacterial activity.

  11. Mammalian aPKC/Par polarity complex mediated regulation of epithelial division orientation and cell fate

    Energy Technology Data Exchange (ETDEWEB)

    Vorhagen, Susanne; Niessen, Carien M., E-mail: carien.niessen@uni-koeln.de

    2014-11-01

    Oriented cell division is a key regulator of tissue architecture and crucial for morphogenesis and homeostasis. Balanced regulation of proliferation and differentiation is an essential property of tissues not only to drive morphogenesis but also to maintain and restore homeostasis. In many tissues orientation of cell division is coupled to the regulation of differentiation producing daughters with similar (symmetric cell division, SCD) or differential fate (asymmetric cell division, ACD). This allows the organism to generate cell lineage diversity from a small pool of stem and progenitor cells. Division orientation and/or the ratio of ACD/SCD need to be tightly controlled. Loss of orientation or an altered ratio can promote overgrowth, alter tissue architecture and induce aberrant differentiation, and have been linked to morphogenetic diseases, cancer and aging. A key requirement for oriented division is the presence of a polarity axis, which can be established through cell intrinsic and/or extrinsic signals. Polarity proteins translate such internal and external cues to drive polarization. In this review we will focus on the role of the polarity complex aPKC/Par3/Par6 in the regulation of division orientation and cell fate in different mammalian epithelia. We will compare the conserved function of this complex in mitotic spindle orientation and distribution of cell fate determinants and highlight common and differential mechanisms in which this complex is used by tissues to adapt division orientation and cell fate to the specific properties of the epithelium.

  12. Xylanase production by a local fungal isolate, Aspergillus niger USM AI 1 via solid state

    Directory of Open Access Journals (Sweden)

    Ibrahim Che Omar

    2005-03-01

    Full Text Available Isolate USM A1 I which was identified to be Aspergillus niger was selected as a potential producer of xylanase via a solid state fermentation system (SSF using palm kernel cake (PKC as substrate. The modification of the physical conditions of the SSF system indicated that the xylanase activity was 23.97 U/g PKC at the moisture ratio of 1:0.75 of PKC: moistening agent with the inoculum size of 1¥104 spores/ml and cultivated at the ambient temperature (28±3ºC. The supplementation of additional carbon and nitrogen sources in the PKC medium could enhance enzyme productivity. The maximum production of xylanase and growth obtained with the supplementation of xylose at 0.75% (w/w were 25.40 U/g and 1.69 mg glucosamine/ g PKC. Moreover, the presence of NaNO3 at 0.075% (w/w as additional nitrogen source further enhanced xylanase production to 33.99 U/g PKC although the growth remained unchanged at about 1.67 mg glucosa- mine/g PKC. The optimized conditions showed an increased xylanase production by 157% compared to before the optimization of the SSF system. The xylanase productivity was 23.12 U/mg glucosamine after optimization and 11.72 U/mg glucosamine before optimization.

  13. The Antioxidant Activities and Total Phenolic of Artemisia Martima, Achillea Millefolium and Matricaria Recutica

    Directory of Open Access Journals (Sweden)

    A Mirzaei

    2010-10-01

    Full Text Available Introduction & Objective: Consumption of plant derived antioxidant contributes to reducing risks of certain chronic and degenerative diseases. The aim of the present study was to study the antioxidant activities and total phenolic of Artemisia Martima, Achillea Millefolium and Matricaria Recutica Materials & Methods: The present study was conducted at Yasuj University of Medical Sciences in 2009. The Stem and flower sample of plants were air-dried, and then grinded and were finally extracted by ethanol: water (70: 30 for 48 h in room temperature. Extracts were filtered and dried under vacuum system. The antioxidant activity of three ethanol extract of the medicinal plants, Artemisia martima, Achillea millefolium and Matricaria recutica, were analyzed by five different methods (1,1-diphenyl-2-picrylhydrazyl (DPPH radical, 2, 20azinobis- (3-ethylbenzthiazoline -6-sulphonic acid (ABTS radical cation,Ferric-reducing antioxidant power assay (FRAP, phosphomolybdenum (PMB and reducing power ( RP. In addition, for determination of antioxidant components, the total phenolic content was also analyzed. The collected data was analyzed by SPSS software. Results: For all antioxidant activity assays, Artemisia martima had the highest antioxidant activity value and also total phenol content. Antioxidant capacity analyses revealed that the FRAP and DPPH had comparable results. Antioxidant activity at 1 mg/mL, in ABTS were in the order Artemisia martima> Achillea millefolium> Matricaria recutica. Similar trend was observed for PMB content. RP, FRAP and DPPH were in the order Artemisia martima> Matricaria recutica > Achillea millefolium . Conclusion: The extracts showed a variety of antioxidant activities in all antioxidant assay system. This study demonstrated that Artemisia martima crude extract exhibit significant antioxidant activity.

  14. Total body-calcium measurements: comparison of two delayed-gamma neutron activation facilities

    International Nuclear Information System (INIS)

    Ma, R.; Ellis, K.J.; Shypailo, R.J.; Pierson, R.N. Jr.

    1999-01-01

    This study compares two independently calibrated delayed-gamma neutron activation (DGNA) facilities, one at the Brookhaven National Laboratory (BNL), Upton, New York, and the other at the Children's Nutrition Research Center (CNRC), Houston, Texas that measure total body calcium (TBCa). A set of BNL phantoms was sent to CNRC for neutron activation analysis, and a set of CNRC phantoms was measured at BNL. Both facilities showed high precision (<2%), and the results were in good agreement, within 5%. (author)

  15. Blockade of IP[subscript 3]-Mediated SK Channel Signaling in the Rat Medial Prefrontal Cortex Improves Spatial Working Memory

    Science.gov (United States)

    Brennan, Avis R.; Dolinsky, Beth; Vu, Mai-Anh T.; Stanley, Marion; Yeckel, Mark F.; Arnsten, Amy F. T.

    2008-01-01

    Planning and directing thought and behavior require the working memory (WM) functions of prefrontal cortex. WM is compromised by stress, which activates phosphatidylinositol (PI)-mediated IP[subscript 3]-PKC intracellular signaling. PKC overactivation impairs WM operations and in vitro studies indicate that IP[subscript 3] receptor (IP[subscript…

  16. Lipid vesicle-mediated affinity chromatography using magnetic activated cell sorting (LIMACS): a novel method to analyze protein-lipid interaction.

    Science.gov (United States)

    Bieberich, Erhard

    2011-04-26

    The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids. To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane

  17. 2,5-hexanedione (HD) treatment alters calmodulin, Ca2+/calmodulin-dependent protein kinase II, and protein kinase C in rats' nerve tissues

    International Nuclear Information System (INIS)

    Wang Qingshan; Hou Liyan; Zhang Cuili; Zhao Xiulan; Yu Sufang; Xie, Ke-Qin

    2008-01-01

    Calcium-dependent mechanisms, particularly those mediated by Ca 2+ /calmodulin (CaM)-dependent protein kinase II (CaMKII), have been implicated in neurotoxicant-induced neuropathy. However, it is unknown whether similar mechanisms exist in 2,5-hexanedione (HD)-induced neuropathy. For that, we investigated the changes of CaM, CaMKII, protein kinase C (PKC) and polymerization ratios (PRs) of NF-L, NF-M and NF-H in cerebral cortex (CC, including total cortex and some gray), spinal cord (SC) and sciatic nerve (SN) of rats treated with HD at a dosage of 1.75 or 3.50 mmol/kg for 8 weeks (five times per week). The results showed that CaM contents in CC, SC and SN were significantly increased, which indicated elevation of Ca 2+ concentrations in nerve tissues. CaMKII contents and activities were also increased in CC and were positively correlated with gait abnormality, but it could not be found in SC and SN. The increases of PKC contents and activities were also observed in SN and were positively correlated with gait abnormality. Except for that of NF-M in CC, the PRs of NF-L, NF-M and NF-H were also elevated in nerve tissues, which was consistent with the activation of protein kinases. The results suggested that CaMKII might be partly (in CC but not in SC and SN) involved in HD-induced neuropathy. CaMKII and PKC might mediate the HD neurotoxicity by altering the NF phosphorylation status and PRs

  18. Qualità totale e mobilità totale Total Quality and Total Mobility

    Directory of Open Access Journals (Sweden)

    Giuseppe Trieste

    2010-05-01

    Full Text Available FIABA ONLUS (Italian Fund for Elimination of Architectural Barriers was founded in 2000 with the aim of promoting a culture of equal opportunities and, above all, it has as its main goal to involve public and private institutions to create a really accessible and usable environment for everyone. Total accessibility, Total usability and Total mobility are key indicators to define quality of life within cities. A supportive environment that is free of architectural, cultural and psychological barriers allows everyone to live with ease and universality. In fact, people who access to goods and services in the urban context can use to their advantage time and space, so they can do their activities and can maintain relationships that are deemed significant for their social life. The main aim of urban accessibility is to raise the comfort of space for citizens, eliminating all barriers that discriminate people, and prevent from an equality of opportunity. “FIABA FUND - City of ... for the removal of architectural barriers” is an idea of FIABA that has already affected many regions of Italy as Lazio, Lombardy, Campania, Abruzzi and Calabria. It is a National project which provides for opening a bank account in the cities of referring, in which for the first time, all together, individuals and private and public institutions can make a donation to fund initiatives for the removal of architectural barriers within its own territory for a real and effective total accessibility. Last February the fund was launched in Rome with the aim of achieving a Capital without barriers and a Town European model of accessibility and usability. Urban mobility is a prerequisite to access to goods and services, and to organize activities related to daily life. FIABA promotes the concept of sustainable mobility for all, supported by the European Commission’s White Paper. We need a cultural change in management and organization of public means, which might focus on

  19. Pharmacologic modulation of protein kinase C isozymes: the role of RACKs and subcellular localisation.

    Science.gov (United States)

    Csukai, M; Mochly-Rosen, D

    1999-04-01

    Protein kinase C (PKC) isozymes are highly homologous kinases and several different isozymes can be present in a cell. Each isozyme is likely to mediate unique functions, but pharmacological tools to explore their isozyme-specific roles have not been available until recently. In this review, we describe the development and application of isozyme-selective inhibitors of PKC. The identification of these inhibitors stems from the observation that PKC isozymes are each localised to unique subcellular locations following activation. Inhibitors of this isozyme-unique localisation have been shown to act as selective inhibitors of the functions of individual isozymes. The identification of isozyme-specific inhibitors should allow the exploration of individual PKC isozyme function in a wide range of cell systems. Copyright 1999 The Italian Pharmacological Society.

  20. The phosphorylation state of CD3gamma influences T cell responsiveness and controls T cell receptor cycling

    DEFF Research Database (Denmark)

    Dietrich, J; Backstrom, T; Lauritsen, JP

    1998-01-01

    The T cell receptor (TCR) is internalized following activation of protein kinase C (PKC) via a leucine (Leu)-based motif in CD3gamma. Some studies have indicated that the TCR is recycled back to the cell surface following PKC-mediated internalization. The functional state of recycled TCR...... the phosphorylation state of CD3gamma and T cell responsiveness. Based on these observations a physiological role of CD3gamma and TCR cycling is proposed....... and the mechanisms involved in the sorting events following PKC-induced internalization are not known. In this study, we demonstrated that following PKC-induced internalization, the TCR is recycled back to the cell surface in a functional state. TCR recycling was dependent on dephosphorylation of CD3gamma, probably...

  1. A gravimetric method for the measurement of total spontaneous activity in rats.

    Science.gov (United States)

    Biesiadecki, B J; Brand, P H; Koch, L G; Britton, S L

    1999-10-01

    Currently available methods for the measurement of spontaneous activity of laboratory animals require expensive, specialized equipment and may not be suitable for use in low light conditions with nocturnal species. We developed a gravimetric method that uses common laboratory equipment to quantify the total spontaneous activity of rats and is suitable for use in the dark. The rat in its home cage is placed on a top-loading electronic balance interfaced to a computer. Movements are recorded by the balance as changes in weight and transmitted to the computer at 10 Hz. Data are analyzed on-line to derive the absolute value of the difference in weight between consecutive samples, and the one-second average of the absolute values is calculated. The averages are written to file for off-line analysis and summed over the desired observation period to provide a measure of total spontaneous activity. The results of in vitro experiments demonstrated that: 1) recorded weight changes were not influenced by position of the weight on the bottom of the cage, 2) values recorded from a series of weight changes were not significantly different from the calculated values, 3) the constantly decreasing force exerted by a swinging pendulum placed on the balance was accurately recorded, 4) the measurement of activity was not influenced by the evaporation of a fluid such as urine, and 5) the method can detect differences in the activity of sleeping and waking rats over a 10-min period, as well as during 4-hr intervals recorded during active (night-time) and inactive (daytime) periods. These results demonstrate that this method provides an inexpensive, accurate, and noninvasive method to quantitate the spontaneous activity of small animals.

  2. A study of extraction process and in vitro antioxidant activity of total ...

    African Journals Online (AJOL)

    The study investigated the extraction method of Rhizoma Imperatae and its antioxidant activity, and provided a basis for its rational development. The extraction method of Rhizoma Imperatae was determined using orthogonal design test and by total phenol content, its hydroxyl radical scavenging ability was measured by ...

  3. Protective Effect of Tempol on Acute Kidney Injury Through PI3K/Akt/Nrf2 Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Gensheng Zhang

    2016-02-01

    Full Text Available Background/Aims: Tempol is a protective antioxidant against ischemic injury in many animal models. The molecular mechanisms are not well understood. Nuclear factor erythroid 2-related factor (Nrf2 is a master transcription factor during oxidative stress, which is enhanced by activation of protein kinase C (PKC pathway. Another factor, tubular epithelial apoptosis, is mediated by activation of phosphoinositide 3-kinase (PI3K/protein kinase B (PKB, Akt signaling pathway during renal ischemic injury. We tested the hypothesis that tempol activates PKC or PI3K/Akt/Nrf2 pathways to transcribe many genes that coordinate endogenous antioxidant defense. Methods: The right renal pedicle was clamped for 45 minutes and the left kidney was removed to study renal ischemia/reperfusion (I/R injury in C57BL/6 mice. The response was assessed from serum parameters, renal morphology and renal expression of PKC, phosphorylated-PKC (p-PKC, Nrf2, heme oxygenase-1 (HO-1, Akt, phosphorylated-Akt (p-Akt, pro-caspase-3 and cleaved caspase-3 in groups of sham and I/R mice given vehicle, or tempol (50 or 100 mg/kg, intraperitoneal injection. Results: The serum malondialdehyde (MDA, marker of reactive oxygen species doubled and the BUN and creatinine increased 5- to 10-fold after I/R injury. Tempol (50 or 100 mg/kg prevented the increases in MDA but only tempol (50 mg/kg lessened the increases in BUN and creatinine and moderated the acute tubular necrosis. I/R did not change expression of PKC or p-PKC but reduced renal expression of Nrf2, p-Akt, HO-1 and pro-caspase-3 and increased cleaved caspase-3. Tempol (50 mg/kg prevented these changes produced by I/R whereas tempol (100 mg/kg had lesser or inconsistent effects. Conclusion: Tempol (50 mg/kg prevents lipid peroxidation and attenuates renal damage after I/R injury. The beneficial pathway apparently is not dependent on upregulation or phosphorylation of PKC, at lower tempol doses, does implicate upregulation of Akt with

  4. Quantitation of the degree of osteoporosis by measure of total-body calcium employing neutron activation

    International Nuclear Information System (INIS)

    Cohn, S.H.; Zanzi, I.; Vaswani, A.; Wallach, S.; Aloia, J.; Ellis, K.J.

    1975-01-01

    Two techniques for measuring the amount of Ca in the total skeleton were employed: total-body neutron activation analysis (TBNAA) and the determination of the mineral content of a bone of the appendicular skeleton (absorptiometric measurement of the radius, BMC). (U.S.)

  5. Heterologous activation of protein kinase C stimulates phosphorylation of delta-opioid receptor at serine 344, resulting in beta-arrestin- and clathrin-mediated receptor internalization

    DEFF Research Database (Denmark)

    Xiang, B; Yu, G H; Guo, J

    2001-01-01

    The purpose of the current study is to investigate the effect of opioid-independent, heterologous activation of protein kinase C (PKC) on the responsiveness of opioid receptor and the underlying molecular mechanisms. Our result showed that removing the C terminus of delta opioid receptor (DOR......) containing six Ser/Thr residues abolished both DPDPE- and phorbol 12-myristate 13-acetate (PMA)-induced DOR phosphorylation. The phosphorylation levels of DOR mutants T352A, T353A, and T358A/T361A/S363S were comparable to that of the wild-type DOR, whereas S344G substitution blocked PMA-induced receptor......, and ionomycin resulted in DOR internalization that required phosphorylation of Ser-344. Expression of dominant negative beta-arrestin and hypertonic sucrose treatment blocked PMA-induced DOR internalization, suggesting that PKC mediates DOR internalization via a beta-arrestin- and clathrin-dependent mechanism...

  6. Regulation of platelet activating factor receptor coupled phosphoinositide-specific phospholipase C activity

    International Nuclear Information System (INIS)

    Morrison, W.J.

    1988-01-01

    The major objectives of this study were two-fold. The first was to establish whether binding of platelet activating factor (PAF) to its receptor was integral to the stimulation of polyphosphoinositide-specific phospholipase C (PLC) in rabbit platelets. The second was to determine regulatory features of this receptor-coupled mechanism. [ 3 H]PAF binding demonstrated two binding sites, a high affinity site with a inhibitory constant (Ki) of 2.65 nM and a low affinity site with a Ki of 0.80 μM. PAF receptor coupled activation of phosphoinositide-specific PLC was studied in platelets which were made refractory, by short term pretreatments, to either PAF or thrombin. Saponin-permeabilized rabbit platelets continue to regulate the mechanism(s) coupling PAF receptors to PLC stimulation. However, TRPγS and GDPβS, which affect guanine nucleotide regulatory protein functions, were unable to modulate the PLC activity to any appreciable extent as compared to PAF. The possible involvement of protein kinase C (PKC) activation in regulating PAF-stimulated PLC activity was studied in rabbit platelets pretreated with staurosporine followed by pretreatments with PAF or phorbol 12-myristate 13-acetate (PMA)

  7. Total phenolic content, antioxidant and antimicrobial activity of Equisetum arvense L.

    Directory of Open Access Journals (Sweden)

    Kukrić Zoran

    2013-01-01

    Full Text Available The antioxidant and antimicrobial activity of Equisetum arvense L. harvested in a polluted urban area were investigated. The total phenolic content determined by a modified Folin-Ciocalteu method was 335.8 mg of GEA/g of the dried ethanol extract. Antioxidant activity determined by FRAP method was 28.7 mM Fe(II/g of extract, whereas antioxidant activity index for stable DPPH free radicals was 3.86. The results were compared with control antioxidants (gallic acid, vitamin C, BHA and BHT. Antimicrobial activity was tested by monitoring the impact of different extract concentrations through measuring the change in optical density of bacterial suspension over time and by determining MIC and MBC using macrodilution method. E. arvense extract has been effective on the tested microorganisms depending on the concentration and exposure time. The extract had the strongest antibacterial activity on Staphylococcus aureus, with MIC and MBC of 11.14 mg/mL and 22.28 mg/mL respectively, and the weakest effect on Bacillus cereus, with MIC of 89.10 mg/mL.

  8. Precise shape reconstruction by active pattern in total-internal-reflection-based tactile sensor.

    Science.gov (United States)

    Saga, Satoshi; Taira, Ryosuke; Deguchi, Koichiro

    2014-03-01

    We are developing a total-internal-reflection-based tactile sensor in which the shape is reconstructed using an optical reflection. This sensor consists of silicone rubber, an image pattern, and a camera. It reconstructs the shape of the sensor surface from an image of a pattern reflected at the inner sensor surface by total internal reflection. In this study, we propose precise real-time reconstruction by employing an optimization method. Furthermore, we propose to use active patterns. Deformation of the reflection image causes reconstruction errors. By controlling the image pattern, the sensor reconstructs the surface deformation more precisely. We implement the proposed optimization and active-pattern-based reconstruction methods in a reflection-based tactile sensor, and perform reconstruction experiments using the system. A precise deformation experiment confirms the linearity and precision of the reconstruction.

  9. Presynaptic muscarinic receptors, calcium channels, and protein kinase C modulate the functional disconnection of weak inputs at polyinnervated neonatal neuromuscular synapses.

    Science.gov (United States)

    Santafe, M M; Garcia, N; Lanuza, M A; Tomàs, M; Besalduch, N; Tomàs, J

    2009-04-01

    We studied the relation among calcium inflows, voltage-dependent calcium channels (VDCC), presynaptic muscarinic acetylcholine receptors (mAChRs), and protein kinase C (PKC) activity in the modulation of synapse elimination. We used intracellular recording to determine the synaptic efficacy in dually innervated endplates of the levator auris longus muscle of newborn rats during axonal competition in the postnatal synaptic elimination period. In these dual junctions, the weak nerve terminal was potentiated by partially reducing calcium entry (P/Q-, N-, or L-type VDCC-specific block or 500 muM magnesium ions), M1- or M4-type selective mAChR block, or PKC block. Moreover, reducing calcium entry or blocking PKC or mAChRs results in unmasking functionally silent nerve endings that now recover neurotransmitter release. Our results show interactions between these molecules and indicate that there is a release inhibition mechanism based on an mAChR-PKC-VDCC intracellular cascade. When it is fully active in certain weak motor axons, it can depress ACh release and even disconnect synapses. We suggest that this mechanism plays a central role in the elimination of redundant neonatal synapses, because functional axonal withdrawal can indeed be reversed by mAChRs, VDCCs, or PKC block.

  10. Sexual activity after total hip replacement in Korean patients: how they do, what they want, and how to improve.

    Science.gov (United States)

    Yoon, Byung-Ho; Lee, Kyung-Hag; Noh, Serae; Ha, Yong-Chan; Lee, Young-Kyun; Koo, Kyung-Hoi

    2013-12-01

    Concerns of patients on sexual activity after total hip arthroplasty have not been well studied in Asian patients. This study aimed to determine the following: (1) what are the concerns of patients related to sexual activity after total hip arthroplasty? (2) what are the changes in sexual activity after total hip replacement in Korean patients? Details of sexual activity and concerns were obtained using a questionnaire designed specifically for the study. The questionnaire was administered to 64 patients in a face-to-face interview at an outpatient clinic. Preoperatively, 53.1% of patients experienced difficulties, primarily due to hip pathology and limitations of motion. The median time to the resumption of sexual activity was 3 months postoperatively, and most patients had no increase in the frequency of sexual activity after the total hip replacement. In 39.1% of patients were seen having difficulties with leg positioning following total hip replacement, and they were likely to change coital positions. The most common concern regarding sexual activity of patients was the fear of dislocation. Furthermore, patients with a higher stress level had lower satisfaction rates. Most patients were unable to obtain information on sexual activity following the total hip arthroplasty, and they did not consult with a physician due to the private nature of the topic. Dislocation was the most common concern of patients during sexual activity following a total hip arthroplasty, and a higher stress level was found to be associated with a lower satisfaction rate. Because most patients were unprepared to consult a physician, the provision of appropriate information before a consultation might be beneficial.

  11. In vitro anthelmintic activity of Barleria buxifolia on Indian adult earthworms and estimation of total flavonoid content

    Directory of Open Access Journals (Sweden)

    Purna A. Chander

    2014-02-01

    Full Text Available Objective: To study the anthelmintic activity of Barleria buxifolia leaf and to estimate the total flavonoid content. Methods: The aqueous and ethanolic leaf extracts were prepared and these were analyzed for total flavonoid content by aluminium chloride colorimetric method and Pheretima posthuma was used for anthelmintic activity by using the different concentrations (10, 20, 40, 80 and 100 mg/mL. Results: All the investigational extracts showed an anthelmintic activity at concentration of 10 mg/mL. The ethanolic extract of 100 mg/mL has produced an significant effect (P<0.001 when compared to aqueous extract. The total flavonoid content was found to be 5.67 mg QE/100 g. Conclusions: From the above study, the leaf extract has shown a good anthelmintic activity.

  12. Mechanisms of pertussis toxin-induced barrier dysfunction in bovine pulmonary artery endothelial cell monolayers.

    Science.gov (United States)

    Patterson, C E; Stasek, J E; Schaphorst, K L; Davis, H W; Garcia, J G

    1995-06-01

    We have previously characterized several G proteins in endothelial cells (EC) as substrates for the ADP-ribosyltransferase activity of both pertussis (PT) and cholera toxin and described the modulation of key EC physiological responses, including gap formation and barrier function, by these toxins. In this study, we investigated the mechanisms involved in PT-mediated regulation of bovine pulmonary artery endothelial cells barrier function. PT caused a dose-dependent increase in albumin transfer, dependent upon action of the holotoxin, since neither the heat-inactivated PT, the isolated oligomer, nor the protomer induced EC permeability. PT-induced gap formation and barrier dysfunction were additive to either thrombin- or thrombin receptor-activating peptide-induced permeability, suggesting that thrombin and PT utilize distinct mechanisms. PT did not result in Ca2+ mobilization or alter either basal or thrombin-induced myosin light chain phosphorylation. However, PT stimulated protein kinase C (PKC) activation, and both PKC downregulation and PKC inhibition attenuated PT-induced permeability, indicating that PKC activity is involved in PT-induced barrier dysfunction. Like thrombin-induced permeability, the PT effect was blocked by prior increases in adenosine 3',5'-cyclic monophosphate. Thus PT-catalyzed ADP-ribosylation of a G protein (possibly other than Gi) may regulate cytoskeletal protein interactions, leading to EC barrier dysfunction.

  13. [Evaluation of the total biological activity and allergenic composition of allergenic extracts].

    Science.gov (United States)

    Lombardero, M; González, R; Duffort, O; Juan, F; Ayuso, R; Ventas, P; Cortés, C; Carreira, J

    1986-01-01

    In the present study, a complete procedure is presented in order to standardize allergenic extracts, the meaning of which is the measurement of the total allergenic activity and the determination of the allergenic composition. The measurement of the biological activity comprises 2 steps: Preparation of Reference Extracts and determination of their "in vivo" activity. Evaluation of the total allergenic activity of extracts for clinical use. Reference extracts were prepared from the main allergens and their "in vivo" biological activity was determined by a quantitative skin prick test in a sample of at least 30 allergic patients. By definition, the protein concentration of Reference Extract that produces, in the allergic population, a geometric mean wheal of 75 mm.2 has an activity of 100 biological units (BUs). The determination of the biological activity of a problem extract is made by RAST inhibition. The sample is compared with the corresponding Reference Extract by this technique and, from this comparison, it is possible to quantify the activity of the problem extract in biologic units (BUs) with clinical significance. Likewise, different techniques have been used to determine the allergenic composition of extracts. These techniques comprise 2 steps: Separation of the components of the extract. Identification of the components that bind specific human IgE. The separation of the components of the extract has been carried out by isoelectric focusing (IEF) and electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE). In order to identify the allergenic components, an immunoblotting technique has been employed. The separated components in the IEF gel or SDS-PAGE gel are transferred to a nitrocellulose sheet and later on, this membrane is overlaid with a serum pool from allergic patients and a mouse monoclonal anti-human IgE, labelled with 125I. Finally, the autoradiography of the nitrocellulose membrane is obtained. In this way it is possible to compare

  14. To total amount of activity ..... and beyond: Perspectives on measuring physical behaviour

    Directory of Open Access Journals (Sweden)

    Johannes B.J. Bussmann

    2013-07-01

    Full Text Available The aim of this paper is to describe and discuss some perspectives on definitions, constructs and outcome parameters of physical behaviour. The paper focuses on the following constructs: Physical activity & active lifestyle vs. sedentary behaviour & sedentary lifestyle; Amount of physical activity vs. amount of walking; Detailed body posture & movement data vs. overall physical activity data; Behavioural context of activities; Quantity vs. quality; Physical behaviour vs. physiological response.Subsequently, the following outcome parameters provided by data reduction procedures are discussed: Distribution of length of bouts; Variability in bout length; Time window; Intensity and intensity threshold.The overview indicates that physical behaviour is a multi-dimensional construct, and it stresses the importance and relevance of constructs and parameters other than total amount of physical activity.It is concluded that the challenge for the future will be to determine which parameters are most relevant, valid and responsive. This is a matter for physical behaviour researchers to consider, that is critical to multi-disciplinary collaboration.

  15. Protein kinase C involvement in the acetylcholine release reduction induced by amyloid-beta(25-35) aggregates on neuromuscular synapses.

    Science.gov (United States)

    Tomàs, Marta; Garcia, Neus; Santafé, Manuel M; Lanuza, Maria; Tomàs, Josep

    2009-01-01

    Using intracellular recording of the diaphragm muscle of adult rats, we have investigated the short-term functional effects of amyloid-beta (Abeta(25-35) peptide aggregates on the modulation of acetylcholine (ACh) release and the involvement of protein kinase C (PKC). The non-aggregated form of this peptide does not change the evoked and spontaneous transmitter release parameters on the neuromuscular synapse. However, the aggregated form of Abeta(25-35) acutely interferes with evoked quantal ACh release (approximately 40% reduction) when synaptic activity in the ex vivo neuromuscular preparation is maintained by low frequency (1 Hz) electrical stimulation. This effect is partially dependent on the activity of PKC that may have a permissive action. The end result of Abeta(25-35) is in opposition to the PKC-dependent maintenance effect on ACh release manifested in active synapses.

  16. The active and passive kinematic difference between primary reverse and total shoulder prostheses

    NARCIS (Netherlands)

    Alta, T.D.; de Toledo, J.S.; Veeger, H.E.J.; Janssen, T.W.J.; Willems, W.J.

    2014-01-01

    Background: Reverse shoulder arthroplasty (RSA) and total shoulder arthroplasty (TSA) effectively decrease pain and improve clinical outcome. However, indications and biomechanical properties vary greatly. Our aim was to analyze both active and passive shoulder motion (thoracohumeral [TH],

  17. High glucose induces inflammatory cytokine through protein kinase C-induced toll-like receptor 2 pathway in gingival fibroblasts

    International Nuclear Information System (INIS)

    Jiang, Shao-Yun; Wei, Cong-Cong; Shang, Ting-Ting; Lian, Qi; Wu, Chen-Xuan; Deng, Jia-Yin

    2012-01-01

    Highlights: ► High glucose significantly induced TLR2 expression in gingival fibroblasts. ► High glucose increased NF-κB p65 nuclear activity, IL-1β and TNF-α levels. ► PKC-α/δ-TLR2 pathway is involved in periodontal inflammation under high glucose. -- Abstract: Toll-like receptors (TLRs) play a key role in innate immune response and inflammation, especially in periodontitis. Meanwhile, hyperglycemia can induce inflammation in diabetes complications. However, the activity of TLRs in periodontitis complicated with hyperglycemia is still unclear. In the present study, high glucose (25 mmol/l) significantly induced TLR2 expression in gingival fibroblasts (p < 0.05). Also, high glucose increased nuclear factor kappa B (NF-κB) p65 nuclear activity, tumor necrosis factor-α (TNF-α) and interleukin-lβ (IL-1β) levels. Protein kinase C (PKC)-α and δ knockdown with siRNA significantly decreased TLR2 and NF-κB p65 expression (p < 0.05), whereas inhibition of PKC-β had no effect on TLR2 and NF-κB p65 under high glucose (p < 0.05). Additional studies revealed that TLR2 knockdown significantly abrogated high-glucose-induced NF-κB expression and inflammatory cytokine secretion. Collectively, these data suggest that high glucose stimulates TNF-α and IL-1β secretion via inducing TLR2 through PKC-α and PKC-δ in human gingival fibroblasts.

  18. High glucose induces inflammatory cytokine through protein kinase C-induced toll-like receptor 2 pathway in gingival fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, Shao-Yun, E-mail: jiangshaoyun@yahoo.com [School of Dentistry, Tianjin Medical University, 12 Qi Xiang Tai Street, Heping District, Tianjin 300070 (China); Wei, Cong-Cong; Shang, Ting-Ting; Lian, Qi; Wu, Chen-Xuan [School of Dentistry, Tianjin Medical University, 12 Qi Xiang Tai Street, Heping District, Tianjin 300070 (China); Deng, Jia-Yin, E-mail: yazhou2991@126.com [School of Dentistry, Tianjin Medical University, 12 Qi Xiang Tai Street, Heping District, Tianjin 300070 (China)

    2012-10-26

    Highlights: Black-Right-Pointing-Pointer High glucose significantly induced TLR2 expression in gingival fibroblasts. Black-Right-Pointing-Pointer High glucose increased NF-{kappa}B p65 nuclear activity, IL-1{beta} and TNF-{alpha} levels. Black-Right-Pointing-Pointer PKC-{alpha}/{delta}-TLR2 pathway is involved in periodontal inflammation under high glucose. -- Abstract: Toll-like receptors (TLRs) play a key role in innate immune response and inflammation, especially in periodontitis. Meanwhile, hyperglycemia can induce inflammation in diabetes complications. However, the activity of TLRs in periodontitis complicated with hyperglycemia is still unclear. In the present study, high glucose (25 mmol/l) significantly induced TLR2 expression in gingival fibroblasts (p < 0.05). Also, high glucose increased nuclear factor kappa B (NF-{kappa}B) p65 nuclear activity, tumor necrosis factor-{alpha} (TNF-{alpha}) and interleukin-l{beta} (IL-1{beta}) levels. Protein kinase C (PKC)-{alpha} and {delta} knockdown with siRNA significantly decreased TLR2 and NF-{kappa}B p65 expression (p < 0.05), whereas inhibition of PKC-{beta} had no effect on TLR2 and NF-{kappa}B p65 under high glucose (p < 0.05). Additional studies revealed that TLR2 knockdown significantly abrogated high-glucose-induced NF-{kappa}B expression and inflammatory cytokine secretion. Collectively, these data suggest that high glucose stimulates TNF-{alpha} and IL-1{beta} secretion via inducing TLR2 through PKC-{alpha} and PKC-{delta} in human gingival fibroblasts.

  19. Patient reported activities after reverse total shoulder arthroplasty in rotator cuff arthropathy patients.

    Science.gov (United States)

    Alcobía-Díaz, B; Lópiz, Y; García-Fernández, C; Rizo de Álvaro, B; Marco, F

    Reverse total shoulder arthroplasty in rotator cuff arthropathy patients, improves anteversion and abduction, but not rotational, outcomes. The main aim of this study is to determine its repercussions on daily life activities in our patients. Between 2009 and 2011 we implanted 210 shoulder arthroplasties, 126 of them were reverse total shoulder arthroplasty in a rotator cuff arthropathy context. About 88% were women, with a mean age at time of surgery of 81 years, 95% were retired. The mean follow up was 53 months. The Constant scale, Visual Analogue Scale, Charlson Comorbidity Index, range of motion were measured for each patient and whether they could manage 40 daily life activities by means of a new questionnaire, classifying them according toshoulder functional demand. Mean normalized by sex and age Constant value was 81.2%. Mean Visual Analogue Scale and Charlson Index were 3.56 and 1.69 respectively. Improvement in anteversion and abduction, not in rotational range of motion. Limitation was found in low and high functional demand activities in 20% and 51% respectively, especially those which involved internal rotation. Reverse total shoulder arthroplasty treatment for RCA in the elderly, achieves adequate pain management and good functional outcomes. Nevertheless, an important risk of DLA limitation must be accepted in those which involve internal rotation or shoulder high functional demand. Copyright © 2017 SECOT. Publicado por Elsevier España, S.L.U. All rights reserved.

  20. Aortic superoxide production at the early hyperglycemic stage in a rat type 2 diabetes model and the effects of pravastatin.

    Science.gov (United States)

    Kikuchi, Chigusa; Kajikuri, Junko; Hori, Eisei; Nagami, Chie; Matsunaga, Tamihide; Kimura, Kazunori; Itoh, Takeo

    2014-01-01

    Endothelium-derived superoxide induces vascular dysfunctions. The aim of this study was to examine the activity of protein kinase C (PKC) isoforms and endothelial nitric oxide synthase (eNOS), which leads to vascular superoxide production in type 2 diabetes, in addition to the effects of pravastatin. We studied these mechanisms in Otsuka Long-Evans Tokushima Fatty (OLETF) rats (type 2 diabetes model) at the early hyperglycemic stage (vs. non-diabetic Long-Evans Tokushima Otsuka [LETO] rats). Superoxide production and catalase activity were measured in aortas, as were the protein expressions of PKCδ and phospho-Ser(1177) eNOS. Superoxide production was increased in OLETF rats, and this increase was inhibited by the selective conventional PKC (cPKC) inhibitor Gö6976 and by the non-selective cPKC and novel PKC inhibitor GF109203X. Phospho-Ser(1177) eNOS was significantly increased in OLETF rats, whereas the protein expressions of PKCδ and phosopho-Thr(505) PKCδ and catalase activity were all greatly reduced. Pravastatin administration to OLETF rats in vivo had normalizing effects on all of these variables. The increment in superoxide production seen in OLETF rats (but not the production in pravastatin-treated OLETF rats) was abolished by high concentration of N(ω)-nitro-L-arginine methyl ester (electron transport inhibitor of eNOS), by sepiapterin (precursor of tetrahydrobiopterin), and by LY294002 (phosphatidylinositol 3-kinase [PI3-kinase] inhibitor). In OLETF rats at the early hyperglycemic stage, aortic superoxide production is increased owing to activation of uncoupled eNOS through phosphorylation by PI3-kinase/Akt. This may be related to the observed reduction in PKCδ/catalase activities. Pravastatin inhibited endothelial superoxide production via normalization of PKCδ/catalase activities.

  1. Determination of chemical composition, total phenolic content and antioxidant activity of xylanthemum macropodum

    International Nuclear Information System (INIS)

    Samiullah, A.; Tareen, R.B.; Khan, N.; Akber, A.; Ali, I.; Khan, A.K.

    2017-01-01

    Evaluation of the phytochemistry, total phenolic content and antioxidant activity of the endemic plant of northern Balochistan Xylanthemum Macropodum of the Asteraceae family, is reported for the first time in this document. Chemical composition of Xylanthemum Macropodum was determined using well-established chemical tests and modern spectroscopic techniques. Extracts were taken from the whole plant using methanol and the extracts were tested for phytochemicals (secondary metabolites), total phenolic content (TPC) and antioxidant activity. The phytochemical (biochemical) examination of Xylanthemum Macropodum exposed the presence of alkaloids, phenols, steroids, flavonoids, tannins, terpenoids, saponins, coumarins, carbohydrates, cardiac glycosides, reducing sugars, and quinines. TPC of crude methanolic extract (CME) of plant was determined using Folin-Ciocalteu's reagent. The TPC determined was 256mg of tannic acid Eq/g of extract. Antioxidant activities were determined spectrophotometrically using the DPPH assay and Ferric ion (Fe/sup +3/) reducing antioxidant power assay. The potency of the DPPH assay of Xylanthemum Macropodum extract was 68% for the 0.10 mg/ml concentration and the FRAP value of the extract was 3.368 mmol Fe/sup +2//g of extract. Xylanthemum Macropodum has proved to be very rich in secondary metabolites, natural phenolics and has a very potent antioxidant activity. (author)

  2. Improvements in knee biomechanics during walking are associated with increased physical activity after total knee arthroplasty.

    Science.gov (United States)

    Arnold, John B; Mackintosh, Shylie; Olds, Timothy S; Jones, Sara; Thewlis, Dominic

    2015-12-01

    Total knee arthroplasty (TKA) in people with knee osteoarthritis increases knee-specific and general physical function, but it has not been established if there is a relationship between changes in these elements of functional ability. This study investigated changes and relationships between knee biomechanics during walking, physical activity, and use of time after TKA. Fifteen people awaiting TKA underwent 3D gait analysis before and six months after surgery. Physical activity and use of time were determined in free-living conditions from a high resolution 24-h activity recall. After surgery, participants displayed significant improvements in sagittal plane knee biomechanics and improved their physical activity profiles, standing for 105 more minutes (p=0.001) and performing 64 min more inside chores on average per day (p=0.008). Changes in sagittal plane knee range of motion (ROM) and peak knee flexion positively correlated with changes in total daily energy expenditure, time spent undertaking moderate to vigorous physical activity, inside chores and passive transport (r=0.52-0.66, p=0.005-0.047). Restoration of knee function occurs in parallel and is associated with improvements in physical activity and use of time after TKA. Increased functional knee ROM is required to support improvements in total and context specific physical activity. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  3. Total's LNG activities from Algeria to Yemen

    International Nuclear Information System (INIS)

    Vedrenne, J.P.

    1997-01-01

    In March 1995, further to an international tender, Total was awarded the leadership of the first LNG project in Yemen. On January 1997 Total announced the extension of the share-holding of the Yemen LNG Co. to include the companies with interests in the Marib area (Hunt-Exxon-Yukong). The Marib area will supply the gas to the future liquefaction plant. The ratification of these agreements confirms the role of Total as lead shareholder with 36% in the share-holding structure and guarantees gas supply from the Marib licence, operated by Hunt-Exxon. (author)

  4. Assessment of uranium exposure from total activity and 234U:238U activity ratios in urine

    International Nuclear Information System (INIS)

    Nicholas, T.; Bingham, D.

    2011-01-01

    Radiation workers at Atomic Weapons Establishment (AWE) are monitored for uranium exposure by routine bioassay sampling (primarily urine sampling). However, the interpretation of uranium in urine and faecal results in terms of occupational intakes is difficult because of the presence of uranium due to intakes from environmental (dietary) sources. For uranium in urine data obtained using current analytical techniques at AWE, the mean, median and standard deviation of excreted uranium concentrations were 0.006, 0.002 and 0.012 μg per g creatinine, respectively. These values are consistent with what might be expected from local dietary intakes and the knowledge that occupational exposures at AWE are likely to be very low. However, some samples do exceed derived investigation levels (DILs), which have been set up taking account of the likely contribution from environmental sources. We investigate how the activity and isotopic composition of uranium in the diet affects the sensitivity of uranium in urine monitoring for occupational exposures. We conclude that DILs based on both total uranium in urine activity and also 234 U: 238 U ratios are useful given the likely variation in dietary contribution for AWE workers. Assuming a background excretion rate and that the enrichment of the likely exposure is known, it is possible to assess exposures using 234 U: 238 U ratios and/or total uranium activity. The health implications of internalised uranium, enriched to 235 U, centre on its nephrotoxicity; the DILs for bioassay samples at AWE are an order of magnitude below the conservative recommendations made by the literature. (authors)

  5. Measurement of total-body oxygen, nitrogen, and carbon in vivo by photon activation analysis

    International Nuclear Information System (INIS)

    Ulin, K.

    1984-01-01

    With the aim of assessing nutritional status, the feasibility of measuring the total body quantities of the major body elements, i.e. oxygen, nitrogen, and carbon, using the photon beam of a 45 MV betatron and a whole-body counter, has been evaluated in detail. Following photon activation a single energy γ-radiation (.511 MeV) is observed from all three elements to be measured. The half-lives of 15 O, 13 N, and 11 C, however, are sufficiently different (20.5 min, 10.0 min, and 20.4 min. respectively) to permit their measurement from an analysis of the measured decay curve. Following corrections for interfering reactions, a computer curve-fitting algorithm is used to resolve the data into 15 O, 13 N, and 11 C components. Measurements of O, N, and C have been made both in phantoms and in live and dead rats. A comparison of the body composition results from this technique with results from chemical analysis indicates that measured carbon can quite accurately predict total body fat. The comparison of the total body nitrogen measurement by photon activation with total body protein by chemical analysis was inconclusive and suggests that further work be done to verify the estimated accuracy of the nitrogen measurement

  6. Oxysterol-binding protein-related protein (ORP) 9 is a PDK-2 substrate and regulates Akt phosphorylation.

    Science.gov (United States)

    Lessmann, Eva; Ngo, Mike; Leitges, Michael; Minguet, Susana; Ridgway, Neale D; Huber, Michael

    2007-02-01

    The oxysterol-binding protein and oxysterol-binding protein-related protein family has been implicated in lipid transport and metabolism, vesicle trafficking and cell signaling. While investigating the phosphorylation of Akt/protein kinase B in stimulated bone marrow-derived mast cells, we observed that a monoclonal antibody directed against phospho-S473 Akt cross-reacted with oxysterol-binding protein-related protein 9 (ORP9). Further analysis revealed that mast cells exclusively express ORP9S, an N-terminal truncated version of full-length ORP9L. A PDK-2 consensus phosphorylation site in ORP9L and OPR9S at S287 (VPEFS(287)Y) was confirmed by site-directed mutagenesis. In contrast to Akt, increased phosphorylation of ORP9S S287 in stimulated mast cells was independent of phosphatidylinositol 3-kinase but sensitive to inhibition of conventional PKC isotypes. PKC-beta dependence was confirmed by lack of ORP9S phosphorylation at S287 in PKC-beta-deficient, but not PKC-alpha-deficient, mast cells. Moreover, co-immunoprecipitation of PKC-beta and ORP9S, and in vitro phosphorylation of ORP9S in this complex, argued for direct phosphorylation of ORP9S by PKC-beta, introducing ORP9S as a novel PKC-beta substrate. Akt was also detected in a PKC-beta/ORP9S immune complex and phosphorylation of Akt on S473 was delayed in PKC-deficient mast cells. In HEK293 cells, RNAi experiments showed that depletion of ORP9L increased Akt S473 phosphorylation 3-fold without affecting T308 phosphorylation in the activation loop. Furthermore, mammalian target of rapamycin was implicated in ORP9L phosphorylation in HEK293 cells. These studies identify ORP9 as a PDK-2 substrate and negative regulator of Akt phosphorylation at the PDK-2 site.

  7. Determination of phytochemicals, antioxidant activity and total phenolic content in Andrographis paniculata using chromatographic methods.

    Science.gov (United States)

    Kurzawa, Marzanna; Filipiak-Szok, Anna; Kłodzińska, Ewa; Szłyk, Edward

    2015-07-15

    Antioxidant activity, total phenolics content and selected phytochemicals (alkaloids and andrographolides) were determined in Andrographis paniculata and in dietary supplements containing this plant. Antioxidant activity was measured by FRAP, CUPRAC and DPPH procedures and ranged from 503.36 to 6164.09μmol TE/100g d.m. depending on methods, part of plant and kind of dietary supplement. The total phenolics (175.13-1723.79mg GAE/100g) and andrographolides content (19.44-85.13mg/g) in the studied samples were correlated with antioxidant activities determined by CUPRAC, FRAP and DPPH (r>0.95, ppaniculata leaves, whereas the lowest in dietary supplement Pn. Moreover principal component analysis, cluster analysis and one-way ANOVA follow by Duncan's tests were also performed. Copyright © 2015. Published by Elsevier B.V.

  8. Protein kinase A and C regulate leak potassium currents in freshly isolated vascular myocytes from the aorta.

    Directory of Open Access Journals (Sweden)

    Sébastien Hayoz

    Full Text Available We tested the hypothesis that protein kinase A (PKA inhibits K2P currents activated by protein kinase C (PKC in freshly isolated aortic myocytes. PDBu, the PKC agonist, applied extracellularly, increased the amplitude of the K2P currents in the presence of the "cocktail" of K(+ channel blockers. Gö 6976 significantly reduced the increase of the K2P currents by PDBu suggesting the involvement of either α or β isoenzymes of PKC. We found that forskolin, or membrane permeable cAMP, did not inhibit K2P currents activated by the PKC. However, when PKA agonists were added prior to PDBu, they produced a strong decrease in the K2P current amplitudes activated by PKC. Inhibition of PDBu-elicited K2P currents by cAMP agonists was not prevented by the treatment of vascular smooth muscle cells with PKA antagonists (H-89 and Rp-cAMPs. Zn(2+ and Hg(2+ inhibited K2P currents in one population of cells, produced biphasic responses in another population, and increased the amplitude of the PDBu-elicited K(+ currents in a third population of myocytes, suggesting expression of several K2P channel types. We found that cAMP agonists inhibited biphasic responses and increase of amplitude of the PDBu-elicited K2P currents produced by Zn(2+ and Hg(2. 6-Bnz-cAMp produced a significantly altered pH sensitivity of PDBu-elicited K2P-currents, suggesting the inhibition of alkaline-activated K2P-currents. These results indicate that 6-Bnz-cAMP and other cAMP analogs may inhibit K2P currents through a PKA-independent mechanism. cAMP analogs may interact with unidentified proteins involved in K2P channel regulation. This novel cellular mechanism could provide insights into the interplay between PKC and PKA pathways that regulate vascular tone.

  9. Total joint replacement: A multiple risk factor analysis of physical activity level 1-2 years postoperatively.

    Science.gov (United States)

    Paxton, Elizabeth W; Torres, Andy; Love, Rebecca M; Barber, Thomas C; Sheth, Dhiren S; Inacio, Maria C S

    2016-07-01

    Background and purpose - The effect of total joint arthroplasty (TJA) on physical activity is not fully understood. We investigated the change in physical activity after TJA and patient factors associated with change. Patients and methods - Using a total joint replacement registry, primary total hip arthroplasty (THA) patients (n = 5,678) and knee arthroplasty (TKA) patients (n = 11,084) between January 1, 2010 and December 31, 2012 were identified. Median age at THA was 68 and median age at TKA was 67. Change in self-reported physical activity (minutes per week) from before TJA (within 1 year of surgery) to after TJA (1-2 years) was the outcome of interest. Patient demographics and comorbidities were evaluated as risk factors. Multiple linear regression was used. Results - Median physical activity before surgery was 50 min/week (IQR: 0-140) for THA patients and 58 (IQR: 3-143) for TKA patients. Median physical activity after surgery was 150 min/week (IQR: 60-280) for both THA patients and TKA patients. Following TJA, 50% of patients met CDC/WHO physical activity guideline criteria. Higher body mass index was associated with lower change in physical activity (THA: -7.1 min/week; TKA: -5.9 min/week). Females had lower change than males (THA: -11 min/week; TKA: -9.1 min/week). In TKA patients, renal failure was associated with lower change (-17 min/week), as were neurological disorders (-30 min/week). Interpretation - Self-reported minutes of physical activity increased from before to after TJA, but 50% of TJA patients did not meet recommended physical activity guideline criteria. Higher body mass index, female sex, and specific comorbidities were found to be associated with low change in physical activity. Patient education on the benefits of physical activity should concentrate on these subgroups of patients.

  10. Molecular mechanisms of responses to radiation through protein kinase C

    International Nuclear Information System (INIS)

    Nakajima, Tetsuo

    2005-01-01

    Described are the activation and cascade of the protein kinase C (PKC) which mediating the control of radiation-induced apoptosis. PKC is a family of c-, n- and a-subtypes and plays a major role in responding to the radiation exposure for DNA repair, cell cycle arrest and apoptosis. The author has conducted studies of mouse thymic lymphoma cells which have a property to respond even to low dose radiation, and has showed that, in the highly radiosensitive cell strain, 3SBH5 where apoptosis occurs in 50 and 90% post 0.5 and 2 Gy exposure, respectively, cPKC works as a surviving signal without intracellular movement after irradiation. In contrast, PKC has been alternatively shown to participate in apoptosis induction, showing that different enzyme species in the subtypes work specifically depending on passing time. Comparison with the radio-resistant cell strain, XR223, has revealed that the difference in the localization controls of PKCδ in the cell determines the radiosensitivity, however, the control mechanism is found to be separate from Atm pathway by which PKCδ is usually regulated. Recent studies have revealed that PKC performs the intracellular cross-talk in various phosphorylation cascades. Studies of PKC can be toward their uses for radiation effect assessment, radiotherapy and medicare for urgent exposure. (S.I.)

  11. Total body neutron activation analysis of calcium: calibration and normalisation

    Energy Technology Data Exchange (ETDEWEB)

    Kennedy, N S.J.; Eastell, R; Ferrington, C M; Simpson, J D; Strong, J A [Western General Hospital, Edinburgh (UK); Smith, M A; Tothill, P [Royal Infirmary, Edinburgh (UK)

    1982-05-01

    An irradiation system has been designed, using a neutron beam from a cyclotron, which optimises the uniformity of activation of calcium. Induced activity is measured in a scanning, shadow-shield whole-body counter. Calibration has been effected and reproducibility assessed with three different types of phantom. Corrections were derived for variations in body height, depth and fat thickness. The coefficient of variation for repeated measurements of an anthropomorphic phantom was 1.8% for an absorbed dose equivalent of 13 mSv (1.3 rem). Measurements of total body calcium in 40 normal adults were used to derive normalisation factors which predict the normal calcium in a subject of given size and age. The coefficient of variation of normalised calcium was 6.2% in men and 6.6% in women, with the demonstration of an annual loss of 1.5% after the menopause. The narrow range should make single measurements useful for diagnostic purposes.

  12. PROTECTIVE EFFECT OF POLYMYXINE B AND NIFEDIPINE ON DIABETIC COMPLICATIONS IN RAT: ROLE OF PROTEIN KINASE C

    Directory of Open Access Journals (Sweden)

    H. Mehrani

    2003-08-01

    Full Text Available Patients with diabetes mellitus (DM, experience significant morbidity and mortality from microvascular retinopathy, nephropathy and neuropathy. Hyperglycemia can induce diabetic complications through multiple pathways. Activation of protein kinase C (PKC by hyperglycemia is one of the pathways which causes diabetic complications. Effect of nifedipine (a calcium channel blocker, and polymyxine B sulphate (a Protein kinase C inhibitor was studied in adult male Sprague- dawley rats, who was made diabetic with streptozotocin. PKC activity was determined in tissues and serum enzymes and metabolite level was measured in all controls, diabetic and drug treated animals. The results showed that, levels of the, urea (two –fold, creatinine (60%, triglyceride (two-fold and liver alanine transaminase (ALT activity (two-fold, were significantly increased in diabetic group. In nifedipine, treated diabetic group, although urea and creatinine level was increased, but liver enzymes were not significantly different from those of control group. In diabetic group which was treated with polymyxine, all the measured metabolites and enzyme levels were the same as the control group, except glucose level which was increased and liver glycogen was decreased significantly. Protein kinase C activity in the cytoplasm of diabetic liver was increased comparing to its control group (5.73 ± 0.56 Vs, 4.00 ± 0.62. The enzyme activity in the plasma membranes of untreated and nifedipine treated diabetic groups was significantly increased (6.2 ± 0.42 and 3.66 ± 0.31 Vs 2.38 ± 0.36. These results show that polymyxine is more effective than nifedipine against protein kinase C activity in diabetic complications. In conclusion our results show that, liver and kidney damage in DM are related to PKC activation. The fact that polymyxine prevents diabetic related increase in PKC activity more than nifedipine, support the hypothesis that different PKC isozymes may play different roles

  13. Bcr-Abl-independent mechanism of resistance to imatinib in K562 cells: Induction of cyclooxygenase-2 (COX-2) by histone deacetylases (HDACs).

    Science.gov (United States)

    Kalle, Arunasree M; Sachchidanand, Sachchidanand; Pallu, Reddanna

    2010-09-01

    Our previous studies have shown that overexpression of MDR1 and cyclooygenase-2 (COX-2) resulted in resistance development to imatinib in chronic myelogenous leukemia (CML) K562 (IR-K562) cells. In the present study, the regulatory mechanism of MDR1 induction by COX-2 was investigated. A gradual overexpression of MDR1 and COX-2 during the process of development was observed. Furthermore, down regulation of MDR1 upon COX-2 knockdown by siRNA showed a decrease in the PKC levels and activation of PKC by addition of PGE(2) to K562 cells, suggesting a role for PKC in the COX-2 mediated induction of MDR1. The present study demonstrates COX-2 induction by HDACs and MDR1 induction by COX-2 via PGE(2)-cAMP-PKC-mediated pathway. Copyright 2010 Elsevier Ltd. All rights reserved.

  14. Protein Kinase C Enzymes in the Hematopoietic and Immune Systems.

    Science.gov (United States)

    Altman, Amnon; Kong, Kok-Fai

    2016-05-20

    The protein kinase C (PKC) family, discovered in the late 1970s, is composed of at least 10 serine/threonine kinases, divided into three groups based on their molecular architecture and cofactor requirements. PKC enzymes have been conserved throughout evolution and are expressed in virtually all cell types; they represent critical signal transducers regulating cell activation, differentiation, proliferation, death, and effector functions. PKC family members play important roles in a diverse array of hematopoietic and immune responses. This review covers the discovery and history of this enzyme family, discusses the roles of PKC enzymes in the development and effector functions of major hematopoietic and immune cell types, and points out gaps in our knowledge, which should ignite interest and further exploration, ultimately leading to better understanding of this enzyme family and, above all, its role in the many facets of the immune system.

  15. Partial purification and characterization of a wortmannin-sensitive and insulin-stimulated protein kinase that activates heart 6-phosphofructo-2-kinase.

    OpenAIRE

    Deprez, J; Bertrand, L; Alessi, D R; Krause, U; Hue, L; Rider, M H

    2000-01-01

    A wortmannin-sensitive and insulin-stimulated protein kinase (WISK), which phosphorylates and activates cardiac 6-phosphofructo-2-kinase (PFK-2), was partially purified from perfused rat hearts. Immunoblotting showed that WISK was devoid of protein kinase B (PKB), serum- and glucocorticoid-regulated protein kinase and protein kinase Czeta (PKCzeta). Comparison of the inhibition of WISK, PKCalpha and PKCzeta by different protein kinase inhibitors suggested that WISK was not a member of the PKC...

  16. Effects of sciatic-conditioned medium on neonatal rat retinal cells in vitro

    Directory of Open Access Journals (Sweden)

    Torres P.M.M.

    1998-01-01

    Full Text Available Schwann cells produce and release trophic factors that induce the regeneration and survival of neurons following lesions in the peripheral nerves. In the present study we examined the in vitro ability of developing rat retinal cells to respond to factors released from fragments of sciatic nerve. Treatment of neonatal rat retinal cells with sciatic-conditioned medium (SCM for 48 h induced an increase of 92.5 ± 8.8% (N = 7 for each group in the amount of total protein. SCM increased cell adhesion, neuronal survival and glial cell proliferation as evaluated by morphological criteria. This effect was completely blocked by 2.5 µM chelerythrine chloride, an inhibitor of protein kinase C (PKC. These data indicate that PKC activation is involved in the effect of SCM on retinal cells and demonstrate that fragments of sciatic nerve release trophic factors having a remarkable effect on neonatal rat retinal cells in culture.

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

    International Nuclear Information System (INIS)

    Carlson, K.E.

    1989-01-01

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

  18. Bladder instillation of Escherichia coli lipopolysaccharide alters the muscle contractions in rat urinary bladder via a protein kinase C-related pathway

    International Nuclear Information System (INIS)

    Weng, T.I.; Chen, W.J.; Liu, S.H.

    2005-01-01

    Uropathogenic Escherichia coli is a common cause of urinary tract infection. We determined the effects of intravesical instillation of E. coli lipopolysaccharide (LPS, endotoxin) on muscle contractions, protein kinase C (PKC) translocation, and inducible nitric oxide synthase (iNOS) expression in rat urinary bladder. The contractions of the isolated rat detrusor muscle evoked by electrical field stimulations were measured short-term (1 h) or long-term (24 h) after intravesical instillation of LPS. One hour after LPS intravesical instillation, bladder PKC-α translocation from cytosolic fraction to membrane fraction and endothelial (e)NOS protein was elevated, and detrusor muscle contractions were significantly increased. PKC inhibitors chelerythrine and Ro32-0432 inhibited this LPS-enhanced contractile response. Application of PKC activator β-phorbol-12,13-dibutyrate enhanced the muscle contractions. Three hours after intravesical instillation of LPS, iNOS mRNA was detected in the bladder. Immunoblotting study also demonstrated that the induction of iNOS proteins is detected in bladder in which LPS was instilled. 24 h after intravesical instillation of LPS, PKC-α translocation was impaired in the bladder; LPS did not affect PKC-δ translocation. Muscle contractions were also decreased 24 h after LPS intravesical instillation. Aminoguanidine, a selective iNOS inhibitor, blocked the decrease in PKC-α translocation and detrusor contractions induced by LPS. These results indicate that there are different mechanisms involved in the alteration of urinary bladder contractions after short-term and long-term treatment of LPS; an iNOS-regulated PKC signaling may participate in causing the inhibition of muscle contractions in urinary bladder induced by long-term LPS treatment

  19. Screening of immunomodulatory activity of total and protein extracts of some Moroccan medicinal plants.

    Science.gov (United States)

    Daoudi, Abdeljlil; Aarab, Lotfi; Abdel-Sattar, Essam

    2013-04-01

    Herbal and traditional medicines are being widely used in practice in many countries for their benefits of treating different ailments. A large number of plants in Morocco were used in folk medicine to treat immune-related disorders. The objective of this study is to evaluate the immunomodulatory activity of protein extracts (PEs) of 14 Moroccan medicinal plants. This activity was tested on the proliferation of immune cells. The prepared total and PEs of the plant samples were tested using MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide) assay on the splenocytes with or without stimulation by concanavalin-A (Con-A), a mitogenic agent used as positive control. The results of this study indicated different activity spectra. Three groups of activities were observed. The first group represented by Citrullus colocynthis, Urtica dioica, Elettaria cardamomum, Capparis spinosa and Piper cubeba showed a significant immunosuppressive activity. The second group that showed a significant immunostimulatory activity was represented by Aristolochia longa, Datura stramonium, Marrubium vulgare, Sinapis nigra, Delphynium staphysagria, Lepidium sativum, Ammi visnaga and Tetraclinis articulata. The rest of the plant extracts did not alter the proliferation induced by Con-A. This result was more important for the PE than for the total extract. In conclusion, this study revealed an interesting immunomodulating action of certain PEs, which could explain their traditional use. The results of this study may also have implications in therapeutic treatment of infections, such as prophylactic and adjuvant with cancer chemotherapy.

  20. Evaluation and comparison of the content of total polyphenols and antioxidant activity of selected species of the genus Allium

    Directory of Open Access Journals (Sweden)

    Marianna LENKOVÁ

    2016-12-01

    Full Text Available The species of the genus Allium are very important crops for human health. They contain many health beneficial substances, such as polyphenols (especially flavonoids, sulphur compounds, vitamins, mineral substances and substances with antioxidant activity. This work has focused on the comparison of total phenolic content and antioxidant activity of selected species of the genus Allium – garlic (Allium sativum L., chives (Allium schoenoprasum L., ramson (Allium ursinum L. and red, yellow and white onion (Allium cepa L.. Samples of plant material were collected at the stage of full maturity in the area of Nitra. Total polyphenols content was determined using the spectrophotometric method of Folin-Ciocalteu agents. Determined the content of total polyphenols were in the range 444.3 - 1591 mg*kg-1. Total polyphenols content in the observed crops declined in the following order: chives > red onion > garlic > yellow onion > ramson > white onion. Antioxidant activity was measured by the spectrophotometric method using a compound DPPH (2.2-diphenyl-1-picryhydrazyl. Determined the value of antioxidant activity ranged 12.29 – 76.57%. Antioxidant activity observed in crops declined in the following order: chives > ramson > red onion > yellow onion > garlic > white onion. In all the analysed crop plants was confirmed by the strong dependence of the antioxidant activity and the total content of polyphenolic substances.

  1. Yeast Interacting Proteins Database: YDR490C, YGR086C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available bait as prey (0) YGR086C PIL1 Primary component of eisosomes, which are large immobile cell cortex struct...ctures associated with endocytosis; null mutants show activation of Pkc1p/Ypk1p str...y (0) Prey ORF YGR086C Prey gene name PIL1 Prey description Primary component of eisosomes, which are large immobile cell cortex stru...ures associated with endocytosis; null mutants show activation of Pkc1p/Ypk1p stres

  2. Physical Activity Behavior of Patients 1 Year After Primary Total Hip Arthroplasty : A Prospective Multicenter Cohort Study

    NARCIS (Netherlands)

    Wagenmakers, Robert; Stevens, Martin; Groothoff, Johan W.; Zijlstra, Wiebren; Bulstra, Sjoerd K.; van Beveren, Jan; van Raaij, Jos J. A. M.; van den Akker-Scheek, Inge

    Background. Besides the important beneficial effects of regular physical activity on general health, some of the musculoskeletal effects of physical activity are of particular interest for older adults after total hip arthroplasty (THA). However, research on physical activity behavior of patients

  3. Population dynamics of active and total ciliate populations in arable soil amended with wheat

    DEFF Research Database (Denmark)

    Ekelund, F.; Frederiksen, Helle B.; Ronn, R.

    2002-01-01

    of the population may be encysted. The factors governing the dynamics of active and encysted cells in the soil are not well understood. Our objective was to determine the dynamics of active and encysted populations of ciliates during the decomposition of freshly added organic material. We monitored, in soil...... microcosms, the active and total populations of cili