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Sample records for activities protein levels

  1. A sensitive and facile assay for the measurement of activated protein C activity levels in vivo.

    PubMed

    Orthner, C L; Kolen, B; Drohan, W N

    1993-05-01

    Activated protein C (APC) is a serine protease which plays an important role as a naturally occurring antithrombotic enzyme. APC, which is formed by thrombin-catalyzed limited proteolysis of the zymogen protein C, functions as an anticoagulant by proteolytic inactivation of the coagulation cofactors VIIIa and Va: APC is inhibited by several members of the serpin family as well a by alpha 2-macroglobulin. APC is being developed as a therapeutic for the prevention and treatment of thrombosis. We have developed an assay to quantify circulating levels of enzymatically active APC during its administration to patients, in healthy individuals, and in various disease states. This assay utilizes an EDTA-dependent anti-protein C monoclonal antibody (Mab) 7D7B10 to capture both APC and protein C from plasma, prepared from blood collected in an anticoagulant supplemented with the reversible inhibitor p-aminobenzamidine. Mab 7D7B10-derivatized agarose beads are added to the wells of a 96-well filtration plate, equilibrated with Tris-buffered saline, and incubated for 10 min with 200 microliters of plasma. After washing, APC and protein C are eluted from the immunosorbent beads with a calcium-containing buffer into the wells of a 96-well microtiter plate containing antithrombin III (ATIII) and heparin. The amidolytic activity of APC is then measured on a kinetic plate reader following the addition of L-pyroglutamyl-L-prolyl-L-arginine-p-nitroanilide (S-2366) substrate. The rate of substrate hydrolysis was proportional to APC concentration over a 200-fold concentration range (5.0 to 1,000 ng/ml) when measured continuously over a 15 to 30 min time period.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Calpain expression in lymphoid cells. Increased mRNA and protein levels after cell activation.

    PubMed

    Deshpande, R V; Goust, J M; Chakrabarti, A K; Barbosa, E; Hogan, E L; Banik, N L

    1995-02-10

    Although calpain is ubiquitously present in human tissues and is thought to play a role in demyelination, its activity is very low in resting normal lymphocytes. To determine the nature of calpain expression at the mRNA and protein levels in human lymphoid cells, we studied human T lymphocytic, B lymphocytic, and monocytic lines as well as peripheral blood mononuclear cells. Stimulation of cells with the phorbol ester phorbol myristate acetate and the calcium ionophore A23187 resulted in increased calpain mRNA and protein expression. Calpain mRNA expression is also increased in human T cells stimulated with anti-CD3. A dissociation between the increases of RNA and protein suggested that calpain could be released from the cells; the subsequent experiments showed its presence in the extracellular environment. 5,6-Dichloro-1b-D-ribofuranosylbenzimidazole, a reversible inhibitor of mRNA synthesis, reduced calpain mRNA levels by 50-67% and protein levels by 72-91%. Its removal resulted in resumption of both calpain mRNA and protein synthesis. Cycloheximide, a translational inhibitor, reduced calpain protein levels by 77-81% and calpain mRNA levels by 96% in activated THP-1 cells. Interferon-gamma induced calpain mRNA and protein in U-937 and THP-1 cells. Dexamethasone increased mRNA expression in THP-1 cells. Our results indicate that activation of lymphoid cells results in de novo synthesis and secretion of calpain. PMID:7852311

  3. Activity and circadian rhythm influence synaptic Shank3 protein levels in mice.

    PubMed

    Sarowar, Tasnuva; Chhabra, Resham; Vilella, Antonietta; Boeckers, Tobias M; Zoli, Michele; Grabrucker, Andreas M

    2016-09-01

    Various recent studies revealed that the proteins of the Shank family act as major scaffold organizing elements in the post-synaptic density of excitatory synapses and that their expression level is able to influence synapse formation, maturation and ultimately brain plasticity. An imbalance in Shank3 protein levels has been associated with a variety of neuropsychological and neurodegenerative disorders including autism spectrum disorders and Phelan-McDermid syndrome. Given that sleep disorders and low melatonin levels are frequently observed in autism spectrum disorders, and that circadian rhythms may be able to modulate Shank3 signaling and thereby synaptic function, here, we performed in vivo studies on CBA mice using protein biochemistry to investigate the synaptic expression levels of Shank3α during the day in different brain regions. Our results show that synaptic Shank3 protein concentrations exhibit minor oscillations during the day in hippocampal and striatal brain regions that correlate with changes in serum melatonin levels. Furthermore, as circadian rhythms are tightly connected to activity levels in mice, we increased physical activity using running wheels. The expression of Shank3α increases rapidly by induced activity in thalamus and cortex, but decreases in striatum, superimposing the circadian rhythms of different brain regions. We conclude that synaptic Shank3 proteins build highly dynamic platforms that are modulated by the light:dark cycles but even more so driven by activity. Using wild-type CBA mice, we show that Shank3 is a highly dynamic and activity-regulated protein at synapses. In the hippocampus, changes in synaptic Shank3 levels are influenced by circadian rhythm/melatonin concentration, while running activity increases and decreases levels of Shank3 in the cortex and striatum respectively. PMID:27329942

  4. SARS-CoV proteins decrease levels and activity of human ENaC via activation of distinct PKC isoforms

    PubMed Central

    Ji, Hong-Long; Song, Weifeng; Gao, Zhiqian; Su, Xue-Feng; Nie, Hong-Guang; Jiang, Yi; Peng, Ji-Bin; He, Yu-Xian; Liao, Ying; Zhou, Yong-Jian; Tousson, Albert; Matalon, Sadis

    2009-01-01

    Among the multiple organ disorders caused by the severe acute respiratory syndrome coronavirus (SARS-CoV), acute lung failure following atypical pneumonia is the most serious and often fatal event. We hypothesized that two of the hydrophilic structural coronoviral proteins (S and E) would regulate alveolar fluid clearance by decreasing the cell surface expression and activity of amiloride-sensitive epithelial sodium (Na+) channels (ENaC), the rate-limiting protein in transepithelial Na+ vectorial transport across distal lung epithelial cells. Coexpression of either S or E protein with human α-, β-, and γ-ENaC in Xenopus oocytes led to significant decreases of both amiloride-sensitive Na+ currents and γ-ENaC protein levels at their plasma membranes. S and E proteins decreased the rate of ENaC exocytosis and either had no effect (S) or decreased (E) rates of endocytosis. No direct interactions among SARS-CoV E protein with either α- or γ-ENaC were indentified. Instead, the downregulation of ENaC activity by SARS proteins was partially or completely restored by administration of inhibitors of PKCα/β1 and PKCζ. Consistent with the whole cell data, expression of S and E proteins decreased ENaC single-channel activity in oocytes, and these effects were partially abrogated by PKCα/β1 inhibitors. Finally, transfection of human airway epithelial (H441) cells with SARS E protein decreased whole cell amiloride-sensitive currents. These findings indicate that lung edema in SARS infection may be due at least in part to activation of PKC by SARS proteins, leading to decreasing levels and activity of ENaC at the apical surfaces of lung epithelial cells. PMID:19112100

  5. Alterations in cyclin-dependent protein kinase 5 (CDK5) protein levels, activity and immunocytochemistry in canine motor neuron disease.

    PubMed

    Green, S L; Vulliet, P R; Pinter, M J; Cork, L C

    1998-11-01

    Hereditary canine spinal muscular atrophy (HCSMA) is a dominantly inherited motor neuron disease in Brittany spaniels that is clinically characterized by progressive muscle weakness leading to paralysis. Histopathologically, degeneration is confined to motor neurons with accumulation of phosphorylated neurofilaments in axonal internodes. Cyclin-dependent kinase 5 (CDK5), a kinase related to the cell cycle kinase cdc2, phosphorylates neurofilaments and regulates neurofilament dynamics. We examined CDK5 activity, protein levels, and cellular immunoreactivity in nervous tissue from dogs with HCSMA, from closely age-matched controls and from dogs with other neurological diseases. On immunoblot analysis, CDK5 protein levels were increased in the HCSMA dogs (by approximately 1.5-fold in both the cytosolic and the particulate fractions). CDK5 activity was significantly increased (by approximately 3-fold) in the particulate fractions in the HCSMA dogs compared to all controls. The finding that CDK5 activity was increased in the young HCSMA homozygotes with the accelerated form of the disease, who do not show axonal swellings histologically, suggests that alterations in CDK5 occurs early in the pathogenesis, prior to the development of significant neurofilament pathology. Immunocytochemically, there was strong CDK5 staining of the nuclei, cytoplasm and axonal processes of the motor neurons in both control dogs and dogs with HCSMA. Further immunocytochemical studies demonstrated CDK5 staining where neurofilaments accumulated, in axonal swellings in the dogs with HCSMA. Our observations suggest phosphorylation-dependent events mediated by CDK5 occur in canine motor neuron disease.

  6. Effect of pumpkin seed (Cucurbita pepo) protein isolate on the activity levels of certain plasma enzymes in CCl4-induced liver injury in low-protein fed rats.

    PubMed

    Nkosi, C Z; Opoku, A R; Terblanche, S E

    2005-04-01

    The effects of pumpkin seed (Cucurbita pepo) protein isolate on the activity levels of lactate dehydrogenase (LD), alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP) against carbon tetrachloride (CCl4)-induced acute liver injury in low-protein fed rats were investigated. A group of male Sprague-Dawley rats maintained on a low-protein diet for 5 days were divided into three subgroups. Two subgroups were injected with carbon tetrachloride and the other group with an equivalent amount of olive oil. Two hours after CCl4 intoxication one of the two subgroups was administered with pumpkin seed protein isolate. All three subgroups of rats were maintained on the low-protein diet for the duration of the investigation. Groups of rats from the different subgroups were killed at 24, 48 and 72 h after their respective treatments. After 5 days on the low-protein diet the activity levels of all four enzymes were significantly higher than their counterparts on a normal balanced diet. CCl4 intoxication resulted in significant increases in the activity levels of all four enzymes investigated. The administration of pumpkin seed protein isolate after CCl4 intoxication resulted in significantly reduced activity levels of all four enzymes. It is concluded that pumpkin seed protein isolate administration was effective in alleviating the detrimental effects associated with protein malnutrition.

  7. Altered CYP2C9 Activity Following Modulation of CYP3A4 Levels in Human Hepatocytes: an Example of Protein-Protein Interactions

    PubMed Central

    Tweedie, Donald J.; Chan, Tom S.; Tracy, Timothy S.

    2014-01-01

    Cytochrome P450 (P450) protein-protein interactions resulting in modulation of enzyme activities have been well documented using recombinant isoforms. This interaction has been less clearly demonstrated in a more physiologic in vitro system such as human hepatocytes. As an expansion of earlier work (Subramanian et al., 2010), in which recombinant CYP2C9 activity decreased with increasing levels of CYP3A4, the current study modulated CYP3A4 content in human hepatocytes to determine the impact on CYP2C9. Modulation of CYP3A4 levels in situ was enabled by the use of a long-term human hepatocyte culture model (HepatoPac) shown to retain phenotypic hepatocyte function over a number of weeks. The extended period of culture allowed time for knockdown of CYP3A4 protein by small interfering RNA (siRNA) with subsequent recovery, as well as upregulation through induction with a recovery period. CYP3A4 gene silencing resulted in a 60% decrease in CYP3A4 activity and protein levels with a concomitant 74% increase in CYP2C9 activity, with no change in CYP2C9 mRNA levels. Upon removal of siRNA, both CYP2C9 and CYP3A4 activities returned to pre-knockdown levels. Importantly, modulation of CYP3A4 protein levels had no impact on cytochrome P450 reductase activities or levels. However, the possibility for competition for limiting reductase cannot be ruled out. Interestingly, lowering CYP3A4 levels also increased UDP-glucuronosyltransferase 2B7 activity. These studies clearly demonstrate that alterations in CYP3A4 levels can modulate CYP2C9 activity in situ and suggest that further studies are warranted to evaluate the possible clinical consequences of these findings. PMID:25157098

  8. High-level expression and characterization of a glycosylated human cementum protein 1 with lectin activity.

    PubMed

    Romo-Arévalo, Enrique; Arzate, Higinio; Montoya-Ayala, Gonzalo; Rodríguez-Romero, Adela

    2016-01-01

    This work aims to contribute to the knowledge of human cementum protein 1 (CEMP1), its conformational characteristics and influence during the biomineralization process. The results revealed that hrCEMP1 expressed in Pichia pastoris is a 2.4% glycosylated, thermostable protein which possesses a molecular mass of 28,770 Da. The circular dichroism spectrum indicated a secondary structure content of 28.6% of alpha-helix, 9.9% of beta-sheet and 61.5% of random-coil forms. Biological activity assays demonstrated that hrCEMP1 nucleates and regulates hydroxyapatite crystal growth. Hereby, it is demonstrated for the first time that CEMP1 has a (C-type) lectin-like activity and specifically recognizes mannopyranoside. The information produced by this biochemical and structural characterization may contribute to understand more fully the biological functions of CEMP1.

  9. Increasing discordant antioxidant protein levels and enzymatic activities contribute to increasing redox imbalance observed during human prostate cancer progression

    PubMed Central

    Chaiswing, Luksana; Zhong, Weixiong; Oberley, Terry D.

    2014-01-01

    A metabolomics study demonstrated a decrease in glutathione and an increase in cysteine (Cys) levels in human prostate cancer (PCa) tissues as Gleason scores increased, indicating redox imbalance with PCa progression. These results were extended in the present study by analyzing redox state of the protein thioredoxin 1 (Trx1) and sulfinylation (SO3) of peroxiredoxins (Prxs) (PrxsSO3) in PCa tissues and cell lines. Lysates of paired human PCa tissues with varying degree of aggressiveness and adjacent benign (BN) tissues were used for analysis. Redox western blot analysis of Trx1 demonstrated low levels of reduced and high levels of oxidized Trx1 (functional and non-functional, respectively) in high grade PCa (Gleason scores 4+4 to 4+5) in comparison to intermediate grade PCa (Gleason scores 3+3 to 3+4) or BN tissues. PrxsSO3 were increased in high grade PCa. Oxidized Trx1 and PrxsSO3 are indicators of oxidative stress. To study whether redox imbalance may potentially affect enzyme activities of antioxidant proteins (AP), we determined levels of selected AP in PCa tissues by western blot analysis and found that mitochondrial manganese superoxide dismutase (MnSOD), Prx 3, and Trx1 were increased in high grade PCa tissues when compared with BN tissues. Enzyme activities of MnSOD in high grade PCa tissues were significantly increased but at a lower magnitude when compared with the levels of MnSOD protein (0.5 folds vs. 2 folds increase). Trx1 activity was not changed in high grade PCa tissues despite a large increase in Trx1 protein expression. Further studies demonstrated a significant increase in posttranslational modifications of tyrosine and lysine residues in MnSOD protein and oxidation of Cys at active site (Cys 32 and Cys 35) and regulatory site (Cys 62 and Cys 69) of Trx1 in high grade PCa compared to BN tissues. These discordant changes between protein levels and enzyme activities are consistent with protein inactivation by redox imbalance and

  10. Gli2 protein expression level is a feasible marker of ligand-dependent hedgehog activation in pancreatic neoplasms.

    PubMed

    Sugiyama, Y; Sasajima, J; Mizukami, Y; Koizumi, K; Kawamoto, T; Ono, Y; Karasaki, H; Tanabe, H; Fujiya, M; Kohgo, Y

    2016-06-01

    The hedgehog pathway is known to promote proliferation of pancreatic ductal adenocarcinoma (PDA) and has been shown to restrain tumor progression. To understand how hedgehog causes these effects, we sought to carefully examine protein expression of hedgehog signaling components during different tumor stages. Genetically engineered mice, Pdx1-Cre;LSL-KrasG12D and Pdx1-Cre;LSL-KrasG12D;p53lox/+, were utilized to model distinct phases of tumorigenesis, pancreatic intraepithelial neoplasm (PanIN) and PDA. Human pancreatic specimens of intraductal papillary mucinous neoplasm (IPMN) and PDA were also employed. PanIN and IPMN lesions highly express Sonic Hedgehog, at a level that is slightly higher than that observed in PDA. GLI2 protein is also expressed in both PanIN/IPMN and PDA. Although there was no difference in the nuclear staining, the cytoplasmic GLI2 level in PDA was modest in comparison to that in PanIN/IPMN. Hedgehog interacting protein was strongly expressed in the precursors, whereas the level in PDA was significantly attenuated. There were no differences in expression of Patched1 at early and late stages. Finally, a strong correlation between Sonic Hedgehog and GLI2 staining was found in both human and murine pancreatic tumors. The results indicate that the GLI2 protein level could serve as a feasible marker of ligand-dependent hedgehog activation in pancreatic neoplasms. PMID:27543868

  11. Relationship between hydrocarbon structure and induction of P450: effects on protein levels and enzyme activities.

    PubMed

    Backes, W L; Sequeira, D J; Cawley, G F; Eyer, C S

    1993-12-01

    1. Treatment of male rat with the small aromatic hydrocarbons, benzene, toluene, ethylbenzene, n-propylbenzene, m-xylene, and p-xylene increased several P450-dependent activities, with ethylbenzene, m-xylene, and n-propylbenzene producing the greatest response. Hydrocarbon treatment differentially affected toluene metabolism, producing a response dependent on the metabolite monitored. In untreated rats, benzyl alcohol was the major hydroxylation product of toluene metabolism, comprising > 99% of the total metabolites formed. Hydrocarbon treatment increased the overall rate of toluene metabolism by dramatically increasing the amount of aromatic hydroxylation. Ethylbenzene, n-propylbenzene and m-xylene were the most effective inducers of aromatic hydroxylation of toluene. In contrast, production of the major toluene metabolite benzyl alcohol was increased only after treatment with m-xylene. 2. P450 2B1/2B2 levels were induced by each of the hydrocarbons examined, with the magnitude of induction increasing with increasing hydrocarbon size. P450 1A1 was also induced after hydrocarbon exposure; however, the degree of induction was smaller than that observed for P450 2B1/2B2. P450 2C11 levels were suppressed after treatment with benzene, ethylbenzene and n-propylbenzene. 3. Taken together these results display two induction patterns. The first generally corresponds to changes in the P450 2B subfamily, where activities (e.g. the aromatic hydroxylations of toluene) were most effectively induced by ethylbenzene, n-propylbenzene and m-xylene. In the second, induction was observed only after m-xylene treatment, a pattern that was found when the metabolism of the substrate was catalysed by both the P450 2B subfamily and P450 2C11. Hydrocarbons that both induced P450 2B1/2B2 and suppressed P450 2C11 (such as ethylbenzene and n-propylbenzene) showed little change in activities catalysed by both isozymes (e.g. aliphatic hydroxylation of toluene, and aniline hydroxylation

  12. Involvement of activated leukocytes in the regulation of plasma levels of acute phase proteins in microgravity simulation experiments

    NASA Astrophysics Data System (ADS)

    Larina, Olga; Bekker, Anna; Turin-Kuzmin, Alexey

    2016-07-01

    Earth-based studies of microgravity effects showed the induction of the mechanisms of acute phase reaction (APR). APR comprises the transition of stress-sensitive protein kinases of macrophages and other responsive cells into the active state and the phosphorylation of transcription factors which in turn stimulate the production of acute-phase reaction cytokines. Leukocyte activation is accompanied by the acceleration of the formation of oxygen radicals which can serve a functional indice of leukocyte cell state. The series of events at acute phase response result in selective changes in the synthesis of a number of secretory blood proteins (acute phase proteins, APPs) in liver cells thus contributing the recovery of homeostasis state in the organism. Earlier experiment with head-down tilt showed the increase in plasma concentrations of two cytokine mediators of acute phase response, tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) being the outcome of the activation of producer cells, foremost, leukocytes. In experiment with 4-day dry immersion chemiluminescent (ChL) reply of the whole blood samples to a test stimulus were studied along with the measurements of plasma levels of APPs, namely, alpha1-antitrypsin (alpha1-AT), alpha1-acid glycoprotein (alpha1-AGP), alpha2-macroglobulin (alpha2-M), ceruloplasmin (Cer), haptoglobin (Hp), C3-complement component (C3), C-reactive protein (CRP). Eight individuals aged 21.2 ± 3.2 years were the test subjects in the investigation. Protein studies showed a noticeable increase in the mean plasma levels of all APPs measured in experiment thus producing the evidence of the activation of acute phase response mechanisms while individual patterns revealed variability during the immersion period. The overall trends were similar to these in the previous immersion series. The augment in the strength of signal in stimulated light emission tests was higher after 1- and 2-day of immersion exposure than before the

  13. Iron regulates cytoplasmic levels of a novel iron-responsive element-binding protein without aconitase activity.

    PubMed

    Guo, B; Yu, Y; Leibold, E A

    1994-09-30

    Iron-responsive element-binding proteins (IRE-BPs) are cytosolic proteins that bind to a conserved RNA stem-loop, termed the iron-responsive element (IRE), that is located in the 5'- or 3'-untranslated regions of mRNAs involved in iron metabolism. Binding of the IRE-BP to 5'-IREs represses translation, whereas binding to 3'-IREs stabilizes the mRNA. The previously identified IRE-BP (BP1) contains a 4Fe-4S cluster and has sequence homology to mitochondrial aconitase. The 4Fe-4S cluster is important for iron-dependent regulation: BP1 containing iron has low affinity for the IRE and contains aconitase activity, whereas BP1 lacking iron has high affinity for the IRE, but lacks aconitase activity. A second IRE-BP (BP2) has been identified in rat tissues and cells and exhibits many of the hallmarks of an IRE-BP, including binding to the IRE and functioning as a translational repressor of IRE-containing RNAs. BP1 and BP2 RNA binding activities are decreased in extracts from cells treated with iron, indicating that BP1 and BP2 are negatively regulated by iron. Although BP1 and BP2 share similar characteristics, they differ in two significant ways. Unlike BP1 levels, which do not change when RNA binding activity decreases in response to iron, BP2 decreases to undetectable levels in extracts from cells treated with iron; and unlike BP1, BP2 does not have aconitase activity. These data indicate that BP1 and BP2 are distinct proteins that have similar specificity for IRE binding and that function similarly in translation, but are regulated by iron via different mechanisms. PMID:7523370

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

    PubMed

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

    2016-08-01

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

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

    PubMed

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

    2016-08-01

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

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

    PubMed Central

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

    2016-01-01

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

  17. Vanadium compounds modulate PPARγ activity primarily by increasing PPARγ protein levels in mouse insulinoma NIT-1 cells.

    PubMed

    Zhao, Pan; Yang, Xiaoda

    2013-06-01

    Vanadium compounds are promising agents in the therapeutic treatment of diabetes; however, their mechanism of action has not been clearly elucidated. The current study investigated the effects of vanadium compounds, vanadyl acetylacetonate [V(IV)O(acac)2] and sodium metavanadate (NaV(V)O3), on peroxisome proliferator-activated receptors (PPARs), especially PPARγ, which are important targets of anti-diabetic drugs. Our experimental results revealed that treatment of NIT-1 β-pancreas cells with vanadium compounds resulted in PPARγ activation and elevation of PPARγ protein levels. Vanadium compounds did not increase PPARγ transcription but ameliorated PPARγ degradation induced by inflammatory stimulators TNF-α/IL-6. Vanadium compounds induced binding of PPARγ to heat shock protein (Hsp60). This PPARγ-Hsp60 interaction might cause inhibition of PPARγ degradation, thus elevating the PPARγ level. In addition, modulation of PPARγ phosphorylation was also observed upon vanadium treatment. The present work demonstrated for the first time that vanadium compounds are novel PPARγ modulators. The results may provide new insights for the mechanism of anti-diabetic action of vanadium compounds.

  18. GC protein-derived macrophage-activating factor decreases α-N-acetylgalactosaminidase levels in advanced cancer patients

    PubMed Central

    Thyer, Lynda; Ward, Emma; Smith, Rodney; Branca, Jacopo JV; Morucci, Gabriele; Gulisano, Massimo; Noakes, David; Eslinger, Robert; Pacini, Stefania

    2013-01-01

    α-N-acetylgalactosaminidase (nagalase) accumulates in the serum of cancer patients and its activity correlates with tumor burden, aggressiveness and clinical disease progression. The administration of GC protein-derived macrophage-activating factor (GcMAF) to cancer patients with elevated levels of nagalase has been associated with a decrease of serum nagalase activity and with significant clinical benefits. Here, we report the results of the administration of GcMAF to a heterogeneous cohort of patients with histologically diverse, advanced neoplasms, generally considered as “incurable” diseases. In most cases, GcMAF therapy was initiated at late stages of tumor progression. As this is an open-label, non-controlled, retrospective analysis, caution must be employed when establishing cause-effect relationships between the administration GcMAF and disease outcome. However, the response to GcMAF was generally robust and some trends emerged. All patients (n = 20) presented with elevated serum nagalase activity, well above normal values. All patients but one showed a significant decrease of serum nagalase activity upon weekly GcMAF injections. Decreased nagalase activity was associated with improved clinical conditions and no adverse side effects were reported. The observations reported here confirm and extend previous results and pave the way to further studies aimed at assessing the precise role and indications for GcMAF-based anticancer immunotherapy. PMID:24179708

  19. GC protein-derived macrophage-activating factor decreases α-N-acetylgalactosaminidase levels in advanced cancer patients.

    PubMed

    Thyer, Lynda; Ward, Emma; Smith, Rodney; Branca, Jacopo Jv; Morucci, Gabriele; Gulisano, Massimo; Noakes, David; Eslinger, Robert; Pacini, Stefania

    2013-08-01

    α-N-acetylgalactosaminidase (nagalase) accumulates in the serum of cancer patients and its activity correlates with tumor burden, aggressiveness and clinical disease progression. The administration of GC protein-derived macrophage-activating factor (GcMAF) to cancer patients with elevated levels of nagalase has been associated with a decrease of serum nagalase activity and with significant clinical benefits. Here, we report the results of the administration of GcMAF to a heterogeneous cohort of patients with histologically diverse, advanced neoplasms, generally considered as "incurable" diseases. In most cases, GcMAF therapy was initiated at late stages of tumor progression. As this is an open-label, non-controlled, retrospective analysis, caution must be employed when establishing cause-effect relationships between the administration GcMAF and disease outcome. However, the response to GcMAF was generally robust and some trends emerged. All patients (n = 20) presented with elevated serum nagalase activity, well above normal values. All patients but one showed a significant decrease of serum nagalase activity upon weekly GcMAF injections. Decreased nagalase activity was associated with improved clinical conditions and no adverse side effects were reported. The observations reported here confirm and extend previous results and pave the way to further studies aimed at assessing the precise role and indications for GcMAF-based anticancer immunotherapy. PMID:24179708

  20. Systemic and lung protein changes in sarcoidosis. Lymphocyte counts, gallium uptake values, and serum angiotensin-converting enzyme levels may reflect different aspects of disease activity

    SciTech Connect

    Check, I.J.; Kidd, M.R.; Staton, G.W. Jr.

    1986-01-01

    BAL lymphocyte percentages, quantitated gallium-67 lung uptake, and SACE levels have all been proposed as measures of disease activity in sarcoidosis. We analyzed 32 paired sera and BAL fluids from sarcoidosis patients by high-resolution agarose electrophoresis to look for protein changes characteristic of systemic or local inflammation and compared the results with those from the above tests. Nine patients (group 1) had serum inflammatory protein changes and increased total protein, albumin, beta 1-globulin (transferrin), and gamma-globulin levels in fluid recovered by BAL. Thirteen patients (group 2) had normal protein levels in sera but abnormal protein levels in BAL specimens. Ten patients (group 3) had normal protein levels in sera and in BAL specimens. Patients in groups 1 and 2 had a disproportionate increase in beta 1-globulin (transferrin) and gamma-globulin levels in their BAL specimens. The BAL lymphocyte percentage changes paralleled the BAL protein level changes, suggesting relationships among the immunoregulatory role of these cells, increased local immunoglobulin synthesis, and the pathogenesis of altered alveolar permeability. Gallium-67 uptake was highest in patients with serum inflammatory protein changes. Thus, systemic inflammation may facilitate pulmonary gallium-67 uptake, possibly by changes in BAL fluid or serum transferrin saturation and/or kinetics. SACE levels showed no relationship to changes in the levels of serum or BAL proteins. These data suggest that the various proposed measures of disease activity reflect different aspects of inflammation in sarcoidosis.

  1. Palmitic acid increase levels of pancreatic duodenal homeobox-1 and p38/stress-activated protein kinase in islets from rats maintained on a low protein diet.

    PubMed

    Arantes, Vanessa C; Reis, Marise A B; Latorraca, Márcia Q; Ferreira, Fabiano; Stoppiglia, Luiz Fabrízio; Carneiro, Everardo M; Boschero, Antonio C

    2006-12-01

    A severe reduction in insulin release in response to glucose is consistently noticed in protein-deprived rats and is attributed partly to the chronic exposure to elevated levels of NEFA. Since the pancreatic and duodenal transcription factor homeobox 1 (PDX-1) is important for the maintenance of beta-cell physiology, and since PDX-1 expression is altered in the islets of rats fed a low protein (LP) diet and that rats show high NEFA levels, we assessed PDX-1 and insulin mRNA expression, as well as PDX-1 and p38/stress activated protein kinase 2 (SAPK2) protein expression, in islets from young rats fed low (6%) or normal (17%; control) protein diets and maintained for 48 h in culture medium containing 5.6 mmol/l glucose, with or without 0.6 mmol/l palmitic acid. We also measured glucose-induced insulin secretion and glucose metabolism. Insulin secretion by isolated islets in response to 16.7 mmol/l glucose was reduced in LP compared with control rats. In the presence of NEFA, there was an increase in insulin secretion in both groups. At 2.8 mmol/l glucose, the metabolism of this sugar was reduced in LP islets, regardless of the presence of this fatty acid. However, when challenged with 16.7 mmol/l glucose, LP and control islets showed a severe reduction in glucose oxidation in the presence of NEFA. The PDX-1 and insulin mRNA were significantly higher when NEFA was added to the culture medium in both groups of islets. The effect of palmitic acid on PDX-1 and p38/SAPK2 protein levels was similar in LP and control islets, but the increase was much more evident in LP islets. These results demonstrate the complex interrelationship between nutrients in the control of insulin release and support the view that fatty acids play an important role in glucose homeostasis by affecting molecular mechanisms and stimulus/secretion coupling pathways. PMID:17181874

  2. Effects of pentylenetetrazole kindling on mitogen-activated protein kinases levels in neocortex and hippocampus of mice.

    PubMed

    Ben, Juliana; de Oliveira, Paulo Alexandre; Gonçalves, Filipe Marques; Peres, Tanara Vieira; Matheus, Filipe Carvalho; Hoeller, Alexandre Ademar; Leal, Rodrigo Bainy; Walz, Roger; Prediger, Rui Daniel

    2014-12-01

    The epileptogenesis process involves cell signaling events associated with neuroplasticity. The mitogen-activated protein kinases (MAPKs) integrate signals originating from a variety of extracellular stimuli and may regulate cell differentiation, survival, cell death and synaptic plasticity. Here we compared the total and phosphorylated MAPKs (ERK1/2, JNK1/2 and p38(MAPK)) levels in the neocortex and hippocampus of adult Swiss male mice quantified by western blotting analysis 48 h after the last injection of pentylenetetrazole (PTZ), according to the kindling protocol (35 mg/kg, i.p., on alternated days, with a total of eight injections). The total levels of the investigated MAPKs and the phospho-p38(MAPK) in the neocortex and hippocampus were not affected by the PTZ injections. The MAPKs phosphorylation levels remain unaltered in PTZ-treated animals without convulsive seizures. The phospho-JNK2 phosphorylation, but not the phospho-JNK1, was increased in the hippocampus of PTZ-treated animals showing 1-3 days with convulsive seizures, whereas no significant changes were observed in those animals with more than 3 days with convulsive seizures. The phospho-ERK1/2 phosphorylation decreased in the neocortex and increased in the hippocampus of animals with 1-4 days with convulsive seizures and became unaltered in mice that showed convulsive seizures for more than 4 days. These findings indicate that resistance to PTZ kindling is associated with unaltered ERK1/2, JNK1/2 and p38(MAPK) phosphorylation levels in the neocortex and hippocampus. Moreover, when the PTZ kindling-induced epileptogenesis manifests behaviorally, the activation of the different MAPKs sub-families shows a variable and non-linear pattern in the neocortex and hippocampus.

  3. Elevated salivary C-reactive protein levels are associated with active and passive smoking in healthy youth: A pilot study

    PubMed Central

    2011-01-01

    Background We examined salivary C-reactive protein (CRP) levels in the context of tobacco smoke exposure (TSE) in healthy youth. We hypothesized that there would be a dose-response relationship between TSE status and salivary CRP levels. Methods This work is a pilot study (N = 45) for a larger investigation in which we aim to validate salivary CRP against serum CRP, the gold standard measurement of low-grade inflammation. Participants were healthy youth with no self-reported periodontal disease, no objectively measured obesity/adiposity, and no clinical depression, based on the Beck Depression Inventory (BDI-II). We assessed tobacco smoking and confirmed smoking status (non-smoking, passive smoking, and active smoking) with salivary cotinine measurement. We measured salivary CRP by the ELISA method. We controlled for several potential confounders. Results We found evidence for the existence of a dose-response relationship between the TSE status and salivary CRP levels. Conclusions Our preliminary findings indicate that salivary CRP seems to have a similar relation to TSE as its widely used serum (systemic inflammatory) biomarker counterpart. PMID:22152006

  4. Effects of a single exposure to UVB radiation on the activities and protein levels of copper-zinc and manganese superoxide dismutase in cultured human keratinocytes.

    PubMed

    Sasaki, H; Akamatsu, H; Horio, T

    1997-04-01

    Ultraviolet B irradiation has been believed to decrease or impair the activity of reactive oxygen species (ROS) scavenging enzymes such as superoxide dismutase (SOD) in the skin. It has been recently reported that two isozymes of SOD, namely copper-zinc SOD (Cu-Zn SOD) and manganese SOD (Mn SOD), exist in mammalian cells and that the two enzymes play different roles in living systems. The aim of this study was to investigate changes in SOD activities and protein levels in cultured human keratinocytes after acute UVB irradiation. In addition, the protein levels of Cu-Zn SOD and Mn SOD were quantified separately. A single exposure to UVB irradiation produced an increase in SOD activity and protein level that peaked immediately after UVB irradiation, after which a decline was observed, with subsequent recovery to baseline levels 24 h after irradiation. In individual assays of Mn SOD and Cu-Zn SOD, the amount of Mn SOD protein decreased and then gradually recovered 24 h after irradiation. In contrast, the amount of Cu-Zn SOD protein increased immediately after UVB irradiation, and then gradually declined. To evaluate the mechanisms of these changes, we examined the effects of the cytokines, interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor-alpha (TNF-alpha), which can be secreted from keratinocytes after UVB irradiation, on the SOD activity and protein levels in keratinocytes. Interleukin-1 alpha and TNF-alpha enhanced both the SOD activity and protein level of Mn SOD, while these cytokines had no effect on Cu-Zn SOD protein levels in cultured human keratinocytes after incubation for 24 h. Furthermore, when neutralizing antibodies against IL-1 alpha and TNF-alpha were added separately or together to the culture medium before UVB irradiation, the recovery of total SOD activity and Mn SOD protein level were markedly inhibited 24 h after irradiation. Our results suggest that significant increases in SOD activity and protein level occur as a cutaneous antioxidant

  5. A protein disulfide-thiol interchange protein with NADH: protein disulfide reductase (NADH oxidase) activity as a molecular target for low levels of exposure to organic solvents in plant growth.

    PubMed

    Morré, D J

    1998-05-01

    A number of solvents including ethyl, amyl, butyl, octyl and benzyl alcohols, ethylene glycol, ethyl acetate, acetone, diethyl ether, propylene oxide, rho-dioxane, benzene, xylene, chloroform and carbon tetrachloride stimulate the growth of plants or plant parts at low concentrations and inhibit at high concentrations. These same solvents, at low dilutions, stimulate the activity of a growth-related protein disulfide-thiol interchange protein (TIP) with NADH: protein disulfide reductase (NADH oxidase) (NOX) activity with plasma membrane vesicles isolated from elongating regions cut from dark grown seedlings of soybeans. Based on these and other findings, we suggest the TIP/NOX protein to be the molecular target of the biological effects of low levels of exposure (hormesis) involved in the stimulation of plant growth.

  6. Studies on the effect of long-term exposure to nitrogen dioxide on serum and liver proteins level and enzyme activity in guinea pigs.

    PubMed

    Drozdz, M; Kucharz, E; Ludyga, K; Molska-Drozda, T

    1976-01-01

    Forty male guinea pigs were exposed to nitrogen dioxide in a concentration of 2 mg/m3, 8 hours daily for a period of 180 days. Forty male animals were used as a control group. The following changes were found in intoxicated animals: the decrease of total protein and seromucoid concentration in blood serum and the decrease of total protein, perchloric acid-soluble proteins, protein-bound hexosamines and sialic acids content, in liver tissue. Electrophoretic examination of the serum proteins showed the increase of alpha 1- and beta 2-globulins and the decrease of albumin concentration. Changes in the level of glycoproteins fractions and protein-bound carbohydrates in blood serum were described also. Estimation of enzymes activity showed the decrease of alanine and aspartate transaminase activity in blood serum caused by the strong decrease of the cytoplasmic fraction of these enzymes. However the simultaneous increase of the mitochondrial fraction of transaminases activity was observed. The decrease of the activity of choline esterase was found also. Similar changes of enzymes activity were found in liver tissue. Histopathological studies were done for the further clearing the influenze of nitrogen dioxide on serum and liver proteins concentration and enzymes activity. It was found that after long-term exposure to nitrogen dioxide the destruction processes may be observed in the liver. The possible mechanism of the nitrogen dioxide-induced damage of protein metabolism is discussed.

  7. Protein kinase C-mediated phosphorylation and activation of PDE3A regulate cAMP levels in human platelets.

    PubMed

    Hunter, Roger W; Mackintosh, Carol; Hers, Ingeborg

    2009-05-01

    The elevation of [cAMP](i) is an important mechanism of platelet inhibition and is regulated by the opposing activity of adenylyl cyclase and phosphodiesterase (PDE). In this study, we demonstrate that a variety of platelet agonists, including thrombin, significantly enhance the activity of PDE3A in a phosphorylation-dependent manner. Stimulation of platelets with the PAR-1 agonist SFLLRN resulted in rapid and transient phosphorylation of PDE3A on Ser(312), Ser(428), Ser(438), Ser(465), and Ser(492), in parallel with the PKC (protein kinase C) substrate, pleckstrin. Furthermore, phosphorylation and activation of PDE3A required the activation of PKC, but not of PI3K/PKB, mTOR/p70S6K, or ERK/RSK. Activation of PKC by phorbol esters also resulted in phosphorylation of the same PDE3A sites in a PKC-dependent, PKB-independent manner. This was further supported by the finding that IGF-1, which strongly activates PI3K/PKB, but not PKC, did not regulate PDE3A. Platelet activation also led to a PKC-dependent association between PDE3A and 14-3-3 proteins. In contrast, cAMP-elevating agents such as PGE(1) and forskolin-induced phosphorylation of Ser(312) and increased PDE3A activity, but did not stimulate 14-3-3 binding. Finally, complete antagonism of PGE(1)-evoked cAMP accumulation by thrombin required both G(i) and PKC activation. Together, these results demonstrate that platelet activation stimulates PKC-dependent phosphorylation of PDE3A on Ser(312), Ser(428), Ser(438), Ser(465), and Ser(492) leading to a subsequent increase in cAMP hydrolysis and 14-3-3 binding. PMID:19261611

  8. Levels of expression of complement regulatory proteins CD46, CD55 and CD59 on resting and activated human peripheral blood leucocytes

    PubMed Central

    Christmas, Stephen E; de la Mata Espinosa, Claudia T; Halliday, Deborah; Buxton, Cheryl A; Cummerson, Joanne A; Johnson, Peter M

    2006-01-01

    The cell surface complement regulatory (CReg) proteins CD46, CD55 and CD59 are widely expressed on human lymphoid and non-lymphoid cells. This study aimed to compare systematically levels of CReg expression by different leucocyte subsets and to determine whether levels were increased following activation in vitro. Levels of each CReg protein were similar on freshly isolated monocytes and all major lymphocyte subsets, except that CD4+ cells expressed significantly less CD46 than CD8+ cells (P < 0·05) while the reverse was observed for CD55 (P < 0·02). CD56+ cells, predominantly natural killer cells, expressed significantly lower levels of CD59 than T cells (P < 0·02). CD45RO+ cells had higher levels of surface CD46 and CD59, but lower levels of CD55, than CD45RO– cells (P < 0·02); CD25+ cells also expressed significantly less CD55 than CD25− cells (P < 0·002). Neutrophils expressed higher levels of CD59, but lower levels of CD55, than monocytes. Following activation with phytohaemagglutinin, CD46 was up-regulated on all leucocyte subsets with the exception of CD56+ cells. Both CD55 and CD59 were also markedly up-regulated on monocytes, and CD55 expression was greater on CD8+ than CD4+ cells following activation (P < 0·02). Lipopolysaccharide treatment did not significantly alter B-cell expression of CReg proteins whereas CD55 and CD59, but not CD46, were significantly up-regulated on monocytes (P < 0·02). These observations that CReg proteins are up-regulated on certain activated leucocyte subsets indicate that levels would be increased following immune responses in vivo. This could enhance both protection against local complement activation at inflammatory sites and also the immunoregulatory properties of these leucocytes. PMID:16999828

  9. Levels of expression of complement regulatory proteins CD46, CD55 and CD59 on resting and activated human peripheral blood leucocytes.

    PubMed

    Christmas, Stephen E; de la Mata Espinosa, Claudia T; Halliday, Deborah; Buxton, Cheryl A; Cummerson, Joanne A; Johnson, Peter M

    2006-12-01

    The cell surface complement regulatory (CReg) proteins CD46, CD55 and CD59 are widely expressed on human lymphoid and non-lymphoid cells. This study aimed to compare systematically levels of CReg expression by different leucocyte subsets and to determine whether levels were increased following activation in vitro. Levels of each CReg protein were similar on freshly isolated monocytes and all major lymphocyte subsets, except that CD4(+) cells expressed significantly less CD46 than CD8(+) cells (P < 0.05) while the reverse was observed for CD55 (P < 0.02). CD56(+) cells, predominantly natural killer cells, expressed significantly lower levels of CD59 than T cells (P < 0.02). CD45RO(+) cells had higher levels of surface CD46 and CD59, but lower levels of CD55, than CD45RO(-) cells (P < 0.02); CD25(+) cells also expressed significantly less CD55 than CD25(-) cells (P < 0.002). Neutrophils expressed higher levels of CD59, but lower levels of CD55, than monocytes. Following activation with phytohaemagglutinin, CD46 was up-regulated on all leucocyte subsets with the exception of CD56(+) cells. Both CD55 and CD59 were also markedly up-regulated on monocytes, and CD55 expression was greater on CD8(+) than CD4(+) cells following activation (P < 0.02). Lipopolysaccharide treatment did not significantly alter B-cell expression of CReg proteins whereas CD55 and CD59, but not CD46, were significantly up-regulated on monocytes (P < 0.02). These observations that CReg proteins are up-regulated on certain activated leucocyte subsets indicate that levels would be increased following immune responses in vivo. This could enhance both protection against local complement activation at inflammatory sites and also the immunoregulatory properties of these leucocytes.

  10. Initial evidence for the link between activities and health: Associations between a balance of activities, functioning and serum levels of cytokines and C-reactive protein.

    PubMed

    Dür, Mona; Steiner, Günter; Stoffer, Michaela Alexandra; Fialka-Moser, Veronika; Kautzky-Willer, Alexandra; Dejaco, Clemens; Ekmekcioglu, Cem; Prodinger, Birgit; Binder, Alexa; Smolen, Josef; Stamm, Tanja Alexandra

    2016-03-01

    Growing evidence shows interrelations of psychological factors, neurological and immunological processes. Therefore, constructs like a balance of activities, the so called "occupational balance", could also have biological correlates. The aim of this study was to investigate potential associations between occupational balance, functioning, cytokines and C-reactive protein (CRP) in patients suffering from a chronic inflammatory disease like rheumatoid arthritis (RA) and healthy people. Moreover, we wanted to explore potential differences in gender and employment status. A descriptive study in patients with RA and healthy people was conducted using the Occupational Balance-Questionnaire (OB-Quest) and the Short-Form 36 Health Survey (SF-36). Serum levels of cytokines, such as interleukin 6 (IL-6) and 8 (IL-8), interferon alpha (INFα), tumour necrosis factor alpha (TNFα), rheumatoid factor (RF) and of CRP were measured. Descriptive statistics, as well as Mann-Whitney U tests and Spearmen's rank correlation coefficients (rs) were calculated. One-hundred-thirty-two patients with RA and 76 healthy people participated. Occupational balance was associated with functioning, cytokines and CRP. The strongest associations were identified in the unemployed healthy-people sample with cytokines and CRP being within the normal range. For example, the OB-Quest item challenging activities was associated with IL-8 (rs=-0.63, p=0.04) and the SF-36 sub-scale bodily pain was associated with IFNα (rs=-0.69, p=0.02). The items rest and sleep (rs=-0.71, p=0.01) and variety of different activities (rs=-0.74, p<0.01) correlated with the SF-36 sub-scale social functioning. Employed and unemployed people differed in their age and CRP levels. Additionally, gender differences were found in two OB-Quest items in that fewer women were able to adapt their activities to changing living conditions and fewer men were overstressed. In conclusion, we found preliminary biological evidence for the link

  11. Initial evidence for the link between activities and health: Associations between a balance of activities, functioning and serum levels of cytokines and C-reactive protein.

    PubMed

    Dür, Mona; Steiner, Günter; Stoffer, Michaela Alexandra; Fialka-Moser, Veronika; Kautzky-Willer, Alexandra; Dejaco, Clemens; Ekmekcioglu, Cem; Prodinger, Birgit; Binder, Alexa; Smolen, Josef; Stamm, Tanja Alexandra

    2016-03-01

    Growing evidence shows interrelations of psychological factors, neurological and immunological processes. Therefore, constructs like a balance of activities, the so called "occupational balance", could also have biological correlates. The aim of this study was to investigate potential associations between occupational balance, functioning, cytokines and C-reactive protein (CRP) in patients suffering from a chronic inflammatory disease like rheumatoid arthritis (RA) and healthy people. Moreover, we wanted to explore potential differences in gender and employment status. A descriptive study in patients with RA and healthy people was conducted using the Occupational Balance-Questionnaire (OB-Quest) and the Short-Form 36 Health Survey (SF-36). Serum levels of cytokines, such as interleukin 6 (IL-6) and 8 (IL-8), interferon alpha (INFα), tumour necrosis factor alpha (TNFα), rheumatoid factor (RF) and of CRP were measured. Descriptive statistics, as well as Mann-Whitney U tests and Spearmen's rank correlation coefficients (rs) were calculated. One-hundred-thirty-two patients with RA and 76 healthy people participated. Occupational balance was associated with functioning, cytokines and CRP. The strongest associations were identified in the unemployed healthy-people sample with cytokines and CRP being within the normal range. For example, the OB-Quest item challenging activities was associated with IL-8 (rs=-0.63, p=0.04) and the SF-36 sub-scale bodily pain was associated with IFNα (rs=-0.69, p=0.02). The items rest and sleep (rs=-0.71, p=0.01) and variety of different activities (rs=-0.74, p<0.01) correlated with the SF-36 sub-scale social functioning. Employed and unemployed people differed in their age and CRP levels. Additionally, gender differences were found in two OB-Quest items in that fewer women were able to adapt their activities to changing living conditions and fewer men were overstressed. In conclusion, we found preliminary biological evidence for the link

  12. The cystine/glutamate antiporter regulates indoleamine 2,3-dioxygenase protein levels and enzymatic activity in human dendritic cells.

    PubMed

    Mattox, Mildred L; D'Angelo, June A; Grimes, Zachary M; Fiebiger, Edda; Dickinson, Bonny L

    2012-11-30

    Indoleamine 2,3-dioxygenase (IDO) is the rate-limiting enzyme in the tryptophan-catabolizing pathway and a key regulator of peripheral immune tolerance. As the suppressive effects of IDO are predominantly mediated by dendritic cells (DCs) and IDO-competent DCs promote long-term immunologic tolerance, a detailed understanding of how IDO expression and activity is regulated in these cells is central to the rational design of therapies to induce robust immune tolerance. We previously reported that the cystine/glutamate antiporter modulates the functional expression of IDO in human monocyte-derived DCs. Specifically, we showed that blocking antiporter uptake of cystine significantly increased both IDO mRNA and IDO enzymatic activity and that this correlated with impaired DC presentation of exogenous antigen to T cells via MHC class II and the cross-presentation pathway. The antiporter regulates intracellular and extracellular redox by transporting cystine into the cell in exchange for glutamate. Intracellular cystine is reduced to cysteine to support biosynthesis of the major cellular antioxidant glutathione and cysteine is exported from the cell where it functions as an extracellular antioxidant. Here we show that antiporter control of IDO expression in DCs is reversible, independent of interferon-γ, regulated by redox, and requires active protein synthesis. These findings highlight a role for antiporter regulation of cellular redox as a critical control point for modulating IDO expression and activity in DCs. Thus, systemic disease and aging, processes that perturb redox homeostasis, may adversely affect immunity by promoting the generation of IDO-competent DCs.

  13. The cystine/glutamate antiporter regulates indoleamine 2,3-dioxygenase protein levels and enzymatic activity in human dendritic cells

    PubMed Central

    Mattox, Mildred L; D’Angelo, June A; Grimes, Zachary M; Fiebiger, Edda; Dickinson, Bonny L

    2012-01-01

    Indoleamine 2,3-dioxygenase (IDO) is the rate-limiting enzyme in the tryptophan-catabolizing pathway and a key regulator of peripheral immune tolerance. As the suppressive effects of IDO are predominantly mediated by dendritic cells (DCs) and IDO-competent DCs promote long-term immunologic tolerance, a detailed understanding of how IDO expression and activity is regulated in these cells is central to the rational design of therapies to induce robust immune tolerance. We previously reported that the cystine/glutamate antiporter modulates the functional expression of IDO in human monocyte-derived DCs. Specifically, we showed that blocking antiporter uptake of cystine significantly increased both IDO mRNA and IDO enzymatic activity and that this correlated with impaired DC presentation of exogenous antigen to T cells via MHC class II and the cross-presentation pathway. The antiporter regulates intracellular and extracellular redox by transporting cystine into the cell in exchange for glutamate. Intracellular cystine is reduced to cysteine to support biosynthesis of the major cellular antioxidant glutathione and cysteine is exported from the cell where it functions as an extracellular antioxidant. Here we show that antiporter control of IDO expression in DCs is reversible, independent of interferon-γ, regulated by redox, and requires active protein synthesis. These findings highlight a role for antiporter regulation of cellular redox as a critical control point for modulating IDO expression and activity in DCs. Thus, systemic disease and aging, processes that perturb redox homeostasis, may adversely affect immunity by promoting the generation of IDO-competent DCs. PMID:23243629

  14. Activation of the PI3K/Akt signal transduction pathway and increased levels of insulin receptor in protein repair-deficient mice.

    PubMed

    Farrar, Christine; Houser, Carolyn R; Clarke, Steven

    2005-02-01

    Protein L-isoaspartate (D-aspartate) O-methyltransferase is an enzyme that catalyses the repair of isoaspartyl damage in proteins. Mice lacking this enzyme (Pcmt1-/- mice) have a progressive increase in brain size compared with wild-type mice (Pcmt1+/+ mice), a phenotype that can be associated with alterations in the PI3K/Akt signal transduction pathway. Here we show that components of this pathway, including Akt, GSK3beta and PDK-1, are more highly phosphorylated in the brains of Pcmt1-/- mice, particularly in cells of the hippocampus, in comparison with Pcmt1+/+ mice. Examination of upstream elements of this pathway in the hippocampus revealed that Pcmt1-/- mice have increased activation of insulin-like growth factor-I (IGF-I) receptor and/or insulin receptor. Western blot analysis revealed an approximate 200% increase in insulin receptor protein levels and an approximate 50% increase in IGF-I receptor protein levels in the hippocampus of Pcmt1-/- mice. Higher levels of the insulin receptor protein were also found in other regions of the adult brain and in whole tissue extracts of brain, liver, heart and testes of both juvenile and adult Pcmt1-/- mice. There were no significant differences in plasma insulin levels for adult Pcmt1-/- mice during glucose tolerance tests. However, they did show higher peak levels of blood glucose, suggesting a mild impairment in glucose tolerance. We propose that Pcmt1-/- mice have altered regulation of the insulin pathway, possibly as a compensatory response to altered glucose uptake or metabolism or as an adaptive response to a general accumulation of isoaspartyl protein damage in the brain and other tissues.

  15. VEGF Gene Polymorphisms Affect Serum Protein Levels and Alter Disease Activity and Synovial Lesions in Rheumatoid Arthritis

    PubMed Central

    Yi, Jin-Ping; Wu, Yu-Zhang; Yu, Nan; Yu, Zhi-Wu; Xie, Fu-Yuan; Yuan, Quan

    2016-01-01

    Background Our study investigated 2 common single-nucleotide polymorphisms (SNPs) of vascular endothelial growth factor (VEGF) for their influences on serum VEGF levels, disease activity, and synovial lesions in rheumatoid arthritis (RA). Material/Methods Clinical information and venous blood samples were collected from 98 RA patients and 100 healthy controls. Genotyping on samples from the subjects was performed using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). Serum VEGF levels were determined using the enzyme-linked immunosorbent assay (ELISA). The synovial thickness and joint effusion of 28 joints were measured in RA patients, and total sharp score (TSS) and disease activity score (DAS) of 28 joints were recorded. Results The genotype and allele frequencies of VEGF rs833070 (G>A) and rs3025030 (G>C) were significantly different between RA group and control group (all P<0.05). VEGF rs833070 and rs3025030 polymorphisms were associated with increasing VEGF serum levels in the RA group (all P<0.01). Statistically significant difference was observed in DAS28 between the different genotypes of VEGF rs833070 in RA patients (P<0.05). Importantly, significant differences in synovial thickening, joint effusion and synovial angiogenesis were observed between the different genotypes of VEGF rs833070 and rs3025030 polymorphisms (all P<0.05). Conclusions Our study provides evidence that VEGF polymorphisms might be important indicators of disease activity and synovial lesions, and prognostic factors in evaluating the treatment effectiveness in RA. PMID:26825024

  16. Protein kinase C activators selectively inhibit insulin-stimulated system A transport activity in skeletal muscle at a post-receptor level.

    PubMed Central

    Gumà, A; Camps, M; Palacín, M; Testar, X; Zorzano, A

    1990-01-01

    We have investigated the role of phorbol esters on different biological effects induced by insulin in muscle, such as activation of system A transport activity, glucose utilization and insulin receptor function. System A transport activity was measured by monitoring the uptake of the system A-specific analogue alpha-(methyl)aminoisobutyric acid (MeAIB), by intact rat extensor digitorum longus muscle. The addition of 12-O-tetradecanoylphorbol 13-acetate (TPA, 0.5 microM) for 60 or 180 min did not modify basal MeAIB uptake by muscle, suggesting that insulin signalling required to stimulate MeAIB transport does not involve protein kinase C activation. However, TPA added 30 min before insulin (100 nM) markedly inhibited insulin-stimulated MeAIB uptake. The addition of polymyxin B (0.1 mM) or H-7 (1 mM), protein kinase C inhibitors, alone or in combination with TPA leads to impairment of insulin-stimulated MeAIB uptake. This paradoxical pattern is incompatible with a unique action of Polymyxin B or H-7 on protein kinase C activity. Therefore these agents are not suitable tools with which to investigate whether a certain insulin effect is mediated by protein kinase C. TPA did not cause a generalized inhibition of insulin action. Thus both TPA and insulin increased 3-O-methylglucose uptake by muscle, and their effects were not additive. Furthermore, TPA did not modify insulin-stimulated lactate production by muscle. In keeping with this selective modification of insulin action, treatment of muscles with TPA did not modify insulin receptor binding or kinase activities. In conclusion, phorbol esters do not mimic insulin action on system A transport activity; however, they markedly inhibit insulin-stimulated amino acid transport, with no modification of insulin receptor function in rat skeletal muscle. It is suggested that protein kinase C activation causes a selective post-receptor modification on the biochemical pathway by which insulin activates system A amino acid

  17. Cocaine differentially regulates activator protein-1 mRNA levels and DNA-binding complexes in the rat striatum and cerebellum.

    PubMed

    Couceyro, P; Pollock, K M; Drews, K; Douglass, J

    1994-10-01

    Cocaine is a psychomotor stimulant that exerts many of its behavioral and physiological effects through alteration of catecholamine reuptake systems. One early cellular response to cocaine administration is a brain region-specific alteration in the transcriptional pattern of immediate early genes belonging to the Fos/Jun family of nucleotide sequence-specific [activator protein-1 (AP-1)] DNA-binding proteins. The work described here compares cocaine-induced transcriptional regulation of immediate early gene mRNA levels, as well as AP-1 DNA-binding activity, within the striatum and cerebellum. In the striatum, acute cocaine administration increases cellular levels of c-fos and jun-B mRNA, whereas transcriptional effects in the cerebellum are limited to c-fos mRNA. After chronic cocaine treatment a desensitization of c-fos mRNA induction is observed in the striatum, with sensitization of the same transcriptional effect occurring in the cerebellum. Pharmacological studies further reveal that the dopamine D1, dopamine D2, gamma-aminobutyric acid type B, and N-methyl-D-aspartate receptor systems mediate the effects of cocaine on cerebellar neurons, whereas striatal effects are modulated through D1 and N-methyl-D-aspartate receptors. Gel retention analysis using antibodies to the various Fos and Jun proteins was used to characterize cocaine-dependent alterations in the composition of striatal and cerebellar AP-1 DNA-binding complexes. In striatum, cocaine increases the relative levels of c-Fos, Fos-B, Jun-B, and Jun-D proteins that bind the AP-1 DNA sequence element, whereas in the cerebellum only c-Fos and Jun-D binding activities are increased. These data suggest two possible neuroanatomical sites where tolerance and sensitization to cocaine can be examined at the genomic level. PMID:7969045

  18. Nobiletin, a flavone from Citrus depressa, induces gene expression and increases the protein level and activity of neprilysin in SK-N-SH cells.

    PubMed

    Fujiwara, Hironori; Kimura, Junko; Sakamoto, Masahiro; Yokosuka, Akihito; Mimaki, Yoshihiro; Murata, Kiyoshi; Yamaguchi, Kikuji; Ohizumi, Yasushi

    2014-05-01

    Neprilysin (NEP) is one of the candidate amyloid β protein (Aβ) degrading enzymes affecting brain Aβ clearance. This enzyme declines in the brain with age, which leads to the increased Aβ deposition in Alzheimer's disease (AD). Pharmacological activation of NEP during the aging process, therefore, represents a potential strategy to prevent the development of AD. To examine the influence of nobiletin on neprilysin activity, we measured cellular NEP activity in SK-N-SH cells. Moreover, NEP expression was examined by using reverse transcription - polymerase chain reaction and Western blotting. Measurement of cellular NEP activity showed that nobiletin stimulated this in a dose- and time-dependent manner in SK-N-SH cells. Moreover, nobiletin increased the expression of NEP mRNA, and then the levels of NEP protein, also in a dose- and time-dependent manner. Our findings showed that nobiletin promoted NEP gene and protein expression, resulting in enhancement of cellular NEP activity in SK-N-SH cells. This compound could be a novel Aβ-degrading compound for use in the development of disease-modifying drugs to prevent and (or) cure AD.

  19. Rapid Diminution in the Level and Activity of DNA-Dependent Protein Kinase in Cancer Cells by a Reactive Nitro-Benzoxadiazole Compound

    PubMed Central

    Silva, Viviane A. O.; Lafont, Florian; Benhelli-Mokrani, Houda; Breton, Magali Le; Hulin, Philippe; Chabot, Thomas; Paris, François; Sakanyan, Vehary; Fleury, Fabrice

    2016-01-01

    The expression and activity of DNA-dependent protein kinase (DNA-PK) is related to DNA repair status in the response of cells to exogenous and endogenous factors. Recent studies indicate that Epidermal Growth Factor Receptor (EGFR) is involved in modulating DNA-PK. It has been shown that a compound 4-nitro-7-[(1-oxidopyridin-2-yl)sulfanyl]-2,1,3-benzoxadiazole (NSC), bearing a nitro-benzoxadiazole (NBD) scaffold, enhances tyrosine phosphorylation of EGFR and triggers downstream signaling pathways. Here, we studied the behavior of DNA-PK and other DNA repair proteins in prostate cancer cells exposed to compound NSC. We showed that both the expression and activity of DNA-PKcs (catalytic subunit of DNA-PK) rapidly decreased upon exposure of cells to the compound. The decline in DNA-PKcs was associated with enhanced protein ubiquitination, indicating the activation of cellular proteasome. However, pretreatment of cells with thioglycerol abolished the action of compound NSC and restored the level of DNA-PKcs. Moreover, the decreased level of DNA-PKcs was associated with the production of intracellular hydrogen peroxide by stable dimeric forms of Cu/Zn SOD1 induced by NSC. Our findings indicate that reactive oxygen species and electrophilic intermediates, generated and accumulated during the redox transformation of NBD compounds, are primarily responsible for the rapid modulation of DNA-PKcs functions in cancer cells. PMID:27187356

  20. Plasma membrane H(+) -ATPase gene expression, protein level and activity in growing and non-growing regions of barley (Hordeum vulgare) leaves.

    PubMed

    Visnovitz, Tamás; Solti, Adám; Csikós, György; Fricke, Wieland

    2012-04-01

    Plasma membrane proton ATPase (PM-H⁺-ATPase) is the key means through which plant cells energize nutrient uptake and acidify the apoplast. Both of these processes aid cell elongation; yet, it is not known how such a suspected role of the PM-H⁺-ATPase in growth is reflected through changes in its transcript level and activity in grass leaves. In the present study on leaf three of barley, the elongation zone and the emerged blade, which contained fully expanded cells were analyzed. Plasma membranes were isolated and used to assay the activity (ATPase assay) and abundance (western blotting) of PM-H⁺-ATPase protein. Expression of mRNA was quantified using real-time polymerase chain reaction (qPCR). PM-H⁺-ATPase transcript and protein level and activity differed little between growing and non-growing leaf regions when values were related to unit extracted total RNA and cell number, respectively. However, when values were related to unit surface area of plasma membrane, they were more than twice as high in growing compared with non-growing leaf tissue. It is concluded that this higher surface density of PM-H⁺-ATPase activity in growing barley leaf tissue aids apoplast acidification and cell expansion. PMID:22257033

  1. Arbuscular mycorrhiza mediates glomalin-related soil protein production and soil enzyme activities in the rhizosphere of trifoliate orange grown under different P levels.

    PubMed

    Wu, Qiang-Sheng; Li, Yan; Zou, Ying-Ning; He, Xin-Hua

    2015-02-01

    Glomalin-related soil protein (GRSP) is beneficial to soil and plants and is affected by various factors. To address whether mycorrhizal-induced GRSP and relevant soil enzymes depend on external P levels, a pot study evaluated effects of the arbuscular mycorrhizal fungus (AMF) Funneliformis mosseae on GRSP production and soil enzyme activities. Three GRSP categories, as easily-extractable GRSP (EE-GRSP), difficultly-extractable GRSP (DE-GRSP), and total (EE-GRSP + DE-GRSP) GRSP (T-GRSP), were analyzed, together with five enzyme activities (β-glucosidase, catalase, peroxidase, phosphatase, polyphenol oxidase) in the rhizosphere of trifoliate orange (Poncirus trifoliata) grown under 0, 3, and 30 mM KH2PO4 in a sand substrate. After 4 months, root AM colonization and substrate hyphal length decreased with increasing P levels. Shoot, root, and total biomass production was significantly increased by AM colonization, regardless of P levels, but more profound under 0 mM P than under 30 mM KH2PO4. In general, production of these three GRSP categories under 0 or 30 mM KH2PO4 was similar in non-mycorrhizosphere but decreased in mycorrhizosphere. Mycorrhization significantly increased the production of EE-GRSP, DE-GRSP and T-GRSP, soil organic carbon (SOC), and activity of substrate β-glucosidase, catalase, peroxidase, and phosphatase, but decreased polyphenol oxidase activity, irrespective of P levels. Production of EE-GRSP, DE-GRSP, and T-GRSP significantly positively correlated with SOC and β-glucosidase, catalase, and peroxidase activity, negatively with polyphenol oxidase activity, but not with hyphal length or phosphatase activity. These results indicate that AM-mediated production of GRSP and relevant soil enzyme activities may not depend on external P concentrations.

  2. Disease Phenotype, Activity and Clinical Course Prediction Based on C-Reactive Protein Levels at Diagnosis in Patients with Crohn’s Disease: Results from the CONNECT Study

    PubMed Central

    Kwon, Jee Hye; Im, Jong Pil; Ye, Byong Duk; Cheon, Jae Hee; Jang, Hyun Joo; Lee, Kang Moon; Kim, You Sun; Kim, Sang Wook; Kim, Young Ho; Song, Geun Am; Han, Dong Soo; Kim, Won Ho; Kim, Joo Sung

    2016-01-01

    Background/Aims C-reactive protein (CRP) is an easily measured index of disease activity, but its ability to predict clinical course is controversial. We therefore designed a study to determine whether the CRP level at Crohn’s disease (CD) diagnosis is a valuable indicator of the disease phenotype, activity, and clinical course. Methods We retrospectively analyzed 705 CD patients from 32 institutions. The patients were classified into two groups according to CRP level. The patients’ demographic and clinical characteristics and their use of immunosuppressive or biological agents were recorded. Disease location and behavior, hospitalization, and surgery were analyzed. Results A high CRP was associated with younger age, steroid use, colonic or ileocolonic location, high CD activity index, and active inflammation at colonoscopy (p<0.001). As the disease progressed, patients with high CRP were more likely to exhibit strictures (p=0.027). There were significant differences in the use of 5-aminosalicylic acid, antibiotics, corticosteroids, azathioprine, and infliximab (p<0.001, p<0.001, p<0.001, p<0.001, and p=0.023, respectively). Hospitalization was also more frequent in patients with high CRP. Conclusions The CRP level at diagnosis is useful for evaluating the phenotype, activity, and clinical course of CD. Closer follow-up strategies, with early aggressive treatment, could be considered for patients with high CRP. PMID:27021506

  3. Elevated serum levels of high mobility group box protein 1 (HMGB1) in patients with ankylosing spondylitis and its association with disease activity and quality of life.

    PubMed

    Oktayoglu, Pelin; Em, Serda; Tahtasiz, Mehmet; Bozkurt, Mehtap; Ucar, Demet; Yazmalar, Levent; Nas, Kemal; Yardımeden, Ibrahim; Cevik, Figen; Celik, Yusuf; Mete, Nuriye

    2013-05-01

    This study was carried out to determine the serum levels of high mobility group box protein 1 (HMGB1) in patients with ankylosing spondylitis (AS) and to evaluate its correlation with disease activity and quality of life. According to our knowledge, it is the first trial evaluating HMGB1 levels in AS. Serum samples of 30 patients (18 males and 12 females) with AS and 29 healthy controls (HC) (15 females and 14 males) were collected. HMGB1 levels were measured by enzyme-linked immunosorbent assay, activity of disease was assessed according to the Bath AS Disease Activity Index (BASDAI), and functional status of patients was evaluated with Bath AS Functional Index (BASFI). Modified Schober, chest expansion values and AS Quality of Life Questionnaire (ASQoL) scores were noted. The serum levels of HMGB1 were obtained significantly increased in AS patients compared to HC (p < 0.05). There was no significant correlation between HMGB1 levels and ESR (p > 0.05), and CRP (p > 0.05) values. BASDAI, BASFI and ASQoL scores were also not correlated with serum levels of HMGB1 (p > 0.05). Our results suggest that HMGB1 might play an important role in the pathogenesis of AS; however, it seems not to be a good candidate for reflecting disease activity, functional abilities and the quality of life in patients with AS; on the other hand, the increased levels of HMGB1 in patients may open a new dimension for targeting this cytokine as a new therapy option in AS.

  4. The effects of graded levels of calorie restriction: V. Impact of short term calorie and protein restriction on physical activity in the C57BL/6 mouse.

    PubMed

    Mitchell, Sharon E; Delville, Camille; Konstantopedos, Penelope; Derous, Davina; Green, Cara L; Wang, Yingchun; Han, Jing-Dong J; Promislow, Daniel E L; Douglas, Alex; Chen, Luonan; Lusseau, David; Speakman, John R

    2016-04-12

    Calorie restriction (CR) delays the onset of age-related disease and extends lifespan in a number of species. When faced with reduced energy supply animals need to lower energy demands, which may be achieved in part by reducing physical activity (PA). We monitored changes in PA using implanted transmitters in male C57BL/6 mice in response to graded levels of CR (10 to 40%) or matched levels of graded protein restriction (PR) for 3 months. Mice were fed at lights out and ad libitum controls were limited to dark-phase feeding (12AL) or 24hr/day. Total daily PA declined in a non-linear manner over the first 30 days of CR or PR, remaining stable thereafter. Total daily PA was not related to the level of CR or PR. Total daily PA over the last 20 days of restriction was related to circulating leptin, insulin, tumor necrosis factor-α (TNF- α) and insulin-like growth factor (IGF)-1 levels, measured after 3 months. Mice under restriction showed a high level of activity in the 2hrs before feeding (food anticipatory activity: FAA). FAA followed a complex pattern, peaking around day 20, falling on ~day 37 then increasing again. FAA was also positively related to the level of restriction and inversely to leptin, insulin, TNF-α and IGF-1. Non-FAA, in contrast, declined over the period of restriction, generally more so in mice under greater restriction, thereby offsetting to some extent the increase in FAA. Mice under PR displayed no changes in PA over time or in comparison to 12AL, and showed no increase in FAA.

  5. The effects of graded levels of calorie restriction: V. Impact of short term calorie and protein restriction on physical activity in the C57BL/6 mouse

    PubMed Central

    Mitchell, Sharon E.; Delville, Camille; Konstantopedos, Penelope; Derous, Davina; Green, Cara L.; Wang, Yingchun; Han, Jing-Dong J.; Promislow, Daniel E.L.; Douglas, Alex; Chen, Luonan; Lusseau, David; Speakman, John R.

    2016-01-01

    Calorie restriction (CR) delays the onset of age-related disease and extends lifespan in a number of species. When faced with reduced energy supply animals need to lower energy demands, which may be achieved in part by reducing physical activity (PA). We monitored changes in PA using implanted transmitters in male C57BL/6 mice in response to graded levels of CR (10 to 40%) or matched levels of graded protein restriction (PR) for 3 months. Mice were fed at lights out and ad libitum controls were limited to dark-phase feeding (12AL) or 24hr/day. Total daily PA declined in a non-linear manner over the first 30 days of CR or PR, remaining stable thereafter. Total daily PA was not related to the level of CR or PR. Total daily PA over the last 20 days of restriction was related to circulating leptin, insulin, tumor necrosis factor-α (TNF-α) and insulin-like growth factor (IGF)-1 levels, measured after 3 months. Mice under restriction showed a high level of activity in the 2hrs before feeding (food anticipatory activity: FAA). FAA followed a complex pattern, peaking around day 20, falling on ∼day 37 then increasing again. FAA was also positively related to the level of restriction and inversely to leptin, insulin, TNF-α and IGF-1. Non-FAA, in contrast, declined over the period of restriction, generally more so in mice under greater restriction, thereby offsetting to some extent the increase in FAA. Mice under PR displayed no changes in PA over time or in comparison to 12AL, and showed no increase in FAA. PMID:27007156

  6. Rare Earth Ion Mediated Fluorescence Accumulation on a Single Microbead: An Ultrasensitive Strategy for the Detection of Protein Kinase Activity at the Single-Cell Level.

    PubMed

    Zhang, Xiaobo; Liu, Chenghui; Wang, Honghong; Wang, Hui; Li, Zhengping

    2015-12-01

    A single microbead-based fluorescence imaging (SBFI) strategy that enables detection of protein kinase activity from single cell lysates is reported. We systematically investigated the ability of various rare earth (RE) ions, immobilized on the microbead, for specific capturing of kinase-induced phosphopeptides, and Dy(3+) was found to be the most prominent one. Through the efficient concentration of kinase-induced fluorescent phosphopeptides on a Dy(3+) -functionalized single microbead, kinase activity can be detected and quantified by reading the fluorescence on the microbead with a confocal fluorescence microscope. Owing to the extremely specific recognition of Dy(3+) towards phosphopeptides and the highly-concentrated fluorescence accumulation on only one microbead, ultrahigh sensitivity has been achieved for the SBFI strategy which allows direct kinase analysis at the single-cell level. PMID:26482714

  7. Rare Earth Ion Mediated Fluorescence Accumulation on a Single Microbead: An Ultrasensitive Strategy for the Detection of Protein Kinase Activity at the Single-Cell Level.

    PubMed

    Zhang, Xiaobo; Liu, Chenghui; Wang, Honghong; Wang, Hui; Li, Zhengping

    2015-12-01

    A single microbead-based fluorescence imaging (SBFI) strategy that enables detection of protein kinase activity from single cell lysates is reported. We systematically investigated the ability of various rare earth (RE) ions, immobilized on the microbead, for specific capturing of kinase-induced phosphopeptides, and Dy(3+) was found to be the most prominent one. Through the efficient concentration of kinase-induced fluorescent phosphopeptides on a Dy(3+) -functionalized single microbead, kinase activity can be detected and quantified by reading the fluorescence on the microbead with a confocal fluorescence microscope. Owing to the extremely specific recognition of Dy(3+) towards phosphopeptides and the highly-concentrated fluorescence accumulation on only one microbead, ultrahigh sensitivity has been achieved for the SBFI strategy which allows direct kinase analysis at the single-cell level.

  8. Cinnamyl alcohol dehydrogenases in the mesocarp of ripening fruit of Prunus persica genotypes with different flesh characteristics: changes in activity and protein and transcript levels.

    PubMed

    Gabotti, Damiano; Negrini, Noemi; Morgutti, Silvia; Nocito, Fabio F; Cocucci, Maurizio

    2015-07-01

    Development of fruit flesh texture quality traits may involve the metabolism of phenolic compounds. This study presents molecular and biochemical results on the possible role played by cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) during ripening [S3, S4 I (pre-climacteric) and S4 III (climacteric) stages] of peach [Prunus persica (L.) Batsch] fruit with different flesh firmness [non-melting flesh (NMF) 'Oro A'/melting flesh (MF) 'Springcrest' and 'Sanguinella'] and color (blood-flesh Sanguinella). A total of 24 putative full-length PRUPE_CAD genes were identified (in silico analysis) in the peach genome. The most abundant CAD isoforms, encoded by genes located on scaffolds 8 and 6, were probed by specifically developed anti-PRUPE_CAD sc8 and by anti-FaCAD (PRUPE_CAD sc6) polyclonal antibodies, respectively. PRUPE_CAD sc8 proteins (SDS-PAGE and native-PAGE/western blot) appeared responsible for the CAD activity (in vitro/in-gel assays) that increased with ripening (parallel to PRUPE_ACO1 transcripts accumulation and ethylene evolution) only in the mesocarp of Oro A and blood-flesh Sanguinella. Accumulation of PRUPE_CAD sc8 transcripts (semi-quantitative RT-PCR) occurred in all three cultivars, but in Oro A and Springcrest it was not always accompanied by that of the related proteins, suggesting possible post-transcriptional regulation. Flesh firmness, as well as levels of lignin, total phenolics and, where present (Sanguinella), anthocyanins, declined with ripening, suggesting that, at least in the studied peach cultivars, CAD activity is related to neither lignification nor differences in flesh firmness (NMF/MF). Further studies are necessary to clarify whether the high levels of CAD activity/expression in Sanguinella play a role in determining the characteristics of this blood-flesh fruit.

  9. Glucagon-like peptide 1 (GLP-1) can reverse AMP-activated protein kinase (AMPK) and S6 kinase (P70S6K) activities induced by fluctuations in glucose levels in hypothalamic areas involved in feeding behaviour.

    PubMed

    Hurtado-Carneiro, Verónica; Sanz, Carmen; Roncero, Isabel; Vazquez, Patricia; Blazquez, Enrique; Alvarez, Elvira

    2012-04-01

    The anorexigenic peptide, glucagon-like peptide-1 (GLP-1), reduces glucose metabolism in the human hypothalamus and brain stem. The brain activity of metabolic sensors such as AMP-activated protein kinase (AMPK) responds to changes in glucose levels. The mammalian target of rapamycin (mTOR) and its downstream target, p70S6 kinase (p70S6K), integrate nutrient and hormonal signals. The hypothalamic mTOR/p70S6K pathway has been implicated in the control of feeding and the regulation of energy balances. Therefore, we investigated the coordinated effects of glucose and GLP-1 on the expression and activity of AMPK and p70S6K in the areas involved in the control of feeding. The effect of GLP-1 on the expression and activities of AMPK and p70S6K was studied in hypothalamic slice explants exposed to low- and high-glucose concentrations by quantitative real-time RT-PCR and by the quantification of active-phosphorylated protein levels by immunoblot. In vivo, the effects of exendin-4 on hypothalamic AMPK and p70S6K activation were analysed in male obese Zucker and lean controls 1 h after exendin-4 injection to rats fasted for 48 h or after re-feeding for 2-4 h. High-glucose levels decreased the expression of Ampk in the lateral hypothalamus and treatment with GLP-1 reversed this effect. GLP-1 treatment inhibited the activities of AMPK and p70S6K when the activation of these protein kinases was maximum in both the ventromedial and lateral hypothalamic areas. Furthermore, in vivo s.c. administration of exendin-4 modulated AMPK and p70S6K activities in those areas, in both fasted and re-fed obese Zucker and lean control rats.

  10. Plasma levels of galectin-3-binding protein reflect type I interferon activity and are increased in patients with systemic lupus erythematosus

    PubMed Central

    Nielsen, Christoffer T; Lood, Christian; Østergaard, Ole; Iversen, Line V; Voss, Anne; Bengtsson, Anders; Jacobsen, Søren; Heegaard, Niels H H

    2014-01-01

    Objective Simple measures of type I interferon (IFN) activity constitute highly attractive biomarkers in systemic lupus erythematosus (SLE). We explore galectin-3-binding protein (G3BP) as a novel measure of type I IFN activity and serum/plasma biomarker in large independent cohorts of patients with SLE and controls. Methods Serum and plasma G3BP concentrations were quantified using ELISA. Type I IFN activity was assessed by Mx1 reporter gene expression assays and correlated to serum G3BP concentrations (SLE-IFN-α, n=26 and healthy controls (HCs), n=10). Plasma G3BP concentrations in the SLE-Denmark (DK) (n=70) and SLE-Sweden (SE) (n=68) cohorts were compared with the HC-DK (n=47) and HC-SE (n=50) cohorts and patients with systemic sclerosis (n=111). In 15 patients with SLE, serum G3BP in consecutive samples was correlated to disease activity. Correlation analysis between G3BP, clinical parameters including disease activity in the four SLE cohorts was performed. Results G3BP concentrations correlated significantly with the IFN-α reporter gene assay (r=0.56, p=0.0005) and with IFN-α gene expression scores (r=0.54, p=0.0002). Plasma concentrations were significantly increased in the SLE-DK and SLE-SE cohorts compared with HCs and patients with systemic sclerosis (p<0.0001 and p=0.0009). G3BP concentrations correlated with disease activity measures in the SLE-DK- and SLE-IFN-α cohorts (p=0.0004 and p=0.05) but not in the SLE-SE cohort (p=0.98). Markedly temporal variation was observed in G3BP levels in the consecutive SLE-samples and was significantly associated with changes in disease activity (r=0.44, p=0.014). Conclusions G3BP plasma levels reflect type I IFN activity and are increased in SLE. Associations with disease activity or clinical manifestations are uncertain. This study highlights G3BP as a convenient measure of type I IFN-dependent gene activation. PMID:25452879

  11. Effect of lowered vitamin D binding protein levels on the biological activity and metabolism of 1,25-dihydroxyvitamin D/sub 3/

    SciTech Connect

    Kost, S.B.; Londowski, J.; Audran, M.; Kumar, R.

    1986-03-01

    The authors studied the effect of lowered vitamin D binding protein levels on the biological activity and metabolism of 1,25-dihydroxyvitamin D/sub 3/ (1,25(OH)/sub 2/D/sub 3/) in vivo. Estrogen administration to vitamin D-deficient rats resulted in decrease of plasma vitamin D binding protein concentrations by about 20%. The authors administered graded doses of 1,25(OH)/sub 2/D/sub 3/ (5 - 5000 pmol intravenously) to vitamin D-deficient rats given estrogen or vehicle, and studied the biological response in intestine and bone. Intestinal calcium transport, following the administration of 1,25(OH)/sub 2/D/sub 3/, was similar in the estrogen or vehicle-treated groups. Serum calcium concentrations were lower in the estrogen-treated rats when compared to rats given vehicle. Serum calcium in both groups, however, increased the same amount over the range of 1,25(OH)/sub 2/D/sub 3/ given. The uptake of (/sup 3/H) 1,25(OH)/sub 2/D/sub 3/ by the intestine and bone 8 hours after the administration of (/sup 3/H) 1,25(OH)/sub 2/D/sub 3/ was similar in estrogen- and vehicle-treated rats. The amount of (/sup 3/H) 1,25(OH)/sub 2/D/sub 3/ in plasma of estrogen-treated rats was the same as in vehicle-treated rats. The authors conclude that in estrogen-treated rats, lowered vitamin D binding protein levels do not alter the effect of 1,25(OH)/sub 2/D/sub 3/ on intestine or bone and do not alter the metabolism of 1,25(OH)/sub 2/D/sub 3/.

  12. Prenyltransferases regulate CD20 protein levels and influence anti-CD20 monoclonal antibody-mediated activation of complement-dependent cytotoxicity.

    PubMed

    Winiarska, Magdalena; Nowis, Dominika; Bil, Jacek; Glodkowska-Mrowka, Eliza; Muchowicz, Angelika; Wanczyk, Malgorzata; Bojarczuk, Kamil; Dwojak, Michal; Firczuk, Malgorzata; Wilczek, Ewa; Wachowska, Malgorzata; Roszczenko, Katarzyna; Miaczynska, Marta; Chlebowska, Justyna; Basak, Grzegorz Wladyslaw; Golab, Jakub

    2012-09-14

    Anti-CD20 monoclonal antibodies (mAbs) are successfully used in the management of non-Hodgkin lymphomas and chronic lymphocytic leukemia. We have reported previously that statins induce conformational changes in CD20 molecules and impair rituximab-mediated complement-dependent cytotoxicity. Here we investigated in more detail the influence of farnesyltransferase inhibitors (FTIs) on CD20 expression and antitumor activity of anti-CD20 mAbs. Among all FTIs studied, L-744,832 had the most significant influence on CD20 levels. It significantly increased rituximab-mediated complement-dependent cytotoxicity against primary tumor cells isolated from patients with non-Hodgkin lymphomas or chronic lymphocytic leukemia and increased CD20 expression in the majority of primary lymphoma/leukemia cells. Incubation of Raji cells with L-744,832 led to up-regulation of CD20 at mRNA and protein levels. Chromatin immunoprecipitation assay revealed that inhibition of farnesyltransferase activity was associated with increased binding of PU.1 and Oct-2 to the CD20 promoter sequences. These studies indicate that CD20 expression can be modulated by FTIs. The combination of FTIs with anti-CD20 mAbs is a promising therapeutic approach, and its efficacy should be examined in patients with B-cell tumors.

  13. Interaction Between Peroxisome Proliferator Activated Receptor δ and Epithelial Membrane Protein 2 Polymorphisms Influences HDL-C Levels in the Chinese Population.

    PubMed

    Ke, Tingjing; Dorajoo, Rajkumar; Han, Yi; Khor, Chiea-Chuen; van Dam, Rob M; Yuan, Jian-Min; Koh, Woon-Puay; Liu, Jianjun; Teo, Yik Ying; Goh, Daniel Y T; Tai, E Shyong; Wong, Tien Yin; Cheng, Ching-Yu; Friedlander, Yechiel; Heng, Chew-Kiat

    2016-09-01

    Peroxisome proliferator activated receptors (PPARs) are transcription factors involved in the regulation of key metabolic pathways. Numerous in vivo and in vitro studies have established their important roles in lipid metabolism. A few SNPs in PPAR genes have been reported to be associated with lipid levels. In this study, we aimed to investigate the interactive effects between single nucleotide polymorphisms (SNPs) in three PPAR isoforms α/δ/γ and other genetic variants across the genome on plasma high-density lipoprotein-cholesterol (HDL-C) levels. Study subjects (N = 2003) were genotyped using Illumina HumanOmniZhongHua-8 Beadchip. Fifty-three tag SNPs ± 100 kb of PPAR α, δ, and γ (r(2) < 0.2) were selected. The effect of interactions between PPAR SNPs and those across the genome on HDL-C was tested using linear regression models. One statistically significant interaction influencing HDL-C was detected between PPARδ SNP rs2267668 and epithelial membrane protein 2 (EMP2) downstream SNP rs7191411 (N = 1993, β = 0.74, adjusted P = 0.022). This interaction was successfully replicated in the meta-analysis of two additional Chinese cohorts (N = 3948, P = 0.01). The present study showed a novel SNP × SNP interaction between rs2267668 in PPARδ and rs7191411 in EMP2 that has significant impact on circulating HDL-C levels in the Singaporean Chinese population. PMID:27530449

  14. Familiar Taste Induces Higher Dendritic Levels of Activity-Regulated Cytoskeleton-Associated Protein in the Insular Cortex than a Novel One

    ERIC Educational Resources Information Center

    Morin, Jean-Pascal; Quiroz, Cesar; Mendoza-Viveros, Lucia; Ramirez-Amaya, Victor; Bermudez-Rattoni, Federico

    2011-01-01

    The immediate early gene (IEG) "Arc" is known to play an important role in synaptic plasticity; its protein is locally translated in the dendrites where it has been involved in several types of plasticity mechanisms. Because of its tight coupling with neuronal activity, "Arc" has been widely used as a tool to tag behaviorally activated networks.…

  15. Separating proteins with activated carbon.

    PubMed

    Stone, Matthew T; Kozlov, Mikhail

    2014-07-15

    Activated carbon is applied to separate proteins based on differences in their size and effective charge. Three guidelines are suggested for the efficient separation of proteins with activated carbon. (1) Activated carbon can be used to efficiently remove smaller proteinaceous impurities from larger proteins. (2) Smaller proteinaceous impurities are most efficiently removed at a solution pH close to the impurity's isoelectric point, where they have a minimal effective charge. (3) The most efficient recovery of a small protein from activated carbon occurs at a solution pH further away from the protein's isoelectric point, where it is strongly charged. Studies measuring the binding capacities of individual polymers and proteins were used to develop these three guidelines, and they were then applied to the separation of several different protein mixtures. The ability of activated carbon to separate proteins was demonstrated to be broadly applicable with three different types of activated carbon by both static treatment and by flowing through a packed column of activated carbon. PMID:24898563

  16. Correlation between CYP1A1 transcript, protein level, enzyme activity and DNA adduct formation in normal human mammary epithelial cell strains exposed to benzo[a]pyrene

    PubMed Central

    Divi, Rao L.; Einem Lindeman, Tracey L.; Shockley, Marie E.; Keshava, Channa; Weston, Ainsley; Poirier, Miriam C.

    2014-01-01

    The polycyclic aromatic hydrocarbon (PAH) benzo(a)pyrene (BP) is thought to bind covalently to DNA, through metabolism by cytochrome P450 1A1 (CYP1A1) and CYP1B1, and other enzymes, to form r7, t8, t9-trihydroxy-c-10-(N 2-deoxyguanosyl)-7,8,9,10-tetrahydro-benzo[a]-pyrene (BPdG). Evaluation of RNA expression data, to understand the contribution of different metabolic enzymes to BPdG formation, is typically presented as fold-change observed upon BP exposure, leaving the actual number of RNA transcripts unknown. Here, we have quantified RNA copies/ng cDNA (RNA cpn) for CYP1A1 and CYP1B1, as well as NAD(P)H:quinone oxidoreductase 1 (NQO1), which may reduce formation of BPdG adducts, using primary normal human mammary epithelial cell (NHMEC) strains, and the MCF-7 breast cancer cell line. In unexposed NHMECs, basal RNA cpn values were 58–836 for CYP1A1, 336–5587 for CYP1B1 and 5943–40112 for NQO1. In cells exposed to 4.0 µM BP for 12h, RNA cpn values were 251–13234 for CYP1A1, 4133–57078 for CYP1B1 and 4456–55887 for NQO1. There were 3.5 (mean, range 0.2–15.8) BPdG adducts/108 nucleotides in the NHMECs (n = 16), and 790 in the MCF-7s. In the NHMECs, BP-induced CYP1A1 RNA cpn was highly associated with BPdG (P = 0.002), but CYP1B1 and NQO1 were not. Western blots of four NHMEC strains, chosen for different levels of BPdG adducts, showed a linear correlation between BPdG and CYP1A1, but not CYP1B1 or NQO1. Ethoxyresorufin-O-deethylase (EROD) activity, which measures CYP1A1 and CYP1B1 together, correlated with BPdG, but NQO1 activity did not. Despite more numerous levels of CYP1B1 and NQO1 RNA cpn in unexposed and BP-exposed NHMECs and MCF-7cells, BPdG formation was only correlated with induction of CYP1A1 RNA cpn. The higher level of BPdG in MCF-7 cells, compared to NHMECs, may have been due to a much increased induction of CYP1A1 and EROD. Overall, BPdG correlation was observed with CYP1A1 protein and CYP1A1/1B1 enzyme activity, but not with CYP1B1 or NQO

  17. THE INFLUENCE OF SERUM BINDING PROTEINS AND FEEDBACK CONTROL OF SERUM ESTRADIOL LEVELS ON THE COMPARATIVE POTENCY OF ENDOCRINE ACTIVE COMPOUNDS

    EPA Science Inventory

    THE INFLUENCE OF SERUM BINDING PROTEINS ON THE COMPARATIVE RECEPTOR BINDING POTENCY OF ENDOCRINE ACTIVE COMPOUNDS. JG Teeguarden1 and HA Barton2. 1ICF Consulting, Research Triangle Park NC; 2US EPA, ORD, NHEERL, ETD, Pharmacokinetics Branch, RTP, NC.

    Accurate comparison of...

  18. [Effects of cell-binding protein A of Staphylococcus aureus on the level of intracellular calcium ions and actomyosin ATP-ase activity in the smooth muscles].

    PubMed

    Melenevs'ka, N V; Miroshnychenko, M S; Filippov, I B; Artemenko, O Iu; Shuba, M F

    2006-01-01

    Immune-active substance of Staphylococcus aureus, cell-bound protein A (CBPA), enhances the acetylcholine- or hyperpotassium (K+) Krebs solution-evoked excitation in Taenia coli smooth muscles. CBPA increases caffeine- and carbachole-evoked Ca2+ signals in smooth muscle cells suspension, loaded with indo-1, and also caffeine- and acetylcholine-evoked contraction in smooth muscles slices. Against a background of CBPA-suppressed action of sodium nitroprusside, ATP evokes the membrane depolarization. CBPA in small concentrations potentiates ATPase (Mg2+,Ca2+-; Mg2+- and Mg2+- in the presence of EGTA) activity of actomyosin in the smooth muscles.

  19. 1,25-Dihydroxyvitamin D3 and its analogues increase catalase at the mRNA, protein and activity level in a canine transitional carcinoma cell line.

    PubMed

    Middleton, R P; Nelson, R; Li, Q; Blanton, A; Labuda, J A; Vitt, J; Inpanbutr, N

    2015-12-01

    Antioxidant enzymes, such as catalase, superoxide dismutases (SOD), MnSOD and Cu/ZnSOD, protect cells by scavenging reactive oxygen species (ROS). Numerous studies have reported the anti-cancer effects of 1,25-dihydroxyvitamin D3 (calcitriol) and its related analogues, seocalcitol and analogue V. In this study, canine bladder transitional cell carcinoma (cbTCC) cells were used to determine effects of calcitriol and its related analogues on antioxidant enzyme gene expression, protein expression and activity. Catalase mRNA was increased in response to calcitriol (10(-7) M), and seocalcitol (10(-7) and 10(-9) M). MnSOD mRNA was decreased in response to calcitriol at 10(-7) M. Catalase was significantly increased in response to calcitriol (10(-7) and 10(-9) M), and seocalcitol (10(-9) M). Catalase enzymatic activity increased in response to calcitriol, seocalcitol and analogue V (10(-9) M). In addition, global gene expression analysis identified the involvement of mitogen-activated protein kinase (MAPK) signalling in cbTCC's response to calcitriol and seocalcitol treatment.

  20. [Abscisic acid level, activities of proteinases and trypsin inhibitory proteins in the germinating seeds of common beans under conditions of water stress].

    PubMed

    Domash, V I; Protsko, R F; Vasiuk, V A; Shumikhin, S V; Ermolitskaia, L V; Sharpio, T P

    2006-01-01

    Specific features of changes in the contents of free and bound abscisic acid (ABA) and activities of neutral and alkaline proteinases and trypsin inhibitory proteins were determined in the embryonic axis and cotyledons of the common bean (Phaseolus vulgaris L.) after drying. The changes in ABA content, observed following the loss of 5% seed weight, were regarded as an adaptive reaction to stress, whereas the corresponding changes after the loss of 10% seed weight, as a result of pathological disturbance of ABA metabolism. Both drying modes had a negative effect on the state of the proteinase-inhibitory system, as was apparent from the disruption of the regular inverse correlation between the activities of proteinases and serine proteinase inhibitory proteins. Comparison of the dynamics of these characteristics with the buildup of water stress demonstrated an inverse correlation between the content of free ABA and the activity of the proteinases studied. This suggests a potential inhibitory effect of this hormone on the function of the hydrolases in question in the germinating seed.

  1. Relative survival of four serotypes of Salmonella enterica in low-water activity whey protein powder held at 36 and 70°C at various water activity levels.

    PubMed

    Farakos, S M Santillana; Hicks, J W; Frye, J G; Frank, J F

    2014-07-01

    Salmonella enterica is not able to grow at water activity (aw) levels below 0.94, but it can survive in low-aw foods for long periods of time. Temperature, aw, substrate, and serotype affect its persistence. The aim of this study was to evaluate the influence of temperature and aw on the relative persistence among four serotypes of Salmonella enterica in low-aw whey protein powder. Whey protein powder was equilibrated to aws 0.18 ± 0.02 and 0.54 ± 0.03, inoculated with a cocktail of Salmonella serovars (Agona, Tennessee, Montevideo, and Typhimurium), vacuum sealed, and stored at 36°C for 6 months and at 70°C for 48 h. Presumptive Salmonella colonies (30 to 32) were randomly picked from each plate at the end of each survival study. PCR multiplex serotyping was used to identify the isolates. A multinomial mixed logistic model with Salmonella Tennessee as a reference was used to test for significant differences in frequency distribution of the surviving serotypes. Salmonella Tennessee and Salmonella Agona were the most prevalent surviving serotypes, followed in decreasing order by Salmonella Montevideo and Salmonella Typhimurium. Statistical analysis indicated that temperature (P = 0.003) and aw (P = 0.012) influenced the relative prevalence of the Salmonella serotypes. If other environmental conditions are equal, Salmonella Tennessee is better able to survive than Salmonella Montevideo and Salmonella Typhimurium at higher temperatures and higher aw levels in low-aw whey protein powder held at 36 and 70°C. The relative prevalence of Salmonella Agona to Salmonella Tennessee did not change with increasing temperature (P = 0.211) or aw (P = 0.453). These results should be considered in risk assessment and when developing predictive models for survival of Salmonella in low-aw foods. PMID:24988029

  2. Phospholipases as GTPase activity accelerating proteins (GAPs) in plants.

    PubMed

    Pandey, Sona

    2016-05-01

    GTPase activity accelerating proteins (GAPs) are key regulators of the G-protein signaling cycle. By facilitating effective hydrolysis of the GTP bound on Gα proteins, GAPs control the timing and amplitude of the signaling cycle and ascertain the availability of the inactive heterotrimer for the next round of activation. Until very recently, the studies of GAPs in plants were focused exclusively on the regulator of G-protein signaling (RGS) protein. We now show that phospholipase Dα1 (PLDα1) is also a bona fide GAP in plants and together with the RGS protein controls the level of activeprotein. PMID:27124090

  3. Plasma protein carbonyl levels and breast cancer risk.

    PubMed

    Rossner, Pavel; Terry, Mary Beth; Gammon, Marilie D; Agrawal, Meenakshi; Zhang, Fang Fang; Ferris, Jennifer S; Teitelbaum, Susan L; Eng, Sybil M; Gaudet, Mia M; Neugut, Alfred I; Santella, Regina M

    2007-01-01

    To study the role of oxidative stress in breast cancer risk, we analysed plasma levels of protein carbonyls in 1050 cases and 1107 controls. We found a statistically significant trend in breast cancer risk in relation to increasing quartiles of plasma protein carbonyl levels (OR = 1.2, 95% CI = 0.9-1.5; OR = 1.5, 95% CI = 1.2-2.0; OR = 1.6, 95% CI = 1.2-2.1, for the 2(nd), 3(rd) and 4(th) quartile relative to the lowest quartile, respectively, P for trend = 0.0001). The increase in risk was similar for younger (<50 years) and older women, more pronounced among women with higher physical activity levels (0.7 hrs/week for 4(th) quartile versus lowest quartile OR = 2.0, 95% CI = 1.4-3.0), higher alcohol consumption (> or = 15 grams/day for 4(th) quartile versus lowest quartile OR = 2.3, 95% CI = 1.1-4.7), and hormone replacement therapy use (HRT, OR = 2.6, 95% CI = 1.6-4.4 for 4(th) quartile versus lowest quartile). The multiplicative interaction terms were statistically significant only for physical activity and HRT. The positive association between plasma protein carbonyl levels and breast cancer risk was also observed when the analysis was restricted to women who had not received chemotherapy or radiation therapy prior to blood collection. Among controls, oxidized protein levels significantly increased with cigarette smoking and higher fruit and vegetable consumption, and decreased with alcohol consumption >30 grams per day. Women with higher levels of plasma protein carbonyl and urinary 15F(2t)-isoprostane had an 80% increase in breast cancer risk (OR = 1.8, 95% CI = 1.2-2.6) compared to women with levels below the median for both markers of oxidative stress. In summary, our results suggest that increased plasma protein carbonyl levels may be associated with breast cancer risk.

  4. Evaluation of Flow Rate, pH, Buffering Capacity, Calcium, Total Proteins and Total Antioxidant Capacity Levels of Saliva in Caries Free and Caries Active Children: An In Vivo Study.

    PubMed

    Preethi, B P; Reshma, Dodawad; Anand, Pyati

    2010-10-01

    The purpose of this study was to evaluate the relationship between the physicochemical properties of saliva such as flow rate, pH, buffering capacity, calcium level, total protein and total antioxidant levels in caries free and caries active children. The present study included one hundred and twenty healthy children who were divided into two groups; group I and group II comprising of age groups 7-10 and 11-14 years, respectively. Both the groups were then sub-divided equally according to gender. They were further divided into caries free and caries active with 15 children in each group. Unstimulated saliva was collected by suction method and flow rates were determined. The samples were then analyzed for pH, buffering capacity, total protein, calcium and total antioxidant capacity. The data was statistically analyzed using student t test (unpaired). The results revealed that when all these parameters were compared among the caries free and caries active children, flow rate, pH, buffering capacity were slightly reduced in caries active children, but total protein and total antioxidant capacity of saliva increased significantly in caries active children and the total calcium decreased significantly in caries active children. Within the limitation of this study, we conclude that, the physicochemical properties of saliva play a major role in the development of caries. PMID:21966118

  5. Residual protein levels on reprocessed dental instruments.

    PubMed

    Smith, A; Letters, S; Lange, A; Perrett, D; McHugh, S; Bagg, J

    2005-11-01

    Reduction of the initial bioburden on instruments, prior to sterilization, is believed to reduce transmission risks of iatrogenic Creutzfeldt-Jakob disease. Endodontic files are used in the preparation of root canals and are likely to have close contact and become contaminated with neural material from branches of the maxillary and mandibular cranial nerves. This study examined methods used by 22 dental practices to clean endodontic files, and scored visible debris and residual protein levels adhering to 220 dental endodontic files that had been used, cleaned, autoclaved and were deemed ready for re-use. Visible debris was scored after examination under a dissecting light microscope. Residual protein was quantified using a fluorescent assay based on reaction of proteins with o-phthaldialdehyde/N-acetyl cysteine. There was wide variation in the methods used by practices to clean endodontic files. The cleaning process varied from a wipe with an alcohol-impregnated cloth to hand scrubbing and/or use of an ultrasonic bath. Surface debris was visually detected on 98% of files. Residual protein was detected on all the files examined (median amount: 5.4 microg; range: 0.5-63.2 microg). These results demonstrate that the cleaning of some instruments reprocessed routinely in primary care is incomplete, and such instruments cannot be excluded as a potential source of cross-infection.

  6. The effect of aluminium-stress and exogenous spermidine on chlorophyll degradation, glutathione reductase activity and the photosystem II D1 protein gene (psbA) transcript level in lichen Xanthoria parietina.

    PubMed

    Sen, Gulseren; Eryilmaz, Isil Ezgi; Ozakca, Dilek

    2014-02-01

    In this study, the effects of short-term aluminium toxicity and the application of spermidine on the lichen Xanthoria parietina were investigated at the physiological and transcriptional levels. Our results suggest that aluminium stress leads to physiological processes in a dose-dependent manner through differences in lipid peroxidation rate, chlorophyll content and glutathione reductase (EC 1.6.4.2) activity in aluminium and spermidine treated samples. The expression of the photosystem II D1 protein (psbA) gene was quantified using semi-quantitative RT-PCR. Increased glutathione reductase activity and psbA mRNA transcript levels were observed in the X. parietina thalli that were treated with spermidine before aluminium-stress. The results showed that the application of spermidine could mitigate aluminium-induced lipid peroxidation and chlorophyll degradation on lichen X. parietina thalli through an increase in psbA transcript levels and activity of glutathione reductase (GR) enzymes.

  7. Allostery in BAX protein activation.

    PubMed

    Jiang, Zhenyan; Zhang, Hansi; Böckmann, Rainer A

    2016-11-01

    BAX is a member of the proapoptotic BCL-2 family of proteins, which is involved in the regulation of the intrinsic pathway of apoptosis. In the process of apoptosis, BH3-only molecules activate cytosolic BAX. Activated BAX molecules insert into the mitochondrial outer membrane with their [Formula: see text]-helix and form oligomers that lead to membrane poration, resulting in the release of apoptogenic factors including cytochrome c. Recently, a novel interaction site for the binding of the BIM SAHB ligand to BAX was reported. BIM SAHB binding was shown to invoke the exposure of the 6A7 epitope (amino acids 13-19) and of the BH3 domain of BAX, followed by mobilization of the BAX [Formula: see text]-helix. However, the intramolecular pathway for signal transmission in BAX, from BIM SAHB binding to mobilization of the [Formula: see text]-helix largely remained elusive. For a molecular understanding of the activation of BAX, and thus the first steps in apoptosis, we performed microsecond atomistic molecular dynamics simulations both of the BAX protein and of the BAX:BIM SAHB complex in aqueous solution. In agreement with experiment, the 6A7 and BH3 domains adopt a more solvent-exposed conformation within the BAX:BIM SAHB complex. BIM SAHB binding was found to stabilize the secondary structure of the [Formula: see text]9-helix. A force distribution analysis revealed a force network of residue-residue interactions responsible for signal transmission from the BIM SAHB binding site predominantly via the [Formula: see text]4- and [Formula: see text]6-helices to the [Formula: see text]9-helix on the opposite site of the protein.

  8. Modeling Protein Folding and Applying It to a Relevant Activity

    ERIC Educational Resources Information Center

    Nelson, Allan; Goetze, Jim

    2004-01-01

    The different levels of protein structure that can be easily understood by creating a model that simulates protein folding, which can then be evaluated by applying it to a relevant activity, is presented. The materials required and the procedure for constructing a protein folding model are mentioned.

  9. Total Cellular RNA Modulates Protein Activity.

    PubMed

    Majumder, Subhabrata; DeMott, Christopher M; Reverdatto, Sergey; Burz, David S; Shekhtman, Alexander

    2016-08-16

    RNA constitutes up to 20% of a cell's dry weight, corresponding to ∼20 mg/mL. This high concentration of RNA facilitates low-affinity protein-RNA quinary interactions, which may play an important role in facilitating and regulating biological processes. In the yeast Pichia pastoris, the level of ubiquitin-RNA colocalization increases when cells are grown in the presence of dextrose and methanol instead of methanol as the sole carbon source. Total RNA isolated from cells grown in methanol increases β-galactosidase activity relative to that seen with RNA isolated from cells grown in the presence of dextrose and methanol. Because the total cellular RNA content changes with growth medium, protein-RNA quinary interactions can alter in-cell protein biochemistry and may play an important role in cell adaptation, critical to many physiological and pathological states. PMID:27456029

  10. The level of intracellular glutathione is a key regulator for the induction of stress-activated signal transduction pathways including Jun N-terminal protein kinases and p38 kinase by alkylating agents.

    PubMed Central

    Wilhelm, D; Bender, K; Knebel, A; Angel, P

    1997-01-01

    Monofunctional alkylating agents like methyl methanesulfonate (MMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) are potent inducers of cellular stress leading to chromosomal aberrations, point mutations, and cell killing. We show that these agents induce a specific cellular stress response program which includes the activation of Jun N-terminal kinases/stress-activated protein kinases (JNK/SAPKs), p38 mitogen-activated protein kinase, and the upstream kinase SEK1/MKK4 and which depends on the reaction mechanism of the alkylating agent in question. Similar to another inducer of cellular stress, UV irradiation, damage of nuclear DNA by alkylation is not involved in the MMS-induced response. However, in contrast to UV and other inducers of the JNK/SAPKs and p38 pathways, activation of growth factor and G-protein-coupled receptors does not play a role in the MMS response. We identified the intracellular glutathione (GSH) level as critical for JNK/SAPK activation by MMS: enhancing the GSH level by pretreatment of the cells with GSH or N-acetylcysteine inhibits, whereas depletion of the cellular GSH pool causes hyperinduction of JNK/SAPK activity by MMS. In light of the JNK/SAPK-dependent induction of c-jun and c-fos transcription, and the Jun/Fos-induced transcription of xenobiotic-metabolizing enzymes, these data provide a potential critical role of JNK/SAPK and p38 in the induction of a cellular defense program against cytotoxic xenobiotics such as MMS. PMID:9234735

  11. Atomic-level analysis of membrane-protein structure.

    PubMed

    Hendrickson, Wayne A

    2016-06-01

    Membrane proteins are substantially more challenging than natively soluble proteins as subjects for structural analysis. Thus, membrane proteins are greatly underrepresented in structural databases. Recently, focused consortium efforts and advances in methodology for protein production, crystallographic analysis and cryo-EM analysis have accelerated the pace of atomic-level structure determination of membrane proteins.

  12. ORMDL proteins regulate ceramide levels during sterile inflammation.

    PubMed

    Cai, Lin; Oyeniran, Clement; Biswas, Debolina D; Allegood, Jeremy; Milstien, Sheldon; Kordula, Tomasz; Maceyka, Michael; Spiegel, Sarah

    2016-08-01

    The bioactive sphingolipid metabolite, ceramide, regulates physiological processes important for inflammation and elevated levels of ceramide have been implicated in IL-1-mediated events. Although much has been learned about ceramide generation by activation of sphingomyelinases in response to IL-1, the contribution of the de novo pathway is not completely understood. Because yeast ORM1 and ORM2 proteins negatively regulate ceramide levels through inhibition of serine palmitoyltransferase, the first committed step in ceramide biosynthesis, we examined the functions of individual mammalian ORM orthologs, ORM (yeast)-like (ORMDL)1-3, in regulation of ceramide levels. In HepG2 liver cells, downregulation of ORMDL3 markedly increased the ceramide precursors, dihydrosphingosine and dihydroceramide, primarily from de novo biosynthesis based on [U-(13)C]palmitate incorporation into base-labeled and dual-labeled dihydroceramides, whereas downregulation of each isoform increased dihydroceramides [(13)C]labeled in only the amide-linked fatty acid. IL-1 and the IL-6 family cytokine, oncostatin M, increased dihydroceramide and ceramide levels in HepG2 cells and concomitantly decreased ORMDL proteins. Moreover, during irritant-induced sterile inflammation in mice leading to induction of the acute-phase response, which is dependent on IL-1, expression of ORMDL proteins in the liver was strongly downregulated and accompanied by increased ceramide levels in the liver and accumulation in the blood. Together, our results suggest that ORMDLs may be involved in regulation of ceramides during IL-1-mediated sterile inflammation. PMID:27313060

  13. ACTIVITY LEVEL AND LEARNING EFFECTIVENESS.

    ERIC Educational Resources Information Center

    SJOGREN, DOUGLAS D.; STAKE, ROBERT E.

    A STUDY OF LEARNING ACTIVITY EXPLORED (1) AN ACTIVITY-ACHIEVEMENT SCALE TO DESCRIBE THE IMPACT OF ACTIVITY ON ACHIEVEMENT AND (2) THE POSSIBLE COMPLEXITY OR DIMENSIONALITY OF THIS IMPACT. TEN GROUPS, OF 10 COLLEGE UNDERGRADUATE STUDENTS EACH, WERE SCHEDULED TO STUDY UNDER EACH OF 10 LEARNING SITUATIONS. THE SITUATIONS CONSISTED OF TWO MODES OF…

  14. Bone morphogenetic protein (BMP) signaling regulates mitotic checkpoint protein levels in human breast cancer cells.

    PubMed

    Yan, Hualong; Zhu, Songcheng; Song, Chenlin; Liu, Naifa; Kang, Jiuhong

    2012-04-01

    Aberrant expression of mitotic checkpoint genes compromises mitotic checkpoint, leads to chromosome instability and tumorigenesis. However, the cell signals that control mitotic checkpoint gene expression have not been reported so far. In the present study we show that, in human breast cancer cells, chemical inhibition of Bone morphogenetic proteins (BMPs), but not Transforming Growth Factor-β (TGF-β), abrogates the mitotic arrest induced by nocodazole. Protein expression analysis reveals that inhibition of BMP signaling dramatically down regulates protein levels of mitotic checkpoint components BUB3, Hec1, TTK and MAD2, but inhibition of TGF-β has relatively minor effect on the expression of these proteins. Activation of BMP signaling specifically up regulates BUB3, and activation of Activin A signaling globally down regulates these proteins level. Furthermore, overexpressing MAD2, TTK, BUB3 or Hec1 significantly rescues the mitotic arrest defect caused by BMP inhibition. Our results demonstrated for the first time that TGF-β family cytokines are cellular signals regulating mitotic checkpoint and perturbations in intrinsic BMP signaling could lead to suppression of mitotic checkpoint signaling by downregulating key checkpoint proteins. The results suggest a possible mechanism by which dysregulation of TGF-β signaling causes mitotic checkpoint defects and drives tumorigenesis. The finding also provides a potential and more specific strategy for cancer prevention by targeting BMP and mitotic checkpoint connection. PMID:22234345

  15. Effect of polysaccharides extract of rhizoma atractylodis macrocephalae on thymus, spleen and cardiac indexes, caspase-3 activity ratio, Smac/DIABLO and HtrA2/Omi protein and mRNA expression levels in aged rats.

    PubMed

    Guo, Ling; Sun, Yong Le; Wang, Ai Hong; Xu, Chong En; Zhang, Meng Yuan

    2012-10-01

    This study was designed to determine the possible protective effect of polysaccharides extract of rhizoma atractylodis macrocephalae on heart function in aged rats. Polysaccharides extract of rhizoma atractylodis macrocephalae was administered to aged rats. Results showed that thymus, spleen and cardiac indexs were significantly increased, whereas caspase-3 activity ratio, Smac/DIABLO and HtrA2/Omi protein expression, Smac/DIABLO and HtrA2/Omi mRNA expression levels were markedly reduced. It can be concluded that polysaccharides extract of rhizoma atractylodis macrocephalae may enhance immunity and improve heart function in aged rats.

  16. Inverse association between gluthathione peroxidase activity and both selenium-binding protein 1 levels and gleason score in human prostate tissue

    Technology Transfer Automated Retrieval System (TEKTRAN)

    BACKGROUND. Data from human epidemiological studies, cultured mammalian cells, and animal models have supported a potentially beneficial role of selenium (Se) in prostate cancer prevention. In addition, Se-containing proteins including members of the gutathione peroxidase (GPx) family and Selenium-B...

  17. Protein-water dynamics in antifreeze protein III activity

    NASA Astrophysics Data System (ADS)

    Xu, Yao; Bäumer, Alexander; Meister, Konrad; Bischak, Connor G.; DeVries, Arthur L.; Leitner, David M.; Havenith, Martina

    2016-03-01

    We combine Terahertz absorption spectroscopy (THz) and molecular dynamics (MD) simulations to investigate the underlying molecular mechanism for the antifreeze activity of one class of antifreeze protein, antifreeze protein type III (AFP-III) with a focus on the collective water hydrogen bond dynamics near the protein. After summarizing our previous work on AFPs, we present a new investigation of the effects of cosolutes on protein antifreeze activity by adding sodium citrate to the protein solution of AFP-III. Our results reveal that for AFP-III, unlike some other AFPs, the addition of the osmolyte sodium citrate does not affect the hydrogen bond dynamics at the protein surface significantly, as indicated by concentration dependent THz measurements. The present data, in combination with our previous THz measurements and molecular simulations, confirm that while long-range solvent perturbation is a necessary condition for the antifreeze activity of AFP-III, the local binding affinity determines the size of the hysteresis.

  18. Yeast prions: Paramutation at the protein level?

    PubMed

    Tuite, Mick F

    2015-08-01

    Prions are proteins that have the potential to refold into a novel conformation that templates the conversion of like molecules to the altered infectious form. In the yeast Saccharomyces cerevisiae, trans-generational epigenetic inheritance can be mediated by a number of structurally and functionally diverse prions. Prionogenesis can confer both loss-of-function and gain-of-function properties to the prion protein and this in turn can have a major impact on host phenotype, short-term adaptation and evolution of new traits. Prionogenesis shares a number of properties in common with paramutation and can be considered as a mitotically and meiotically heritable change in protein conformation induced by trans-interactions between homologous proteins. PMID:26386407

  19. Endogenous protein phosphorylation and protein kinase activity in winged bean.

    PubMed

    Mukhopadhyay, K; Singh, M

    1997-10-01

    In winged bean (Psophocarpus tetragonolobus) protein kinases (E.C. 2.7.1.37) were found in all tissues studied. There was a significant increase in kinase activity during seed development, with a concomitant enhancement in the phosphorylation of a number of polypeptides; this was reversed in germinating seed cotyledons. Protein phosphorylation was apparently correlated with the increase in the protein content of the developing seed and the growing axis. At least three distinct autophosphorylating proteins could be distinguished in the developing seeds after SDS-PAGE, indicating the presence of different types of protein kinases in winged bean.

  20. The peroxisome proliferator-activated receptor α agonist, AZD4619, induces alanine aminotransferase-1 gene and protein expression in human, but not in rat hepatocytes: Correlation with serum ALT levels.

    PubMed

    Thulin, Petra; Bamberg, Krister; Buler, Marcin; Dahl, Björn; Glinghammar, Björn

    2016-09-01

    Alanine aminotransferase (ALT) in serum is the standard biomarker for liver injury. We have previously described a clinical trial with a novel selective peroxisome proliferator-activated receptor α (PPARα) agonist (AZD4619), which unexpectedly caused increased serum levels of ALT in treated individuals without any other evidence of liver injury. We pinpointed a plausible mechanism through which AZD4619 could increase serum ALT levels; namely through the PPARα-specific activation of the human ALT1 gene at the transcriptional level. In the present study, we present data from the preceding rat toxicity study, demonstrating that AZD4619 had no effect on rat serum ALT activity levels, and further experiments were performed to elucidate the mechanisms responsible for this species-related difference. Our results revealed that AZD4619 increased ALT1 protein expression in a dose-dependent manner in human, but not in rat primary hepatocytes. Cloning of the human and rat ALT1 promoters into luciferase vectors confirmed that AZD4619 induced only the human, but not the rat ALT1 gene promoter in a dose-dependent manner. In PPARα-GAL4 reporter gene assays, AZD4619 was >100-fold more potent on the human vs. rat PPARα levels, explaining the differences in induction of the ALT1 gene between the species at the concentration range tested. These data demonstrate the usefulness of the human and rat ALT1 reporter gene assays for testing future drug candidates at the preclinical stage. In drug discovery projects, these assays elucidate whether elevations in ALT levels observed in vivo or in the clinic are due to metabolic effects rather than a toxic event in the liver. PMID:27430334

  1. Birth Order and Activity Level in Children.

    ERIC Educational Resources Information Center

    Eaton, Warren O.; And Others

    1989-01-01

    Studied 7,018 children between birth and 7 years and 81 children of 5-8 years to test the hypothesis that birth order is negatively related to motor activity level. Activity level declined linearly across birth position, so that early-borns were rated as more active than later-borns. (RJC)

  2. Heat dissipation guides activation in signaling proteins

    PubMed Central

    Weber, Jeffrey K.; Shukla, Diwakar; Pande, Vijay S.

    2015-01-01

    Life is fundamentally a nonequilibrium phenomenon. At the expense of dissipated energy, living things perform irreversible processes that allow them to propagate and reproduce. Within cells, evolution has designed nanoscale machines to do meaningful work with energy harnessed from a continuous flux of heat and particles. As dictated by the Second Law of Thermodynamics and its fluctuation theorem corollaries, irreversibility in nonequilibrium processes can be quantified in terms of how much entropy such dynamics produce. In this work, we seek to address a fundamental question linking biology and nonequilibrium physics: can the evolved dissipative pathways that facilitate biomolecular function be identified by their extent of entropy production in general relaxation processes? We here synthesize massive molecular dynamics simulations, Markov state models (MSMs), and nonequilibrium statistical mechanical theory to probe dissipation in two key classes of signaling proteins: kinases and G-protein–coupled receptors (GPCRs). Applying machinery from large deviation theory, we use MSMs constructed from protein simulations to generate dynamics conforming to positive levels of entropy production. We note the emergence of an array of peaks in the dynamical response (transient analogs of phase transitions) that draw the proteins between distinct levels of dissipation, and we see that the binding of ATP and agonist molecules modifies the observed dissipative landscapes. Overall, we find that dissipation is tightly coupled to activation in these signaling systems: dominant entropy-producing trajectories become localized near important barriers along known biological activation pathways. We go on to classify an array of equilibrium and nonequilibrium molecular switches that harmonize to promote functional dynamics. PMID:26240354

  3. 4-Anilino-6-phenyl-quinoline inhibitors of mitogen activated protein kinase-activated protein kinase 2 (MK2).

    PubMed

    Olsson, Henric; Sjö, Peter; Ersoy, Oguz; Kristoffersson, Anna; Larsson, Joakim; Nordén, Bo

    2010-08-15

    A class of inhibitors of mitogen activated protein kinase-activated kinase 2 (MK2) was discovered via high-throughput screening. This compound class demonstrates activity against the enzyme with sub-microM IC(50) values, and suppresses LPS-induced TNFalpha levels in THP-1 cells. MK2 inhibition kinetic measurements indicated mixed binding approaching non-ATP competitive inhibition.

  4. Adenosine A1 receptor protein levels and activity is increased in the cerebral cortex in Creutzfeldt-Jakob disease and in bovine spongiform encephalopathy-infected bovine-PrP mice.

    PubMed

    Rodríguez, Agustín; Martín, Mairena; Albasanz, José Luís; Barrachina, Marta; Espinosa, Juan Carlos; Torres, Juan María; Ferrer, Isidro

    2006-10-01

    Prion diseases are characterized by neuronal loss, astrocytic gliosis, spongiform change, and abnormal protease-resistant prion protein (PrP) deposition. Creutzfeldt-Jakob disease (CJD) is the most prevalent human prion disease, whereas scrapie and bovine spongiform encephalopathy (BSE) are the most common animal prion diseases. Several candidates have been proposed as mediators of degeneration in prion diseases, one of them glutamate. Recent studies have shown reduced metabotropic glutamate receptor/phospholipase C signaling in the cerebral cortex in CJD, suggesting that this important neuromodulator and neuroprotector pathway is attenuated in CJD. Adenosine is involved in the regulation of different metabolic processes under physiological and pathologic conditions. Adenosine function is mediated by adenosine receptors, which are categorized into 4 types: A1, A2A, A2B, and A3. A1Rs are G-protein-coupled receptors that induce the inhibition of adenylyl cyclase activity. The most dramatic inhibitory actions of adenosine receptors are on the glutamatergic system. For these reasons, we examined the levels of A1Rs in the frontal cortex of 12 patients with CJD and 6 age-matched controls and in BSE-infected bovine-PrP transgenic mice (BoPrP-Tg110 mice) at different postincubation times to address modifications in A1Rs with disease progression. A significant increase in the protein levels of A1Rs was found in the cerebral cortex in CJD and in the murine BSE model at advanced stages of the disease and coincidental with the appearance of PrP expression. In addition, the activity of A1Rs was analyzed by in vitro assays with isolated membranes of the frontal cortex in CJD. Increased activity of the receptor, as revealed by the decreased forskolin-stimulated cAMP production in response to the A1R agonists cyclohexyl adenosine and cyclopentyl adenosine, was observed in CJD cases when compared with controls. Finally, mRNA A1R levels were similar in CJD and control cases, thus

  5. Dietary protein source and level alters growth in neon tetras.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nutritional studies for aquarium fish like the neon tetra are sparse in comparison with those for food fish. To determine the optimum dietary protein level and source for growth of neon tetras, diets were formulated to contain 25, 35, 45 and 55% dietary protein from either marine animal protein or ...

  6. Development of Potent Adenosine Monophosphate Activated Protein Kinase (AMPK) Activators.

    PubMed

    Dokla, Eman M E; Fang, Chun-Sheng; Lai, Po-Ting; Kulp, Samuel K; Serya, Rabah A T; Ismail, Nasser S M; Abouzid, Khaled A M; Chen, Ching-Shih

    2015-11-01

    Previously, we reported the identification of a thiazolidinedione-based adenosine monophosphate activated protein kinase (AMPK) activator, compound 1 (N-[4-({3-[(1-methylcyclohexyl)methyl]-2,4-dioxothiazolidin-5-ylidene}methyl)phenyl]-4-nitro-3-(trifluoromethyl)benzenesulfonamide), which provided a proof of concept to delineate the intricate role of AMPK in regulating oncogenic signaling pathways associated with cell proliferation and epithelial-mesenchymal transition (EMT) in cancer cells. In this study, we used 1 as a scaffold to conduct lead optimization, which generated a series of derivatives. Analysis of the antiproliferative and AMPK-activating activities of individual derivatives revealed a distinct structure-activity relationship and identified 59 (N-(3-nitrophenyl)-N'-{4-[(3-{[3,5-bis(trifluoromethyl)phenyl]methyl}-2,4-dioxothiazolidin-5-ylidene)methyl]phenyl}urea) as the optimal agent. Relative to 1, compound 59 exhibits multifold higher potency in upregulating AMPK phosphorylation in various cell lines irrespective of their liver kinase B1 (LKB1) functional status, accompanied by parallel changes in the phosphorylation/expression levels of p70S6K, Akt, Foxo3a, and EMT-associated markers. Consistent with its predicted activity against tumors with activated Akt status, orally administered 59 was efficacious in suppressing the growth of phosphatase and tensin homologue (PTEN)-null PC-3 xenograft tumors in nude mice. Together, these findings suggest that 59 has clinical value in therapeutic strategies for PTEN-negative cancer and warrants continued investigation in this regard.

  7. Small Molecule Control of Intracellular Protein Levels Through Modulation of the Ubiquitin Proteasome System

    PubMed Central

    Buckley, Dennis L.

    2015-01-01

    Traditionally, biological probes and drugs have targeted the activities of proteins (such as enzymes and receptors) that can be easily controlled by small molecules. The remaining majority of the proteome has been deemed “undruggable”. By using small molecule modulators of the ubiquitin proteasome, protein levels, rather than protein activities can be targeted instead, increasing the number of druggable targets. While targeting the proteasome itself can lead to a global increase in protein levels, targeting other components of the UPS (e.g., the hundreds of E3 ubiquitin ligases) can lead to an increase in protein levels in a more targeted fashion. Alternatively, multiple strategies for inducing protein degradation with small molecule probes are emerging. With the ability to induce and inhibit the degradation of targeted proteins, small molecule modulators of the UPS have the potential to significantly expand the druggable portion of the proteome beyond traditional targets such as enzymes and receptors. PMID:24459094

  8. Identification of intracellular receptor proteins for activated protein kinase C.

    PubMed Central

    Mochly-Rosen, D; Khaner, H; Lopez, J

    1991-01-01

    Protein kinase C (PKC) translocates from the cytosol to the particulate fraction on activation. This activation-induced translocation of PKC is thought to reflect PKC binding to the membrane lipids. However, immunological and biochemical data suggest that PKC may bind to proteins in the cytoskeletal elements in the particulate fraction and in the nuclei. Here we describe evidence for the presence of intracellular receptor proteins that bind activated PKC. Several proteins from the detergent-insoluble material of the particulate fraction bound PKC in the presence of phosphatidylserine and calcium; binding was further increased with the addition of diacylglycerol. Binding of PKC to two of these proteins was concentration-dependent, saturable, and specific, suggesting that these binding proteins are receptors for activated C-kinase, termed here "RACKs." PKC binds to RACKs via a site on PKC distinct from the substrate binding site. We suggest that binding to RACKs may play a role in activation-induced translocation of PKC. Images PMID:1850844

  9. Activity-Based Protein Profiling of Microbes

    SciTech Connect

    Sadler, Natalie C.; Wright, Aaron T.

    2015-02-01

    Activity-Based Protein Profiling (ABPP) in conjunction with multimodal characterization techniques has yielded impactful findings in microbiology, particularly in pathogen, bioenergy, drug discovery, and environmental research. Using small molecule chemical probes that react irreversibly with specific proteins or protein families in complex systems has provided insights in enzyme functions in central metabolic pathways, drug-protein interactions, and regulatory protein redox, for systems ranging from photoautotrophic cyanobacteria to mycobacteria, and combining live cell or cell extract ABPP with proteomics, molecular biology, modeling, and other techniques has greatly expanded our understanding of these systems. New opportunities for application of ABPP to microbial systems include: enhancing protein annotation, characterizing protein activities in myriad environments, and reveal signal transduction and regulatory mechanisms in microbial systems.

  10. Factors Influencing Cypriot Children's Physical Activity Levels

    ERIC Educational Resources Information Center

    Loucaides, Constantinos A.; Chedzoy, Sue M.

    2005-01-01

    The purpose of this paper is to present selected findings from a larger study, which set out to examine the physical activity levels of Cypriot primary school children and determinants of their activity. Twenty parents of children who obtained high and low activity scores based on pedometer counts and self-reports scores were interviewed. By…

  11. C-reactive protein levels in hereditary angioedema

    PubMed Central

    Hofman, Z L M; Relan, A; Hack, C E

    2014-01-01

    Hereditary angioedema (HAE) patients experience recurrent episodes of angioedema attacks that can be painful, disfiguring and even life-threatening. The disorder results from a mutation in the gene that controls the synthesis of C1-inhibitor (C1INH). C1INH is a major regulator of activation of the contact system. It is often assumed that attacks results from uncontrolled local activation of the contact system with subsequent formation of bradykinin. To evaluate the involvement of inflammatory reactions in HAE, we analysed C-reactive protein (CRP) levels. HAE patients included in a clinical database of recombinant human C1-inhibitor (rhC1INH) studies were evaluated. For the current study we analysed CRP levels when patients were asymptomatic, during a clinical attack and in a follow-up period, and correlated these with the clinical manifestations of the attack. Data from 68 HAE patients were analysed and included CRP levels on 273 occasions. While asymptomatic, 20% of the patients analysed had increased CRP. At the onset of the attack (P = 0·049) and during the next 24 h CRP rose significantly (P = 0·002) in patients with an abdominal location, and post-attack levels were significantly higher in these patients than in patients with attacks at other locations (P = 0·034). In conclusion, CRP levels are elevated in a substantial proportion of asymptomatic HAE patients. Levels of CRP increase significantly during an abdominal attack. These data suggest low-grade systemic inflammatory reactions in HAE patients as well as a triggering event for attacks that starts prior to symptom onset. PMID:24588117

  12. C-reactive protein levels in hereditary angioedema.

    PubMed

    Hofman, Z L M; Relan, A; Hack, C E

    2014-07-01

    Hereditary angioedema (HAE) patients experience recurrent episodes of angioedema attacks that can be painful, disfiguring and even life-threatening. The disorder results from a mutation in the gene that controls the synthesis of C1-inhibitor (C1INH). C1INH is a major regulator of activation of the contact system. It is often assumed that attacks results from uncontrolled local activation of the contact system with subsequent formation of bradykinin. To evaluate the involvement of inflammatory reactions in HAE, we analysed C-reactive protein (CRP) levels. HAE patients included in a clinical database of recombinant human C1-inhibitor (rhC1INH) studies were evaluated. For the current study we analysed CRP levels when patients were asymptomatic, during a clinical attack and in a follow-up period, and correlated these with the clinical manifestations of the attack. Data from 68 HAE patients were analysed and included CRP levels on 273 occasions. While asymptomatic, 20% of the patients analysed had increased CRP. At the onset of the attack (P = 0·049) and during the next 24 h CRP rose significantly (P = 0·002) in patients with an abdominal location, and post-attack levels were significantly higher in these patients than in patients with attacks at other locations (P = 0·034). In conclusion, CRP levels are elevated in a substantial proportion of asymptomatic HAE patients. Levels of CRP increase significantly during an abdominal attack. These data suggest low-grade systemic inflammatory reactions in HAE patients as well as a triggering event for attacks that starts prior to symptom onset.

  13. Hydrogen peroxide activates activator protein-1 and mitogen-activated protein kinases in pancreatic stellate cells.

    PubMed

    Kikuta, Kazuhiro; Masamune, Atsushi; Satoh, Masahiro; Suzuki, Noriaki; Satoh, Kennichi; Shimosegawa, Tooru

    2006-10-01

    Activated pancreatic stellate cells (PSCs) are implicated in the pathogenesis of pancreatic inflammation and fibrosis, where oxidative stress is thought to play a key role. Reactive oxygen species such as hydrogen peroxide (H(2)O(2)) may act as a second messenger to mediate the actions of growth factors and cytokines. But the role of reactive oxygen species in the activation and regulation of cell functions in PSCs remains largely unknown. We here examined the effects of H(2)O(2) on the activation of signal transduction pathways and cell functions in PSCs. PSCs were isolated from the pancreas of male Wistar rats, and used in their culture-activated, myofibroblast-like phenotype unless otherwise stated. Activation of transcription factors was examined by electrophoretic mobility shift assay and luciferase assay. Activation of mitogen-activated protein (MAP) kinases was assessed by Western blotting using anti-phosphospecific antibodies. The effects of H(2)O(2) on proliferation, alpha(1)(I)procollagen gene expression, and monocyte chemoattractant protein-1 production were evaluated. The effect of H(2)O(2) on the transformation of freshly isolated PSCs in culture was also assessed. H(2)O(2) at non-cytotoxic concentrations (up to 100 microM) induced oxidative stress in PSCs. H(2)O(2) activated activator protein-1, but not nuclear factor kappaB. In addition, H(2)O(2) activated three classes of MAP kinases: extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 MAP kinase. H(2)O(2) induced alpha(1)(I)procollagen gene expression but did not induce proliferation or monocyte chemoattractant protein-1 production. H(2)O(2) did not initiate the transformation of freshly isolated PSCs to myofibroblast-like phenotype. Specific activation of these signal transduction pathways and collagen gene expression by H(2)O(2) may play a role in the pathogenesis of pancreatic fibrosis.

  14. Computational Introduction of Catalytic Activity into Proteins.

    PubMed

    Bertolani, Steve J; Carlin, Dylan Alexander; Siegel, Justin B

    2016-01-01

    Recently, there have been several successful cases of introducing catalytic activity into proteins. One method that has been used successfully to achieve this is the theozyme placement and enzyme design algorithms implemented in Rosetta Molecular Modeling Suite. Here, we illustrate how to use this software to recapitulate the placement of catalytic residues and ligand into a protein using a theozyme, protein scaffold, and catalytic constraints as input. PMID:27094294

  15. Counteracting Protein Kinase Activity in the Heart: The Multiple Roles of Protein Phosphatases

    PubMed Central

    Weber, Silvio; Meyer-Roxlau, Stefanie; Wagner, Michael; Dobrev, Dobromir; El-Armouche, Ali

    2015-01-01

    Decades of cardiovascular research have shown that variable and flexible levels of protein phosphorylation are necessary to maintain cardiac function. A delicate balance between phosphorylated and dephosphorylated states of proteins is guaranteed by a complex interplay of protein kinases (PKs) and phosphatases. Serine/threonine phosphatases, in particular members of the protein phosphatase (PP) family govern dephosphorylation of the majority of these cardiac proteins. Recent findings have however shown that PPs do not only dephosphorylate previously phosphorylated proteins as a passive control mechanism but are capable to actively control PK activity via different direct and indirect signaling pathways. These control mechanisms can take place on (epi-)genetic, (post-)transcriptional, and (post-)translational levels. In addition PPs themselves are targets of a plethora of proteinaceous interaction partner regulating their endogenous activity, thus adding another level of complexity and feedback control toward this system. Finally, novel approaches are underway to achieve spatiotemporal pharmacologic control of PPs which in turn can be used to fine-tune misleaded PK activity in heart disease. Taken together, this review comprehensively summarizes the major aspects of PP-mediated PK regulation and discusses the subsequent consequences of deregulated PP activity for cardiovascular diseases in depth. PMID:26617522

  16. Probing Protein Channel Dynamics At The Single Molecule Level.

    NASA Astrophysics Data System (ADS)

    Lee, M. Ann; Dunn, Robert C.

    1997-03-01

    It would be difficult to overstate the importance played by protein ion channels in cellular function. These macromolecular pores allow the passage of ions across the cellular membrane and play indispensable roles in all aspects of neurophysiology. While the patch-clamp technique continues to provide elegant descriptions of the kinetic processes involved in ion channel gating, the associated conformational changes remain a mystery. We are using the spectroscopic capabilities and single molecule fluorescence sensitivity of near-field scanning optical microscopy (NSOM) to probe these dynamics at the single channel level. Using a newly developed cantilevered NSOM probe capable of probing soft biological samples with single molecule fluorescence sensitivity, we have begun mapping the location of single NMDA receptors in intact rat cortical neurons with <100 nm spatial resolution. We will also present recent results exploring the conformational changes accompanying activation of nuclear pore channels located in the nuclear membrane of Xenopus oocytes. Our recent NSOM and AFM measurements on single nuclear pore complexes reveal large conformational changes taking place upon activation, providing rich, new molecular level details of channel function.

  17. DNA-based control of protein activity

    PubMed Central

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

    2016-01-01

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

  18. Increased flexibility decreases antifreeze protein activity

    PubMed Central

    Patel, Shruti N; Graether, Steffen P

    2010-01-01

    Antifreeze proteins protect several cold-blooded organisms from subzero environments by preventing death from freezing. The Type I antifreeze protein (AFP) isoform from Pseudopleuronectes americanus, named HPLC6, is a 37-residue protein that is a single α-helix. Mutational analysis of the protein showed that its alanine-rich face is important for binding to and inhibiting the growth of macromolecular ice. Almost all structural studies of HPLC6 involve the use of chemically synthesized protein as it requires a native N-terminal aspartate and an amidated C-terminus for full activity. Here, we examine the role of C-terminal amide and C-terminal arginine side chain in the activity, structure, and dynamics of nonamidated Arg37 HPLC6, nonamidated HPLC6 Ala37, amidated HPLC6 Ala37, and fully native HPLC6 using a recombinant bacterial system. The thermal hysteresis (TH) activities of the nonamidated mutants are 35% lower compared with amidated proteins, but analysis of the NMR data and circular dichroism spectra shows that they are all still α-helical. Relaxation data from the two nonamidated mutants indicate that the C-terminal residues are considerably more flexible than the rest of the protein because of the loss of the amide group, whereas the amidated Ala37 mutant has a C-terminus that is as rigid as the wild-type protein and has high TH activity. We propose that an increase in flexibility of the AFP causes it to lose activity because its dynamic nature prevents it from binding strongly to the ice surface. PMID:20936690

  19. Dietary protein level and performance of growing Baladi kids.

    PubMed

    Abdelrahman, M M; Aljumaah, R S

    2014-01-01

    A study was conducted to evaluate the effect of feeding different levels of protein to black Baladi breed kids. Weanling Baladi kids (n=18; 75 to 90 days old) were selected and individually housed at our experimental farm. Kids were divided randomly to one of the three treatments for 12 weeks. The three dietary treatments were: T1: control ration, formulated according to NRC to cover the protein (level 1) and other nutrients requirements. T2: ration formulated to cover only 75% of protein (level 2) recommended by NRC. T3: control diet + 2.4 g undegradable methionine (Smartamine®)/day/kid (level 3). Feed intake, initial and monthly body weights were recorded. Blood samples were collected monthly and analyzed for metabolites and Co, Zn and Cu levels. Decreasing the dietary level of protein (T2) negatively affected (P<0.05) the total live weight gain, average daily gain and feed conversion ratio when compared with the control and T3 groups. Moreover, treatment, time and time × treatment caused a significant change on Co concentration in blood serum with higher value at the end of the experiment. Treatments had a significant effect (P<0.05) on blood serum cholesterol and protein levels. Undegradable methionine supplementation (T3) significantly increased longissimus dorsi weight, fat thickness and omental fat%. In conclusion, feeding Baladi kids below the NRC requirements of protein negatively affect the growth performance and feed efficiency. The recommended protein level by NRC for growing kids cover the requirements of growing black Baladi kids for maximum growth and productivity.

  20. Dietary protein level and performance of growing Baladi kids

    PubMed Central

    Abdelrahman, M. M.; Aljumaah, R. S.

    2014-01-01

    A study was conducted to evaluate the effect of feeding different levels of protein to black Baladi breed kids. Weanling Baladi kids (n=18; 75 to 90 days old) were selected and individually housed at our experimental farm. Kids were divided randomly to one of the three treatments for 12 weeks. The three dietary treatments were: T1: control ration, formulated according to NRC to cover the protein (level 1) and other nutrients requirements. T2: ration formulated to cover only 75% of protein (level 2) recommended by NRC. T3: control diet + 2.4 g undegradable methionine (Smartamine®)/day/kid (level 3). Feed intake, initial and monthly body weights were recorded. Blood samples were collected monthly and analyzed for metabolites and Co, Zn and Cu levels. Decreasing the dietary level of protein (T2) negatively affected (P<0.05) the total live weight gain, average daily gain and feed conversion ratio when compared with the control and T3 groups. Moreover, treatment, time and time × treatment caused a significant change on Co concentration in blood serum with higher value at the end of the experiment. Treatments had a significant effect (P<0.05) on blood serum cholesterol and protein levels. Undegradable methionine supplementation (T3) significantly increased longissimus dorsi weight, fat thickness and omental fat%. In conclusion, feeding Baladi kids below the NRC requirements of protein negatively affect the growth performance and feed efficiency. The recommended protein level by NRC for growing kids cover the requirements of growing black Baladi kids for maximum growth and productivity. PMID:27175130

  1. Real-time quantification of protein expression at the single-cell level via dynamic protein synthesis translocation reporters.

    PubMed

    Aymoz, Delphine; Wosika, Victoria; Durandau, Eric; Pelet, Serge

    2016-01-01

    Protein expression is a dynamic process, which can be rapidly induced by extracellular signals. It is widely appreciated that single cells can display large variations in the level of gene induction. However, the variability in the dynamics of this process in individual cells is difficult to quantify using standard fluorescent protein (FP) expression assays, due to the slow maturation of their fluorophore. Here we have developed expression reporters that accurately measure both the levels and dynamics of protein synthesis in live single cells with a temporal resolution under a minute. Our system relies on the quantification of the translocation of a constitutively expressed FP into the nucleus. As a proof of concept, we used these reporters to measure the transient protein synthesis arising from two promoters responding to the yeast hyper osmolarity glycerol mitogen-activated protein kinase pathway (pSTL1 and pGPD1). They display distinct expression dynamics giving rise to strikingly different instantaneous expression noise. PMID:27098003

  2. Real-time quantification of protein expression at the single-cell level via dynamic protein synthesis translocation reporters

    PubMed Central

    Aymoz, Delphine; Wosika, Victoria; Durandau, Eric; Pelet, Serge

    2016-01-01

    Protein expression is a dynamic process, which can be rapidly induced by extracellular signals. It is widely appreciated that single cells can display large variations in the level of gene induction. However, the variability in the dynamics of this process in individual cells is difficult to quantify using standard fluorescent protein (FP) expression assays, due to the slow maturation of their fluorophore. Here we have developed expression reporters that accurately measure both the levels and dynamics of protein synthesis in live single cells with a temporal resolution under a minute. Our system relies on the quantification of the translocation of a constitutively expressed FP into the nucleus. As a proof of concept, we used these reporters to measure the transient protein synthesis arising from two promoters responding to the yeast hyper osmolarity glycerol mitogen-activated protein kinase pathway (pSTL1 and pGPD1). They display distinct expression dynamics giving rise to strikingly different instantaneous expression noise. PMID:27098003

  3. Physical activity level, waist circumference, and mortality

    PubMed Central

    Staiano, Amanda E.; Reeder, Bruce A.; Elliott, Susan; Joffres, Michel R.; Pahwa, Punam; Kirkland, Susan A.; Paradis, Gilles; Katzmarzyk, Peter T.

    2014-01-01

    This study predicted all-cause mortality based on physical activity level (active or inactive) and waist circumference (WC) in 8208 Canadian adults in Alberta, Manitoba, Nova Scotia, and Saskatchewan, surveyed between 1986–1995 and followed through 2004. Physically inactive adults had higher mortality risk than active adults overall (hazard ratio, 95% confidence interval = 1.20, 1.05–1.37) and within the low WC category (1.51, 1.19–1.92). Detrimental effects of physical inactivity and high WC demonstrate the need for physical activity promotion. PMID:22703160

  4. Sterol carrier protein2-like activity in rat intestine.

    PubMed

    Kharroubi, A; Wadsworth, J A; Chanderbhan, R; Wiesenfeld, P; Noland, B; Scallen, T; Vahouny, G V; Gallo, L L

    1988-03-01

    A sterol carrier protein2 (SCP2)-like activity has been demonstrated in rat intestinal mucosal homogenates and in isolated intestinal cells from both crypt and villus zones. The results indicate the presence of a protein with similar molecular weight and antigenicity to that of authentic SCP2 purified from rat liver cytosol. Like liver SCP2, mucosal cytosol stimulates pregnenolone production in rat adrenal mitochondria and acyl coenzyme A:cholesterol acyltransferase activity of liver and mucosal microsomes. The distribution of SCP2-like activity as determined by radioimmunoassay indicates high levels in mitochondria and cytosol and relatively lower levels in microsomes and in brush-border membranes. The widespread distribution of SCP2-like protein in the intestine is consistent with potential transfer functions in all phases of cholesterol processing. PMID:3379341

  5. Detecting protein complexes from active protein interaction networks constructed with dynamic gene expression profiles

    PubMed Central

    2013-01-01

    Background Protein interaction networks (PINs) are known to be useful to detect protein complexes. However, most available PINs are static, which cannot reflect the dynamic changes in real networks. At present, some researchers have tried to construct dynamic networks by incorporating time-course (dynamic) gene expression data with PINs. However, the inevitable background noise exists in the gene expression array, which could degrade the quality of dynamic networkds. Therefore, it is needed to filter out contaminated gene expression data before further data integration and analysis. Results Firstly, we adopt a dynamic model-based method to filter noisy data from dynamic expression profiles. Then a new method is proposed for identifying active proteins from dynamic gene expression profiles. An active protein at a time point is defined as the protein the expression level of whose corresponding gene at that time point is higher than a threshold determined by a standard variance involved threshold function. Furthermore, a noise-filtered active protein interaction network (NF-APIN) is constructed. To demonstrate the efficiency of our method, we detect protein complexes from the NF-APIN, compared with those from other dynamic PINs. Conclusion A dynamic model based method can effectively filter out noises in dynamic gene expression data. Our method to compute a threshold for determining the active time points of noise-filtered genes can make the dynamic construction more accuracy and provide a high quality framework for network analysis, such as protein complex prediction. PMID:24565281

  6. Cellular reprogramming through mitogen-activated protein kinases

    PubMed Central

    Lee, Justin; Eschen-Lippold, Lennart; Lassowskat, Ines; Böttcher, Christoph; Scheel, Dierk

    2015-01-01

    Mitogen-activated protein kinase (MAPK) cascades are conserved eukaryote signaling modules where MAPKs, as the final kinases in the cascade, phosphorylate protein substrates to regulate cellular processes. While some progress in the identification of MAPK substrates has been made in plants, the knowledge on the spectrum of substrates and their mechanistic action is still fragmentary. In this focused review, we discuss the biological implications of the data in our original paper (Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana; Frontiers in Plant Science 5: 554) in the context of related research. In our work, we mimicked in vivo activation of two stress-activated MAPKs, MPK3 and MPK6, through transgenic manipulation of Arabidopsis thaliana and used phosphoproteomics analysis to identify potential novel MAPK substrates. Here, we plotted the identified putative MAPK substrates (and downstream phosphoproteins) as a global protein clustering network. Based on a highly stringent selection confidence level, the core networks highlighted a MAPK-induced cellular reprogramming at multiple levels of gene and protein expression—including transcriptional, post-transcriptional, translational, post-translational (such as protein modification, folding, and degradation) steps, and also protein re-compartmentalization. Additionally, the increase in putative substrates/phosphoproteins of energy metabolism and various secondary metabolite biosynthesis pathways coincides with the observed accumulation of defense antimicrobial substances as detected by metabolome analysis. Furthermore, detection of protein networks in phospholipid or redox elements suggests activation of downstream signaling events. Taken in context with other studies, MAPKs are key regulators that reprogram cellular events to orchestrate defense signaling in eukaryotes. PMID:26579181

  7. Phosphorylation of platelet actin-binding protein during platelet activation

    SciTech Connect

    Carroll, R.C.; Gerrard, J.M.

    1982-03-01

    In this study we have followed the 32P-labeling of actin-binding protein as a function of platelet activation. Utilizing polyacrylamide-sodium dodecyl sulfate gel electrophoresis to resolve total platelet protein samples, we found 2 to 3-fold labeling increases in actin-binding protein 30 to 60 sec after thrombin stimulation. Somewhat larger increases were observed for 40,000 and 20,000 apparent molecular weight peptides. The actin-binding protein was identified on the gels by coelectrophoresis with purified actin-binding protein, its presence in cytoskeletal cores prepared by detergent extraction of activated 32P-labeled platelets, and by direct immunoprecipitation with antibodies against guinea pig vas deferens filamin (actin-binding protein). In addition, these cytoskeletal cores indicated that the 32P-labeled actin-binding protein was closely associated with the activated platelet's cytoskeleton. Following the 32P-labeling of actin-binding protein over an 8-min time course revealed that in aggregating platelet samples rapid dephosphorylation to almost initial levels occurred between 3 and 5 min. A similar curve was obtained for the 20,000 apparent molecular weight peptide. However, rapid dephosphorylation was not observed if platelet aggregation was prevented by chelating external calcium or by using thrombasthenic platelets lacking the aggregation response. Thus, cell-cell contact would seem to be crucial in initiating the rapid dephosphorylation response.

  8. Active Sites Environmental Monitoring Program: Action levels

    SciTech Connect

    Ashwood, J.S.; Ashwood, T.L.

    1991-10-01

    The Active Sites Environmental Monitoring Program (ASEMP) was established at Oak Ridge National Laboratory to provide for early leak detection and to monitor performance of the active low-level waste disposal facilities in Solid Waste Storage Area (SWSA) 6 and the transuranic waste storage areas in SWSA 5 North. Early leak detection is accomplished by sampling runoff, groundwater, and perched water in burial trenches. Sample results are compared to action levels that represent background contamination by naturally occurring and fallout-derived radionuclides. 15 refs., 3 figs., 12 tabs.

  9. The Hsp90-Dependent Proteome Is Conserved and Enriched for Hub Proteins with High Levels of Protein–Protein Connectivity

    PubMed Central

    Swamy, Krishna B.S.; Yu, Jau-Song; Schuyler, Scott C.; Leu, Jun-Yi

    2014-01-01

    Hsp90 is one of the most abundant and conserved proteins in the cell. Reduced levels or activity of Hsp90 causes defects in many cellular processes and also reveals genetic and nongenetic variation within a population. Despite information about Hsp90 protein–protein interactions, a global view of the Hsp90-regulated proteome in yeast is unavailable. To investigate the degree of dependency of individual yeast proteins on Hsp90, we used the “stable isotope labeling by amino acids in cell culture” method coupled with mass spectrometry to quantify around 4,000 proteins in low-Hsp90 cells. We observed that 904 proteins changed in their abundance by more than 1.5-fold. When compared with the transcriptome of the same population of cells, two-thirds of the misregulated proteins were observed to be affected posttranscriptionally, of which the majority were downregulated. Further analyses indicated that the downregulated proteins are highly conserved and assume central roles in cellular networks with a high number of protein interacting partners, suggesting that Hsp90 buffers genetic and nongenetic variation through regulating protein network hubs. The downregulated proteins were enriched for essential proteins previously not known to be Hsp90-dependent. Finally, we observed that downregulation of transcription factors and mating pathway components by attenuating Hsp90 function led to decreased target gene expression and pheromone response, respectively, providing a direct link between observed proteome regulation and cellular phenotypes. PMID:25316598

  10. A Cul-3-BTB ubiquitylation pathway regulates junctional levels and asymmetry of core planar polarity proteins

    PubMed Central

    Strutt, Helen; Searle, Elizabeth; Thomas-MacArthur, Victoria; Brookfield, Rosalind; Strutt, David

    2013-01-01

    The asymmetric localisation of core planar polarity proteins at apicolateral junctions is required to specify cell polarity in the plane of epithelia. This asymmetric distribution of the core proteins is proposed to require amplification of an initial asymmetry by feedback loops. In addition, generation of asymmetry appears to require the regulation of core protein levels, but the importance of such regulation and the underlying mechanisms is unknown. Here we show that ubiquitylation acts through more than one mechanism to control core protein levels in Drosophila, and that without this regulation cellular asymmetry is compromised. Levels of Dishevelled at junctions are regulated by a Cullin-3-Diablo/Kelch ubiquitin ligase complex, the activity of which is most likely controlled by neddylation. Furthermore, activity of the deubiquitylating enzyme Fat facets is required to maintain Flamingo levels at junctions. Notably, ubiquitylation does not alter the total cellular levels of Dishevelled or Flamingo, but only that of the junctional population. When junctional core protein levels are either increased or decreased by disruption of the ubiquitylation machinery, their asymmetric localisation is reduced and this leads to disruption of planar polarity at the tissue level. Loss of asymmetry by altered core protein levels can be explained by reference to feedback models for amplification of asymmetry. PMID:23487316

  11. Soy protein isolate molecular level contributions to bulk adhesive properties

    NASA Astrophysics Data System (ADS)

    Shera, Jeanne Norton

    Increasing environmental awareness and the recognized health hazards of formaldehyde-based resins has prompted a strong demand for environmentally-responsible adhesives for wood composites. Soy protein-based adhesives have been shown to be commercially viable with 90-day shelf stability and composite physical properties comparable to those of commercial formaldehyde-based particleboards. The main research focus is to isolate and characterize the molecular level features in soy protein isolate responsible for providing mechanical properties, storage stability, and water resistance during adhesive formulation, processing, and wood composite fabrication. Commercial composite board will be reviewed to enhance our understanding of the individual components and processes required for particleboard production. The levels of protein structure will be defined and an overview of current bio-based technology will be presented. In the process, the logic for utilizing soy protein as a sole binder in the adhesive will be reinforced. Variables such as adhesive components, pH, divalent ions, blend aging, protein molecular weight, formulation solids content, and soy protein functionalization will relate the bulk properties of soy protein adhesives to the molecular configuration of the soybean protein. This work has demonstrated that when intermolecular beta-sheet interactions and protein long-range order is disrupted, viscosity and mechanical properties decrease. Storage stability can be maintained through the stabilization of intermolecular beta-sheet interactions. When molecular weight is reduced through enzymatic digestion, long-range order is disrupted and viscosity and mechanical properties decrease accordingly. Processibility and physical properties must be balanced to increase solids while maintaining low viscosity, desirable mechanical properties, and adequate storage stability. The structure of the soybean protein must be related to the particleboard bulk mechanical

  12. Active Nuclear Import of Membrane Proteins Revisited.

    PubMed

    Laba, Justyna K; Steen, Anton; Popken, Petra; Chernova, Alina; Poolman, Bert; Veenhoff, Liesbeth M

    2015-01-01

    It is poorly understood how membrane proteins destined for the inner nuclear membrane pass the crowded environment of the Nuclear Pore Complex (NPC). For the Saccharomyces cerevisiae proteins Src1/Heh1 and Heh2, a transport mechanism was proposed where the transmembrane domains diffuse through the membrane while the extralumenal domains encoding a nuclear localization signal (NLS) and intrinsically disordered linker (L) are accompanied by transport factors and travel through the NPC. Here, we validate the proposed mechanism and explore and discuss alternative interpretations of the data. First, to disprove an interpretation where the membrane proteins become membrane embedded only after nuclear import, we present biochemical and localization data to support that the previously used, as well as newly designed reporter proteins are membrane-embedded irrespective of the presence of the sorting signals, the specific transmembrane domain (multipass or tail anchored), independent of GET, and also under conditions that the proteins are trapped in the NPC. Second, using the recently established size limit for passive diffusion of membrane proteins in yeast, and using an improved assay, we confirm active import of polytopic membrane protein with extralumenal soluble domains larger than those that can pass by diffusion on similar timescales. This reinforces that NLS-L dependent active transport is distinct from passive diffusion. Thirdly, we revisit the proposed route through the center of the NPC and conclude that the previously used trapping assay is, unfortunately, poorly suited to address the route through the NPC, and the route thus remains unresolved. Apart from the uncertainty about the route through the NPC, the data confirm active, transport factor dependent, nuclear transport of membrane-embedded mono- and polytopic membrane proteins in baker's yeast. PMID:26473931

  13. Active Nuclear Import of Membrane Proteins Revisited

    PubMed Central

    Laba, Justyna K.; Steen, Anton; Popken, Petra; Chernova, Alina; Poolman, Bert; Veenhoff, Liesbeth M.

    2015-01-01

    It is poorly understood how membrane proteins destined for the inner nuclear membrane pass the crowded environment of the Nuclear Pore Complex (NPC). For the Saccharomyces cerevisiae proteins Src1/Heh1 and Heh2, a transport mechanism was proposed where the transmembrane domains diffuse through the membrane while the extralumenal domains encoding a nuclear localization signal (NLS) and intrinsically disordered linker (L) are accompanied by transport factors and travel through the NPC. Here, we validate the proposed mechanism and explore and discuss alternative interpretations of the data. First, to disprove an interpretation where the membrane proteins become membrane embedded only after nuclear import, we present biochemical and localization data to support that the previously used, as well as newly designed reporter proteins are membrane-embedded irrespective of the presence of the sorting signals, the specific transmembrane domain (multipass or tail anchored), independent of GET, and also under conditions that the proteins are trapped in the NPC. Second, using the recently established size limit for passive diffusion of membrane proteins in yeast, and using an improved assay, we confirm active import of polytopic membrane protein with extralumenal soluble domains larger than those that can pass by diffusion on similar timescales. This reinforces that NLS-L dependent active transport is distinct from passive diffusion. Thirdly, we revisit the proposed route through the center of the NPC and conclude that the previously used trapping assay is, unfortunately, poorly suited to address the route through the NPC, and the route thus remains unresolved. Apart from the uncertainty about the route through the NPC, the data confirm active, transport factor dependent, nuclear transport of membrane-embedded mono- and polytopic membrane proteins in baker’s yeast. PMID:26473931

  14. Effect of N-acetylcysteine administration on homocysteine level, oxidative damage to proteins, and levels of iron (Fe) and Fe-related proteins in lead-exposed workers.

    PubMed

    Kasperczyk, Sławomir; Dobrakowski, Michał; Kasperczyk, Aleksandra; Romuk, Ewa; Rykaczewska-Czerwińska, Monika; Pawlas, Natalia; Birkner, Ewa

    2016-09-01

    N-Acetylcysteine (NAC) could be included in protocols designed for the treatment of lead toxicity. Therefore, in this study, we decided to investigate the influence of NAC administration on homocysteine (Hcy) levels, oxidative damage to proteins, and the levels of iron (Fe), transferrin (TRF), and haptoglobin (HPG) in lead (Pb)-exposed workers. The examined population (n = 171) was composed of male employees who worked with Pb. They were randomized into four groups. Workers who were not administered any antioxidants, drugs, vitamins, or dietary supplements were classified as the reference group (n = 49). The remaining three groups consisted of workers who were treated orally with NAC at three different doses (1 × 200, 2 × 200, or 2 × 400 mg) for 12 weeks. After the treatment, blood Pb levels significantly decreased in the groups receiving NAC compared with the reference group. The protein concentration was not affected by NAC administration. In contrast, Hcy levels significantly decreased or showed a strong tendency toward lower values depending on the NAC dose. Levels of the protein carbonyl groups were significantly decreased in all of the groups receiving NAC. Conversely, glutamate dehydrogenase activity was significantly elevated in all of the groups receiving NAC, while the level of protein thiol groups was significantly elevated only in the group receiving 200 mg of NAC. Treatment with NAC did not significantly affect Fe and TRF levels, whereas HPG levels showed a tendency toward lower values. Treatment with NAC normalized the level of Hcy and decreased oxidative stress as measured by the protein carbonyl content; this effect occurred in a dose-dependent manner. Moreover, small doses of NAC elevated the levels of protein thiol groups. Therefore, NAC could be introduced as an alternative therapy for chronic Pb toxicity in humans. PMID:25731901

  15. Protein kinase activators alter glial cholesterol esterification

    SciTech Connect

    Jeng, I.; Dills, C.; Klemm, N.; Wu, C.

    1986-05-01

    Similar to nonneural tissues, the activity of glial acyl-CoA cholesterol acyltransferase is controlled by a phosphorylation and dephosphorylation mechanism. Manipulation of cyclic AMP content did not alter the cellular cholesterol esterification, suggesting that cyclic AMP is not a bioregulator in this case. Therefore, the authors tested the effect of phorbol-12-myristate 13-acetate (PMA) on cellular cholesterol esterification to determine the involvement of protein kinase C. PMA has a potent effect on cellular cholesterol esterification. PMA depresses cholesterol esterification initially, but cells recover from inhibition and the result was higher cholesterol esterification, suggesting dual effects of protein kinase C. Studies of other phorbol analogues and other protein kinase C activators such as merezein indicate the involvement of protein kinase C. Oleoyl-acetyl glycerol duplicates the effect of PMA. This observation is consistent with a diacyl-glycerol-protein kinase-dependent reaction. Calcium ionophore A23187 was ineffective in promoting the effect of PMA. They concluded that a calcium-independent and protein C-dependent pathway regulated glial cholesterol esterification.

  16. DNA-dependent protein phosphorylation activity in Xenopus is coupled to a Ku-like protein.

    PubMed

    Kanungo, J; Cameron, R S; Takeda, Y; Hardin, J A

    1997-10-01

    DNA-dependent protein kinase (DNA-PK) is a nuclear enzyme and functions as a serine/threonine kinase that has been well characterized in both the human and the mouse. The regulatory subunit of DNA-PK is the Ku autoantigen. To demonstrate that a Ku-like protein is present in Xenopus oocytes, we used immunoprecipitation analysis with a monoclonal antibody raised against human Ku antigen and autoimmune serum containing anti-Ku antibodies. Metabolic labeling studies indicate that the Ku-like protein is synthesized mainly in late vitellogenic oocytes. By using a specific peptide substrate for DNA-PK, we demonstrate the activity of a DNA-dependent protein kinase in oocyte extracts. The kinase activity requires the Ku-like protein, since extracts depleted of Ku protein by immunoadsorption with human anti-Ku antibodies fail to demonstrate the DNA-dependent phosphorylation activity. The increased enzyme activity in vitellogenic oocytes may be correlated to the increased levels of Ku protein observed in these oocytes compared to the pre- and early vitellogenic oocytes.

  17. Phytochrome activation of two nuclear genes requires cytoplasmic protein synthesis.

    PubMed Central

    Lam, E; Green, P J; Wong, M; Chua, N H

    1989-01-01

    We have investigated the effects of protein synthesis inhibitors on light-induced expression of two plant nuclear genes, Cab and rbcS, in wheat, pea and transgenic tobacco. Light activation of these two genes is very sensitive to cycloheximide, an inhibitor of cytoplasmic protein synthesis but not to chloramphenicol, an inhibitor of organellar protein synthesis. Studies with chimeric gene constructs in transgenic tobacco seedlings show that cycloheximide exerts its effect at the transcriptional level. As a control, we show that the expression of the cauliflower mosaic virus (CaMV) 35S promoter is enhanced by cycloheximide treatment, irrespective of the coding sequence used. Escape-time analyses with green wheat seedlings show that the cycloheximide block for Cab gene expression is after the primary signal transduction step linked to phytochrome photoconversion. Our results suggest that phytochrome activation of Cab and rbcS is mediated by a labile protein factor(s) synthesized on cytoplasmic ribosomes. Images PMID:2583082

  18. Multi-level machine learning prediction of protein-protein interactions in Saccharomyces cerevisiae.

    PubMed

    Zubek, Julian; Tatjewski, Marcin; Boniecki, Adam; Mnich, Maciej; Basu, Subhadip; Plewczynski, Dariusz

    2015-01-01

    Accurate identification of protein-protein interactions (PPI) is the key step in understanding proteins' biological functions, which are typically context-dependent. Many existing PPI predictors rely on aggregated features from protein sequences, however only a few methods exploit local information about specific residue contacts. In this work we present a two-stage machine learning approach for prediction of protein-protein interactions. We start with the carefully filtered data on protein complexes available for Saccharomyces cerevisiae in the Protein Data Bank (PDB) database. First, we build linear descriptions of interacting and non-interacting sequence segment pairs based on their inter-residue distances. Secondly, we train machine learning classifiers to predict binary segment interactions for any two short sequence fragments. The final prediction of the protein-protein interaction is done using the 2D matrix representation of all-against-all possible interacting sequence segments of both analysed proteins. The level-I predictor achieves 0.88 AUC for micro-scale, i.e., residue-level prediction. The level-II predictor improves the results further by a more complex learning paradigm. We perform 30-fold macro-scale, i.e., protein-level cross-validation experiment. The level-II predictor using PSIPRED-predicted secondary structure reaches 0.70 precision, 0.68 recall, and 0.70 AUC, whereas other popular methods provide results below 0.6 threshold (recall, precision, AUC). Our results demonstrate that multi-scale sequence features aggregation procedure is able to improve the machine learning results by more than 10% as compared to other sequence representations. Prepared datasets and source code for our experimental pipeline are freely available for download from: http://zubekj.github.io/mlppi/ (open source Python implementation, OS independent).

  19. Multi-level machine learning prediction of protein-protein interactions in Saccharomyces cerevisiae.

    PubMed

    Zubek, Julian; Tatjewski, Marcin; Boniecki, Adam; Mnich, Maciej; Basu, Subhadip; Plewczynski, Dariusz

    2015-01-01

    Accurate identification of protein-protein interactions (PPI) is the key step in understanding proteins' biological functions, which are typically context-dependent. Many existing PPI predictors rely on aggregated features from protein sequences, however only a few methods exploit local information about specific residue contacts. In this work we present a two-stage machine learning approach for prediction of protein-protein interactions. We start with the carefully filtered data on protein complexes available for Saccharomyces cerevisiae in the Protein Data Bank (PDB) database. First, we build linear descriptions of interacting and non-interacting sequence segment pairs based on their inter-residue distances. Secondly, we train machine learning classifiers to predict binary segment interactions for any two short sequence fragments. The final prediction of the protein-protein interaction is done using the 2D matrix representation of all-against-all possible interacting sequence segments of both analysed proteins. The level-I predictor achieves 0.88 AUC for micro-scale, i.e., residue-level prediction. The level-II predictor improves the results further by a more complex learning paradigm. We perform 30-fold macro-scale, i.e., protein-level cross-validation experiment. The level-II predictor using PSIPRED-predicted secondary structure reaches 0.70 precision, 0.68 recall, and 0.70 AUC, whereas other popular methods provide results below 0.6 threshold (recall, precision, AUC). Our results demonstrate that multi-scale sequence features aggregation procedure is able to improve the machine learning results by more than 10% as compared to other sequence representations. Prepared datasets and source code for our experimental pipeline are freely available for download from: http://zubekj.github.io/mlppi/ (open source Python implementation, OS independent). PMID:26157620

  20. Steap4 plays a critical role in osteoclastogenesis in vitro by regulating cellular iron/reactive oxygen species (ROS) levels and cAMP response element-binding protein (CREB) activation.

    PubMed

    Zhou, Jian; Ye, Shiqiao; Fujiwara, Toshifumi; Manolagas, Stavros C; Zhao, Haibo

    2013-10-18

    Iron is essential for osteoclast differentiation, and iron overload in a variety of hematologic diseases is associated with excessive bone resorption. Iron uptake by osteoclast precursors via the transferrin cycle increases mitochondrial biogenesis, reactive oxygen species production, and activation of cAMP response element-binding protein, a critical transcription factor downstream of receptor activator of NF-κB-ligand-induced calcium signaling. These changes are required for the differentiation of osteoclast precursors to mature bone-resorbing osteoclasts. However, the molecular mechanisms regulating cellular iron metabolism in osteoclasts remain largely unknown. In this report, we provide evidence that Steap4, a member of the six-transmembrane epithelial antigen of prostate (Steap) family proteins, is an endosomal ferrireductase with a critical role in cellular iron utilization in osteoclasts. Specifically, we show that Steap4 is the only Steap family protein that is up-regulated during osteoclast differentiation. Knocking down Steap4 expression in vitro by lentivirus-mediated short hairpin RNAs inhibits osteoclast formation and decreases cellular ferrous iron, reactive oxygen species, and the activation of cAMP response element-binding protein. These results demonstrate that Steap4 is a critical enzyme for cellular iron uptake and utilization in osteoclasts and, thus, indispensable for osteoclast development and function.

  1. Serum Renalase Levels Correlate with Disease Activity in Lupus Nephritis

    PubMed Central

    Zhang, Minfang; Shao, Xinghua; Chang, Xinbei; Fan, Zhuping; Cao, Qin; Mou, Shan; Wang, Qin; Yan, Yucheng; Desir, Gary; Ni, Zhaohui

    2015-01-01

    Introduction Lupus nephritis (LN) is among the most serious complications of systemic lupus erythematosus (SLE), which causes significant morbidity and mortality. Renalase is a novel, kidney-secreted cytokine-like protein that promotes cell survival. Here, we aimed to investigate the relationship of serum renalase levels with LN and its role in the disease progression of LN. Methods For this cross-sectional study, 67 LN patients and 35 healthy controls were enrolled. Seventeen active LN patients who received standard therapies were followed up for six months. Disease activity was determined by the SLE Disease Activity–2000 (SLEDAI-2K) scoring system and serum renalase amounts were determined by ELISA. Predictive value of renalase for disease activity was assessed. Furthermore, the expression of renalase in the kidneys of patients and macrophage infiltration was assessed by immunohistochemistry. Results Serum renalase amounts were significantly higher in LN patients than in healthy controls. Moreover, patients with proliferative LN had more elevated serum renalase levels than Class V LN patients. In proliferative LN patients, serum renalase levels were significantly higher in patients with active LN than those with inactive LN. Serum renalase levels were positively correlated with SLEDAI-2K, 24-h urine protein excretion, ds-DNA and ESR but inversely correlated with serum albumin and C3. Renalase amounts decreased significantly after six-months of standard therapy. The performance of renalase as a marker for diagnosis of active LN was 0.906 with a cutoff value of 66.67 μg/ml. We also observed that the amount of renalase was significantly higher in glomerular of proliferative LN along with the co-expression of macrophages. Conclusion Serum renalase levels were correlated with disease activity in LN. Serum renalase might serve as a potential indicator for disease activity in LN. The marked increase of glomerular renalase and its association with macrophages suggest

  2. Protein kinase A activity and Hedgehog signaling pathway.

    PubMed

    Kotani, Tomoya

    2012-01-01

    Protein kinase A (PKA) is a well-known kinase that plays fundamental roles in a variety of biological processes. In Hedgehog-responsive cells, PKA plays key roles in proliferation and fate specification by modulating the transduction of Hedgehog signaling. In the absence of Hedgehog, a basal level of PKA activity represses the transcription of Hedgehog target genes. The main substrates of PKA in this process are the Ci/Gli family of bipotential transcription factors, which activate and repress Hedgehog target gene expression. PKA phosphorylates Ci/Gli, promoting the production of the repressor forms of Ci/Gli and thus repressing Hedgehog target gene expression. In contrast, the activation of Hedgehog signaling in response to Hedgehog increases the active forms of Ci/Gli, resulting in Hedgehog target gene expression. Because both decreased and increased levels of PKA activity cause abnormal cell proliferation and alter cell fate specification, the basal level of PKA activity in Hedgehog-responsive cells should be precisely regulated. However, the mechanism by which PKA activity is regulated remains obscure and appears to vary between cell types, tissues, and organisms. To date, two mechanisms have been proposed. One is a classical mechanism in which PKA activity is regulated by a small second messenger, cAMP; the other is a novel mechanism in which PKA activity is regulated by a protein, Misty somites. PMID:22391308

  3. Subunits of the Drosophila actin-capping protein heterodimer regulate each other at multiple levels.

    PubMed

    Amândio, Ana Rita; Gaspar, Pedro; Whited, Jessica L; Janody, Florence

    2014-01-01

    The actin-Capping Protein heterodimer, composed of the α and β subunits, is a master F-actin regulator. In addition to its role in many cellular processes, Capping Protein acts as a main tumor suppressor module in Drosophila and in humans, in part, by restricting the activity of Yorkie/YAP/TAZ oncogenes. We aimed in this report to understand how both subunits regulate each other in vivo. We show that the levels and capping activities of both subunits must be tightly regulated to control F-actin levels and consequently growth of the Drosophila wing. Overexpressing capping protein α and β decreases both F-actin levels and tissue growth, while expressing forms of Capping Protein that have dominant negative effects on F-actin promote tissue growth. Both subunits regulate each other's protein levels. In addition, overexpressing one of the subunit in tissues knocked-down for the other increases the mRNA and protein levels of the subunit knocked-down and compensates for its loss. We propose that the ability of the α and β subunits to control each other's levels assures that a pool of functional heterodimer is produced in sufficient quantities to restrict the development of tumor but not in excess to sustain normal tissue growth.

  4. Effect of phosphorus levels on the protein profiles of secreted protein and root surface protein of rice.

    PubMed

    Shinano, Takuro; Yoshimura, Tomoko; Watanabe, Toshihiro; Unno, Yusuke; Osaki, Mitsuru; Nanjo, Yohei; Komatsu, Setsuko

    2013-11-01

    Plant roots are complicated organs that absorb water and nutrients from the soil. Roots also play an essential role in protecting plants from attack by soil pathogens and develop a beneficial role with some soil microorganisms. Plant-derived rhizosphere proteins (e.g., root secretory proteins and root surface binding proteins) are considered to play important roles in developing mutual relationships in the rhizosphere. In the rhizosphere, where plant roots meet the surrounding environment, it has been suggested that root secretory protein and root surface binding protein are important factors. Furthermore, it is not known how the physiological status of the plant affects the profile of these proteins. In this study, rice plants were grown aseptically, with or without phosphorus nutrition, and proteins were obtained from root bathing solution (designated as root secretory proteins) and obtained using 0.2 M CaCl2 solution (designated as root surface binding proteins). The total number of identified proteins in the root bathing solution was 458, and the number of root surface binding proteins was 256. More than half of the proteins were observed in both fractions. Most of the proteins were categorized as either having signal peptides or no membrane transport helix sites. The functional categorization suggested that most of the proteins seemed to have secretory pathways and were involved in defense/disease-related functions. These characteristics seem to be unique to rhizosphere proteins, and the latter might be part of the plants strategy to defeat pathogens in the soil. The low phosphorus treatment significantly increased the number of pathogenesis-related proteins in the root secretory proteins, whereas the change was small in the case of the root surface binding proteins. The results suggested that the roots are actively and selectively secreting protein into the rhizosphere. PMID:24083427

  5. Smoking, COPD and 3-Nitrotyrosine Levels of Plasma Proteins

    SciTech Connect

    Jin, Hongjun; Webb-Robertson, Bobbie-Jo M.; Peterson, Elena S.; Tan, Ruimin; Bigelow, Diana J.; Scholand, Mary Beth; Hoidal, John R.; Pounds, Joel G.; Zangar, Richard C.

    2011-09-01

    BACKGROUND: Nitric oxide is a physiologically regulator of endothelial function and hemodynamics. Oxidized products of nitric oxide can form nitrotyrosine, which is a marker of nitrative stress. Cigarette smoking decreases exhaled nitric oxide, and the underlying mechanism may be important in the cardiovascular toxicity of cigarette smoke, although it is not clear if this effect results from decreased nitric oxide production or oxidation of nitric oxide to reactive, nitrating, species. These processes would be expected to have opposite effects on nitrotyrosine levels, a marker of nitrative stress. OBJECTIVE: In this study, we determine the effects of smoking and chronic obstructive pulmonary disease (COPD) on circulating levels of nitrotyrosine, and thereby gain insight into the processes regulating nitrotyrosine formation. METHODS: A custom antibody microarray platform was used to analyze the levels of 3-nitrotyrosine modifications on 24 proteins in plasma. Plasma samples from 458 individuals were analyzed. RESULTS: Nitrotyrosine levels in circulating proteins were uniformly reduced in smokers but increased in COPD patients. We also observed a persistent suppression of nitrotyrosine in former smokers. CONCLUSIONS: Smoking broadly suppresses the levels of 3-nitrotyrosine in plasma proteins, suggesting that cigarette smoke suppresses endothelial nitric oxide production. In contrast, the increase in nitrotyrosine levels in COPD patients most likely results from inflammatory processes. This study provides the first evidence that smoking has irreversible effects on endothelial production of nitric oxide, and provides insight into how smoking could induce a loss of elasticity in the vasculature and a long-term increase in the risk of cardiovascular disease.

  6. Relative survival of four serotypes of Salmonella enterica in low-water activity whey protein powder held at 36 and 70°C at various water activity levels

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Salmonella enterica is the leading cause of health burdens in the United States. Although the pathogen is not able to grow at aw levels below 0.94, it can survive in low-moisture foods for long periods of time. Temperature, aw, substrate and serotype affect its persistence. The aim of this study was...

  7. Importin-β facilitates nuclear import of human GW proteins and balances cytoplasmic gene silencing protein levels.

    PubMed

    Schraivogel, Daniel; Schindler, Susann G; Danner, Johannes; Kremmer, Elisabeth; Pfaff, Janina; Hannus, Stefan; Depping, Reinhard; Meister, Gunter

    2015-09-01

    MicroRNAs (miRNAs) guide Argonaute (Ago) proteins to distinct target mRNAs leading to translational repression and mRNA decay. Ago proteins interact with a member of the GW protein family, referred to as TNRC6A-C in mammals, which coordinate downstream gene-silencing processes. The cytoplasmic functions of TNRC6 and Ago proteins are reasonably well established. Both protein families are found in the nucleus as well. Their detailed nuclear functions, however, remain elusive. Furthermore, it is not clear which import routes Ago and TNRC6 proteins take into the nucleus. Using different nuclear transport assays, we find that Ago as well as TNRC6 proteins shuttle between the cytoplasm and the nucleus. While import receptors might function redundantly to transport Ago2, we demonstrate that TNRC6 proteins are imported by the Importin-β pathway. Finally, we show that nuclear localization of both Ago2 and TNRC6 proteins can depend on each other suggesting actively balanced cytoplasmic Ago - TNRC6 levels.

  8. Active Wnt proteins are secreted on exosomes.

    PubMed

    Gross, Julia Christina; Chaudhary, Varun; Bartscherer, Kerstin; Boutros, Michael

    2012-10-01

    Wnt signalling has important roles during development and in many diseases. As morphogens, hydrophobic Wnt proteins exert their function over a distance to induce patterning and cell differentiation decisions. Recent studies have identified several factors that are required for the secretion of Wnt proteins; however, how Wnts travel in the extracellular space remains a largely unresolved question. Here we show that Wnts are secreted on exosomes both during Drosophila development and in human cells. We demonstrate that exosomes carry Wnts on their surface to induce Wnt signalling activity in target cells. Together with the cargo receptor Evi/WIs, Wnts are transported through endosomal compartments onto exosomes, a process that requires the R-SNARE Ykt6. Our study demonstrates an evolutionarily conserved functional role of extracellular vesicular transport of Wnt proteins.

  9. Electrochemical Activation of Engineered Protein Switches

    PubMed Central

    Choi, Jay H.; Zayats, Maya; Searson, Peter C.; Ostermeier, Marc

    2016-01-01

    Engineered protein switches have a large dynamic range, high specificity for the activating ligand, and a modular architecture, and have been explored for a wide range of applications including biosensors and therapeutics. The ability to externally control switch function is important in extending applications for protein switches. We recently demonstrated that the on/off state could be controlled by the redox state of disulfide bonds introduced into the switches at select locations. Here, we demonstrate that an electrochemical signal can be used as an exogenous input to control switch function via reduction of the engineered disulfide bonds. This study suggests that disulfide-containing protein switch is a potentially useful platform for bioelectronic sensors with remote control of the sensing ability. PMID:26241391

  10. GUN1 Controls Accumulation of the Plastid Ribosomal Protein S1 at the Protein Level and Interacts with Proteins Involved in Plastid Protein Homeostasis.

    PubMed

    Tadini, Luca; Pesaresi, Paolo; Kleine, Tatjana; Rossi, Fabio; Guljamow, Arthur; Sommer, Frederik; Mühlhaus, Timo; Schroda, Michael; Masiero, Simona; Pribil, Mathias; Rothbart, Maxi; Hedtke, Boris; Grimm, Bernhard; Leister, Dario

    2016-03-01

    Developmental or metabolic changes in chloroplasts can have profound effects on the rest of the plant cell. Such intracellular responses are associated with signals that originate in chloroplasts and convey information on their physiological status to the nucleus, which leads to large-scale changes in gene expression (retrograde signaling). A screen designed to identify components of retrograde signaling resulted in the discovery of the so-called genomes uncoupled (gun) mutants. Genetic evidence suggests that the chloroplast protein GUN1 integrates signals derived from perturbations in plastid redox state, plastid gene expression, and tetrapyrrole biosynthesis (TPB) in Arabidopsis (Arabidopsis thaliana) seedlings, exerting biogenic control of chloroplast functions. However, the molecular mechanism by which GUN1 integrates retrograde signaling in the chloroplast is unclear. Here we show that GUN1 also operates in adult plants, contributing to operational control of chloroplasts. The gun1 mutation genetically interacts with mutations of genes for the chloroplast ribosomal proteins S1 (PRPS1) and L11. Analysis of gun1 prps1 lines indicates that GUN1 controls PRPS1 accumulation at the protein level. The GUN1 protein physically interacts with proteins involved in chloroplast protein homeostasis based on coimmunoprecipitation experiments. Furthermore, yeast two-hybrid and bimolecular fluorescence complementation experiments suggest that GUN1 might transiently interact with several TPB enzymes, including Mg-chelatase subunit D (CHLD) and two other TPB enzymes known to activate retrograde signaling. Moreover, the association of PRPS1 and CHLD with protein complexes is modulated by GUN1. These findings allow us to speculate that retrograde signaling might involve GUN1-dependent formation of protein complexes. PMID:26823545

  11. Response of protein and urea kinetics in burn patients to different levels of protein intake.

    PubMed Central

    Wolfe, R R; Goodenough, R D; Burke, J F; Wolfe, M H

    1983-01-01

    The effects of two levels of protein intake on protein metabolism in six severely burned adult patients were studied (means of 70% BSA burned). A crossover experimental design enabled the authors to study each patient at the end of two three-day dietary regimens. All diets were isocaloric and provided approximately 25% more calories than the measured energy expenditure (means = 40.8 Kcal/kg X day). In one regimen, each patient received 2.2 g protein/kg X day, while during the other treatment period they received 1.4 g protein/kg X day. The patients were studied in the fed state and after 10 to 12 hours of fasting. Leucine kinetics were determined by means of the primed-constant infusion of [1--13C]--leucine. The authors were able to distinguish the oxidation of plasma leucine from the oxidation of leucine derived from intracellular protein at the site of the deamination of leucine (predominantly muscle) by simultaneously determining both leucine and alpha-ketoisocaproic acid enrichment. Also, rates of whole-body protein synthesis and catabolism were calculated from the leucine flux and oxidation data. Net protein synthesis was also calculated by means of another stable-isotope technique involving the infusion of [15N2]--urea. Finally, a third means of estimating net protein catabolism based on urinary N-excretion data was used at the same time that the isotopic studies were performed. The 13C leucine-data and the N-excretion data indicated that a balance between protein synthesis and catabolism could be achieved with a protein intake of 1.4 protein/kg X day. When protein intake was increased to 2.2 g protein/kg X day, neither isotopic method indicated a further beneficial effect on net protein synthesis, although the absolute rates of protein synthesis and catabolism were stimulated. The N-excretion data, on the other hand, indicated a significant improvement in net protein synthesis with higher protein intake. Regardless of the level of protein intake, the

  12. Analysis of mitogen-activated protein kinase activity in yeast.

    PubMed

    Elion, Elaine A; Sahoo, Rupam

    2010-01-01

    Mitogen-activated protein (MAP) kinases play central roles in transmitting extracellular and intracellular information in a wide variety of situations in eukaryotic cells. Their activities are perturbed in a large number of diseases, and their activating kinases are currently therapeutic targets in cancer. MAPKs are highly conserved among all eukaryotes. MAPKs were first cloned from the yeast Saccharomyces cerevisiae. Yeast has five MAPKs and one MAPK-like kinase. The mating MAPK Fus3 is the best characterized yeast MAPK. Members of all subfamilies of human MAPKs can functionally substitute S. cerevisiae MAPKs, providing systems to use genetic approaches to study the functions of either yeast or human MAPKs and to identify functionally relevant amino acid residues that enhance or reduce the effects of therapeutically relevant inhibitors and regulatory proteins. Here, we describe an assay to measure Fus3 activity in immune complexes prepared from S. cerevisiae extracts. The assay conditions are applicable to other MAPKs, as well. PMID:20811996

  13. De Novo Construction of Redox Active Proteins.

    PubMed

    Moser, C C; Sheehan, M M; Ennist, N M; Kodali, G; Bialas, C; Englander, M T; Discher, B M; Dutton, P L

    2016-01-01

    Relatively simple principles can be used to plan and construct de novo proteins that bind redox cofactors and participate in a range of electron-transfer reactions analogous to those seen in natural oxidoreductase proteins. These designed redox proteins are called maquettes. Hydrophobic/hydrophilic binary patterning of heptad repeats of amino acids linked together in a single-chain self-assemble into 4-alpha-helix bundles. These bundles form a robust and adaptable frame for uncovering the default properties of protein embedded cofactors independent of the complexities introduced by generations of natural selection and allow us to better understand what factors can be exploited by man or nature to manipulate the physical chemical properties of these cofactors. Anchoring of redox cofactors such as hemes, light active tetrapyrroles, FeS clusters, and flavins by His and Cys residues allow cofactors to be placed at positions in which electron-tunneling rates between cofactors within or between proteins can be predicted in advance. The modularity of heptad repeat designs facilitates the construction of electron-transfer chains and novel combinations of redox cofactors and new redox cofactor assisted functions. Developing de novo designs that can support cofactor incorporation upon expression in a cell is needed to support a synthetic biology advance that integrates with natural bioenergetic pathways. PMID:27586341

  14. Synaptic Vesicle Proteins and Active Zone Plasticity.

    PubMed

    Kittel, Robert J; Heckmann, Manfred

    2016-01-01

    Neurotransmitter is released from synaptic vesicles at the highly specialized presynaptic active zone (AZ). The complex molecular architecture of AZs mediates the speed, precision and plasticity of synaptic transmission. Importantly, structural and functional properties of AZs vary significantly, even for a given connection. Thus, there appear to be distinct AZ states, which fundamentally influence neuronal communication by controlling the positioning and release of synaptic vesicles. Vice versa, recent evidence has revealed that synaptic vesicle components also modulate organizational states of the AZ. The protein-rich cytomatrix at the active zone (CAZ) provides a structural platform for molecular interactions guiding vesicle exocytosis. Studies in Drosophila have now demonstrated that the vesicle proteins Synaptotagmin-1 (Syt1) and Rab3 also regulate glutamate release by shaping differentiation of the CAZ ultrastructure. We review these unexpected findings and discuss mechanistic interpretations of the reciprocal relationship between synaptic vesicles and AZ states, which has heretofore received little attention.

  15. Synaptic Vesicle Proteins and Active Zone Plasticity

    PubMed Central

    Kittel, Robert J.; Heckmann, Manfred

    2016-01-01

    Neurotransmitter is released from synaptic vesicles at the highly specialized presynaptic active zone (AZ). The complex molecular architecture of AZs mediates the speed, precision and plasticity of synaptic transmission. Importantly, structural and functional properties of AZs vary significantly, even for a given connection. Thus, there appear to be distinct AZ states, which fundamentally influence neuronal communication by controlling the positioning and release of synaptic vesicles. Vice versa, recent evidence has revealed that synaptic vesicle components also modulate organizational states of the AZ. The protein-rich cytomatrix at the active zone (CAZ) provides a structural platform for molecular interactions guiding vesicle exocytosis. Studies in Drosophila have now demonstrated that the vesicle proteins Synaptotagmin-1 (Syt1) and Rab3 also regulate glutamate release by shaping differentiation of the CAZ ultrastructure. We review these unexpected findings and discuss mechanistic interpretations of the reciprocal relationship between synaptic vesicles and AZ states, which has heretofore received little attention. PMID:27148040

  16. Less is More: Membrane Protein Digestion Beyond Urea–Trypsin Solution for Next-level Proteomics*

    PubMed Central

    Zhang, Xi

    2015-01-01

    The goal of next-level bottom-up membrane proteomics is protein function investigation, via high-coverage high-throughput peptide-centric quantitation of expression, modifications and dynamic structures at systems scale. Yet efficient digestion of mammalian membrane proteins presents a daunting barrier, and prevalent day-long urea–trypsin in-solution digestion proved insufficient to reach this goal. Many efforts contributed incremental advances over past years, but involved protein denaturation that disconnected measurement from functional states. Beyond denaturation, the recent discovery of structure/proteomics omni-compatible detergent n-dodecyl-β-d-maltopyranoside, combined with pepsin and PNGase F columns, enabled breakthroughs in membrane protein digestion: a 2010 DDM-low-TCEP (DLT) method for H/D-exchange (HDX) using human G protein-coupled receptor, and a 2015 flow/detergent-facilitated protease and de-PTM digestions (FDD) for integrative deep sequencing and quantitation using full-length human ion channel complex. Distinguishing protein solubilization from denaturation, protease digestion reliability from theoretical specificity, and reduction from alkylation, these methods shifted day(s)-long paradigms into minutes, and afforded fully automatable (HDX)-protein-peptide-(tandem mass tag)-HPLC pipelines to instantly measure functional proteins at deep coverage, high peptide reproducibility, low artifacts and minimal leakage. Promoting—not destroying—structures and activities harnessed membrane proteins for the next-level streamlined functional proteomics. This review analyzes recent advances in membrane protein digestion methods and highlights critical discoveries for future proteomics. PMID:26081834

  17. Predicting Protein-Protein Interactions from the Molecular to the Proteome Level.

    PubMed

    Keskin, Ozlem; Tuncbag, Nurcan; Gursoy, Attila

    2016-04-27

    Identification of protein-protein interactions (PPIs) is at the center of molecular biology considering the unquestionable role of proteins in cells. Combinatorial interactions result in a repertoire of multiple functions; hence, knowledge of PPI and binding regions naturally serve to functional proteomics and drug discovery. Given experimental limitations to find all interactions in a proteome, computational prediction/modeling of protein interactions is a prerequisite to proceed on the way to complete interactions at the proteome level. This review aims to provide a background on PPIs and their types. Computational methods for PPI predictions can use a variety of biological data including sequence-, evolution-, expression-, and structure-based data. Physical and statistical modeling are commonly used to integrate these data and infer PPI predictions. We review and list the state-of-the-art methods, servers, databases, and tools for protein-protein interaction prediction. PMID:27074302

  18. Development of Novel Adenosine Monophosphate-Activated Protein Kinase Activators

    PubMed Central

    Guh, Jih-Hwa; Chang, Wei-Ling; Yang, Jian; Lee, Su-Lin; Wei, Shuo; Wang, Dasheng; Kulp, Samuel K.; Chen, Ching-Shih

    2010-01-01

    In light of the unique ability of thiazolidinediones to mediate peroxisome proliferator-activated receptor (PPAR)γ-independent activation of adenosine monophosphate-activated protein kinase (AMPK) and suppression of interleukin (IL)-6 production, we conducted a screening of an in-house, thiazolidinedione-based focused compound library to identify novel agents with these dual pharmacological activities. Cell-based assays pertinent to the activation status of AMPK and mammalian homolog of target of rapamycin (i.e., phosphorylation of AMPK and p70 ribosomal protein S6 kinase, respectively), and IL-6/IL-6 receptor signaling (i.e., IL-6 production and signal transducer and activator of transcription 3 phosphorylation, respectively) in lipopolysaccharide (LPS)-stimulated THP-1 human macrophages were used to screen this compound library, which led to the identification of compound 53 (N-{4-[3-(1-Methylcyclohexylmethyl)-2,4-dioxo-thiazolidin-5-ylidene-methyl]-phenyl}-4-nitro-3-trifluoromethyl-benzenesulfonamide) as the lead agent. Evidence indicates that this drug-induced suppression of LPS-stimulated IL-6 production was attributable to AMPK activation. Furthermore, compound 53-mediated AMPK activation was demonstrated in C-26 colon adenocarcinoma cells, indicating that it is not a cell line-specific event. PMID:20170185

  19. Butachlor impact on protein, free amino acid and glutamine contents, and on activity levels of aminotransferases, glutamate dehydrogenase and glutamine synthetase in the fresh water snail, Pila globosa (Swainson).

    PubMed

    Rajyalakshmi, T; Srinivas, T; Swamy, K V; Mohan, P M

    1996-08-01

    Biochemical changes followed in the freshwater snail Pila globosa (Swainson) during exposure to sublethal concentrations of the herbicide butachlor (26.6 ppm) in the ambient medium, at 3,6,12,24 and 48 h intervals, were marked by a significant decrease in total and soluble proteins, and an increase in free amino acids in foot and hepatopancreas up to 12 h before gradually recovering. Aminotransferase activities and glutamine content decreased during the early periods of exposure, while glutamate dehydrogenase activity increased. After an initial elevation, glutamate synthetase activity decreased at later intervals. Maximum effect of butachlor on the enzymes was seen after 12 h exposure. The extent of increase or decrease in different parameters examined varied between the two tissues studied. These changes are discussed in relation to the toxic stress of butachlor.

  20. UCP2, a mitochondrial protein regulated at multiple levels.

    PubMed

    Donadelli, Massimo; Dando, Ilaria; Fiorini, Claudia; Palmieri, Marta

    2014-04-01

    An ever-increasing number of studies highlight the role of uncoupling protein 2 (UCP2) in a broad range of physiological and pathological processes. The knowledge of the molecular mechanisms of UCP2 regulation is becoming fundamental in both the comprehension of UCP2-related physiological events and the identification of novel therapeutic strategies based on UCP2 modulation. The study of UCP2 regulation is a fast-moving field. Recently, several research groups have made a great effort to thoroughly understand the various molecular mechanisms at the basis of UCP2 regulation. In this review, we describe novel findings concerning events that can occur in a concerted manner at various levels: Ucp2 gene mutation (single nucleotide polymorphisms), UCP2 mRNA and protein expression (transcriptional, translational, and protein turn-over regulation), UCP2 proton conductance (ligands and post-transcriptional modifications), and nutritional and pharmacological regulation of UCP2.

  1. Low copper and high manganese levels in prion protein plaques

    USGS Publications Warehouse

    Johnson, Christopher J.; Gilbert, P.U.P.A.; Abrecth, Mike; Baldwin, Katherine L.; Russell, Robin E.; Pedersen, Joel A.; McKenzie, Debbie

    2013-01-01

    Accumulation of aggregates rich in an abnormally folded form of the prion protein characterize the neurodegeneration caused by transmissible spongiform encephalopathies (TSEs). The molecular triggers of plaque formation and neurodegeneration remain unknown, but analyses of TSE-infected brain homogenates and preparations enriched for abnormal prion protein suggest that reduced levels of copper and increased levels of manganese are associated with disease. The objectives of this study were to: (1) assess copper and manganese levels in healthy and TSE-infected Syrian hamster brain homogenates; (2) determine if the distribution of these metals can be mapped in TSE-infected brain tissue using X-ray photoelectron emission microscopy (X-PEEM) with synchrotron radiation; and (3) use X-PEEM to assess the relative amounts of copper and manganese in prion plaques in situ. In agreement with studies of other TSEs and species, we found reduced brain levels of copper and increased levels of manganese associated with disease in our hamster model. We also found that the in situ levels of these metals in brainstem were sufficient to image by X-PEEM. Using immunolabeled prion plaques in directly adjacent tissue sections to identify regions to image by X-PEEM, we found a statistically significant relationship of copper-manganese dysregulation in prion plaques: copper was depleted whereas manganese was enriched. These data provide evidence for prion plaques altering local transition metal distribution in the TSE-infected central nervous system.

  2. Low Copper and High Manganese Levels in Prion Protein Plaques

    PubMed Central

    Johnson, Christopher J.; Gilbert, P.U.P.A.; Abrecht, Mike; Baldwin, Katherine L.; Russell, Robin E.; Pedersen, Joel A.; Aiken, Judd M.; McKenzie, Debbie

    2013-01-01

    Accumulation of aggregates rich in an abnormally folded form of the prion protein characterize the neurodegeneration caused by transmissible spongiform encephalopathies (TSEs). The molecular triggers of plaque formation and neurodegeneration remain unknown, but analyses of TSE-infected brain homogenates and preparations enriched for abnormal prion protein suggest that reduced levels of copper and increased levels of manganese are associated with disease. The objectives of this study were to: (1) assess copper and manganese levels in healthy and TSE-infected Syrian hamster brain homogenates; (2) determine if the distribution of these metals can be mapped in TSE-infected brain tissue using X-ray photoelectron emission microscopy (X-PEEM) with synchrotron radiation; and (3) use X-PEEM to assess the relative amounts of copper and manganese in prion plaques in situ. In agreement with studies of other TSEs and species, we found reduced brain levels of copper and increased levels of manganese associated with disease in our hamster model. We also found that the in situ levels of these metals in brainstem were sufficient to image by X-PEEM. Using immunolabeled prion plaques in directly adjacent tissue sections to identify regions to image by X-PEEM, we found a statistically significant relationship of copper-manganese dysregulation in prion plaques: copper was depleted whereas manganese was enriched. These data provide evidence for prion plaques altering local transition metal distribution in the TSE-infected central nervous system. PMID:23435237

  3. Low copper and high manganese levels in prion protein plaques.

    PubMed

    Johnson, Christopher J; Gilbert, P U P A; Abrecht, Mike; Baldwin, Katherine L; Russell, Robin E; Pedersen, Joel A; Aiken, Judd M; McKenzie, Debbie

    2013-02-01

    Accumulation of aggregates rich in an abnormally folded form of the prion protein characterize the neurodegeneration caused by transmissible spongiform encephalopathies (TSEs). The molecular triggers of plaque formation and neurodegeneration remain unknown, but analyses of TSE-infected brain homogenates and preparations enriched for abnormal prion protein suggest that reduced levels of copper and increased levels of manganese are associated with disease. The objectives of this study were to: (1) assess copper and manganese levels in healthy and TSE-infected Syrian hamster brain homogenates; (2) determine if the distribution of these metals can be mapped in TSE-infected brain tissue using X-ray photoelectron emission microscopy (X-PEEM) with synchrotron radiation; and (3) use X-PEEM to assess the relative amounts of copper and manganese in prion plaques in situ. In agreement with studies of other TSEs and species, we found reduced brain levels of copper and increased levels of manganese associated with disease in our hamster model. We also found that the in situ levels of these metals in brainstem were sufficient to image by X-PEEM. Using immunolabeled prion plaques in directly adjacent tissue sections to identify regions to image by X-PEEM, we found a statistically significant relationship of copper-manganese dysregulation in prion plaques: copper was depleted whereas manganese was enriched. These data provide evidence for prion plaques altering local transition metal distribution in the TSE-infected central nervous system.

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

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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

  5. RNF4-Dependent Oncogene Activation by Protein Stabilization.

    PubMed

    Thomas, Jane J; Abed, Mona; Heuberger, Julian; Novak, Rostislav; Zohar, Yaniv; Beltran Lopez, Angela P; Trausch-Azar, Julie S; Ilagan, Ma Xenia G; Benhamou, David; Dittmar, Gunnar; Kopan, Raphael; Birchmeier, Walter; Schwartz, Alan L; Orian, Amir

    2016-09-20

    Ubiquitylation regulates signaling pathways critical for cancer development and, in many cases, targets proteins for degradation. Here, we report that ubiquitylation by RNF4 stabilizes otherwise short-lived oncogenic transcription factors, including β-catenin, Myc, c-Jun, and the Notch intracellular-domain (N-ICD) protein. RNF4 enhances the transcriptional activity of these factors, as well as Wnt- and Notch-dependent gene expression. While RNF4 is a SUMO-targeted ubiquitin ligase, protein stabilization requires the substrate's phosphorylation, rather than SUMOylation, and binding to RNF4's arginine-rich motif domain. Stabilization also involves generation of unusual polyubiquitin chains and docking of RNF4 to chromatin. Biologically, RNF4 enhances the tumor phenotype and is essential for cancer cell survival. High levels of RNF4 mRNA correlate with poor survival of a subgroup of breast cancer patients, and RNF4 protein levels are elevated in 30% of human colon adenocarcinomas. Thus, RNF4-dependent ubiquitylation translates transient phosphorylation signal(s) into long-term protein stabilization, resulting in enhanced oncoprotein activation. PMID:27653698

  6. C-reactive protein serum level in patients with psoriasis before and after treatment with narrow-band ultraviolet B*

    PubMed Central

    Farshchian, Mahmoud; Ansar, Akram; Sobhan, Mohammadreza; Hoseinpoor, Valiollah

    2016-01-01

    Background C-reactive protein is an inflammatory biomarker and its level increases in the serum of psoriatic patients. Its level is also associated with Psoriasis Area and Severity Index score. Objective The aim of this study was to assess the decrement of serum C-reactive protein level with narrow-band ultraviolet B (NB-UVB) therapy. Methods C-reactive protein serum levels in psoriasis patients were measured before and after treatment with NB-UVB and the data were analyzed in relation to the Psoriasis Area and Severity Index score improvement. Results Baseline C-reactive protein levels among psoriatic patients were higher than normal. These levels decreased significantly after treatment (P<0.001). At the beginning of the study, patients with higher levels of C-reactive protein also had more extensive and severe skin involvement. The highest decrease in C-reactive protein was observed in patients who responded better to the treatment and achieved a higher Psoriasis Area and Severity Index 75%. There was an association between baseline Psoriasis Area and Severity Index scores and C-reactive protein levels. Conclusion Patients with moderate to severe plaque-type psoriasis had active systemic inflammation, which was demonstrated by increased levels of C-reactive protein. Furthermore, skin disease severity was correlated with C-reactive protein levels. Phototherapy healed the psoriatic skin lesions and reduced inflammation, while decreasing C-reactive protein levels.

  7. Residue level quantification of protein stability in living cells.

    PubMed

    Monteith, William B; Pielak, Gary J

    2014-08-01

    The intracellular milieu differs from the dilute conditions in which most biophysical and biochemical studies are performed. This difference has led both experimentalists and theoreticians to tackle the challenging task of understanding how the intracellular environment affects the properties of biopolymers. Despite a growing number of in-cell studies, there is a lack of quantitative, residue-level information about equilibrium thermodynamic protein stability under nonperturbing conditions. We report the use of NMR-detected hydrogen-deuterium exchange of quenched cell lysates to measure individual opening free energies of the 56-aa B1 domain of protein G (GB1) in living Escherichia coli cells without adding destabilizing cosolutes or heat. Comparisons to dilute solution data (pH 7.6 and 37 °C) show that opening free energies increase by as much as 1.14 ± 0.05 kcal/mol in cells. Importantly, we also show that homogeneous protein crowders destabilize GB1, highlighting the challenge of recreating the cellular interior. We discuss our findings in terms of hard-core excluded volume effects, charge-charge GB1-crowder interactions, and other factors. The quenched lysate method identifies the residues most important for folding GB1 in cells, and should prove useful for quantifying the stability of other globular proteins in cells to gain a more complete understanding of the effects of the intracellular environment on protein chemistry.

  8. Comparison of Metalloproteinase Protein and Activity Profiling

    PubMed Central

    Giricz, Orsi; Lauer, Janelle L.; Fields, Gregg B.

    2010-01-01

    Proteolytic enzymes play fundamental roles in many biological processes. Members of the matrix metalloproteinase (MMP) family have been shown to take part in processes crucial in disease progression. The present study used the ExcelArray Human MMP/TIMP Array to quantify MMP and tissue inhibitor of metalloproteinase (TIMP) production in the lysates and media of 14 cancer and one normal cell line. The overall patterns were very similar in terms of which MMPs and TIMPs were secreted in the media versus associated with the cells in the individual samples. However, more MMP was found in the media, both in amount and in variety. TIMP-1 was produced in all cell lines. MMP activity assays with three different FRET substrates were then utilized to determine if protein production correlated with function for the WM-266-4 and BJ cell lines. Metalloproteinase activity was observed for both cell lines with a general MMP substrate (Knight SSP), consistent with protein production data. However, although both cell lines promoted the hydrolysis of a more selective MMP substrate (NFF-3), metalloproteinase activity was only confirmed in the BJ cell line. The use of inhibitors to confirm metalloproteinase activities pointed to the strengths and weaknesses of in situ FRET substrate assays. PMID:20920458

  9. Crowding Activates Heat Shock Protein 90.

    PubMed

    Halpin, Jackson C; Huang, Bin; Sun, Ming; Street, Timothy O

    2016-03-18

    Hsp90 is a dimeric ATP-dependent chaperone involved in the folding, maturation, and activation of diverse target proteins. Extensive in vitro structural analysis has led to a working model of Hsp90's ATP-driven conformational cycle. An implicit assumption is that dilute experimental conditions do not significantly perturb Hsp90 structure and function. However, Hsp90 undergoes a dramatic open/closed conformational change, which raises the possibility that this assumption may not be valid for this chaperone. Indeed, here we show that the ATPase activity of Hsp90 is highly sensitive to molecular crowding, whereas the ATPase activities of Hsp60 and Hsp70 chaperones are insensitive to crowding conditions. Polymer crowders activate Hsp90 in a non-saturable manner, with increasing efficacy at increasing concentration. Crowders exhibit a non-linear relationship between their radius of gyration and the extent to which they activate Hsp90. This experimental relationship can be qualitatively recapitulated with simple structure-based volume calculations comparing open/closed configurations of Hsp90. Thermodynamic analysis indicates that crowding activation of Hsp90 is entropically driven, which is consistent with a model in which excluded volume provides a driving force that favors the closed active state of Hsp90. Multiple Hsp90 homologs are activated by crowders, with the endoplasmic reticulum-specific Hsp90, Grp94, exhibiting the highest sensitivity. Finally, we find that crowding activation works by a different mechanism than co-chaperone activation and that these mechanisms are independent. We hypothesize that Hsp90 has a higher intrinsic activity in the cell than in vitro. PMID:26797120

  10. Physiological enzymology: The next frontier in understanding protein structure and function at the cellular level.

    PubMed

    Lee, Irene; Berdis, Anthony J

    2016-01-01

    Historically, the study of proteins has relied heavily on characterizing the activity of a single purified protein isolated from other cellular components. This classic approach allowed scientists to unambiguously define the intrinsic kinetic and chemical properties of that protein. The ultimate hope was to extrapolate this information toward understanding how the enzyme or receptor behaves within its native cellular context. These types of detailed in vitro analyses were necessary to reduce the innate complexities of measuring the singular activity and biochemical properties of a specific enzyme without interference from other enzymes and potential competing substrates. However, recent developments in fields encompassing cell biology, molecular imaging, and chemical biology now provide the unique chemical tools and instrumentation to study protein structure, function, and regulation in their native cellular environment. These advancements provide the foundation for a new field, coined physiological enzymology, which quantifies the function and regulation of enzymes and proteins at the cellular level. In this Special Edition, we explore the area of Physiological Enzymology and Protein Function through a series of review articles that focus on the tools and techniques used to measure the cellular activity of proteins inside living cells. This article is part of a Special Issue entitled: Physiological Enzymology and Protein Functions.

  11. C-reactive protein activates complement in infarcted human myocardium.

    PubMed

    Nijmeijer, Remco; Lagrand, Wim K; Lubbers, Yvonne T P; Visser, Cees A; Meijer, Chris J L M; Niessen, Hans W M; Hack, C Erik

    2003-07-01

    Circulating levels of C-reactive protein (CRP) constitute a cardiovascular risk marker. Immunohistochemical studies have revealed co-localization of CRP and activated complement in human infarcted myocardium suggesting CRP to enhance inflammation in ischemic myocardium by inducing local complement activation. The aim was to establish whether CRP activates complement in infarcted human myocardium and to assess the relationship between this activation and the duration of infarction. Myocardial tissue samples from 56 patients that had died from acute myocardial infarction were evaluated. Specimens were taken from infarcted as well as noninfarcted sites of the heart. CRP-mediated complement activation was assessed by immunohistochemistry and by measuring levels of complement, CRP, and CRP-complement complexes, specific markers for CRP-mediated activation, in homogenates of the heart. Infarctions of 12 hours to 5 days had significantly more extensive depositions of complement and CRP and contained significantly more CRP, activated complement, and CRP-complement complexes than infarctions that were less than 12 hours old. Levels of CRP complexes correlated significantly with CRP and complement concentrations in the infarctions, as well as with the extent of complement and CRP depositions as measured via immunohistochemistry. Specific activation products of CRP-mediated activation of complement are increased in infarcts of more than 12 hours in duration and correlate with the extent of complement depositions. Hence, CRP seems to enhance local inflammatory reactions ensuing in human myocardial infarcts of more than 12 hours duration.

  12. Polycarboxylates Enhance Beetle Antifreeze Protein Activity

    PubMed Central

    Amornwittawat, Natapol; Wang, Sen; Duman, John G.; Wen, Xin

    2008-01-01

    Summary Antifreeze proteins (AFPs) lower the noncolligative freezing point of water in the presence of ice below the ice melting point. The temperature difference between the melting point and the noncolligative freezing point is termed thermal hysteresis (TH). The magnitude of the TH depends on the specific activity and the concentration of AFP, and the concentration of enhancers in the solution. Known enhancers are certain low molecular mass molecules and proteins. Here, we investigated a series of polycarboxylates that enhance the TH activity of an AFP from the beetle Dendroides canadensis (DAFP) using differential scanning calorimetry (DSC). Triethylenetetramine-N,N,N′,N″,N‴,N‴-hexaacetate, the most efficient enhancer identified in this work, can increase the TH of DAFP by nearly 1.5 fold over than that of the published best enhancer, citrate. The Zn2+ coordinated carboxylate results in loss of the enhancement ability of the carboxylate on antifreeze activity. There is not an additional increase in TH when a weaker enhancer is added to a stronger enhancer solution. These observations suggest that the more carboxylate groups per enhancer molecule the better the efficiency of the enhancer and that the freedom of motion of these molecules is necessary for them to serve as enhancers for AFP. The hydroxyl groups in the enhancer molecules can also positively affect their TH enhancement efficiency, though not as strongly as carboxylate groups. Mechanisms are discussed. PMID:18620083

  13. Accumulated p53 protein and UVA protection level of sunscreens.

    PubMed

    Seité, S; Moyal, D; Verdier, M P; Hourseau, C; Fourtanier, A

    2000-02-01

    Nuclear p53 expression is a sensitive parameter for the detection of ultraviolet (UV)-induced skin damage, and it has been used as an endpoint to evaluate the effectiveness of sunscreens. In this study, we compared the protection provided by two sunscreens having identical sun protection factors (SPF) but different UVA protection factors (UVA-PF) measured by the persistent pigment darkening method (PPD). The SPF of the sunscreens was 7 and the UVA-PF were respectively 7 and 3. Nuclear p53 protein was quantified in human skin biopsies treated with sunscreens and exposed 8 times to 5 MED of solar simulated radiation (SSR). The results showed that both sunscreens offered only partial protection against the increased expression of nuclear p53 protein induced by repetitive SSR exposures. However, a significantly lower level of p53-positive cells was found in areas protected with the sunscreen having the higher UVA-PF compared to the other sunscreen protected areas. In order to verify whether the difference in efficacy of these products was due to the difference in UVA absorption capacity, we quantified epidermal p53 protein accumulation after 8 exposures to either UVA (320-400 nm) or UVA1 (340-400 nm). We showed that as with SSR, repetitive exposures to 12.5 and 25 J/cm2 of UVA or UVA1 induced a significant increase in p53-positive cells in the human epidermis. These results confirmed that SPF determined on the basis of an acute erythemal reaction does not predict the level of protection against cumulative damage. They also showed that the protection provided by two sunscreens with different UVA protection factors is different (based on nuclear p53 protein accumulation), and that the PPD method can distinguish varying levels of sunscreen efficacy against UVA-induced cell damage. PMID:10721857

  14. Effect of protein level and protein source on zinc absorption in humans

    SciTech Connect

    Sandstroem, B.A.; Almgren, A.; Kivistoe, B.C.; Cederblad, A.

    1989-01-01

    The effect of increasing levels of various protein sources on zinc absorption from a legume-based meal was studied in humans with the use of a radionuclide technique. The meals were extrinsically labelled with 65Zn and absorption was determined from measurements of the whole-body retention of the isotope. The mean fractional zinc absorption for the 13 meals was 24.7 +/- 6.9% and was only influenced by the protein content of the meal to a limited extent (r = 0.45). However, the amount of zinc absorbed from the meals was strongly correlated with both the protein (r = 0.85) and zinc content (r = 0.86): 5.9 +/- 1.7 mumol of zinc was absorbed from the basal bean meal which had the lowest protein content; the addition of low zinc chicken doubled the protein content and increased zinc absorption to 10.3 +/- 2.0 mumol; the addition of zinc-rich beef also doubled the protein content, however, zinc absorption was increased to 15.9 +/- 4.7 mumol. It is concluded that the zinc content of the main protein source of the diet determines the amount of zinc absorbed to a large extent. However, relatively small amounts of animal protein can significantly improve the value of a legume-based meal as a source of zinc.

  15. Ascertaining effects of nanoscale polymeric interfaces on competitive protein adsorption at the individual protein level.

    PubMed

    Song, Sheng; Xie, Tian; Ravensbergen, Kristina; Hahm, Jong-in

    2016-02-14

    With the recent development of biomaterials and biodevices with reduced dimensionality, it is critical to comprehend protein adhesion processes to nanoscale solid surfaces, especially those occurring in a competitive adsorption environment. Complex sequences of adhesion events in competitive adsorption involving multicomponent protein systems have been extensively investigated, but our understanding is still limited primarily to macroscopic adhesion onto chemically simple surfaces. We examine the competitive adsorption behavior from a binary protein mixture containing bovine serum albumin and fibrinogen at the single protein level. We subsequently evaluate a series of adsorption and displacement processes occurring on both the macroscopic homopolymer and nanoscopic diblock copolymer surfaces, while systematically varying the protein concentration and incubation time. We identify the similarities and dissimilarities in competitive protein adsorption behavior between the two polymeric surfaces, the former presenting chemical uniformity at macroscale versus the latter exhibiting periodic nanointerfaces of chemically alternating polymeric segments. We then present our novel experimental finding of a large increase in the nanointerface-engaged residence time of the initially bound proteins and further explain the origin of this phenomenon manifested on nanoscale diblock copolymer surfaces. The outcomes of this study may provide timely insight into nanoscale competitive protein adsorption that is much needed in designing bioimplant and tissue engineering materials. In addition, the fundamental understanding gained from this study can be beneficial for the development of highly miniaturized biodevices and biomaterials fabricated by using nanoscale polymeric materials and interfaces.

  16. Influence of ACE I/D Polymorphism on Circulating Levels of Plasminogen Activator Inhibitor 1, D-Dimer, Ultrasensitive C-Reactive Protein and Transforming Growth Factor β1 in Patients Undergoing Hemodialysis

    PubMed Central

    de Carvalho, Sara Santos; Simões e Silva, Ana Cristina; Sabino, Adriano de Paula; Evangelista, Fernanda Cristina Gontijo; Gomes, Karina Braga; Dusse, Luci Maria SantAna; Rios, Danyelle Romana Alves

    2016-01-01

    Background There is substantial evidence that chronic renal and cardiovascular diseases are associated with coagulation disorders, endothelial dysfunction, inflammation and fibrosis. Angiotensin-Converting Enzyme Insertion/Deletion polymorphism (ACE I/D polymorphism) has also be linked to cardiovascular diseases. Therefore, this study aimed to compare plasma levels of ultrassensible C-reactive protein (usCRP), PAI-1, D-dimer and TGF-β1 in patients undergoing HD with different ACE I/D polymorphisms. Methods The study was performed in 138 patients at ESRD under hemodialysis therapy for more than six months. The patients were divided into three groups according to the genotype. Genomic DNA was extracted from blood cells (leukocytes). ACE I/D polymorphism was investigated by single polymerase chain reaction (PCR). Plasma levels of D-dimer, PAI-1 and TGF-β1 were measured by enzyme-linked immunosorbent assay (ELISA), and the determination of plasma levels of usCRP was performed by immunonephelometry. Data were analyzed by the software SigmaStat 2.03. Results Clinical characteristics were similar in patients with these three ACE I/D polymorphisms, except for interdialytic weight gain. I allele could be associated with higher interdialytic weight gain (P = 0.017). Patients genotyped as DD and as ID had significantly higher levels of PAI-1 than those with II genotype. Other laboratory parameters did not significantly differ among the three subgroups (P = 0.033). Despite not reaching statistical significance, plasma levels of usCRP were higher in patients carrying the D allele. Conclusion ACE I/D polymorphisms could be associated with changes in the regulation of sodium, fibrinolytic system, and possibly, inflammation. Our data showed that high levels of PAI-1 are detected when D allele is present, whereas greater interdialytic gain is associated with the presence of I allele. However, further studies with different experimental designs are necessary to elucidate the

  17. Genipin Suppresses NLRP3 Inflammasome Activation Through Uncoupling Protein-2

    PubMed Central

    Rajanbabu, Venugopal; Galam, Lakshmi; Fukumoto, Jutaro; Enciso, Juan; Tadikonda, Pratima; Lane, Troy N.; Bandyopadhyay, Sayantani; Parthasarathy, Prasanna Tamarapu; Cho, Young; Cho, Seong Ho; Lee, Yong Chul; Lockey, Richard F.; Kolliputi, Narasaiah

    2015-01-01

    Incomplete clearance of apoptotic cells and reactive oxygen species (ROS) release are known to trigger inflammasome activation causing severe inflammation in acute lung injury and various metabolic and autoimmune diseases. Moreover, it has been reported that apoptotic cell clearance and ROS-mediated apoptosis critically depend on mitochondrial uncoupling protein-2 (UCP2). However, the relationship between UCP2 and inflammasome activation has not been studied. This report investigates the role of UCP2 in the expression and activation of NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome in human macrophages. We found that UCP2 overexpression significantly enhanced the expression levels of NLRP3. The NLRP3 expression levels were significantly suppressed in THP1 cells treated with genipin, a UCP2 inhibitor, compared to controls. In addition, genipin altered adenosine triphosphate (ATP)- and hydrogen peroxide (H2O2)-mediated interleukin-1 beta (IL-1β) secretion and significantly suppressed caspase-1 activity in inflammasome-activated human macrophages. Taken together, our results suggest that genipin modulates NLRP3 inflammasome activation and ATP- or H2O2-mediated IL-1β release. PMID:26123077

  18. [Protein kinase C activation induces platelet apoptosis].

    PubMed

    Zhao, Li-Li; Chen, Meng-Xing; Zhang, Ming-Yi; Dai, Ke-Sheng

    2013-10-01

    Platelet apoptosis elucidated by either physical or chemical compound or platelet storage occurs wildly, which might play important roles in controlling the numbers and functions of circulated platelets, or in the development of some platelet-related diseases. However, up to now, a little is known about the regulatory mechanisms of platelet apoptosis. Protein kinase C (PKC) is highly expressed in platelets and plays central roles in regulating platelet functions. Although there is evidence indicating that PKC is involved in the regulation of apoptosis of nucleated cells, it is still unclear whether PKC plays a role in platelet apoptosis. The aim of this study was to investigate the role of PKC in platelet apoptosis. The effects of PKC on mitochondrial membrane potential (ΔΨm), phosphatidylserine (PS) exposure, and caspase-3 activation of platelets were analyzed by flow cytometry and Western blot. The results showed that the ΔΨm depolarization in platelets was induced by PKC activator in time-dependent manner, and the caspase-3 activation in platelets was induced by PKC in concentration-dependent manner. However, the platelets incubated with PKC inhibitor did not results in ΔΨm depolarization and PS exposure. It is concluded that the PKC activation induces platelet apoptosis through influencing the mitochondrial functions and activating caspase 3. The finds suggest a novel mechanism for PKC in regulating platelet numbers and functions, which has important pathophysiological implications for thrombosis and hemostasis.

  19. Dysregulation of TAp63 mRNA and protein levels in psoriasis.

    PubMed

    Gu, Xiaolian; Lundqvist, Elisabet N; Coates, Philip J; Thurfjell, Niklas; Wettersand, Emma; Nylander, Karin

    2006-01-01

    Psoriasis is a chronic and excessive inflammation of the skin and is currently incurable. The cause of psoriasis remains poorly understood and a central and cooperative role for keratinocytes and T-cells in triggering the disease is highlighted. The p63 gene encodes six different proteins with homology to the tumor suppressor protein p53 that are crucial for normal development of ectodermally derived structures such as skin and oral mucosa. In this study, we have analyzed levels of the different p63 isoforms using quantitative reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry in 15 patients diagnosed with psoriasis. Quantitative RT-PCR results showed downregulation of the full-length TAp63 in psoriatic lesions compared to both clinically normal skin from patients (P<0.001) and matched healthy controls (P<0.001); however, p63 protein levels detected by immunohistochemistry were similar. All psoriasis lesions also had detectable levels of activated Stat3, a protein indicated in development of the disease, whereas control tissue lacked this protein. The present data show a different regulation of TAp63 in psoriasis, where the discrepancy between mRNA levels and protein expression indicates a post-transcriptional regulation analogous to that seen in p53.

  20. Physical activity and high-sensitivity C-reactive protein.

    PubMed

    Plaisance, Eric P; Grandjean, Peter W

    2006-01-01

    Cardiovascular disease (CVD) remains one of the leading causes of death and disability in developed countries around the world despite the documented success of lifestyle and pharmacological interventions. This illustrates the multifactorial nature of atherosclerosis and the use of novel inflammatory markers as an adjunct to risk factor reduction strategies. As evidence continues to accumulate that inflammation is involved in all stages of the development and progression of atherosclerosis, markers of inflammation such as high-sensitivity C-reactive protein (CRP) may provide additional information regarding the biological status of the atherosclerotic lesion. Recent investigations suggest that physical activity reduces CRP levels. Higher levels of physical activity and cardiorespiratory fitness are consistently associated with 6-35% lower CRP levels. Longitudinal training studies that have demonstrated reductions in CRP concentrations range from 16% to 41%, an effect that may be independent of baseline levels of CRP, body composition or weight loss. The average change in CRP associated with physical activity appears to be at least as good, if not better, than currently prescribed pharmacological interventions in similar populations. The primary purpose of this review will be to present evidence from both cross-sectional and longitudinal investigations that physical activity lowers CRP levels in a dose-response manner. Finally, this review will examine factors such as body composition, sex, blood sample timing, diet and smoking, which may influence the CRP response to physical activity. PMID:16646631

  1. Classification of G-protein coupled receptors at four levels.

    PubMed

    Gao, Qing-Bin; Wang, Zheng-Zhi

    2006-11-01

    G-protein coupled receptors (GPCRs) are transmembrane proteins which via G-proteins initiate some of the important signaling pathways in a cell and are involved in various physiological processes. Thus, computational prediction and classification of GPCRs can supply significant information for the development of novel drugs in pharmaceutical industry. In this paper, a nearest neighbor method has been introduced to discriminate GPCRs from non-GPCRs and subsequently classify GPCRs at four levels on the basis of amino acid composition and dipeptide composition of proteins. Its performance is evaluated on a non-redundant dataset consisted of 1406 GPCRs for six families and 1406 globular proteins using the jackknife test. The present method based on amino acid composition achieved an overall accuracy of 96.4% and Matthew's correlation coefficient (MCC) of 0.930 for correctly picking out the GPCRs from globular proteins. The overall accuracy and MCC were further enhanced to 99.8% and 0.996 by dipeptide composition-based method. On the other hand, the present method has successfully classified 1406 GPCRs into six families with an overall accuracy of 89.6 and 98.8% using amino acid composition and dipeptide composition, respectively. For the subfamily prediction of 1181 GPCRs of rhodopsin-like family, the present method achieved an overall accuracy of 76.7 and 94.5% based on the amino acid composition and dipeptide composition, respectively. Finally, GPCRs belonging to the amine subfamily and olfactory subfamily of rhodopsin-like family were further analyzed at the type level. The overall accuracy of dipeptide composition-based method for the classification of amine type and olfactory type of GPCRs reached 94.5 and 86.9%, respectively, while the overall accuracy of amino acid composition-based method was very low for both subfamilies. In comparison with existing methods in the literature, the present method also displayed great competitiveness. These results demonstrate

  2. Redox Control of Protein Arginine Methyltransferase 1 (PRMT1) Activity*

    PubMed Central

    Morales, Yalemi; Nitzel, Damon V.; Price, Owen M.; Gui, Shanying; Li, Jun; Qu, Jun; Hevel, Joan M.

    2015-01-01

    Elevated levels of asymmetric dimethylarginine (ADMA) correlate with risk factors for cardiovascular disease. ADMA is generated by the catabolism of proteins methylated on arginine residues by protein arginine methyltransferases (PRMTs) and is degraded by dimethylarginine dimethylaminohydrolase. Reports have shown that dimethylarginine dimethylaminohydrolase activity is down-regulated and PRMT1 protein expression is up-regulated under oxidative stress conditions, leading many to conclude that ADMA accumulation occurs via increased synthesis by PRMTs and decreased degradation. However, we now report that the methyltransferase activity of PRMT1, the major PRMT isoform in humans, is impaired under oxidative conditions. Oxidized PRMT1 displays decreased activity, which can be rescued by reduction. This oxidation event involves one or more cysteine residues that become oxidized to sulfenic acid (-SOH). We demonstrate a hydrogen peroxide concentration-dependent inhibition of PRMT1 activity that is readily reversed under physiological H2O2 concentrations. Our results challenge the unilateral view that increased PRMT1 expression necessarily results in increased ADMA synthesis and demonstrate that enzymatic activity can be regulated in a redox-sensitive manner. PMID:25911106

  3. Protein kinase C controls activation of the DNA integrity checkpoint

    PubMed Central

    Soriano-Carot, María; Quilis, Inma; Bañó, M. Carmen; Igual, J. Carlos

    2014-01-01

    The protein kinase C (PKC) superfamily plays key regulatory roles in numerous cellular processes. Saccharomyces cerevisiae contains a single PKC, Pkc1, whose main function is cell wall integrity maintenance. In this work, we connect the Pkc1 protein to the maintenance of genome integrity in response to genotoxic stresses. Pkc1 and its kinase activity are necessary for the phosphorylation of checkpoint kinase Rad53, histone H2A and Xrs2 protein after deoxyribonucleic acid (DNA) damage, indicating that Pkc1 is required for activation of checkpoint kinases Mec1 and Tel1. Furthermore, Pkc1 electrophoretic mobility is delayed after inducing DNA damage, which reflects that Pkc1 is post-translationally modified. This modification is a phosphorylation event mediated by Tel1. The expression of different mammalian PKC isoforms at the endogenous level in yeast pkc1 mutant cells revealed that PKCδ is able to activate the DNA integrity checkpoint. Finally, downregulation of PKCδ activity in HeLa cells caused a defective activation of checkpoint kinase Chk2 when DNA damage was induced. Our results indicate that the control of the DNA integrity checkpoint by PKC is a mechanism conserved from yeast to humans. PMID:24792164

  4. Xylazine Activates Adenosine Monophosphate-Activated Protein Kinase Pathway in the Central Nervous System of Rats.

    PubMed

    Shi, Xing-Xing; Yin, Bai-Shuang; Yang, Peng; Chen, Hao; Li, Xin; Su, Li-Xue; Fan, Hong-Gang; Wang, Hong-Bin

    2016-01-01

    Xylazine is a potent analgesic extensively used in veterinary and animal experimentation. Evidence exists that the analgesic effect can be inhibited using adenosine 5'-monophosphate activated protein kinase (AMPK) inhibitors. Considering this idea, the aim of this study was to investigate whether the AMPK signaling pathway is involved in the central analgesic mechanism of xylazine in the rat. Xylazine was administrated via the intraperitoneal route. Sprague-Dawley rats were sacrificed and the cerebral cortex, cerebellum, hippocampus, thalamus and brainstem were collected for determination of liver kinase B1 (LKB1) and AMPKα mRNA expression using quantitative real-time polymerase chain reaction (qPCR), and phosphorylated LKB1 and AMPKα levels using western blot. The results of our study showed that compared with the control group, xylazine induced significant increases in AMPK activity in the cerebral cortex, hippocampus, thalamus and cerebellum after rats received xylazine (P < 0.01). Increased AMPK activities were accompanied with increased phosphorylation levels of LKB1 in corresponding regions of rats. The protein levels of phosphorylated LKB1 and AMPKα in these regions returned or tended to return to control group levels. However, in the brainstem, phosphorylated LKB1 and AMPKα protein levels were decreased by xylazine compared with the control (P < 0.05). In conclusion, our data indicates that xylazine alters the activities of LKB1 and AMPK in the central nervous system of rats, which suggests that xylazine affects the regulatory signaling pathway of the analgesic mechanism in the rat brain. PMID:27049320

  5. Proteasome-mediated degradation antagonizes critical levels of the apoptosis-inducing C1D protein

    PubMed Central

    Rothbarth, Karsten; Stammer, Hermann; Werner, Dieter

    2002-01-01

    The C1D gene is expressed in a broad spectrum of mammalian cells and tissues but its product induces apoptotic cell death when exceeding a critical level. Critical levels are achieved in a fraction of cells by transient transfection with EGFP-tagged C1D expression constructs. However, transfected cells expressing sub-critical levels of C1D(EGFP) escape apoptotic cell death by activation of a proteasome-mediated rescue mechanism. Inhibition of the proteasome-dependent degradation of the C1D(EGFP) protein results in a parallel increase of the intracellular C1D level and in the fraction of apoptotic cells. PMID:12379155

  6. Relationship between protein C antigen and anticoagulant activity during oral anticoagulation and in selected disease states.

    PubMed Central

    Vigano D'Angelo, S; Comp, P C; Esmon, C T; D'Angelo, A

    1986-01-01

    Protein C is a natural vitamin K-dependent plasma anticoagulant, deficiencies of which have been found in patients with recurrent thrombosis and warfarin-induced skin necrosis. To appreciate more fully the role of protein C in disease states and during oral anticoagulation, a new functional assay for protein C involving adsorption of plasma protein C on a Ca+2-dependent monoclonal antibody, elution, quantitative activation, and assessment of plasma anticoagulant activity, has been developed. When oral anticoagulation is initiated, the anticoagulant activity of protein C decreases to a greater extent than either the amidolytic or immunologic levels. During stabilized warfarin treatment, there is no correlation between either amidolytic or antigenic levels and the functional protein C activity, suggesting that measurement of protein C anticoagulant activity may be necessary to reflect adequately the anticoagulant protection afforded by this protein. In contrast, there was a strong correlation between anticoagulant and amidolytic and immunologic levels in liver failure and disseminated intravascular coagulation. Two patients with thromboembolic disease have been identified who exhibit a marked decrease in anticoagulant activity, but who have normal immunologic and amidolytic levels. Thus, this assay permits assessment of protein C in individuals who have received anticoagulant treatment and identification of a new class of protein C-deficient individuals. PMID:3511097

  7. Receptor activity-modifying proteins; multifunctional G protein-coupled receptor accessory proteins.

    PubMed

    Hay, Debbie L; Walker, Christopher S; Gingell, Joseph J; Ladds, Graham; Reynolds, Christopher A; Poyner, David R

    2016-04-15

    Receptor activity-modifying proteins (RAMPs) are single pass membrane proteins initially identified by their ability to determine the pharmacology of the calcitonin receptor-like receptor (CLR), a family B G protein-coupled receptor (GPCR). It is now known that RAMPs can interact with a much wider range of GPCRs. This review considers recent developments on the structure of the complexes formed between the extracellular domains (ECDs) of CLR and RAMP1 or RAMP2 as these provide insights as to how the RAMPs direct ligand binding. The range of RAMP interactions is also considered; RAMPs can interact with numerous family B GPCRs as well as examples of family A and family C GPCRs. They influence receptor expression at the cell surface, trafficking, ligand binding and G protein coupling. The GPCR-RAMP interface offers opportunities for drug targeting, illustrated by examples of drugs developed for migraine. PMID:27068971

  8. Pyrrolopyridine inhibitors of mitogen-activated protein kinase-activated protein kinase 2 (MK-2).

    PubMed

    Anderson, David R; Meyers, Marvin J; Vernier, William F; Mahoney, Matthew W; Kurumbail, Ravi G; Caspers, Nicole; Poda, Gennadiy I; Schindler, John F; Reitz, David B; Mourey, Robert J

    2007-05-31

    A new class of potent kinase inhibitors selective for mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP-K2 or MK-2) for the treatment of rheumatoid arthritis has been prepared and evaluated. These inhibitors have IC50 values as low as 10 nM against the target and have good selectivity profiles against a number of kinases including CDK2, ERK, JNK, and p38. These MK-2 inhibitors have been shown to suppress TNFalpha production in U397 cells and to be efficacious in an acute inflammation model. The structure-activity relationships of this series, the selectivity for MK-2 and their activity in both in vitro and in vivo models are discussed. The observed selectivity is discussed with the aid of an MK-2/inhibitor crystal structure.

  9. An inducible expression system for high-level expression of recombinant proteins in slow growing mycobacteria.

    PubMed

    Leotta, Lisa; Spratt, Joanne M; Kong, Carlyn U; Triccas, James A

    2015-09-01

    A novel protein expression vector utilising the inducible hspX promoter of Mycobacterium tuberculosis was constructed and evaluated in this study. High-level induction of three mycobacterial antigens, comprising up to 9% of bacterial sonicate, was demonstrated in recombinant Mycobacterium bovis BCG when grown under low-oxygen tension, which serves to enhance hspX promoter activity. Recombinant proteins were efficiently purified from bacterial lysates in a soluble form by virtue of a C-terminal 6-histidine tag. Purification of the immunodominant M. tuberculosis Ag85B antigen using this system resulted in a recombinant protein that stimulated significant IFN-γ release from Ag85B-reactive T cells generated after vaccination of mice with an Ag85B-expressing vaccine. Further, the M. tuberculosis L-alanine dehydrogenase (Ald) protein purified from recombinant BCG displayed strong enzymatic activity in recombinant form. This study demonstrated that high levels of native-like recombinant mycobacterial proteins can be produced in mycobacterial hosts, and this may aid the analysis of mycobacterial protein function and the development of new treatments. PMID:26021569

  10. Activation of G protein by opioid receptors: role of receptor number and G-protein concentration.

    PubMed

    Remmers, A E; Clark, M J; Alt, A; Medzihradsky, F; Woods, J H; Traynor, J R

    2000-05-19

    The collision-coupling model for receptor-G-protein interaction predicts that the rate of G-protein activation is dependent on receptor density, but not G-protein levels. C6 cells expressing mu- or delta-opioid receptors, or SH-SY5Y cells, were treated with beta-funaltrexamine (mu) or naltrindole-5'-isothiocyanate (delta) to decrease receptor number. The time course of full or partial agonist-stimulated ¿35SGTPgammaS binding did not vary in C6 cell membranes containing <1-25 pmol/mg mu-opioid receptor, or 1. 4-4.3 pmol/mg delta-opioid receptor, or in SHSY5Y cells containing 0. 16-0.39 pmol/mg receptor. The association of ¿35SGTPgammaS binding was faster in membranes from C6mu cells than from C6delta cells. A 10-fold reduction in functional G-protein, following pertussis toxin treatment, lowered the maximal level of ¿35SGTPgammaS binding but not the association rate. These data indicate a compartmentalization of opioid receptors and G protein at the cell membrane. PMID:10822058

  11. The total protein content, protein fractions and proteases activities of drone prepupae of Apis mellifera due to varrosis.

    PubMed

    Zółtowska, Krystyna; Lipiński, Zbigniew; Dmitryjuk, Małgorzata

    2005-01-01

    The proteins level and activities of acid and alkaline proteases in whole body extracts of drone prepupae of Apis mellifera naturally infested with Varroa destructor were studied. The infested and a non-infested group did not differ significantly in their total protein content. However, some differences in protein profiles were found. A lack of three protein fractions of moderate and lower molecular weight in infested prepupae was noted. Moreover, some differences in the quantity of protein in most of the fractions were observed. The activity of acid proteases from infested prepupae was lower (p < 0.05) compared with the activity of these proteases from the non-infested one group. The infested drone had higher activity of alkaline proteases than non-infested but this difference was not statisticaly significant.

  12. The total protein content, protein fractions and proteases activities of drone prepupae of Apis mellifera due to varrosis.

    PubMed

    Zółtowska, Krystyna; Lipiński, Zbigniew; Dmitryjuk, Małgorzata

    2005-01-01

    The proteins level and activities of acid and alkaline proteases in whole body extracts of drone prepupae of Apis mellifera naturally infested with Varroa destructor were studied. The infested and a non-infested group did not differ significantly in their total protein content. However, some differences in protein profiles were found. A lack of three protein fractions of moderate and lower molecular weight in infested prepupae was noted. Moreover, some differences in the quantity of protein in most of the fractions were observed. The activity of acid proteases from infested prepupae was lower (p < 0.05) compared with the activity of these proteases from the non-infested one group. The infested drone had higher activity of alkaline proteases than non-infested but this difference was not statisticaly significant. PMID:16841690

  13. Ascertaining effects of nanoscale polymeric interfaces on competitive protein adsorption at the individual protein level

    NASA Astrophysics Data System (ADS)

    Song, Sheng; Xie, Tian; Ravensbergen, Kristina; Hahm, Jong-In

    2016-02-01

    With the recent development of biomaterials and biodevices with reduced dimensionality, it is critical to comprehend protein adhesion processes to nanoscale solid surfaces, especially those occurring in a competitive adsorption environment. Complex sequences of adhesion events in competitive adsorption involving multicomponent protein systems have been extensively investigated, but our understanding is still limited primarily to macroscopic adhesion onto chemically simple surfaces. We examine the competitive adsorption behavior from a binary protein mixture containing bovine serum albumin and fibrinogen at the single protein level. We subsequently evaluate a series of adsorption and displacement processes occurring on both the macroscopic homopolymer and nanoscopic diblock copolymer surfaces, while systematically varying the protein concentration and incubation time. We identify the similarities and dissimilarities in competitive protein adsorption behavior between the two polymeric surfaces, the former presenting chemical uniformity at macroscale versus the latter exhibiting periodic nanointerfaces of chemically alternating polymeric segments. We then present our novel experimental finding of a large increase in the nanointerface-engaged residence time of the initially bound proteins and further explain the origin of this phenomenon manifested on nanoscale diblock copolymer surfaces. The outcomes of this study may provide timely insight into nanoscale competitive protein adsorption that is much needed in designing bioimplant and tissue engineering materials. In addition, the fundamental understanding gained from this study can be beneficial for the development of highly miniaturized biodevices and biomaterials fabricated by using nanoscale polymeric materials and interfaces.With the recent development of biomaterials and biodevices with reduced dimensionality, it is critical to comprehend protein adhesion processes to nanoscale solid surfaces, especially those

  14. Nestling activity levels during begging behaviour predicts activity level and body mass in adulthood

    PubMed Central

    Griffith, Simon C.

    2014-01-01

    Across a range of species including humans, personality traits, or differences in behaviour between individuals that are consistent over time, have been demonstrated. However, few studies have measured whether these consistent differences are evident in very young animals, and whether they persist over an individual’s entire lifespan. Here we investigated the begging behaviour of very young cross-fostered zebra finch nestlings and the relationship between that and adult activity levels. We found a link between the nestling activity behaviour head movements during begging, measured at just five and seven days after hatching, and adult activity levels, measured when individuals were between three and three and a half years old. Moreover, body mass was found to be negatively correlated with both nestling and adult activity levels, suggesting that individuals which carry less body fat as adults are less active both as adults and during begging as nestlings. Our work suggests that the personality traits identified here in both very young nestlings and adults may be linked to physiological factors such as metabolism or environmental sources of variation. Moreover, our work suggests it may be possible to predict an individual’s future adult personality at a very young age, opening up new avenues for future work to explore the relationship between personality and a number of aspects of individual life history and survival. PMID:25279258

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

    PubMed

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

    2015-07-01

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

  16. Detection of expansin proteins and activity during tomato fruit ontogeny.

    PubMed

    Rose, J K; Cosgrove, D J; Albersheim, P; Darvill, A G; Bennett, A B

    2000-08-01

    Expansins are plant proteins that have the capacity to induce extension in isolated cell walls and are thought to mediate pH-dependent cell expansion. J.K.C. Rose, H.H. Lee, and A.B. Bennett ([1997] Proc Natl Acad Sci USA 94: 5955-5960) reported the identification of an expansin gene (LeExp1) that is specifically expressed in ripening tomato (Lycopersicon esculentum) fruit where cell wall disassembly, but not cell expansion, is prominent. Expansin expression during fruit ontogeny was examined using antibodies raised to recombinant LeExp1 or a cell elongation-related expansin from cucumber (CsExp1). The LeExp1 antiserum detected expansins in extracts from ripe, but not preripe tomato fruit, in agreement with the pattern of LeExp1 mRNA accumulation. In contrast, antibodies to CsExp1 cross-reacted with expansins in early fruit development and the onset of ripening, but not at a later ripening stage. These data suggest that ripening-related and expansion-related expansin proteins have distinct antigenic epitopes despite overall high sequence identity. Expansin proteins were detected in a range of fruit species and showed considerable variation in abundance; however, appreciable levels of expansin were not present in fruit of the rin or Nr tomato mutants that exhibit delayed and reduced softening. LeExp1 protein accumulation was ethylene-regulated and matched the previously described expression of mRNA, suggesting that expression is not regulated at the level of translation. We report the first detection of expansin activity in several stages of fruit development and while characteristic creep activity was detected in young and developing tomato fruit and in ripe pear, avocado, and pepper, creep activity in ripe tomato showed qualitative differences, suggesting both hydrolytic and expansin activities.

  17. Identification of maize embryo-preferred promoters suitable for high-level heterologous protein production.

    PubMed

    Streatfield, Stephen J; Bray, Jeffrey; Love, Robert T; Horn, Michael E; Lane, Jeffrey R; Drees, Carol F; Egelkrout, Erin M; Howard, John A

    2010-01-01

    The production of heterologous proteins in plants at levels consistent with commercialization of protein products requires molecular tools to ensure high-level transgene expression. The identification of strong promoters, preferably specific to the target expression tissue, is a focus for improving foreign protein yields using transgenic cereals as a production system. Thus, there is a requirement for strong embryo preferred monocot promoters. We obtained the sequences of 500 randomly selected maize cDNA clones to determine gene expression profiles in embryo tissues at multiple stages during development. Promoters corresponding to the most abundant clones were identified and isolated. These promoters were fused to the b-glucuronidase reporter and their tissue specificity and developmental expression characteristics assessed in transgenic maize. All of the isolated promoters tested drove transgene expression predominantly in the embryo and were most active late in embryogenesis during storage protein deposition. One of the most active promoters assessed by transgene expression was associated with the globulin-1 protein. Sequence identified here extended approximately 1.6 kb distal to the previously identified extent of the globulin-1 promoter, and this additional sequence boosted expression over two-fold. The extended globulin-1 promoter sequence isolated in this study has the potential for driving transgene expression at higher levels than those previously reported for cereals. Also, other highly active embryo promoters identified here offer opportunities to express multiple foreign proteins simultaneously at high levels in embryo tissues, while avoiding concerns over gene silencing due to the repeated use of a single promoter. PMID:21844671

  18. Characterization of protein expression levels with label-free detected reverse phase protein arrays.

    PubMed

    Guo, Xuexue; Deng, Yihong; Zhu, Chenggang; Cai, Junlong; Zhu, Xiangdong; Landry, James P; Zheng, Fengyun; Cheng, Xunjia; Fei, Yiyan

    2016-09-15

    In reverse-phase protein arrays (RPPA), one immobilizes complex samples (e.g., cellular lysate, tissue lysate or serum etc.) on solid supports and performs parallel reactions of antibodies with immobilized protein targets from the complex samples. In this work, we describe a label-free detection of RPPA that enables quantification of RPPA data and thus facilitates comparison of studies performed on different samples and on different solid supports. We applied this detection platform to characterization of phosphoserine aminotransferase (PSAT) expression levels in Acanthamoeba lysates treated with artemether and the results were confirmed by Western blot studies. PMID:27372609

  19. Arabinogalactan proteins: focus on carbohydrate active enzymes

    PubMed Central

    Knoch, Eva; Dilokpimol, Adiphol; Geshi, Naomi

    2014-01-01

    Arabinogalactan proteins (AGPs) are a highly diverse class of cell surface proteoglycans that are commonly found in most plant species. AGPs play important roles in many cellular processes during plant development, such as reproduction, cell proliferation, pattern formation and growth, and in plant-microbe interaction. However, little is known about the molecular mechanisms of their function. Numerous studies using monoclonal antibodies that recognize different AGP glycan epitopes have shown the appearance of a slightly altered AGP glycan in a specific stage of development in plant cells. Therefore, it is anticipated that the biosynthesis and degradation of AGP glycan is tightly regulated during development. Until recently, however, little was known about the enzymes involved in the metabolism of AGP glycans. In this review, we summarize recent discoveries of carbohydrate active enzymes (CAZy; http://www.cazy.org/) involved in the biosynthesis and degradation of AGP glycans, and we discuss the biological role of these enzymes in plant development. PMID:24966860

  20. Abscisic Acid Induces Mitogen-Activated Protein Kinase Activation in Barley Aleurone Protoplasts.

    PubMed

    Knetsch, MLW.; Wang, M.; Snaar-Jagalska, B. E.; Heimovaara-Dijkstra, S.

    1996-06-01

    Abscisic acid (ABA) induces a rapid and transient mitogen-activated protein (MAP) kinase activation in barley aleurone protoplasts. MAP kinase activity, measured as myelin basic protein phosphorylation by MAP kinase immunoprecipitates, increased after 1 min, peaked after 3 min, and decreased to basal levels after ~5 min of ABA treatment in vivo. Antibodies recognizing phosphorylated tyrosine residues precipitate with myelin basic protein kinase activity that has identical ABA activation characteristics and demonstrate that tyrosine phosphorylation of MAP kinase occurs during activation. The half-maximal concentration of ABA required for MAP kinase activation, 3 x 10-7 M, is very similar to that required for ABA-induced rab16 gene expression. The tyrosine phosphatase inhibitor phenylarsine oxide can completely block ABA-induced MAP kinase activation and rab16 gene expression. These results lead us to conclude that ABA activates MAP kinase via a tyrosine phosphatase and that these steps are a prerequisite for ABA induction of rab16 gene expression.

  1. Abscisic Acid Induces Mitogen-Activated Protein Kinase Activation in Barley Aleurone Protoplasts.

    PubMed Central

    Knetsch, MLW.; Wang, M.; Snaar-Jagalska, B. E.; Heimovaara-Dijkstra, S.

    1996-01-01

    Abscisic acid (ABA) induces a rapid and transient mitogen-activated protein (MAP) kinase activation in barley aleurone protoplasts. MAP kinase activity, measured as myelin basic protein phosphorylation by MAP kinase immunoprecipitates, increased after 1 min, peaked after 3 min, and decreased to basal levels after ~5 min of ABA treatment in vivo. Antibodies recognizing phosphorylated tyrosine residues precipitate with myelin basic protein kinase activity that has identical ABA activation characteristics and demonstrate that tyrosine phosphorylation of MAP kinase occurs during activation. The half-maximal concentration of ABA required for MAP kinase activation, 3 x 10-7 M, is very similar to that required for ABA-induced rab16 gene expression. The tyrosine phosphatase inhibitor phenylarsine oxide can completely block ABA-induced MAP kinase activation and rab16 gene expression. These results lead us to conclude that ABA activates MAP kinase via a tyrosine phosphatase and that these steps are a prerequisite for ABA induction of rab16 gene expression. PMID:12239411

  2. Differential Acute and Chronic Effects of Leptin on Hypothalamic Astrocyte Morphology and Synaptic Protein Levels

    PubMed Central

    García-Cáceres, Cristina; Fuente-Martín, Esther; Burgos-Ramos, Emma; Granado, Miriam; Frago, Laura M.; Barrios, Vicente; Horvath, Tamas

    2011-01-01

    Astrocytes participate in neuroendocrine functions partially through modulation of synaptic input density in the hypothalamus. Indeed, glial ensheathing of neurons is modified by specific hormones, thus determining the availability of neuronal membrane space for synaptic inputs, with the loss of this plasticity possibly being involved in pathological processes. Leptin modulates synaptic inputs in the hypothalamus, but whether astrocytes participate in this action is unknown. Here we report that astrocyte structural proteins, such as glial fibrillary acidic protein (GFAP) and vimentin, are induced and astrocyte morphology modified by chronic leptin administration (intracerebroventricular, 2 wk), with these changes being inversely related to modifications in synaptic protein densities. Similar changes in glial structural proteins were observed in adult male rats that had increased body weight and circulating leptin levels due to neonatal overnutrition (overnutrition: four pups/litter vs. control: 12 pups/litter). However, acute leptin treatment reduced hypothalamic GFAP levels and induced synaptic protein levels 1 h after administration, with no effect on vimentin. In primary hypothalamic astrocyte cultures leptin also reduced GFAP levels at 1 h, with an induction at 24 h, indicating a possible direct effect of leptin. Hence, one mechanism by which leptin may affect metabolism is by modifying hypothalamic astrocyte morphology, which in turn could alter synaptic inputs to hypothalamic neurons. Furthermore, the responses to acute and chronic leptin exposure are inverse, raising the possibility that increased glial activation in response to chronic leptin exposure could be involved in central leptin resistance. PMID:21343257

  3. Observation of microtubule-based motor protein activity.

    PubMed

    Sloboda, Roger D

    2015-02-01

    It is possible to detect the presence of motor proteins that have the ability to translocate particles along microtubules. The two procedures described here were developed to detect microtubule-dependent motor protein activity in cell lysates or of purified proteins. In the first procedure, latex beads bound to the putative motor protein are assayed for their ability to translocate along microtubules in an ATP-dependent fashion. If motor protein activity is present, it will bind to the beads and translocate them unidirectionally along the microtubules. In the second procedure, motor proteins induce microtubule gliding over a glass coverslip surface that is coated with active motor protein. Because the mass of a microtubule is negligible compared to that of a coverslip or slide, the microtubule glides over the glass surface when the surface is coated with active motor protein. Also included here are descriptions of assays designed to determine the directionality of movement of microtubule-based motor proteins. PMID:25646501

  4. Mys protein regulates protein kinase A activity by interacting with regulatory type Ialpha subunit during vertebrate development.

    PubMed

    Kotani, Tomoya; Iemura, Shun-ichiro; Natsume, Tohru; Kawakami, Koichi; Yamashita, Masakane

    2010-02-12

    During embryonic development, protein kinase A (PKA) plays a key role in cell fate specification by antagonizing the Hedgehog (Hh) signaling pathway. However, the mechanism by which PKA activity is regulated remains unknown. Here we show that the Misty somites (Mys) protein regulates the level of PKA activity during embryonic development in zebrafish. We isolate PKA regulatory type Ialpha subunit (Prkar1a) as a protein interacting with Mys by pulldown assay in HEK293 cells followed by mass spectrometry analysis. We show an interaction between endogenous Mys and Prkar1a in the zebrafish embryo. Mys binds to Prkar1a in its C terminus region, termed PRB domain, and activates PKA in vitro. Conversely, knockdown of Mys in zebrafish embryos results in reduction in PKA activity. We also show that knockdown of Mys induces ectopic activation of Hh target genes in the eyes, neural tube, and somites downstream of Smoothened, a protein essential for transduction of Hh signaling activity. The altered patterning of gene expression is rescued by activation of PKA. Together, our results reveal a molecular mechanism of regulation of PKA activity that is dependent on a protein-protein interaction and demonstrate that PKA activity regulated by Mys is indispensable for negative regulation of the Hh signaling pathway in Hh-responsive cells. PMID:20018846

  5. Honey bee protein atlas at organ-level resolution.

    PubMed

    Chan, Queenie W T; Chan, Man Yi; Logan, Michelle; Fang, Yuan; Higo, Heather; Foster, Leonard J

    2013-11-01

    Genome sequencing has provided us with gene lists but cannot tell us where and how their encoded products work together to support life. Complex organisms rely on differential expression of subsets of genes/proteins in organs and tissues, and, in concert, evolved to their present state as they function together to improve an organism's overall reproductive fitness. Proteomics studies of individual organs help us understand their basic functions, but this reductionist approach misses the larger context of the whole organism. This problem could be circumvented if all the organs in an organism were comprehensively studied by the same methodology and analyzed together. Using honey bees (Apis mellifera L.) as a model system, we report here an initial whole proteome of a complex organism, measuring 29 different organ/tissue types among the three honey bee castes: queen, drone, and worker. The data reveal that, e.g., workers have a heightened capacity to deal with environmental toxins and queens have a far more robust pheromone detection system than their nestmates. The data also suggest that workers altruistically sacrifice not only their own reproductive capacity but also their immune potential in favor of their queen. Finally, organ-level resolution of protein expression offers a systematic insight into how organs may have developed.

  6. Honey bee protein atlas at organ-level resolution.

    PubMed

    Chan, Queenie W T; Chan, Man Yi; Logan, Michelle; Fang, Yuan; Higo, Heather; Foster, Leonard J

    2013-11-01

    Genome sequencing has provided us with gene lists but cannot tell us where and how their encoded products work together to support life. Complex organisms rely on differential expression of subsets of genes/proteins in organs and tissues, and, in concert, evolved to their present state as they function together to improve an organism's overall reproductive fitness. Proteomics studies of individual organs help us understand their basic functions, but this reductionist approach misses the larger context of the whole organism. This problem could be circumvented if all the organs in an organism were comprehensively studied by the same methodology and analyzed together. Using honey bees (Apis mellifera L.) as a model system, we report here an initial whole proteome of a complex organism, measuring 29 different organ/tissue types among the three honey bee castes: queen, drone, and worker. The data reveal that, e.g., workers have a heightened capacity to deal with environmental toxins and queens have a far more robust pheromone detection system than their nestmates. The data also suggest that workers altruistically sacrifice not only their own reproductive capacity but also their immune potential in favor of their queen. Finally, organ-level resolution of protein expression offers a systematic insight into how organs may have developed. PMID:23878156

  7. Cullin-3 protein expression levels correlate with breast cancer progression

    PubMed Central

    Haagenson, Kelly K.; Tait, Larry; Wang, Juan; Shekhar, Malathy P.; Polin, Lisa; Chen, Wei; Wu, Gen Sheng

    2012-01-01

    Cullin-3 is a component of the Cullin-Ring ubiquitin ligase (CRL) family that plays an important role in mediating protein degradation. Deregulation of Cullin-3 expression has been observed in human cancers; however, a role for Cullin-3 in tumor progression has not been previously recognized. Using the MCF10DCIS.com human breast cancer xenograft model, we show that Cullin-3 is increasingly expressed during progression from comedo ductal carcinoma in situ (DCIS) to invasive carcinomas. Cullin-3 protein is not detected in early lesions but is noticeably increased in DCIS tumors and significantly overexpressed in invasive cancers. In experimental metastasis assays, high expression of Cullin-3 was observed in the lung site. Importantly, Cullin-3 staining is detected in human breast cancer tissues, not in normal breast tissues and its expression level positively correlates with tumor stage. These data suggest that Cullin-3 may play an important role in tumor progression from DCIS to invasive cancer and may serve as a biomarker for the diagnosis of aggressive breast cancer. PMID:22825334

  8. Protein inhibitor of activated STAT3 inhibits adipogenic gene expression

    SciTech Connect

    Deng Jianbei; Hua Kunjie; Caveney, Erica J.; Takahashi, Nobuyuki; Harp, Joyce B. . E-mail: jharp@unc.edu

    2006-01-20

    Protein inhibitor of activated STAT3 (PIAS3), a cytokine-induced repressor of signal transducer and activator of transcription 3 (STAT3) and a modulator of a broad array of nuclear proteins, is expressed in white adipose tissue, but its role in adipogenesis is not known. Here, we determined that PIAS3 was constitutively expressed in 3T3-L1 cells at all stages of adipogenesis. However, it translocated from the nucleus to the cytoplasm 4 days after induction of differentiation by isobutylmethylxanthine, dexamethasone, and insulin (MDI). In ob/ob mice, PIAS3 expression was increased in white adipose tissue depots compared to lean mice and was found in the cytoplasm of adipocytes. Overexpression of PIAS3 in differentiating preadipocytes, which localized primarily to the nucleus, inhibited mRNA level gene expression of adipogenic transcription factors C/EBP{alpha} and PPAR{gamma}, as well as their downstream target genes aP2 and adiponectin. PIAS3 also inhibited C/EBP{alpha} promoter activation mediated specifically by insulin, but not dexamethasone or isobutylmethylxanthine. Taken together, these data suggest that PIAS3 may play an inhibitory role in adipogenesis by modulating insulin-activated transcriptional activation events. Increased PIAS3 expression in adipose tissue may play a role in the metabolic disturbances of obesity.

  9. Job level risk assessment using task level ACGIH hand activity level TLV scores: a pilot study.

    PubMed

    Drinkaus, Phillip; Sesek, Richard; Bloswick, Donald S; Mann, Clay; Bernard, Thomas

    2005-01-01

    Existing upper extremity musculoskeletal disorder analytical tools are primarily intended for single or mono-task jobs. However, many jobs contain more than 1 task and some include job rotation. This case/control study investigates methods of modifying an existing tool, the American Conference of Governmental Industrial Hygienists (ACGIH) Hand Activity Level (HAL) Threshold Limit Value (TLV), to assess the upper extremity risk of multi-task jobs. Various methods of combining the task differences and ratios into a job level assessment were explored. Two methods returned significant odds ratios, (p < .05) of 18.0 (95% CI 1.8-172) and 12.0 (95% CI 1.2-120). These results indicate that a modified ACGIH HAL TLV may provide insight into the work-related risk of multi-task jobs. Further research is needed to optimize this process. PMID:16219155

  10. Role of Deleted in Breast Cancer 1 (DBC1) Protein in SIRT1 Deacetylase Activation Induced by Protein Kinase A and AMP-activated Protein Kinase*

    PubMed Central

    Nin, Veronica; Escande, Carlos; Chini, Claudia C.; Giri, Shailendra; Camacho-Pereira, Juliana; Matalonga, Jonathan; Lou, Zhenkun; Chini, Eduardo N.

    2012-01-01

    The NAD+-dependent deacetylase SIRT1 is a key regulator of several aspects of metabolism and aging. SIRT1 activation is beneficial for several human diseases, including metabolic syndrome, diabetes, obesity, liver steatosis, and Alzheimer disease. We have recently shown that the protein deleted in breast cancer 1 (DBC1) is a key regulator of SIRT1 activity in vivo. Furthermore, SIRT1 and DBC1 form a dynamic complex that is regulated by the energetic state of the organism. Understanding how the interaction between SIRT1 and DBC1 is regulated is therefore essential to design strategies aimed to activate SIRT1. Here, we investigated which pathways can lead to the dissociation of SIRT1 and DBC1 and consequently to SIRT1 activation. We observed that PKA activation leads to a fast and transient activation of SIRT1 that is DBC1-dependent. In fact, an increase in cAMP/PKA activity resulted in the dissociation of SIRT1 and DBC1 in an AMP-activated protein kinase (AMPK)-dependent manner. Pharmacological AMPK activation led to SIRT1 activation by a DBC1-dependent mechanism. Indeed, we found that AMPK activators promote SIRT1-DBC1 dissociation in cells, resulting in an increase in SIRT1 activity. In addition, we observed that the SIRT1 activation promoted by PKA and AMPK occurs without changes in the intracellular levels of NAD+. We propose that PKA and AMPK can acutely activate SIRT1 by inducing dissociation of SIRT1 from its endogenous inhibitor DBC1. Our experiments provide new insight on the in vivo mechanism of SIRT1 regulation and a new avenue for the development of pharmacological SIRT1 activators targeted at the dissociation of the SIRT1-DBC1 complex. PMID:22553202

  11. Activating AMP-activated protein kinase (AMPK) slows renal cystogenesis.

    PubMed

    Takiar, Vinita; Nishio, Saori; Seo-Mayer, Patricia; King, J Darwin; Li, Hui; Zhang, Li; Karihaloo, Anil; Hallows, Kenneth R; Somlo, Stefan; Caplan, Michael J

    2011-02-01

    Renal cyst development and expansion in autosomal dominant polycystic kidney disease (ADPKD) involves both fluid secretion and abnormal proliferation of cyst-lining epithelial cells. The chloride channel of the cystic fibrosis transmembrane conductance regulator (CFTR) participates in secretion of cyst fluid, and the mammalian target of rapamycin (mTOR) pathway may drive proliferation of cyst epithelial cells. CFTR and mTOR are both negatively regulated by AMP-activated protein kinase (AMPK). Metformin, a drug in wide clinical use, is a pharmacological activator of AMPK. We find that metformin stimulates AMPK, resulting in inhibition of both CFTR and the mTOR pathways. Metformin induces significant arrest of cystic growth in both in vitro and ex vivo models of renal cystogenesis. In addition, metformin administration produces a significant decrease in the cystic index in two mouse models of ADPKD. Our results suggest a possible role for AMPK activation in slowing renal cystogenesis as well as the potential for therapeutic application of metformin in the context of ADPKD. PMID:21262823

  12. The human phenolsulphotransferase polymorphism is determined by the level of expression of the enzyme protein.

    PubMed Central

    Jones, A L; Roberts, R C; Coughtrie, M W

    1993-01-01

    We have examined the expression of platelet phenolsulphotransferase (PST) in 60 individuals. Using an antibody which recognizes both forms of PST present in man (P-PST and M-PST), we determined that the polymorphism of platelet P-PST activity is determined by the level of expression of the enzyme protein. The implications for susceptibility to adverse drug reactions and chemical carcinogenesis are discussed. Images Figure 1 Figure 2 Figure 3 PMID:8257413

  13. Activation of 5' adenosine monophosphate-activated protein kinase blocks cumulus cell expansion through inhibition of protein synthesis during in vitro maturation in Swine.

    PubMed

    Santiquet, Nicolas; Sasseville, Maxime; Laforest, Martin; Guillemette, Christine; Gilchrist, Robert B; Richard, François J

    2014-08-01

    The serine/threonine kinase 5' adenosine monophosphate-activated protein kinase (AMPK), a heterotrimeric protein known as a metabolic switch, is involved in oocyte nuclear maturation in mice, cattle, and swine. The present study analyzed AMPK activation in cumulus cell expansion during in vitro maturation (IVM) of porcine cumulus-oocyte complexes (COC). 5-Aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) is a well-known activator of AMPK. It inhibited oocyte meiotic resumption in COC. Moreover, cumulus cell expansion did not occur in the presence of AICAR, demonstrating its marked impact on cumulus cells. Activation of AMPK was supported by AICAR-mediated phosphorylation of alpha AMPK subunits. Furthermore, the presence of AICAR increased glucose uptake, a classical response to activation of this metabolic switch in response to depleted cellular energy levels. Neither nuclear maturation nor cumulus expansion was reversed by glucosamine, an alternative substrate in hyaluronic acid synthesis, through the hexosamine biosynthetic pathway, which ruled out possible depletion of substrates. Both increased gap junction communication and phosphodiesterase activity in COC are dependent on protein synthesis during the initial hours of IVM; however, both were inhibited in the presence of AICAR, which supports the finding that activation of AMPK by AICAR mediated inhibition of protein synthesis. Moreover, this protein synthesis inhibition was equivalent to that of the well-known protein synthesis inhibitor cycloheximide, as observed on cumulus expansion and protein concentration. Finally, the phosphorylation level of selected kinases was investigated. The pattern of raptor phosphorylation is supportive of activation of AMPK-mediated inhibition of protein synthesis. In conclusion, AICAR-mediated AMPK activation in porcine COC inhibited cumulus cell expansion and protein synthesis. These results bring new considerations to the importance of this kinase in ovarian

  14. Positive feedback of protein kinase C proteolytic activation during apoptosis.

    PubMed Central

    Leverrier, Sabrina; Vallentin, Alice; Joubert, Dominique

    2002-01-01

    In contrast with protein kinase Calpha (PKCalpha) and PKCepsilon, which are better known for promoting cell survival, PKCdelta is known for its pro-apoptotic function, which is exerted mainly through a caspase-3-dependent proteolytic activation pathway. In the present study, we used the rat GH3B6 pituitary adenoma cell line to show that PKCalpha and PKCepsilon are activated and relocalized together with PKCdelta when apoptosis is induced by a genotoxic stress. Proteolytic activation is a crucial step used by the three isoforms since: (1) the catalytic domains of the PKCalpha, PKCepsilon or PKCdelta isoforms (CDalpha, CDepsilon and CDdelta respectively) accumulated, and this accumulation was dependent on the activity of both calpain and caspase; and (2) transient expression of CDalpha, CDepsilon or CDdelta sufficed to induce apoptosis. However, following this initial step of proteolytic activation, the pathways diverge; cytochrome c release and caspase-3 activation are induced by CDepsilon and CDdelta, but not by CDalpha. Another interesting finding of the present study is the proteolysis of PKCdelta induced by CDepsilon expression that revealed the existence of a cross-talk between PKC isoforms during apoptosis. Hence the PKC family may participate in the apoptotic process of pituitary adenoma cells at two levels: downstream of caspase and calpain, and via retro-activation of caspase-3, resulting in the amplification of its own proteolytic activation. PMID:12238950

  15. SIRT1 Suppresses Activator Protein-1 Transcriptional Activity and Cyclooxygenase-2 Expression in Macrophages*

    PubMed Central

    Zhang, Ran; Chen, Hou-Zao; Liu, Jin-Jing; Jia, Yu-Yan; Zhang, Zhu-Qin; Yang, Rui-Feng; Zhang, Yuan; Xu, Jing; Wei, Yu-Sheng; Liu, De-Pei; Liang, Chih-Chuan

    2010-01-01

    SIRT1 (Sirtuin type 1), a mammalian orthologue of yeast SIR2 (silent information regulator 2), has been shown to mediate a variety of calorie restriction (CR)-induced physiological events, such as cell fate regulation via deacetylation of the substrate proteins. However, whether SIRT1 deacetylates activator protein-1 (AP-1) to influence its transcriptional activity and target gene expression is still unknown. Here we demonstrate that SIRT1 directly interacts with the basic leucine zipper domains of c-Fos and c-Jun, the major components of AP-1, by which SIRT1 suppressed the transcriptional activity of AP-1. This process requires the deacetylase activity of SIRT1. Notably, SIRT1 reduced the expression of COX-2, a typical AP-1 target gene, and decreased prostaglandin E2 (PGE2) production of peritoneal macrophages (pMΦs). pMΦs with SIRT1 overexpression displayed improved phagocytosis and tumoricidal functions, which are associated with depressed PGE2. Furthermore, SIRT1 protein level was up-regulated in CR mouse pMΦs, whereas elevated SIRT1 decreased COX-2 expression and improved PGE2-related macrophage functions that were reversed following inhibition of SIRT1 deacetylase activity. Thus, our results indicate that SIRT1 may be a mediator of CR-induced macrophage regulation, and its deacetylase activity contributes to the inhibition of AP-1 transcriptional activity and COX-2 expression leading to amelioration of macrophage function. PMID:20042607

  16. Metals in the active site of native protein phosphatase-1.

    PubMed

    Heroes, Ewald; Rip, Jens; Beullens, Monique; Van Meervelt, Luc; De Gendt, Stefan; Bollen, Mathieu

    2015-08-01

    Protein phosphatase-1 (PP1) is a major protein Ser/Thr phosphatase in eukaryotic cells. Its activity depends on two metal ions in the catalytic site, which were identified as manganese in the bacterially expressed phosphatase. However, the identity of the metal ions in native PP1 is unknown. In this study, total reflection X-ray fluorescence (TXRF) was used to detect iron and zinc in PP1 that was purified from rabbit skeletal muscle. Metal exchange experiments confirmed that the distinct substrate specificity of recombinant and native PP1 is determined by the nature of their associated metals. We also found that the iron level associated with native PP1 is decreased by incubation with inhibitor-2, consistent with a function of inhibitor-2 as a PP1 chaperone. PMID:25890482

  17. SPX proteins regulate Pi homeostasis and signaling in different subcellular level.

    PubMed

    Zhou, Zhipeng; Wang, Zhiye; Lv, Qundan; Shi, Jing; Zhong, Yongjia; Wu, Ping; Mao, Chuanzao

    2015-01-01

    To cope with low phosphate (Pi) availability, plants have to adjust its gene expression profile to facilitate Pi acquisition and remobilization. Sensing the levels of Pi is essential for reprogramming the gene expression profile to adapt to the fluctuating Pi environment. AtPHR1 in Arabidopsis and OsPHR2 in rice are central regulators of Pi signaling, which regulates the expression of phosphate starvation-induced (PSI) genes by binding to the P1BS elements in the promoter of PSI genes. However, how the Pi level affects the central regulator to regulate the PSI genes have puzzled us for a decade. Recent progress in SPX proteins indicated that the SPX proteins play important role in regulating the activity of central regulator AtPHR1/OsPHR2 in a Pi dependent manner at different subcellular levels.

  18. 34 CFR 300.704 - State-level activities.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 34 Education 2 2011-07-01 2010-07-01 true State-level activities. 300.704 Section 300.704... Allotments, Grants, and Use of Funds § 300.704 State-level activities. (a) State administration. (1) For the... under that Part. (b) Other State-level activities. (1) States may reserve a portion of their...

  19. 34 CFR 300.704 - State-level activities.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 34 Education 2 2014-07-01 2013-07-01 true State-level activities. 300.704 Section 300.704... Allotments, Grants, and Use of Funds § 300.704 State-level activities. (a) State administration. (1) For the... under that Part. (b) Other State-level activities. (1) States may reserve a portion of their...

  20. 34 CFR 300.704 - State-level activities.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 34 Education 2 2013-07-01 2013-07-01 false State-level activities. 300.704 Section 300.704... Allotments, Grants, and Use of Funds § 300.704 State-level activities. (a) State administration. (1) For the... under that Part. (b) Other State-level activities. (1) States may reserve a portion of their...

  1. 34 CFR 300.704 - State-level activities.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 34 Education 2 2012-07-01 2012-07-01 false State-level activities. 300.704 Section 300.704... Allotments, Grants, and Use of Funds § 300.704 State-level activities. (a) State administration. (1) For the... under that Part. (b) Other State-level activities. (1) States may reserve a portion of their...

  2. Circulating FGF21 proteolytic processing mediated by fibroblast activation protein

    PubMed Central

    Zhen, Eugene Y.; Jin, Zhaoyan; Ackermann, Bradley L.; Thomas, Melissa K.; Gutierrez, Jesus A.

    2015-01-01

    Fibroblast growth factor 21 (FGF21), a hormone implicated in the regulation of glucose homoeostasis, insulin sensitivity, lipid metabolism and body weight, is considered to be a promising therapeutic target for the treatment of metabolic disorders. Despite observations that FGF21 is rapidly proteolysed in circulation rending it potentially inactive, little is known regarding mechanisms by which FGF21 protein levels are regulated. We systematically investigated human FGF21 protein processing using mass spectrometry. In agreement with previous reports, circulating human FGF21 was found to be cleaved primarily after three proline residues at positions 2, 4 and 171. The extent of FGF21 processing was quantified in a small cohort of healthy human volunteers. Relative abundance of FGF21 proteins cleaved after Pro-2, Pro-4 and Pro-171 ranged from 16 to 30%, 10 to 25% and 10 to 34%, respectively. Dipeptidyl peptidase IV (DPP-IV) was found to be the primary protease responsible for N-terminal cleavages after residues Pro-2 and Pro-4. Importantly, fibroblast activation protein (FAP) was implicated as the protease responsible for C-terminal cleavage after Pro-171, rendering the protein inactive. The requirement of FAP for FGF21 proteolysis at the C-terminus was independently demonstrated by in vitro digestion, immunodepletion of FAP in human plasma, administration of an FAP-specific inhibitor and by human FGF21 protein processing patterns in FAP knockout mouse plasma. The discovery that FAP is responsible for FGF21 inactivation extends the FGF21 signalling pathway and may enable novel approaches to augment FGF21 actions for therapeutic applications. PMID:26635356

  3. Monocyte chemoattractant protein-1 serum levels in ovarian cancer patients

    PubMed Central

    Hefler, L; Tempfer, C; Heinze, G; Mayerhofer, K; Breitenecker, G; Leodolter, S; Reinthaller, A; Kainz, C

    1999-01-01

    The chemokine monocyte chemoattractant protein (MCP)-1 is an important mediator of monocyte infiltration in various solid tumours of epithelial origin. The aim of the present study was to evaluate the role of MCP-1 in the natural history of ovarian cancer and to determine its value as differentiation marker and prognostic marker regarding disease free and overall survival. This retrospective study comprises 86 patients with ovarian cancer, 48 with primary ovarian cancer and 38 with recurrent ovarian cancer, 67 patients with benign ovarian cysts and 42 healthy women. Median serum levels in patients with primary ovarian cancer, recurrent ovarian cancer, benign ovarian cysts and in healthy women were 535.6 (range 129.6–1200) pg ml–1, 427.3 (range 193.4–1101) pg ml–1, 371.2 (range 222–986.8) pg ml–1 and 318.7 (range 241.3–681.4) pg ml–1 respectively (Mann–Whitney U-test, P < 0.001). Univariate logistic regression models revealed a significant influence of MCP-1 serum levels on the odds of presenting with primary ovarian cancer versus benign cysts and versus healthy women respectively (univariate logistic regression, P < 0.001 and P < 0.001 respectively). In a multivariate logistic regression model considering MCP-1 and CA 125 serum levels simultaneously, both MCP-1 and CA 125 revealed statistical significance on the odds of presenting with primary ovarian cancer versus benign cysts (multivariate logistic regression, P = 0.05 and P < 0.001 respectively). In ovarian cancer patients, MCP-1 serum levels showed a statistically significant correlation with histological grade (Mann–Whitney U-test, P = 0.02) and age at the time of diagnosis (Mann–Whitney U-test, P = 0.03). Elevated MCP-1 serum levels prior to therapy were not associated with disease-free and overall survival (log-rank test, P = 0.2 and P = 0.7 respectively). In summary these data indicate that MCP-1 might play a functional role in the natural history of ovarian cancer and might serve as

  4. Biologically active LIL proteins built with minimal chemical diversity

    PubMed Central

    Heim, Erin N.; Marston, Jez L.; Federman, Ross S.; Edwards, Anne P. B.; Karabadzhak, Alexander G.; Petti, Lisa M.; Engelman, Donald M.; DiMaio, Daniel

    2015-01-01

    We have constructed 26-amino acid transmembrane proteins that specifically transform cells but consist of only two different amino acids. Most proteins are long polymers of amino acids with 20 or more chemically distinct side-chains. The artificial transmembrane proteins reported here are the simplest known proteins with specific biological activity, consisting solely of an initiating methionine followed by specific sequences of leucines and isoleucines, two hydrophobic amino acids that differ only by the position of a methyl group. We designate these proteins containing leucine (L) and isoleucine (I) as LIL proteins. These proteins functionally interact with the transmembrane domain of the platelet-derived growth factor β-receptor and specifically activate the receptor to transform cells. Complete mutagenesis of these proteins identified individual amino acids required for activity, and a protein consisting solely of leucines, except for a single isoleucine at a particular position, transformed cells. These surprisingly simple proteins define the minimal chemical diversity sufficient to construct proteins with specific biological activity and change our view of what can constitute an active protein in a cellular context. PMID:26261320

  5. G protein activation by G protein coupled receptors: ternary complex formation or catalyzed reaction?

    PubMed

    Roberts, David J; Waelbroeck, Magali

    2004-09-01

    G protein coupled receptors catalyze the GDP/GTP exchange on G proteins, thereby activating them. The ternary complex model, designed to describe agonist binding in the absence of GTP, is often extended to G protein activation. This is logically unsatisfactory as the ternary complex does not accumulate when G proteins are activated by GTP. Extended models taking into account nucleotide binding exist, but fail to explain catalytic G protein activation. This review puts forward an enzymatic model of G protein activation and compares its predictions with the ternary complex model and with observed receptor phenomenon. This alternative model does not merely provide a new set of formulae but leads to a new philosophical outlook and more readily accommodates experimental observations. The ternary complex model implies that, HRG being responsible for efficient G protein activation, it should be as stable as possible. In contrast, the enzyme model suggests that although a limited stabilization of HRG facilitates GDP release, HRG should not be "too stable" as this might trap the G protein in an inactive state and actually hinder G protein activation. The two models also differ completely in the definition of the receptor "active state": the ternary complex model implies that the active state corresponds to a single active receptor conformation (HRG); in contrast, the catalytic model predicts that the active receptor state is mobile, switching smoothly through various conformations with high and low affinities for agonists (HR, HRG, HRGGDP, HRGGTP, etc.).

  6. Characterization of murine BATF: a negative regulator of activator protein-1 activity in the thymus.

    PubMed

    Williams, K L; Nanda, I; Lyons, G E; Kuo, C T; Schmid, M; Leiden, J M; Kaplan, M H; Taparowsky, E J

    2001-05-01

    BATF belongs to the AP-1/ATF superfamily of transcription factors and forms heterodimers with Jun proteins to bind AP-1 consensus DNA. Unlike Fos/Jun heterodimers which stimulate gene transcription, BATF/Jun heterodimers are transcriptionally inert and inhibit biological processes that are associated with the overstimulation of AP-1 activity. Here, we describe the murine BATF cDNA and genomic clones and map the BATF locus to chromosome 12 D2-3. Using in situ hybridization of BATF mRNA, we show that BATF gene expression is highly restricted, with the most prominent signals detected in the thymus. BATF mRNA levels are regulated differentially during discrete stages of T cell development and are up-regulated following activation of T cells in the periphery. To demonstrate the impact of BATF on AP-1 activity in vivo, AP-1 luciferase reporter mice were crossed to transgenic mice overexpressing BATF exclusively in thymic T cells. Results show that elevated levels of BATF protein correlate with reduced transactivation by AP-1. Since the differential regulation of AP-1 activity is linked to key transitions in the developing immune system, our observations support a critical role for BATF in determining the overall level of AP-1 activity, and thus AP-1 target gene expression, in specific T cell subtypes.

  7. A case for protein-level and site-level specificity in glycoproteomic studies of disease.

    PubMed

    Schumacher, Katherine N; Dodds, Eric D

    2016-06-01

    Abnormal glycosylation of proteins is known to be either resultant or causative of a variety of diseases. This makes glycoproteins appealing targets as potential biomarkers and focal points of molecular studies on the development and progression of human ailment. To date, a majority of efforts in disease glycoproteomics have tended to center on either determining the concentration of a given glycoprotein, or on profiling the total population of glycans released from a mixture of glycoproteins. While these approaches have demonstrated some diagnostic potential, they are inherently insensitive to the fine molecular detail which distinguishes unique and possibly disease relevant glycoforms of specific proteins. As a consequence, such analyses can be of limited sensitivity, specificity, and accuracy because they do not comprehensively consider the glycosylation status of any particular glycoprotein, or of any particular glycosylation site. Therefore, significant opportunities exist to improve glycoproteomic inquiry into disease by engaging in these studies at the level of individual glycoproteins and their exact loci of glycosylation. In this concise review, the rationale for glycoprotein and glycosylation site specificity is developed in the context of human disease glycoproteomics with an emphasis on N-glycosylation. Recent examples highlighting disease-related perturbations in glycosylation will be presented, including those involving alterations in the overall glycosylation of a specific protein, alterations in the occupancy of a given glycosylation site, and alterations in the compositional heterogeneity of glycans occurring at a given glycosylation site. Each will be discussed with particular emphasis on how protein-specific and site-specific approaches can contribute to improved discrimination between glycoproteomes and glycoproteins associated with healthy and unhealthy states.

  8. Entry-Level Activities in System Consultation

    ERIC Educational Resources Information Center

    Hylander, Ingrid

    2014-01-01

    System-level consultation or organizational development in schools is an area in great need of theoretical models and definitions. The three articles in this special issue provide a unique learning opportunity not only for consultation across borders but also for consultation within the same nation. In my commentary, I limit my remarks to a few…

  9. Stimulation of protein phosphatase activity by insulin and growth factors in 3T3 cells

    SciTech Connect

    Chan, C.P.; McNall, S.J.; Krebs, E.G.; Fischer, E.H. )

    1988-09-01

    Incubation of Swiss mouse 3T3-D1 cells with physiological concentrations of insulin resulted in a rapid and transient activation of protein phosphatase activity as measure by using ({sup 32}P)phosphorylase {alpha} as substrate. Activation reached a maximum level (140% of control value) within 5 min of addition and returned to control levels within 20 min. The effect of insulin was dose-dependent with half-maximal activation occurring at {approx}5 nM insulin. This activity could be completely inhibited by addition of the heat-stable protein inhibitor 2, which suggests the presence of an activated type-1 phosphatase. Similar effects on phosphatase activity were seen when epidermal growth factor and platelet-derived growth factor were tested. These results suggest that some of the intracellular effects caused by insulin and growth factors are mediated through the activation of a protein phosphatase.

  10. Receptor activity modifying protein-3 mediates the protumorigenic activity of lysyl oxidase-like protein-2.

    PubMed

    Brekhman, Vera; Lugassie, Jennie; Zaffryar-Eilot, Shelly; Sabo, Edmond; Kessler, Ofra; Smith, Victoria; Golding, Hana; Neufeld, Gera

    2011-01-01

    Lysyl oxidase-like protein-2 (LOXL2) induces epithelial to mesenchymal transition and promotes invasiveness. To understand the mechanisms involved, we examined the effect of LOXL2 overexpression in MCF-7 cells on gene expression. We found that LOXL2 up-regulated the expression of receptor activity modifying protein-3 (RAMP3). Expression of RAMP3 in MDA-MB-231 cells in which LOXL2 expression was inhibited restored vimentin expression, invasiveness, and tumor development. Inhibition of RAMP3 expression in MDA-MB-231 cells mimicked the effects produced by inhibition of LOXL2 expression and was accompanied by inhibition of p38 phosphorylation. LOXL2 overexpression in these cells did not restore invasiveness, suggesting that RAMP3 functions downstream to LOXL2. LOXL2 and RAMP3 are strongly coexpressed in human colon, breast, and gastric carcinomas but not in normal colon or gastric epithelial cells. RAMP3 associates with several G-protein-coupled receptors forming receptors for peptides, such as adrenomedullin and amylin. We hypothesized that RAMP3 could function as a transducer of autocrine signals induced by such peptides. However, the proinvasive effects of RAMP3 could not be abrogated following inhibition of the expression or activity of these peptides. Our experiments suggest that the protumorigenic effects of LOXL2 are partially mediated by RAMP3 and that RAMP3 inhibitors may function as antitumorigenic agents. -

  11. Quantitative plasma proteome analysis reveals aberrant level of blood coagulation-related proteins in nasopharyngeal carcinoma.

    PubMed

    Peng, Pei-Hua; Wu, Chih-Ching; Liu, Shu-Chen; Chang, Kai-Ping; Chen, Chi-De; Chang, Ya-Ting; Hsu, Chia-Wei; Chang, Yu-Sun; Yu, Jau-Song

    2011-05-01

    Nasopharyngeal carcinoma (NPC), one of the most common cancers in Southeast Asia, is not easily diagnosed until advanced stages. To discover potential biomarkers for improving NPC diagnosis, we herein identified the aberrant plasma proteins in NPC patients. We first removed the top-seven abundant proteins from plasma samples of healthy controls and NPC patients, and then labeled the samples with different fluorescent cyanine dyes. The labeled samples were then mixed equally and fractionated with ion-exchange chromatography followed by SDS-PAGE. Proteins showing altered levels in NPC patients were identified by in-gel tryptic digestion and LC-MS/MS. When the biological roles of the 45 identified proteins were assessed via MetaCore™ analysis, the blood coagulation pathway emerged as the most significantly altered pathway in NPC plasma. Plasma kallikrein (KLKB1) and thrombin-antithrombin III complex (TAT) were chosen for evaluation as the candidate NPC biomarkers because of their involvement in blood coagulation. ELISAs confirmed the elevation of their plasma levels in NPC patients versus healthy controls. Western blot and activity assays further showed that the KLKB1 active form was significantly increased in NPC plasma. Collectively, our results identified the significant alteration of blood coagulation pathway in NPC patients, and KLKB1 and TAT may represent the potential NPC biomarkers.

  12. Xylazine Activates Adenosine Monophosphate-Activated Protein Kinase Pathway in the Central Nervous System of Rats

    PubMed Central

    Shi, Xing-Xing; Yin, Bai-Shuang; Yang, Peng; Chen, Hao; Li, Xin; Su, Li-Xue; Fan, Hong-Gang; Wang, Hong-Bin

    2016-01-01

    Xylazine is a potent analgesic extensively used in veterinary and animal experimentation. Evidence exists that the analgesic effect can be inhibited using adenosine 5’-monophosphate activated protein kinase (AMPK) inhibitors. Considering this idea, the aim of this study was to investigate whether the AMPK signaling pathway is involved in the central analgesic mechanism of xylazine in the rat. Xylazine was administrated via the intraperitoneal route. Sprague-Dawley rats were sacrificed and the cerebral cortex, cerebellum, hippocampus, thalamus and brainstem were collected for determination of liver kinase B1 (LKB1) and AMPKα mRNA expression using quantitative real-time polymerase chain reaction (qPCR), and phosphorylated LKB1 and AMPKα levels using western blot. The results of our study showed that compared with the control group, xylazine induced significant increases in AMPK activity in the cerebral cortex, hippocampus, thalamus and cerebellum after rats received xylazine (P < 0.01). Increased AMPK activities were accompanied with increased phosphorylation levels of LKB1 in corresponding regions of rats. The protein levels of phosphorylated LKB1 and AMPKα in these regions returned or tended to return to control group levels. However, in the brainstem, phosphorylated LKB1 and AMPKα protein levels were decreased by xylazine compared with the control (P < 0.05). In conclusion, our data indicates that xylazine alters the activities of LKB1 and AMPK in the central nervous system of rats, which suggests that xylazine affects the regulatory signaling pathway of the analgesic mechanism in the rat brain. PMID:27049320

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

    PubMed

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

    2015-07-01

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

  14. Auxin acts independently of DELLA proteins in regulating gibberellin levels.

    PubMed

    Reid, James B; Davidson, Sandra E; Ross, John J

    2011-03-01

    Shoot elongation is a vital process for plant development and productivity, in both ecological and economic contexts. Auxin and bioactive gibberellins (GAs), such as GA1, play critical roles in the control of elongation, along with environmental and endogenous factors, including other hormones such as the brassinosteroids. The effect of auxins, such as indole-3-acetic acid (IAA), is at least in part mediated by its effect on GA metabolism, since auxin up-regulates biosynthesis genes such as GA 3-oxidase and GA 20-oxidase and down regulates GA catabolism genes such as GA 2-oxidases, leading to elevated levels of bioactive GA 1. In our recent paper, we have provided evidence that this action of IAA is largely independent of DELLA proteins, the negative regulators of GA action, since the auxin effects are still present in the DELLA-deficient la cry-s genotype of pea. This was a crucial issue to resolve, since like auxin, the DELLAs also promote GA 1 synthesis and inhibit its deactivation. DELLAs are deactivated by GA, and thereby mediate a feedback system by which bioactive GA regulates its own level. However, our recent results, in themselves, do not show the generality of the auxin-GA relationship across species and phylogenetic groups or across different tissue types and responses. Further, they do not touch on the ecological benefits of the auxin-GA interaction. These issues are discussed below as well as the need for the development of suitable experimental systems to allow this process to be examined. PMID:21358281

  15. Expression level tuning for optimal heterologous protein secretion in Saccharomyces cerevisiae.

    PubMed

    Parekh, R N; Wittrup, K D

    1997-01-01

    The relationship between expression level and secretion of bovine pancreatic trypsin inhibitor (BPTI) was determined in Saccharomyces cerevisiae using a tunable amplifiable delta integration vector. Optimal secretory productivity of 15 mg of BPTI/g cell dry weight yields 180 mg/L secreted active BPTI in test-tube cultures, an order of magnitude increase over 2 mu plasmid-directed secretion. Maximum productivity is determined by the protein folding capacity of the endoplasmic reticulum (ER). Unfolded protein accumulates in the ER as synthesis increases, until a physiological instability is reached and secretion decreases precipitously despite high BPTI mRNA levels. Optimal specific productivity of a standard laboratory strain of S. cerevisiae is double that reported for secretion of BPTI by Pichia pastoris, indicating that efficient utilization of S. cerevisiae's available secretory capacity can eliminate apparent differences among yeast species in their capacity for heterologous protein secretion. Although not generally recognized, the existence of an optimum synthesis level for secretion is apparently a general feature of eucaryotic expression systems and could be of substantial significance for maximization of protein secretion in mammalian and insect cell culture. PMID:9104035

  16. High level protein expression in mammalian cells using a safe viral vector: modified vaccinia virus Ankara.

    PubMed

    Hebben, Matthias; Brants, Jan; Birck, Catherine; Samama, Jean-Pierre; Wasylyk, Bohdan; Spehner, Danièle; Pradeau, Karine; Domi, Arban; Moss, Bernard; Schultz, Patrick; Drillien, Robert

    2007-12-01

    Vaccinia virus vectors are attractive tools to direct high level protein synthesis in mammalian cells. In one of the most efficient strategies developed so far, the gene to be expressed is positioned downstream of a bacteriophage T7 promoter within the vaccinia genome and transcribed by the T7 RNA polymerase, also encoded by the vaccinia virus genome. Tight regulation of transcription and efficient translation are ensured by control elements of the Escherichia coli lactose operon and the encephalomyocarditis virus leader sequence, respectively. We have integrated such a stringently controlled expression system, previously used successfully in a standard vaccinia virus backbone, into the modified vaccinia virus Ankara strain (MVA). In this manner, proteins of interest can be produced in mammalian cells under standard laboratory conditions because of the inherent safety of the MVA strain. Using this system for expression of beta-galactosidase, about 15 mg protein could be produced from 10(8) BHK21 cells over a 24-h period, a value 4-fold higher than the amount produced from an identical expression system based on a standard vaccinia virus strain. In another application, we employed the MVA vector to produce human tubulin tyrosine ligase and demonstrate that this protein becomes a major cellular protein upon induction conditions and displays its characteristic enzymatic activity. The MVA vector should prove useful for many other applications in which mammalian cells are required for protein production. PMID:17892951

  17. Dissecting the active site of a photoreceptor protein

    NASA Astrophysics Data System (ADS)

    Hoff, Wouter; Hara, Miwa; Ren, Jie; Moghadam, Farzaneh; Xie, Aihua; Kumauchi, Masato

    While enzymes are quite large molecules, functionally important chemical events are often limited to a small region of the protein: the active site. The physical and chemical properties of residues at such active sites are often strongly altered compared to the same groups dissolved in water. Understanding such effects is important for unraveling the mechanisms underlying protein function and for protein engineering, but has proven challenging. Here we report on our ongoing efforts on using photoactive yellow protein (PYP), a bacterial photoreceptor, as a model system for such effects. We will report on the following questions: How many residues affect active site properties? Are these residues in direct physical contact with the active site? Can functionally important residues be recognized in the crystal structure of a protein? What structural resolution is needed to understand active sites? What spectroscopic techniques are most informative? Which weak interactions dominate active site properties?

  18. Determination of Protein Carbonylation and Proteasome Activity in Seeds.

    PubMed

    Xia, Qiong; El-Maarouf-Bouteau, Hayat; Bailly, Christophe; Meimoun, Patrice

    2016-01-01

    Reactive oxygen species (ROS) have been shown to be toxic but also function as signaling molecules in a process called redox signaling. In seeds, ROS are produced at different developmental stages including dormancy release and germination. Main targets of oxidation events by ROS in cell are lipids, nucleic acids, and proteins. Protein oxidation has various effects on their function, stability, location, and degradation. Carbonylation represents an irreversible and unrepairable modification that can lead to protein degradation through the action of the 20S proteasome. Here, we present techniques which allow the quantification of protein carbonyls in complex protein samples after derivatization by 2,4-dinitrophenylhydrazine (DNPH) and the determination proteasome activity by an activity-based protein profiling (ABPP) using the probe MV151. These techniques, routinely easy to handle, allow the rapid assessment of protein carbonyls and proteasome activity in seeds in various physiological conditions where ROS may act as signaling or toxic elements. PMID:27424756

  19. Egg Activation at Fertilization by a Soluble Sperm Protein.

    PubMed

    Swann, Karl; Lai, F Anthony

    2016-01-01

    The most fundamental unresolved issue of fertilization is to define how the sperm activates the egg to begin embryo development. Egg activation at fertilization in all species thus far examined is caused by some form of transient increase in the cytoplasmic free Ca(2+) concentration. What has not been clear, however, is precisely how the sperm triggers the large changes in Ca(2+) observed within the egg cytoplasm. Here, we review the studies indicating that the fertilizing sperm stimulates a cytosolic Ca(2+) increase in the egg specifically by delivering a soluble factor that diffuses into the cytosolic space of the egg upon gamete membrane fusion. Evidence is primarily considered in species of eggs where the sperm has been shown to elicit a cytosolic Ca(2+) increase by initiating Ca(2+) release from intracellular Ca(2+) stores. We suggest that our best understanding of these signaling events is in mammals, where the sperm triggers a prolonged series of intracellular Ca(2+) oscillations. The strongest empirical studies to date suggest that mammalian sperm-triggered Ca(2+) oscillations are caused by the introduction of a sperm-specific protein, called phospholipase C-zeta (PLCζ) that generates inositol trisphosphate within the egg. We will discuss the role and mechanism of action of PLCζ in detail at a molecular and cellular level. We will also consider some of the evidence that a soluble sperm protein might be involved in egg activation in nonmammalian species.

  20. Activated protein C anticoagulant system dysfunction and thrombophilia in Asia.

    PubMed

    Hamasaki, Naotaka; Kuma, Hiroyuki; Tsuda, Hiroko

    2013-01-01

    Thrombophilia that is common among Caucasians is caused by genetic polymorphisms of coagulation factor V Leiden (R506Q) and prothrombin G20210A. Unlike that in Caucasians, thrombophilia that is common in the Japanese and Chinese involve dysfunction of the activated protein C (APC) anticoagulant system caused by abnormal protein S and protein C molecules. Approximately 50% of Japanese and Chinese individuals who develop venous thrombosis have reduced activities of protein S. The abnormal sites causing the protein S molecule abnormalities are distributed throughout the protein S gene, PROS1. One of the most common abnormalities is protein S Tokushima (K155E), which accounts for about 30% of the protein S molecule abnormalities in the Japanese. Whether APC dysfunction occurs in other Asian countries is an important aspect of mapping thrombophilia among Asians. International surveys using an accurate assay system are needed to determine this.

  1. Visna virus Tat protein: a potent transcription factor with both activator and suppressor domains.

    PubMed Central

    Carruth, L M; Hardwick, J M; Morse, B A; Clements, J E

    1994-01-01

    Visna virus is a pathogenic lentivirus of sheep tat is distantly related to the primate lentiviruses, including human immunodeficiency virus type 1. The visna virus genome encodes a small regulatory protein, Tat, which is necessary for efficient viral replication and enhanced viral transcription. To investigate the mechanism of action of the visna Tat protein and to localize the protein domain(s) responsible for transcriptional activation, chimeric proteins containing visna virus Tat sequences fused to the DNA binding domain of the yeast transactivation factor GAL4 (residues 1 to 147) were made. The GAL4-Tat fusion proteins were transfected into cells and tested for the ability to activate the adenovirus E1b promoter via upstream GAL4 DNA binding sites. Full-length GAL4-Tat fusion proteins were weak transactivators in this system, giving only a two- to fourfold increase in transcription in several cell types, including HeLa and sheep choroid plexus cells. In contrast, fusion of the N-terminal region of the Tat protein to GAL4 revealed a potent activation domain. Amino acids 13 to 38 appeared to be the most critical for activation. No other region of the protein showed any activation in the GAL4 system. This N-terminal region of the visna virus Tat protein has a large number of acidic and hydrophobic residues, suggesting that Tat has an acidic activation domain common to many transcriptional transactivators. Mutations in hydrophobic and bulky aromatic residues dramatically reduced the activity of the chimeric protein. Competition experiments suggest that mechanism of the visna virus Tat activation domain may closely resemble that of the herpesvirus activator VP16 and human immunodeficiency virus Tat, a related lentivirus activator, since both significantly reduce the level of visna virus Tat activation. Finally, a domain between residues 39 and 53 was identified in the Tat protein that, in the GAL4 system, negatively regulates activation by Tat. Images PMID:8083955

  2. Global Analysis of Protein Activities Using Proteome Chips

    NASA Astrophysics Data System (ADS)

    Zhu, Heng; Bilgin, Metin; Bangham, Rhonda; Hall, David; Casamayor, Antonio; Bertone, Paul; Lan, Ning; Jansen, Ronald; Bidlingmaier, Scott; Houfek, Thomas; Mitchell, Tom; Miller, Perry; Dean, Ralph A.; Gerstein, Mark; Snyder, Michael

    2001-09-01

    To facilitate studies of the yeast proteome, we cloned 5800 open reading frames and overexpressed and purified their corresponding proteins. The proteins were printed onto slides at high spatial density to form a yeast proteome microarray and screened for their ability to interact with proteins and phospholipids. We identified many new calmodulin- and phospholipid-interacting proteins; a common potential binding motif was identified for many of the calmodulin-binding proteins. Thus, microarrays of an entire eukaryotic proteome can be prepared and screened for diverse biochemical activities. The microarrays can also be used to screen protein-drug interactions and to detect posttranslational modifications.

  3. Expression levels of apoptosis-related proteins predict clinical outcome in anaplastic large cell lymphoma.

    PubMed

    ten Berge, Rosita L; Meijer, Chris J L M; Dukers, Danny F; Kummer, J Alain; Bladergroen, Bellinda A; Vos, Wim; Hack, C Erik; Ossenkoppele, Gert J; Oudejans, Joost J

    2002-06-15

    In vitro studies suggest that resistance to chemotherapy-induced apoptosis might explain poor response to therapy in fatal cases. Actual execution of apoptosis depends on proper functioning of effector caspases, particularly caspase 3, and on the expression levels of apoptosis-regulating proteins, including Bcl-2 and the recently identified granzyme B- specific protease inhibitor 9 (PI9). Thus, high levels of caspase 3 activation should reflect proper functioning of the apoptosis pathways, resulting in chemotherapy-sensitive neoplastic cells and a favorable prognosis. We tested this hypothesis by quantifying numbers of tumor cells positive for active caspase 3, Bcl-2, and PI9, respectively, in pretreatment biopsies of systemic anaplastic large cell lymphoma (ALCL) patients and by comparing these numbers with clinical outcome. Activation of caspase 3 in more than 5% of the tumor cells was strongly correlated with a highly favorable outcome. High numbers of Bcl-2- and PI9-positive tumor cells were found to predict unfavorable prognosis. This prognostic effect was strongly related to anaplastic lymphoma kinase (ALK) status: ALK-positive ALCL had significantly higher levels of active caspase 3, while high expression of the antiapoptotic proteins Bcl-2 and PI9 was almost completely restricted to ALK-negative cases. In conclusion, high numbers of active caspase 3-positive tumor cells predict a highly favorable prognosis in systemic ALCL patients. Poor prognosis is strongly related to high numbers of Bcl-2- and PI9-positive neoplastic cells. These data support the notion that a favorable response to chemotherapy depends on an intact apoptosis cascade. Moreover, these data indicate that differences in prognosis between ALK-positive and ALK-negative ALCL might be explained by differences in expression of apoptosis-inhibiting proteins.

  4. Suppression of cellular proliferation and invasion by the concerted lipid and protein phosphatase activities of PTEN

    PubMed Central

    Davidson, Lindsay; Maccario, Helene; Perera, Nevin M.; Yang, Xuesong; Spinelli, Laura; Tibarewal, Priyanka; Glancy, Ben; Gray, Alex; Weijer, Cornelis J.; Downes, C. Peter; Leslie, Nick R.

    2009-01-01

    PTEN is a tumour suppressor with phosphatase activity in vitro against both lipids and proteins and other potential non-enzymatic mechanisms of action. Although the importance of PTEN’s lipid phosphatase activity in regulating the PI3K signalling pathway is recognised, the significance of PTEN’s other mechanisms of action is currently unclear. Here, we describe the systematic identification of a PTEN mutant, PTEN Y138L, with activity against lipid, but not soluble substrates. Using this mutant we provide evidence for the interfacial activation of PTEN against lipid substrates. We also show that when re-expressed at physiological levels in PTEN null U87MG glioblastoma cells the protein phosphatase activity of PTEN is not required to regulate cellular PtdInsP3 levels or the downstream protein kinase Akt/PKB. Finally, in 3D Matrigel cultures of U87MG cells similarly re-expressing PTEN mutants, both the protein and lipid phosphatase activities were required to inhibit invasion, but either activity alone significantly inhibited proliferation, albeit only weakly for the protein phosphatase activity. Our data provides a novel tool to address the significance of PTEN’s separable lipid and protein phosphatase activities and suggest that both activities act to suppress proliferation and act together to suppress invasion. PMID:19915616

  5. Snow-mold-induced apoplastic proteins in winter rye leaves lack antifreeze activity

    PubMed

    Hiilovaara-Teijo; Hannukkala; Griffith; Yu; Pihakaski-Maunsbach

    1999-10-01

    During cold acclimation, winter rye (Secale cereale L.) plants secrete antifreeze proteins that are similar to pathogenesis-related (PR) proteins. In this experiment, the secretion of PR proteins was induced at warm temperatures by infection with pink snow mold (Microdochium nivale), a pathogen of overwintering cereals. A comparison of cold-induced and pathogen-induced proteins showed that PR proteins accumulated in the leaf apoplast to a greater level in response to cold. The PR proteins induced by cold and by snow mold were similar when separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and examined by immunoblotting. Both groups of PR proteins contained glucanase-like, chitinase-like, and thaumatin-like proteins, and both groups exhibited similar levels of glucanase and chitinase activities. However, only the PR proteins induced by cold exhibited antifreeze activity. Our findings suggest that the cold-induced PR proteins may be isoforms that function as antifreeze proteins to modify the growth of ice during freezing while also providing resistance to the growth of low-temperature pathogens in advance of infection. Both functions of the cold-induced PR proteins may improve the survival of overwintering cereals.

  6. Activation of AMP-activated protein kinase by tributyltin induces neuronal cell death

    SciTech Connect

    Nakatsu, Yusuke; Kotake, Yaichiro Hino, Atsuko; Ohta, Shigeru

    2008-08-01

    AMP-activated protein kinase (AMPK), a member of the metabolite-sensing protein kinase family, is activated by energy deficiency and is abundantly expressed in neurons. The environmental pollutant, tributyltin chloride (TBT), is a neurotoxin, and has been reported to decrease cellular ATP in some types of cells. Therefore, we investigated whether TBT activates AMPK, and whether its activation contributes to neuronal cell death, using primary cultures of cortical neurons. Cellular ATP levels were decreased 0.5 h after exposure to 500 nM TBT, and the reduction was time-dependent. It was confirmed that most neurons in our culture system express AMPK, and that TBT induced phosphorylation of AMPK. Compound C, an AMPK inhibitor, reduced the neurotoxicity of TBT, suggesting that AMPK is involved in TBT-induced cell death. Next, the downstream target of AMPK activation was investigated. Nitric oxide synthase, p38 phosphorylation and Akt dephosphorylation were not downstream of TBT-induced AMPK activation because these factors were not affected by compound C, but glutamate release was suggested to be controlled by AMPK. Our results suggest that activation of AMPK by TBT causes neuronal death through mediating glutamate release.

  7. Total protein, animal protein and physical activity in relation to muscle mass in middle-aged and older Americans.

    PubMed

    Morris, Martha Savaria; Jacques, Paul F

    2013-04-14

    Resistance training is recognised as a good strategy for retarding age-related declines in muscle mass and strength. Recent studies have also highlighted the potential value of protein intakes in excess of present recommendations. The roles that leisure-time physical activity and protein quality play in the preservation of skeletal muscle during ageing, and how such influences interact in free-living people are unclear. We sought to clarify these issues using data collected on 2425 participants aged ≥ 50 years in the US National Health and Nutrition Examination Survey (2003-2006). We estimated subjects' usual intakes of total protein and beef from two 24 h diet recalls and computed the appendicular skeletal muscle mass index from anthropometric measures. Participants self-reported their physical activity levels. Analyses accounted for demographic factors and smoking. The association between muscle-strengthening activity and the appendicular skeletal muscle mass index varied with protein intake. Furthermore, among obese subjects with protein intakes < 70 g/d, those who performed such activities had a lower appendicular skeletal muscle mass index than those who were physically inactive. Protein intakes above the present recommendations were associated with benefits to obese subjects only. The appendicular skeletal muscle mass index of non-obese subjects who performed vigorous aerobic activities was consistently high; in obese subjects, it varied with protein intake. High-protein intake was associated with a modest increase in the appendicular skeletal muscle mass index in non-obese, physically inactive subjects. The present findings reinforce the idea that muscle-strengthening exercise preserves muscle when combined with adequate dietary protein. Vigorous aerobic activity may also help.

  8. Development, characterization, and optimization of protein level in date bars using response surface methodology.

    PubMed

    Nadeem, Muhammad; Salim-ur-Rehman; Muhammad Anjum, Faqir; Murtaza, Mian Anjum; Mueen-ud-Din, Ghulam

    2012-01-01

    This project was designed to produce a nourishing date bar with commercial value especially for school going children to meet their body development requirements. Protein level of date bars was optimized using response surface methodology (RSM). Economical and underutilized sources, that is, whey protein concentrate and vetch protein isolates, were explored for protein supplementation. Fourteen date bar treatments were produced using a central composite design (CCD) with 2 variables and 3 levels for each variable. Date bars were then analyzed for nutritional profile. Proximate composition revealed that addition of whey protein concentrate and vetch protein isolates improved the nutritional profile of date bars. Protein level, texture, and taste were considerably improved by incorporating 6.05% whey protein concentrate and 4.35% vetch protein isolates in date bar without affecting any sensory characteristics during storage. Response surface methodology was observed as an economical and effective tool to optimize the ingredient level and to discriminate the interactive effects of independent variables. PMID:22792044

  9. Microsecond protein dynamics observed at the single-molecule level

    PubMed Central

    Otosu, Takuhiro; Ishii, Kunihiko; Tahara, Tahei

    2015-01-01

    How polypeptide chains acquire specific conformations to realize unique biological functions is a central problem of protein science. Single-molecule spectroscopy, combined with fluorescence resonance energy transfer, is utilized to study the conformational heterogeneity and the state-to-state transition dynamics of proteins on the submillisecond to second timescales. However, observation of the dynamics on the microsecond timescale is still very challenging. This timescale is important because the elementary processes of protein dynamics take place and direct comparison between experiment and simulation is possible. Here we report a new single-molecule technique to reveal the microsecond structural dynamics of proteins through correlation of the fluorescence lifetime. This method, two-dimensional fluorescence lifetime correlation spectroscopy, is applied to clarify the conformational dynamics of cytochrome c. Three conformational ensembles and the microsecond transitions in each ensemble are indicated from the correlation signal, demonstrating the importance of quantifying microsecond dynamics of proteins on the folding free energy landscape. PMID:26151767

  10. Microsecond protein dynamics observed at the single-molecule level

    NASA Astrophysics Data System (ADS)

    Otosu, Takuhiro; Ishii, Kunihiko; Tahara, Tahei

    2015-07-01

    How polypeptide chains acquire specific conformations to realize unique biological functions is a central problem of protein science. Single-molecule spectroscopy, combined with fluorescence resonance energy transfer, is utilized to study the conformational heterogeneity and the state-to-state transition dynamics of proteins on the submillisecond to second timescales. However, observation of the dynamics on the microsecond timescale is still very challenging. This timescale is important because the elementary processes of protein dynamics take place and direct comparison between experiment and simulation is possible. Here we report a new single-molecule technique to reveal the microsecond structural dynamics of proteins through correlation of the fluorescence lifetime. This method, two-dimensional fluorescence lifetime correlation spectroscopy, is applied to clarify the conformational dynamics of cytochrome c. Three conformational ensembles and the microsecond transitions in each ensemble are indicated from the correlation signal, demonstrating the importance of quantifying microsecond dynamics of proteins on the folding free energy landscape.

  11. Calcium-Oxidant Signaling Network Regulates AMP-activated Protein Kinase (AMPK) Activation upon Matrix Deprivation*

    PubMed Central

    Sundararaman, Ananthalakshmy; Amirtham, Usha; Rangarajan, Annapoorni

    2016-01-01

    The AMP-activated protein kinase (AMPK) has recently been implicated in anoikis resistance. However, the molecular mechanisms that activate AMPK upon matrix detachment remain unexplored. In this study, we show that AMPK activation is a rapid and sustained phenomenon upon matrix deprivation, whereas re-attachment to the matrix leads to its dephosphorylation and inactivation. Because matrix detachment leads to loss of integrin signaling, we investigated whether integrin signaling negatively regulates AMPK activation. However, modulation of focal adhesion kinase or Src, the major downstream components of integrin signaling, failed to cause a corresponding change in AMPK signaling. Further investigations revealed that the upstream AMPK kinases liver kinase B1 (LKB1) and Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) contribute to AMPK activation upon detachment. In LKB1-deficient cells, we found AMPK activation to be predominantly dependent on CaMKKβ. We observed no change in ATP levels under detached conditions at early time points suggesting that rapid AMPK activation upon detachment was not triggered by energy stress. We demonstrate that matrix deprivation leads to a spike in intracellular calcium as well as oxidant signaling, and both these intracellular messengers contribute to rapid AMPK activation upon detachment. We further show that endoplasmic reticulum calcium release-induced store-operated calcium entry contributes to intracellular calcium increase, leading to reactive oxygen species production, and AMPK activation. We additionally show that the LKB1/CaMKK-AMPK axis and intracellular calcium levels play a critical role in anchorage-independent cancer sphere formation. Thus, the Ca2+/reactive oxygen species-triggered LKB1/CaMKK-AMPK signaling cascade may provide a quick, adaptable switch to promote survival of metastasizing cancer cells. PMID:27226623

  12. Identification of a functional SNP in the 3'-UTR of caprine MTHFR gene that is associated with milk protein levels.

    PubMed

    An, Xiaopeng; Song, Yuxuan; Hou, Jinxing; Wang, Shan; Gao, Kexin; Cao, Binyun

    2016-08-01

    Xinong Saanen (n = 305) and Guanzhong (n = 317) dairy goats were used to detect SNPs in the caprine MTHFR 3'-UTR by DNA sequencing. One novel SNP (c.*2494G>A) was identified in the said region. Individuals with the AA genotype had greater milk protein levels than did those with the GG genotype at the c.*2494 G>A locus in both dairy goat breeds (P < 0.05). Functional assays indicated that the MTHFR:c.2494G>A substitution could increase the binding activity of bta-miR-370 with the MTHFR 3'-UTR. In addition, we observed a significant increase in the MTHFR protein level of AA carriers relative to that of GG carriers. These altered levels of MTHFR protein may account for the association of the SNP with milk protein level. PMID:27062401

  13. Identification of a functional SNP in the 3'-UTR of caprine MTHFR gene that is associated with milk protein levels.

    PubMed

    An, Xiaopeng; Song, Yuxuan; Hou, Jinxing; Wang, Shan; Gao, Kexin; Cao, Binyun

    2016-08-01

    Xinong Saanen (n = 305) and Guanzhong (n = 317) dairy goats were used to detect SNPs in the caprine MTHFR 3'-UTR by DNA sequencing. One novel SNP (c.*2494G>A) was identified in the said region. Individuals with the AA genotype had greater milk protein levels than did those with the GG genotype at the c.*2494 G>A locus in both dairy goat breeds (P < 0.05). Functional assays indicated that the MTHFR:c.2494G>A substitution could increase the binding activity of bta-miR-370 with the MTHFR 3'-UTR. In addition, we observed a significant increase in the MTHFR protein level of AA carriers relative to that of GG carriers. These altered levels of MTHFR protein may account for the association of the SNP with milk protein level.

  14. Role of AMP-activated protein kinase and carbohydrate response element binding protein in the regulation of energy balance in chickens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    AMP-activated protein kinase (AMPK) is a heterotrimeric enzyme complex playing a key role in maintaining intracellular energy balance and, on the whole animal level, regulating energy expenditure and food intake. Once activated by phosphorylation, AMPK phosphorylates a variety of protein targets tha...

  15. Double-Stranded RNA-Induced Activation of Activating Protein-1 Promoter Is Differentially Regulated by the Non-structural Protein 1 of Avian Influenza A Viruses

    PubMed Central

    Zohari, Siamak; Belák, Sándor; Berg, Mikael

    2012-01-01

    Abstract Non-structural protein 1 (NS1) of influenza A viruses is a multifunctional protein that antagonizes the host immune response by interfering with several host signaling pathways. Based on putative amino acid sequences, NS1 proteins are categorized into two gene pools, allele A and allele B. Here we identified that allele A NS1 proteins of H6N8 and H4N6 are able to inhibit double-stranded RNA (dsRNA)-induced activating protein-1 (AP-1) promoter in cultured cell lines (human A549 and mink lung cells). Allele B NS1 proteins from corresponding subtypes of influenza A viruses are weak in this inhibition, despite significant levels of expression of each NS1 protein in human A549 cells. Furthermore, the capability to inhibit AP-1 promoter was mapped in the effector domain, since RNA binding domain alone lost its ability to inhibit this promoter activation. Chimeric forms of NS1 protein, composed of either RNA binding domain of allele A or B and effector domain of allele A or B, showed comparable inhibition to that of their wild-type NS1 proteins, or to the effector domain of corresponding NS1 proteins. Both alleles A and B NS1 proteins of H6N8 and H4N6 were expressed to significant levels, and were localized predominantly in the nucleus of human A549 cells. These results underscore the importance of the effector domain in inhibiting AP-1 promoter activation, and the biological function of the effector domain in stabilizing the RNA binding domain. Further, we revealed the versatile nature of NS1 in inhibiting the AP-1 transcription factor, in a manner dependent on allele type. Comprehensive studies, focusing on the molecular mechanisms behind this differential inhibition, may facilitate exploration of the zoonotic and pathogenic potential of influenza A viruses. PMID:22239235

  16. Testicular hyperthermia induces Unfolded Protein Response signaling activation in spermatocyte.

    PubMed

    Kim, Jung-Hak; Park, Sun-Ji; Kim, Tae-Shin; Park, Hyo-Jin; Park, Junghyung; Kim, Bo Kyung; Kim, Gyeong-Ryul; Kim, Jin-Man; Huang, Song Mei; Chae, Jung-Il; Park, Choon-Keun; Lee, Dong-Seok

    2013-05-17

    The testes of most mammals are sensitive to temperature. To survive and adapt under conditions that promote endoplasmic reticulum (ER) stress such as heat shock, cells have a self-protective mechanism against ER stress that has been termed the "Unfolded Protein Response" (UPR). However, the cellular and molecular events underlying spermatogenesis with testicular hyperthermia involved in the UPR signaling pathway under ER stress remain poorly understood. In the present study, we verified that UPR signaling via phospho-eIF2α/ATF4/GADD34, p90ATF6, and phospho-IRE1α/XBP-1 is activated with testicular hyperthermia (43 °C, 15 min/day) and induced ER stress-mediated apoptosis associated with CHOP, phospho-JNK, and caspase-3 after repetitive periods of hyperthermia. Levels of phospho-eIF2α protein of mouse spermatocytes in the testis were rapidly increased by one cycle of testicular hyperthermia. ATF4/GADD34 and p90ATF6 expression gradually increased and decreased, respectively, with repetitive cycles of hyperthermia. Spliced XBP1 mRNA as a marker of IRE1 activity was increased after one, three cycles of hyperthermia and decreased by five cycles of hyperthermia. Although the levels of anti-apoptotic phospho-JNK (p54) were gradually decreased after three cycles of hyperthermia, CHOP expression was rapidly increased. After five cycles of testicular hyperthermia, the levels of cleaved caspase-3 and TUNEL-positive apoptotic spermatocytes cells were significantly increased. Our data demonstrated that testicular hyperthermia induces UPR signaling and repetitive cycles of hyperthermia lead to apoptosis of spermatocytes in mouse testis. These results suggest a link between the UPR signaling pathway and testicular hyperthermia.

  17. Dormancy alleviation by NO or HCN leading to decline of protein carbonylation levels in apple (Malus domestica Borkh.) embryos.

    PubMed

    Krasuska, Urszula; Ciacka, Katarzyna; Dębska, Karolina; Bogatek, Renata; Gniazdowska, Agnieszka

    2014-08-15

    Deep dormancy of apple (Malus domestica Borkh.) embryos can be overcome by short-term pre-treatment with nitric oxide (NO) or hydrogen cyanide (HCN). Dormancy alleviation of embryos modulated by NO or HCN and the first step of germination depend on temporary increased production of reactive oxygen species (ROS). Direct oxidative attack on some amino acid residues or secondary reactions via reactive carbohydrates and lipids can lead to the formation of protein carbonyl derivatives. Protein carbonylation is a widely accepted covalent and irreversible modification resulting in inhibition or alteration of enzyme/protein activities. It also increases the susceptibility of proteins to proteolytic degradation. The aim of this work was to investigate protein carbonylation in germinating apple embryos, the dormancy of which was removed by pre-treatment with NO or HCN donors. It was performed using a quantitative spectrophotometric method, while patterns of carbonylated protein in embryo axes were analyzed by immunochemical techniques. The highest concentration of protein carbonyl groups was observed in dormant embryos. It declined in germinating embryos pre-treated with NO or HCN, suggesting elevated degradation of modified proteins during seedling formation. A decrease in the concentration of carbonylated proteins was accompanied by modification in proteolytic activity in germinating apple embryos. A strict correlation between the level of protein carbonyl groups and cotyledon growth and greening was detected. Moreover, direct in vitro carbonylation of BSA treated with NO or HCN donors was analyzed, showing action of both signaling molecules as protein oxidation agents. PMID:24973585

  18. Dormancy alleviation by NO or HCN leading to decline of protein carbonylation levels in apple (Malus domestica Borkh.) embryos.

    PubMed

    Krasuska, Urszula; Ciacka, Katarzyna; Dębska, Karolina; Bogatek, Renata; Gniazdowska, Agnieszka

    2014-08-15

    Deep dormancy of apple (Malus domestica Borkh.) embryos can be overcome by short-term pre-treatment with nitric oxide (NO) or hydrogen cyanide (HCN). Dormancy alleviation of embryos modulated by NO or HCN and the first step of germination depend on temporary increased production of reactive oxygen species (ROS). Direct oxidative attack on some amino acid residues or secondary reactions via reactive carbohydrates and lipids can lead to the formation of protein carbonyl derivatives. Protein carbonylation is a widely accepted covalent and irreversible modification resulting in inhibition or alteration of enzyme/protein activities. It also increases the susceptibility of proteins to proteolytic degradation. The aim of this work was to investigate protein carbonylation in germinating apple embryos, the dormancy of which was removed by pre-treatment with NO or HCN donors. It was performed using a quantitative spectrophotometric method, while patterns of carbonylated protein in embryo axes were analyzed by immunochemical techniques. The highest concentration of protein carbonyl groups was observed in dormant embryos. It declined in germinating embryos pre-treated with NO or HCN, suggesting elevated degradation of modified proteins during seedling formation. A decrease in the concentration of carbonylated proteins was accompanied by modification in proteolytic activity in germinating apple embryos. A strict correlation between the level of protein carbonyl groups and cotyledon growth and greening was detected. Moreover, direct in vitro carbonylation of BSA treated with NO or HCN donors was analyzed, showing action of both signaling molecules as protein oxidation agents.

  19. Ethylene regulates monomeric GTP-binding protein gene expression and activity in Arabidopsis.

    PubMed

    Moshkov, Igor E; Mur, Luis A J; Novikova, Galina V; Smith, Aileen R; Hall, Michael A

    2003-04-01

    Ethylene rapidly and transiently up-regulates the activity of several monomeric GTP-binding proteins (monomeric G proteins) in leaves of Arabidopsis as determined by two-dimensional gel electrophoresis and autoradiographic analyses. The activation is suppressed by the receptor-directed inhibitor 1-methylcyclopropene. In the etr1-1 mutant, constitutive activity of all the monomeric G proteins activated by ethylene is down-regulated relative to wild type, and ethylene treatment has no effect on the levels of activity. Conversely, in the ctr1-1 mutant, several of the monomeric G proteins activated by ethylene are constitutively up-regulated. However, the activation profile of ctr1-1 does not exactly mimic that of ethylene-treated wild type. Biochemical and molecular evidence suggested that some of these monomeric G proteins are of the Rab class. Expression of the genes for a number of monomeric G proteins in response to ethylene was investigated by reverse transcriptase-PCR. Rab8 and Ara3 expression was increased within 10 min of ethylene treatment, although levels fell back significantly by 40 min. In the etr1-1 mutant, expression of Rab8 was lower than wild type and unaffected by ethylene; in ctr1-1, expression of Rab8 was much higher than wild type and comparable with that seen in ethylene treatments. Expression in ctr1-1 was also unaffected by ethylene. Thus, the data indicate a role for monomeric G proteins in ethylene signal transduction.

  20. Nitric oxide stress and activation of AMP-activated protein kinase impair β-cell sarcoendoplasmic reticulum calcium ATPase 2b activity and protein stability

    PubMed Central

    Tong, X; Kono, T; Evans-Molina, C

    2015-01-01

    The sarcoendoplasmic reticulum Ca2+ ATPase 2b (SERCA2b) pump maintains a steep Ca2+ concentration gradient between the cytosol and ER lumen in the pancreatic β-cell, and the integrity of this gradient has a central role in regulated insulin production and secretion, maintenance of ER function and β-cell survival. We have previously demonstrated loss of β-cell SERCA2b expression under diabetic conditions. To define the mechanisms underlying this, INS-1 cells and rat islets were treated with the proinflammatory cytokine interleukin-1β (IL-1β) combined with or without cycloheximide or actinomycin D. IL-1β treatment led to increased inducible nitric oxide synthase (iNOS) gene and protein expression, which occurred concurrently with the activation of AMP-activated protein kinase (AMPK). IL-1β led to decreased SERCA2b mRNA and protein expression, whereas time-course experiments revealed a reduction in protein half-life with no change in mRNA stability. Moreover, SERCA2b protein but not mRNA levels were rescued by treatment with the NOS inhibitor l-NMMA (NG-monomethyl l-arginine), whereas the NO donor SNAP (S-nitroso-N-acetyl-d,l-penicillamine) and the AMPK activator AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) recapitulated the effects of IL-1β on SERCA2b protein stability. Similarly, IL-1β-induced reductions in SERCA2b expression were rescued by pharmacological inhibition of AMPK with compound C or by transduction of a dominant-negative form of AMPK, whereas β-cell death was prevented in parallel. Finally, to determine a functional relationship between NO and AMPK signaling and SERCA2b activity, fura-2/AM (fura-2-acetoxymethylester) Ca2+ imaging experiments were performed in INS-1 cells. Consistent with observed changes in SERCA2b expression, IL-1β, SNAP and AICAR increased cytosolic Ca2+ and decreased ER Ca2+ levels, suggesting congruent modulation of SERCA activity under these conditions. In aggregate, these results show that SERCA2b protein

  1. How Integrated Management Strategies Promote Protein Quality of Cotton Embryos: High Levels of Soil Available N, N Assimilation and Protein Accumulation Rate

    PubMed Central

    Yang, HongKun; Meng, YaLi; Chen, BingLin; Zhang, XingYue; Wang, YouHua; Zhao, WenQing; Zhou, ZhiGuo

    2016-01-01

    Cottonseed is widely used as a source of ruminant feed and for industrial purposes. Therefore, there is a tremendous need to improve the nutritional value of cotton embryos. In this study, a conventional management (CM) and two integrated cotton management strategies (IMS1, IMS2) were performed at two soil fertility levels to study the relationships among soil N, N assimilation, embryonic protein accumulation and protein quality. The levels of proteins, essential amino acids, and semi-essential amino acids, especially those of glutamate, lysine, and methionine, were higher in IMS1 and IMS2 embryos than in CM embryos. These changes were significantly positively correlated with the soil-available N content, glutamine synthetase activity and peak value of protein accumulation rate and were negatively correlated with the free amino acid level. These results illustrated that integrated management strategies, especially the rates and timing of N application, raise the level of soil available N, which is beneficial for N assimilation in developing cotton embryos. The protein content was limited by the rate of protein accumulation rather than by the free amino acid content. The combination of target yield fertilization, a growth-driven N application schedule, a high plant density and the seedling raising with bio-organic fertilizer can substantially improve protein quality in cotton embryos, especially at a soil with low soil organic matter and total nitrogen. PMID:27532007

  2. How Integrated Management Strategies Promote Protein Quality of Cotton Embryos: High Levels of Soil Available N, N Assimilation and Protein Accumulation Rate.

    PubMed

    Yang, HongKun; Meng, YaLi; Chen, BingLin; Zhang, XingYue; Wang, YouHua; Zhao, WenQing; Zhou, ZhiGuo

    2016-01-01

    Cottonseed is widely used as a source of ruminant feed and for industrial purposes. Therefore, there is a tremendous need to improve the nutritional value of cotton embryos. In this study, a conventional management (CM) and two integrated cotton management strategies (IMS1, IMS2) were performed at two soil fertility levels to study the relationships among soil N, N assimilation, embryonic protein accumulation and protein quality. The levels of proteins, essential amino acids, and semi-essential amino acids, especially those of glutamate, lysine, and methionine, were higher in IMS1 and IMS2 embryos than in CM embryos. These changes were significantly positively correlated with the soil-available N content, glutamine synthetase activity and peak value of protein accumulation rate and were negatively correlated with the free amino acid level. These results illustrated that integrated management strategies, especially the rates and timing of N application, raise the level of soil available N, which is beneficial for N assimilation in developing cotton embryos. The protein content was limited by the rate of protein accumulation rather than by the free amino acid content. The combination of target yield fertilization, a growth-driven N application schedule, a high plant density and the seedling raising with bio-organic fertilizer can substantially improve protein quality in cotton embryos, especially at a soil with low soil organic matter and total nitrogen. PMID:27532007

  3. How Integrated Management Strategies Promote Protein Quality of Cotton Embryos: High Levels of Soil Available N, N Assimilation and Protein Accumulation Rate.

    PubMed

    Yang, HongKun; Meng, YaLi; Chen, BingLin; Zhang, XingYue; Wang, YouHua; Zhao, WenQing; Zhou, ZhiGuo

    2016-01-01

    Cottonseed is widely used as a source of ruminant feed and for industrial purposes. Therefore, there is a tremendous need to improve the nutritional value of cotton embryos. In this study, a conventional management (CM) and two integrated cotton management strategies (IMS1, IMS2) were performed at two soil fertility levels to study the relationships among soil N, N assimilation, embryonic protein accumulation and protein quality. The levels of proteins, essential amino acids, and semi-essential amino acids, especially those of glutamate, lysine, and methionine, were higher in IMS1 and IMS2 embryos than in CM embryos. These changes were significantly positively correlated with the soil-available N content, glutamine synthetase activity and peak value of protein accumulation rate and were negatively correlated with the free amino acid level. These results illustrated that integrated management strategies, especially the rates and timing of N application, raise the level of soil available N, which is beneficial for N assimilation in developing cotton embryos. The protein content was limited by the rate of protein accumulation rather than by the free amino acid content. The combination of target yield fertilization, a growth-driven N application schedule, a high plant density and the seedling raising with bio-organic fertilizer can substantially improve protein quality in cotton embryos, especially at a soil with low soil organic matter and total nitrogen.

  4. Atomic-level Snapshot Catches Protein Motor in Action

    SciTech Connect

    2009-01-01

    Using a state-of-the-art protein crystallography beamline at Berkeley Labs Advanced Light Source, researchers have captured a critical action shapshot of an enzyme that is vital to the survival of all biological cells.

  5. Activation of mitogen-activated protein kinase by membrane-targeted Raf chimeras is independent of raft localization.

    PubMed

    Chen, X; Resh, M D

    2001-09-14

    Binding of proteins to the plasma membrane can be achieved with various membrane targeting motifs, including combinations of fatty acids, isoprenoids, and basic domains. In this study, we investigate whether attachment of different membrane targeting motifs influences the signaling capacity of membrane-bound signal transduction proteins by directing the proteins to different membrane microdomains. We used c-Raf-1 as a model for a signaling protein that is activated when membrane-bound. Three different membrane targeting motifs from K-Ras, Fyn, and Src proteins were fused to the N or C terminus of Raf-1. The ability of the modified Rafs to initiate MAPK signaling was then investigated. All three modified Raf-1 constructs activated MAPK to nearly equivalent levels. The extent of localization of the Raf-1 constructs to membrane microdomains known as rafts did not correlate with the level of MAPK activation. Moreover, treatment of cells with the raft disrupting drug methyl-beta-cyclodextrin (MbetaCD) caused activation of MAPK to levels equivalent to those achieved with membrane-targeted Raf constructs. The use of pharmacological agents as well as dominant negative mutants revealed that MAPK activation by MbetaCD proceeds via a phosphoinositide 3-kinase-dependent mechanism that is Ras/Raf-independent. We conclude that cholesterol depletion from the plasma membrane by MbetaCD constitutes an alternative pathway for activating MAPK.

  6. Protein C activity in dogs envenomed by Vipera palaestinae.

    PubMed

    Hadar, Gil; Kelmer, Efrat; Segev, Gilad; Bruchim, Yaron; Aroch, Itamar

    2014-09-01

    Vipera palaestinae is responsible for most envenomations in humans and domestic animal in Israel. Its venom has pro- and anticoagulant properties. Protein C is a major natural anticoagulant, preventing excess clotting and thrombosis. This study investigated protein C activity and its prognostic value, as well as several other hemostatic analytes in dogs (Canis familiaris) accidently envenomed by V. palaestinae. Protein C activity was compared between envenomed dogs and 33 healthy control dogs. Mean protein C was lower in dogs envenomed by V. palaestinae compared to controls (12.9% vs. 22.9%, respectively; P < 0.01). It was positively correlated with antithrombin activity (r = 0.3, P = 0.04), but not with other hemostatic analytes. The overall mortality rate was 13%, and at presentation no significant protein C activity difference was noted between survivors and non-survivors. A receiver operator characteristics analysis of protein C activity as a predictor of mortality had an area under the curve of 0.7 (95% confidence interval 0.52-0.87). A protein C cutoff point of 8% corresponded to sensitivity and specificity of 70% and 57%, respectively. Dogs diagnosed with consumptive coagulopathy (14%) tended to have lower protein C activity compared to others; however, their mortality did differ from that of other dogs. This is the first study assessing protein C activity in V. palaestinae victims. Decreased protein C activity in such dogs may play a role in formation of thrombosis and hemostatic derangement as well as inflammation in V. palaestinae envenomations.

  7. Fluctuations in Species-Level Protein Expression Occur during Element and Nutrient Cycling in the Subsurface

    PubMed Central

    Wilkins, Michael J.; Wrighton, Kelly C.; Nicora, Carrie D.; Williams, Kenneth H.; McCue, Lee Ann; Handley, Kim M.; Miller, Chris S.; Giloteaux, Ludovic; Montgomery, Alison P.; Lovley, Derek R.; Banfield, Jillian F.; Long, Philip E.; Lipton, Mary S.

    2013-01-01

    While microbial activities in environmental systems play a key role in the utilization and cycling of essential elements and compounds, microbial activity and growth frequently fluctuates in response to environmental stimuli and perturbations. To investigate these fluctuations within a saturated aquifer system, we monitored a carbon-stimulated in situ Geobacter population while iron reduction was occurring, using 16S rRNA abundances and high-resolution tandem mass spectrometry proteome measurements. Following carbon amendment, 16S rRNA analysis of temporally separated samples revealed the rapid enrichment of Geobacter-like environmental strains with strong similarity to G. bemidjiensis. Tandem mass spectrometry proteomics measurements suggest high carbon flux through Geobacter respiratory pathways, and the synthesis of anapleurotic four carbon compounds from acetyl-CoA via pyruvate ferredoxin oxidoreductase activity. Across a 40-day period where Fe(III) reduction was occurring, fluctuations in protein expression reflected changes in anabolic versus catabolic reactions, with increased levels of biosynthesis occurring soon after acetate arrival in the aquifer. In addition, localized shifts in nutrient limitation were inferred based on expression of nitrogenase enzymes and phosphate uptake proteins. These temporal data offer the first example of differing microbial protein expression associated with changing geochemical conditions in a subsurface environment. PMID:23472107

  8. Fluctuations in Species-Level Protein Expression Occur during Element and Nutrient Cycling in the Subsurface

    SciTech Connect

    Wilkins, Michael J.; Wrighton, Kelly C.; Nicora, Carrie D.; Williams, Kenneth H.; McCue, Lee Ann; Handley, Kim M.; Miller, C. S.; Giloteaux, L.; Montgomery, A. P.; Lovley, Derek R.; Banfield, Jillian F.; Long, Philip E.; Lipton, Mary S.

    2013-03-05

    While microbial activities in environmental systems play a key role in the utilization and cycling of essential elements and compounds, microbial activity and growth frequently fluctuates in response to environmental stimuli and perturbations. To investigate these fluctuations within a saturated aquifer system, we monitored a carbon-stimulated in situ Geobacter population while iron reduction was occurring, using 16S rRNA abundances and high-resolution tandem mass spectrometry proteome measurements. Following carbon amendment, 16S rRNA analysis of temporally separated samples revealed the rapid enrichment of Geobacter-like environmental strains with strong similarity to G. bemidjiensis. Tandem mass spectrometry proteomics measurements suggest high carbon flux through Geobacter respiratory pathways, and the synthesis of anapleurotic four carbon compounds from acetyl-CoA via pyruvate ferredoxin oxidoreductase activity. Across a 40-day period where Fe(III) reduction was occurring, fluctuations in protein expression reflected changes in anabolic versus catabolic reactions, with increased levels of biosynthesis occurring soon after acetate arrival in the aquifer. In addition, localized shifts in nutrient limitation were inferred based on expression of nitrogenase enzymes and phosphate uptake proteins. These temporal data offer the first example of differing microbial protein expression associated with changing geochemical conditions in a subsurface environment.

  9. Immersion freezing of ice nucleation active protein complexes

    NASA Astrophysics Data System (ADS)

    Hartmann, S.; Augustin, S.; Clauss, T.; Wex, H.; Šantl-Temkiv, T.; Voigtländer, J.; Niedermeier, D.; Stratmann, F.

    2013-06-01

    Utilising the Leipzig Aerosol Cloud Interaction Simulator (LACIS), the immersion freezing behaviour of droplet ensembles containing monodisperse particles, generated from a Snomax™ solution/suspension, was investigated. Thereto ice fractions were measured in the temperature range between -5 °C to -38 °C. Snomax™ is an industrial product applied for artificial snow production and contains Pseudomonas syringae} bacteria which have long been used as model organism for atmospheric relevant ice nucleation active (INA) bacteria. The ice nucleation activity of such bacteria is controlled by INA protein complexes in their outer membrane. In our experiments, ice fractions increased steeply in the temperature range from about -6 °C to about -10 °C and then levelled off at ice fractions smaller than one. The plateau implies that not all examined droplets contained an INA protein complex. Assuming the INA protein complexes to be Poisson distributed over the investigated droplet populations, we developed the CHESS model (stoCHastic modEl of similar and poiSSon distributed ice nuclei) which allows for the calculation of ice fractions as function of temperature and time for a given nucleation rate. Matching calculated and measured ice fractions, we determined and parameterised the nucleation rate of INA protein complexes exhibiting class III ice nucleation behaviour. Utilising the CHESS model, together with the determined nucleation rate, we compared predictions from the model to experimental data from the literature and found good agreement. We found that (a) the heterogeneous ice nucleation rate expression quantifying the ice nucleation behaviour of the INA protein complex is capable of describing the ice nucleation behaviour observed in various experiments for both, Snomax™ and P. syringae bacteria, (b) the ice nucleation rate, and its temperature dependence, seem to be very similar regardless of whether the INA protein complexes inducing ice nucleation are attached

  10. Anthocyanidins inhibit activator protein 1 activity and cell transformation: structure-activity relationship and molecular mechanisms.

    PubMed

    Hou, De-Xing; Kai, Keiko; Li, Jian-Jian; Lin, Shigang; Terahara, Norihiko; Wakamatsu, Mika; Fujii, Makoto; Young, Mattew R; Colburn, Nancy

    2004-01-01

    Anthocyanins are the chemical components that give the intense color to many fruits and vegetables, such as blueberries, red cabbages and purple sweet potatoes. Extensive studies have indicated that anthocyanins have strong antioxidant activities. To investigate the mechanism of anthocyanidins as an anticancer food source, six kinds of anthocyanidins representing the aglycons of most anthocyanins, were used to examine their effects on tumor promotion in mouse JB6 cells, a validated model for screening cancer chemopreventive agents and elucidating the molecular mechanisms. Of the six anthocyanins tested, only those with an ortho-dihydroxyphenyl structure on the B-ring suppressed 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cell transformation and activator protein-1 transactivation, suggesting that the ortho-dihydroxyphenyl may contribute to the inhibitory action. Delphinidin, but not peonidin, blocked the phosphorylation of protein kinases in the extracellular signal-regulated protein kinase (ERK) pathway at early times and the c-Jun N-terminal kinase (JNK) signaling pathway at later times. p38 kinase was not inhibited by delphinidin. Furthermore, two mitogen-activated protein kinase (MAPK) specific inhibitors (SP600125 for JNK and UO126 for ERK) could specifically block the activation of JNK and ERK and cell transformation. Those results demonstrate that anthocyanidins contribute to the inhibition of tumorigenesis by blocking activation of the MAPK pathway. These findings provide the first molecular basis for the anticarcinogenic action of anthocyanidins. PMID:14514663

  11. Anthocyanidins inhibit activator protein 1 activity and cell transformation: structure-activity relationship and molecular mechanisms.

    PubMed

    Hou, De-Xing; Kai, Keiko; Li, Jian-Jian; Lin, Shigang; Terahara, Norihiko; Wakamatsu, Mika; Fujii, Makoto; Young, Mattew R; Colburn, Nancy

    2004-01-01

    Anthocyanins are the chemical components that give the intense color to many fruits and vegetables, such as blueberries, red cabbages and purple sweet potatoes. Extensive studies have indicated that anthocyanins have strong antioxidant activities. To investigate the mechanism of anthocyanidins as an anticancer food source, six kinds of anthocyanidins representing the aglycons of most anthocyanins, were used to examine their effects on tumor promotion in mouse JB6 cells, a validated model for screening cancer chemopreventive agents and elucidating the molecular mechanisms. Of the six anthocyanins tested, only those with an ortho-dihydroxyphenyl structure on the B-ring suppressed 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cell transformation and activator protein-1 transactivation, suggesting that the ortho-dihydroxyphenyl may contribute to the inhibitory action. Delphinidin, but not peonidin, blocked the phosphorylation of protein kinases in the extracellular signal-regulated protein kinase (ERK) pathway at early times and the c-Jun N-terminal kinase (JNK) signaling pathway at later times. p38 kinase was not inhibited by delphinidin. Furthermore, two mitogen-activated protein kinase (MAPK) specific inhibitors (SP600125 for JNK and UO126 for ERK) could specifically block the activation of JNK and ERK and cell transformation. Those results demonstrate that anthocyanidins contribute to the inhibition of tumorigenesis by blocking activation of the MAPK pathway. These findings provide the first molecular basis for the anticarcinogenic action of anthocyanidins.

  12. Breadboard activities for advanced protein crystal growth

    NASA Technical Reports Server (NTRS)

    Rosenberger, Franz; Banish, Michael

    1993-01-01

    The proposed work entails the design, assembly, testing, and delivery of a turn-key system for the semi-automated determination of protein solubilities as a function of temperature. The system will utilize optical scintillation as a means of detecting and monitoring nucleation and crystallite growth during temperature lowering (or raising, with retrograde solubility systems). The deliverables of this contract are: (1) turn-key scintillation system for the semi-automatic determination of protein solubilities as a function of temperature, (2) instructions and software package for the operation of the scintillation system, and (3) one semi-annual and one final report including the test results obtained for ovostatin with the above scintillation system.

  13. The activation sequence of cellular protein handling systems after proteasomal inhibition in dopaminergic cells

    PubMed Central

    Xiong, Rui; Siegel, David; Ross, David

    2013-01-01

    Dysfunction of protein handling has been implicated in many neurodegenerative diseases and inhibition of the ubiquitin-proteasome system (UPS) has been linked to the formation of protein aggregates and proteinopathies in such diseases. While proteasomal inhibition could trigger an array of downstream protein handling changes including up-regulation of heat shock proteins (HSPs), induction of molecular chaperones, activation of the ER stress/unfolded protein response (UPR), autophagy and aggresome formation, little is known of the relationship of proteasomal inhibition to the sequence of activation of these diverse protein handling systems. In this study we utilized the reversible proteasome inhibitor MG132 and examined the activity of several major protein handling systems in the immortalized dopaminergic neuronal N27 cell line. In the early phase (up to 6 hours after proteasomal inhibition), MG132 induced time-dependent proteasomal inhibition which resulted in stimulation of the UPR, increased autophagic flux and stimulated heat shock protein response as determined by increased levels of phosphorylation of the eukaryotic translation initiation factor 2 alpha (eIF2α), C/EBP homologous protein (CHOP)/GADD153, turnover of autophagy related microtubule-associated protein 1 light chain 3 (LC3) and increased levels of Hsp70 respectively. After prolonged proteasomal inhibition induced by MG132, we observed the formation of vimentin-caged aggresome-like inclusion bodies. A recovery study after MG132-induced proteasomal inhibition indicated that the autophagy-lysosomal pathway participated in the clearance of aggresomes. Our data characterizes the relationship between proteasome inhibition and activation of other protein handling systems. These data also indicated that the induction of alternate protein handling systems and their temporal relationships may be important factors that determine the extent of accumulation of misfolded proteins in cells as a result of

  14. Physical Activity Levels during Adventure-Physical Education Lessons

    ERIC Educational Resources Information Center

    Gehris, Jeffrey; Myers, Elizabeth; Whitaker, Robert

    2012-01-01

    Adventure-physical education has been proposed to promote adolescents' physical development, but little is known about physical activity levels during such lessons. Using the System for Observing Fitness Instruction Time, we observed students' (ages 11-14 years) physical activity levels in co-educational classes during 43 adventure-physical…

  15. Visualizing active membrane protein complexes by electron cryotomography

    PubMed Central

    Gold, Vicki A.M.; Ieva, Raffaele; Walter, Andreas; Pfanner, Nikolaus; van der Laan, Martin; Kühlbrandt, Werner

    2014-01-01

    Unravelling the structural organization of membrane protein machines in their active state and native lipid environment is a major challenge in modern cell biology research. Here we develop the STAMP (Specifically TArgeted Membrane nanoParticle) technique as a strategy to localize protein complexes in situ by electron cryotomography (cryo-ET). STAMP selects active membrane protein complexes and marks them with quantum dots. Taking advantage of new electron detector technology that is currently revolutionizing cryotomography in terms of achievable resolution, this approach enables us to visualize the three-dimensional distribution and organization of protein import sites in mitochondria. We show that import sites cluster together in the vicinity of crista membranes, and we reveal unique details of the mitochondrial protein import machinery in action. STAMP can be used as a tool for site-specific labelling of a multitude of membrane proteins by cryo-ET in the future. PMID:24942077

  16. Effect of hyperoxaluria on the inhibitory activity of a 45-kD urinary protein.

    PubMed

    Selvam, Ramasamy; Balakrishnan, Selvakumar; Kalaiselvi, Periandavan

    2002-02-01

    Proteins are thought to play a major role in stone formation and structurally abnormal proteins have been reported to be present in the urine of stone formers. This study was aimed to determine whether hyperoxaluria modifies the kinetic properties of urinary inhibitory proteins. Hyperoxaluria was induced by feeding 1% ethylene glycol to rats. Oxalate, uric acid and calcium excretion were increased progressively during hyperoxaluria, while magnesium level was decreased. Urinary proteins were separated on a DEAE-cellulose column by eluting with stepwise increasing salt concentration in 0.05 M Tris-HCl buffer (pH 7.0). Each protein fraction was studied for its crystallization inhibitory potential by the spectrophotometric method. The protein eluted in 0.3 M NaCl containing buffer had the maximal nucleation as well as inhibitory activity. The protein had a molecular weight of 45 kD. In hyperoxaluria, the urinary excretion of this protein significantly increased. In the crystal growth assay, the control rat 45-kD protein inhibited nucleation by 75% and aggregation by 100%. In contrast, it is very interesting to note that the protein derived from 28th day hyperoxaluric urine, behaved as a promoter of nucleation (-113%, percentage inhibition) and weak inhibitor of aggregation (28%). A significantly high negative correlation (r = -0.97) between oxalate excretion and the inhibitory activity of the 45-kD protein was observed suggesting a modification of the protein by oxalate. PMID:11818706

  17. Exposure to Hydrogen Peroxide Induces Oxidation and Activation of AMP-activated Protein Kinase*

    PubMed Central

    Zmijewski, Jaroslaw W.; Banerjee, Sami; Bae, Hongbeom; Friggeri, Arnaud; Lazarowski, Eduardo R.; Abraham, Edward

    2010-01-01

    Although metabolic conditions associated with an increased AMP/ATP ratio are primary factors in the activation of 5′-adenosine monophosphate-activated protein kinase (AMPK), a number of recent studies have shown that increased intracellular levels of reactive oxygen species can stimulate AMPK activity, even without a decrease in cellular levels of ATP. We found that exposure of recombinant AMPKαβγ complex or HEK 293 cells to H2O2 was associated with increased kinase activity and also resulted in oxidative modification of AMPK, including S-glutathionylation of the AMPKα and AMPKβ subunits. In experiments using C-terminal truncation mutants of AMPKα (amino acids 1–312), we found that mutation of cysteine 299 to alanine diminished the ability of H2O2 to induce kinase activation, and mutation of cysteine 304 to alanine totally abrogated the enhancing effect of H2O2 on kinase activity. Similar to the results obtained with H2O2-treated HEK 293 cells, activation and S-glutathionylation of the AMPKα subunit were present in the lungs of acatalasemic mice or mice treated with the catalase inhibitor aminotriazole, conditions in which intracellular steady state levels of H2O2 are increased. These results demonstrate that physiologically relevant concentrations of H2O2 can activate AMPK through oxidative modification of the AMPKα subunit. The present findings also imply that AMPK activation, in addition to being a response to alterations in intracellular metabolic pathways, is directly influenced by cellular redox status. PMID:20729205

  18. Antioxidant activities of protein hydrolysates obtained from the housefly larvae.

    PubMed

    Zhang, Huan; Wang, Pan; Zhang, Ai-Jun; Li, Xuan; Zhang, Ji-Hong; Qin, Qi-Lian; Wu, Yi-Jun

    2016-09-01

    The housefly is an important resource insect and the housefly larvae are ideal source of food additives. The housefly larvae protein hydrolysates were obtained by enzymatic hydrolysis by alcalase and neutral proteinase. Their antioxidant activities were investigated, including the superoxide and hydroxyl radicalscavenging activity, 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity, reducing power and metal chelating activity. The antioxidant activities of both hydrolysates increased with their increasing concentrations. The alcalase hydrolysate (AH) showed higher scavenging activities against hydroxyl radical and superoxide anion radical at low concentrations and higher metal-chelating activity than the neutral proteinase hydrolysate (NPH). The NPH exhibited higher scavenging activity against DPPH free radical and higher reducing power than the AH. Both hydrolysates showed more than 50% superoxide anion radical-scavenging activity at 10 μg/mL. These results indicate that both housefly larvae protein hydrolysates display high antioxidant activities and they could serve as potential natural antioxidant food additives. PMID:27630047

  19. Amyloid precursor protein controls cholesterol turnover needed for neuronal activity

    PubMed Central

    Pierrot, Nathalie; Tyteca, Donatienne; D'auria, Ludovic; Dewachter, Ilse; Gailly, Philippe; Hendrickx, Aurélie; Tasiaux, Bernadette; Haylani, Laetitia El; Muls, Nathalie; N'Kuli, Francisca; Laquerrière, Annie; Demoulin, Jean-Baptiste; Campion, Dominique; Brion, Jean-Pierre; Courtoy, Pierre J; Kienlen-Campard, Pascal; Octave, Jean-Noël

    2013-01-01

    Perturbation of lipid metabolism favours progression of Alzheimer disease, in which processing of Amyloid Precursor Protein (APP) has important implications. APP cleavage is tightly regulated by cholesterol and APP fragments regulate lipid homeostasis. Here, we investigated whether up or down regulation of full-length APP expression affected neuronal lipid metabolism. Expression of APP decreased HMG-CoA reductase (HMGCR)-mediated cholesterol biosynthesis and SREBP mRNA levels, while its down regulation had opposite effects. APP and SREBP1 co-immunoprecipitated and co-localized in the Golgi. This interaction prevented Site-2 protease-mediated processing of SREBP1, leading to inhibition of transcription of its target genes. A GXXXG motif in APP sequence was critical for regulation of HMGCR expression. In astrocytes, APP and SREBP1 did not interact nor did APP affect cholesterol biosynthesis. Neuronal expression of APP decreased both HMGCR and cholesterol 24-hydroxylase mRNA levels and consequently cholesterol turnover, leading to inhibition of neuronal activity, which was rescued by geranylgeraniol, generated in the mevalonate pathway, in both APP expressing and mevastatin treated neurons. We conclude that APP controls cholesterol turnover needed for neuronal activity. PMID:23554170

  20. Solar Activity, Different Geomagnetic Activity Levels and Acute Myocardial Infarction

    NASA Astrophysics Data System (ADS)

    Dimitrova, Svetla; Jordanova, Malina; Stoilova, Irina; Taseva, Tatiana; Maslarov, Dimitar

    Results on revealing a possible relationship between solar activity (SA) and geomagnetic activity (GMA) and acute myocardial infarction (AMI) morbidity are presented. Studies were based on medical data covering the period from 1.12.1995 to 31.12.2004 and concerned daily distribution of patients with AMI diagnose (in total 1192 cases) from Sofia region on the day of admission at the hospital. Analysis of variance (ANOVA) was applied to check the significance of GMA intensity effect and the type of geomagnetic storms, those caused by Magnetic Clouds (MC) and by High Speed Solar Wind Streams (HSSWS), on AMI morbidity. Relevant correlation coefficients were calculated. Results revealed statistically significant positive correlation between considered GMA indices and AMI. ANOVA revealed that AMI number was signifi- cantly increased from the day before (-1st) till the day after (+1st) geomagnetic storms with different intensities. Geomagnetic storms caused by MC were related to significant increase of AMI number in comparison with the storms caused by HSSWS. There was a trend for such different effects even on -1st and +1st day.

  1. Controlled Activation of Protein Rotational Dynamics Using Smart Hydrogel Tethering

    SciTech Connect

    Beech, Brenda M.; Xiong, Yijia; Boschek, Curt B.; Baird, Cheryl L.; Bigelow, Diana J.; Mcateer, Kathleen; Squier, Thomas C.

    2014-09-05

    Stimulus-responsive hydrogel materials that stabilize and control protein dynamics have the potential to enable a range of applications to take advantage of the inherent specificity and catalytic efficiencies of proteins. Here we describe the modular construction of a hydrogel using an engineered calmodulin (CaM) within a polyethylene glycol (PEG) matrix that involves the reversible tethering of proteins through an engineered CaM-binding sequence. For these measurements, maltose binding protein (MBP) was isotopically labeled with [13C] and [15N], permitting dynamic structural measurements using TROSY-HSQC NMR spectroscopy. Upon initial formation of hydrogels protein dynamics are suppressed, with concomitant increases in protein stability. Relaxation of the hydrogel matrix following transient heating results in the activation of protein dynamics and restoration of substrate-induced large-amplitude domain motions necessary for substrate binding.

  2. Severe acute respiratory syndrome coronavirus envelope protein ion channel activity promotes virus fitness and pathogenesis.

    PubMed

    Nieto-Torres, Jose L; DeDiego, Marta L; Verdiá-Báguena, Carmina; Jimenez-Guardeño, Jose M; Regla-Nava, Jose A; Fernandez-Delgado, Raul; Castaño-Rodriguez, Carlos; Alcaraz, Antonio; Torres, Jaume; Aguilella, Vicente M; Enjuanes, Luis

    2014-05-01

    Deletion of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) envelope (E) gene attenuates the virus. E gene encodes a small multifunctional protein that possesses ion channel (IC) activity, an important function in virus-host interaction. To test the contribution of E protein IC activity in virus pathogenesis, two recombinant mouse-adapted SARS-CoVs, each containing one single amino acid mutation that suppressed ion conductivity, were engineered. After serial infections, mutant viruses, in general, incorporated compensatory mutations within E gene that rendered active ion channels. Furthermore, IC activity conferred better fitness in competition assays, suggesting that ion conductivity represents an advantage for the virus. Interestingly, mice infected with viruses displaying E protein IC activity, either with the wild-type E protein sequence or with the revertants that restored ion transport, rapidly lost weight and died. In contrast, mice infected with mutants lacking IC activity, which did not incorporate mutations within E gene during the experiment, recovered from disease and most survived. Knocking down E protein IC activity did not significantly affect virus growth in infected mice but decreased edema accumulation, the major determinant of acute respiratory distress syndrome (ARDS) leading to death. Reduced edema correlated with lung epithelia integrity and proper localization of Na+/K+ ATPase, which participates in edema resolution. Levels of inflammasome-activated IL-1β were reduced in the lung airways of the animals infected with viruses lacking E protein IC activity, indicating that E protein IC function is required for inflammasome activation. Reduction of IL-1β was accompanied by diminished amounts of TNF and IL-6 in the absence of E protein ion conductivity. All these key cytokines promote the progression of lung damage and ARDS pathology. In conclusion, E protein IC activity represents a new determinant for SARS-CoV virulence. PMID:24788150

  3. Towards a new paradigm: Activity level balanced sustainability reporting.

    PubMed

    Samudhram, Ananda; Siew, Eu-Gene; Sinnakkannu, Jothee; Yeow, Paul H P

    2016-11-01

    Technoeconomic paradigms based economic growth theories suggest that waves of technological innovations drove the economic growth of advanced economies. Widespread economic degradation and pollution is an unintended consequence of such growth. Tackling environmental and social issues at firm levels would help us to overcome such issues at macro-levels. Consequently, the Triple Bottom Line (TBL) reporting approach promotes firm level economic, environmental and social performances. Incorporating Zink's (2014) 3-pillar presentation model, this paper indicates that economic, social and environmental performances tend to be reported at firm level. All three pillars are not covered evenly at the activity levels. Thus, a loophole is identified whereby excellent environmental performance at activity levels could potentially leave poor social performance undisclosed. A refinement of the TBL paradigm, whereby all three pillars are covered at the activity level, is suggested, to enhance sustainability reporting.

  4. Towards a new paradigm: Activity level balanced sustainability reporting.

    PubMed

    Samudhram, Ananda; Siew, Eu-Gene; Sinnakkannu, Jothee; Yeow, Paul H P

    2016-11-01

    Technoeconomic paradigms based economic growth theories suggest that waves of technological innovations drove the economic growth of advanced economies. Widespread economic degradation and pollution is an unintended consequence of such growth. Tackling environmental and social issues at firm levels would help us to overcome such issues at macro-levels. Consequently, the Triple Bottom Line (TBL) reporting approach promotes firm level economic, environmental and social performances. Incorporating Zink's (2014) 3-pillar presentation model, this paper indicates that economic, social and environmental performances tend to be reported at firm level. All three pillars are not covered evenly at the activity levels. Thus, a loophole is identified whereby excellent environmental performance at activity levels could potentially leave poor social performance undisclosed. A refinement of the TBL paradigm, whereby all three pillars are covered at the activity level, is suggested, to enhance sustainability reporting. PMID:27029522

  5. Regulation of AMP-activated protein kinase by natural and synthetic activators

    PubMed Central

    Grahame Hardie, David

    2015-01-01

    The AMP-activated protein kinase (AMPK) is a sensor of cellular energy status that is almost universally expressed in eukaryotic cells. While it appears to have evolved in single-celled eukaryotes to regulate energy balance in a cell-autonomous manner, during the evolution of multicellular animals its role has become adapted so that it also regulates energy balance at the whole body level, by responding to hormones that act primarily on the hypothalamus. AMPK monitors energy balance at the cellular level by sensing the ratios of AMP/ATP and ADP/ATP, and recent structural analyses of the AMPK heterotrimer that have provided insight into the complex mechanisms for these effects will be discussed. Given the central importance of energy balance in diseases that are major causes of morbidity or death in humans, such as type 2 diabetes, cancer and inflammatory disorders, there has been a major drive to develop pharmacological activators of AMPK. Many such activators have been described, and the various mechanisms by which these activate AMPK will be discussed. A particularly large class of AMPK activators are natural products of plants derived from traditional herbal medicines. While the mechanism by which most of these activate AMPK has not yet been addressed, I will argue that many of them may be defensive compounds produced by plants to deter infection by pathogens or grazing by insects or herbivores, and that many of them will turn out to be inhibitors of mitochondrial function. PMID:26904394

  6. Regulation of AMP-activated protein kinase by natural and synthetic activators.

    PubMed

    Grahame Hardie, David

    2016-01-01

    The AMP-activated protein kinase (AMPK) is a sensor of cellular energy status that is almost universally expressed in eukaryotic cells. While it appears to have evolved in single-celled eukaryotes to regulate energy balance in a cell-autonomous manner, during the evolution of multicellular animals its role has become adapted so that it also regulates energy balance at the whole body level, by responding to hormones that act primarily on the hypothalamus. AMPK monitors energy balance at the cellular level by sensing the ratios of AMP/ATP and ADP/ATP, and recent structural analyses of the AMPK heterotrimer that have provided insight into the complex mechanisms for these effects will be discussed. Given the central importance of energy balance in diseases that are major causes of morbidity or death in humans, such as type 2 diabetes, cancer and inflammatory disorders, there has been a major drive to develop pharmacological activators of AMPK. Many such activators have been described, and the various mechanisms by which these activate AMPK will be discussed. A particularly large class of AMPK activators are natural products of plants derived from traditional herbal medicines. While the mechanism by which most of these activate AMPK has not yet been addressed, I will argue that many of them may be defensive compounds produced by plants to deter infection by pathogens or grazing by insects or herbivores, and that many of them will turn out to be inhibitors of mitochondrial function. PMID:26904394

  7. The effects of dietary protein levels on the population growth, performance, and physiology of honey bee workers during early spring.

    PubMed

    Zheng, Benle; Wu, Zaifu; Xu, Baohua

    2014-01-01

    This study was conducted to investigate the effects of dietary protein levels on honey bee colonies, specifically the population growth, physiology, and longevity of honey bee workers during early spring. Diets containing four different levels of crude protein (25.0, 29.5, 34.0, or 38.5%) and pure pollen (control) were evaluated. Twenty-five colonies of honey bees with sister queens were used in the study. We compared the effects of the different bee diets by measuring population growth, emergent worker weight, midgut proteolytic enzyme activity, hypopharyngeal gland development, and survival. After 48 d, the cumulative number of workers produced by the colonies ranged from 22,420 to 29,519, providing a significant fit to a quadratic equation that predicts the maximum population growth when the diet contains 31.7% crude protein. Significantly greater emergent worker weight, midgut proteolytic enzyme activity, hypopharyngeal gland acini, and survival were observed in the colonies that were fed diets containing 34.0% crude protein compared with the other crude protein levels. Although higher emergent worker weight and survival were observed in the colonies that were fed the control diet, there were no significant differences between the control colonies and the colonies that were fed 34.0% crude protein. Based on these results, we concluded that a dietary crude protein content of 29.5-34.0% is recommended to maximize the reproduction rate of honey bee colonies in early spring.

  8. The effects of dietary protein levels on the population growth, performance, and physiology of honey bee workers during early spring.

    PubMed

    Zheng, Benle; Wu, Zaifu; Xu, Baohua

    2014-01-01

    This study was conducted to investigate the effects of dietary protein levels on honey bee colonies, specifically the population growth, physiology, and longevity of honey bee workers during early spring. Diets containing four different levels of crude protein (25.0, 29.5, 34.0, or 38.5%) and pure pollen (control) were evaluated. Twenty-five colonies of honey bees with sister queens were used in the study. We compared the effects of the different bee diets by measuring population growth, emergent worker weight, midgut proteolytic enzyme activity, hypopharyngeal gland development, and survival. After 48 d, the cumulative number of workers produced by the colonies ranged from 22,420 to 29,519, providing a significant fit to a quadratic equation that predicts the maximum population growth when the diet contains 31.7% crude protein. Significantly greater emergent worker weight, midgut proteolytic enzyme activity, hypopharyngeal gland acini, and survival were observed in the colonies that were fed diets containing 34.0% crude protein compared with the other crude protein levels. Although higher emergent worker weight and survival were observed in the colonies that were fed the control diet, there were no significant differences between the control colonies and the colonies that were fed 34.0% crude protein. Based on these results, we concluded that a dietary crude protein content of 29.5-34.0% is recommended to maximize the reproduction rate of honey bee colonies in early spring. PMID:25368092

  9. Lack of hepatic enzymatic adaptation to low and high levels of dietary protein in the adult cat.

    PubMed

    Rogers, Q R; Morris, J G; Freedland, R A

    1977-01-01

    The activities of three urea cycle enzymes, several nitrogen catabolic, gluconeogenic, and lipogenic enzymes were measured in the liver of adult cats fed: a commercial kibble; a 17.5 or 70% protein purified diet, or starved for 5 days. Except for an increase in tyrosine transaminase (EC 2.6.1.5) after feeding the high protein diet, there were no changes in the activities of the hepatic enzymes as influenced by dietary protein level. Likewise, starvation had a minimal effect on the activities of these enzymes as compared to that found in similar experiments in rats. These results indicate that the cat may have only minimal capabilities for enzyme adaptation as compared to that found in many herbivores and omnivores and may provide an explanation as to why cats have an unusually high protein requirement as compared to many other mammals.

  10. Recombinant protein production technology

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recombinant protein production is an important technology for antibody production, biochemical activity study, and structural determination during the post-genomic era. Limiting factors in recombinant protein production include low-level protein expression, protein precipitation, and loss of protein...

  11. A proteogenomic approach for protein-level evidence of genomic variants in cancer cells

    PubMed Central

    Yeom, Jeonghun; Kabir, Mohammad Humayun; Lim, Byungho; Ahn, Hee-Sung; Kim, Seon-Young; Lee, Cheolju

    2016-01-01

    Variations in protein coding sequence may sometimes play important roles in cancer development. However, since variants may not express into proteins due to various cellular quality control systems, it is important to get protein-level evidence of the genomic variations. We present a proteogenomic strategy getting protein-level evidence of genomic variants, which we call sequential targeted LC-MS/MS based on prediction of peptide pI and Retention time (STaLPIR). Our approach shows improved peptide identification, and has the potential for the unbiased analysis of variant sequence as well as corresponding reference sequence. Integrated analysis of DNA, mRNA and protein suggests that protein expression level of the nonsynonymous variant is regulated either before or after translation, according to influence of the variant on protein function. In conclusion, our data provides an excellent approach getting direct evidence for the expression of variant protein forms from genome sequence data. PMID:27734975

  12. Methods to alter levels of a DNA repair protein

    DOEpatents

    Petrini, John H.; Morgan, William Francis; Maser, Richard Scott; Carney, James Patrick

    2006-10-17

    An isolated and purified DNA molecule encoding a DNA repair protein, p95, is provided, as is isolated and purified p95. Also provided are methods of detecting p95 and DNA encoding p95. The invention further provides p95 knock-out mice.

  13. Effects of ethanol on protein kinase C alpha activity induced by association with Rho GTPases.

    PubMed

    Slater, Simon J; Cook, Anthony C; Seiz, Jodie L; Malinowski, Steve A; Stagliano, Brigid A; Stubbs, Christopher D

    2003-10-21

    Previous studies have shown that n-alkanols have biphasic chain length-dependent effects on protein kinase C (PKC) activity induced by association with membranes or with filamentous actin [Slater, S. J., et al. (1997) J. Biol. Chem. 272, 6167-6173; Slater, S. J., et al. (2001) Biochim. Biophys. Acta 1544, 207-216]. Recently, we showed that PKCalpha is also activated by a direct membrane lipid-independent interaction with Rho GTPases. Here, the effects of ethanol and 1-hexanol on Rho GTPase-induced activity were investigated using an in vitro assay system to provide further insight into the mechanism of the effects of n-alkanols on PKC activity. Both ethanol and 1-hexanol were found to have two competing concentration-dependent effects on the Ca(2+)- and phorbol ester- or diacylglycerol-dependent activities of PKCalpha associated with either RhoA or Cdc42, consisting of a potentiation at low alcohol levels and an attenuation of activity at higher levels. Measurements of the Ca(2+), phorbol ester, and diacylglycerol concentration-response curves for Cdc42-induced activation indicated that the activating effect corresponded to a shift in the midpoints of each of the curves to lower activator concentrations, while the attenuating effect corresponded to a decrease in the level of activity induced by maximal activator levels. The presence of ethanol enhanced the interaction of PKCalpha with Cdc42 within a concentration range corresponding to the potentiating effect, whereas the level of binding was unaffected by higher ethanol levels that were found to attenuate activity. Thus, ethanol may either enhance activation of PKCalpha by Rho GTPases by enhancing the interaction between the two proteins or attenuate the level of activity of Rho GTPase-associated PKCalpha by inhibiting the ensuing activating conformational change. The results also suggest that the effects of ethanol on Rho GTPase-induced activity may switch between an activation and inhibition depending on the

  14. Hox proteins: sculpting body parts by activating localized cell death.

    PubMed

    Alonso, Claudio R

    2002-11-19

    Hox proteins shape animal structures by eliciting different developmental programs along the anteroposterior body axis. A recent study reveals that the Drosophila Hox protein Deformed directly activates the cell-death-promoting gene reaper to maintain the boundaries between distinct head segments.

  15. Modeling the SHG activities of diverse protein crystals

    SciTech Connect

    Haupert, Levi M.; DeWalt, Emma L.; Simpson, Garth J.

    2012-11-01

    The origins of the diversity in the SHG signal from protein crystals are investigated and potential protein-crystal coverage by SHG microscopy is assessed. A symmetry-additive ab initio model for second-harmonic generation (SHG) activity of protein crystals was applied to assess the likely protein-crystal coverage of SHG microscopy. Calculations were performed for 250 proteins in nine point-group symmetries: a total of 2250 crystals. The model suggests that the crystal symmetry and the limit of detection of the instrument are expected to be the strongest predictors of coverage of the factors considered, which also included secondary-structural content and protein size. Much of the diversity in SHG activity is expected to arise primarily from the variability in the intrinsic protein response as well as the orientation within the crystal lattice. Two or more orders-of-magnitude variation in intensity are expected even within protein crystals of the same symmetry. SHG measurements of tetragonal lysozyme crystals confirmed detection, from which a protein coverage of ∼84% was estimated based on the proportion of proteins calculated to produce SHG responses greater than that of tetragonal lysozyme. Good agreement was observed between the measured and calculated ratios of the SHG intensity from lysozyme in tetragonal and monoclinic lattices.

  16. Hemagglutinating activity of proteins from Parkia speciosa seeds.

    PubMed

    Chankhamjon, Kanokwan; Petsom, Amorn; Sawasdipuksa, Narumon; Sangvanich, Polkit

    2010-01-01

    Proteins from Parkia speciosa Hassk. (Fabaceae) seeds were extracted and stepwise precipitated using ammonium sulfate. Proteins precipitated with 25% ammonium sulfate were separated by affinity chromatography on Affi-Gel Blue gel followed by protein liquid chromatography on Superdex 200. The protein Gj, which was identified as a protein similar to putative aristolochene synthase, 3'-partial from Oryza sativa L. (Poaceae), had hemagglutinating activity of 0.39 mug/muL. Moreover, fraction C2 from the proteins precipitated with 60% ammonium sulfate, separated by lectin-specific adsorption chromatography using Con A Sepharose, had hemagglutinating activity of 1.17 mug/muL. Using gel electrophoresis, two proteins C2a and C2b were separated, having molecular weights of 45 kDa and 23 kDa, respectively. From protein identification, C2a was found to be similar to the hypothetical protein B1342F01.11 from Oryza sativa, and C2b was similar to the hypothetical protein At1g51560 from Arabidopsis thaliana (L.) Heynh. (Brassicaceae). PMID:20645760

  17. TOPICAL REVIEW: Protein stability and enzyme activity at extreme biological temperatures

    NASA Astrophysics Data System (ADS)

    Feller, Georges

    2010-08-01

    Psychrophilic microorganisms thrive in permanently cold environments, even at subzero temperatures. To maintain metabolic rates compatible with sustained life, they have improved the dynamics of their protein structures, thereby enabling appropriate molecular motions required for biological activity at low temperatures. As a consequence of this structural flexibility, psychrophilic proteins are unstable and heat-labile. In the upper range of biological temperatures, thermophiles and hyperthermophiles grow at temperatures > 100 °C and synthesize ultra-stable proteins. However, thermophilic enzymes are nearly inactive at room temperature as a result of their compactness and rigidity. At the molecular level, both types of extremophilic proteins have adapted the same structural factors, but in opposite directions, to address either activity at low temperatures or stability in hot environments. A model based on folding funnels is proposed accounting for the stability-activity relationships in extremophilic proteins.

  18. Fibroblast growth factor, but not activin, is a potent activator of mitogen-activated protein kinase in Xenopus explants.

    PubMed Central

    Graves, L M; Northrop, J L; Potts, B C; Krebs, E G; Kimelman, D

    1994-01-01

    Isolated explants from the animal hemisphere of Xenopus embryos were incubated with Xenopus basic fibroblast growth factor (XbFGF) or human activin A. XbFGF incubation resulted in the rapid activation of mitogen-activated protein kinase (MAPK) and ribosomal S6 protein kinase (pp90rsk) in a dose-dependent manner with the highest levels of activation occurring at 50 ng/ml. Maximal activation occurred within 6-10 min after the addition of growth factor, and the activity of both kinases declined to unstimulated levels after 30 min. Activin was unable to activate either MAPK or pp90rsk in the Xenopus explants to a substantial level, although it induced dorsal mesoderm better than XbFGF under the same experimental conditions. The regulatory protein Xwnt-8 did not activate MAPK, nor did it enhance the activation of MAPK by XbFGF. XbFGF was able to activate MAPK through at least the midgastrula stage, suggesting that this family of growth factors may have a role in gastrula-stage events. Images PMID:7510404

  19. Differential expression of active zone proteins in neuromuscular junctions suggests functional diversification.

    PubMed

    Juranek, Judyta; Mukherjee, Konark; Rickmann, Michael; Martens, Henrik; Calka, Jaroslaw; Südhof, Thomas C; Jahn, Reinhard

    2006-12-01

    Nerve terminals of the central nervous system (CNS) contain specialized release sites for synaptic vesicles, referred to as active zones. They are characterized by electron-dense structures that are tightly associated with the presynaptic plasma membrane and organize vesicle docking and priming sites. Recently, major protein constituents of active zones have been identified, including the proteins Piccolo, Bassoon, RIM, Munc13, ERCs/ELKs/CASTs and liprins. While it is becoming apparent that each of these proteins is essential for synaptic function in the CNS, it is not known to what extent these proteins are involved in synaptic function of the peripheral nervous system. Somatic neuromuscular junctions contain morphologically and functionally defined active zones with similarities to CNS synapses. In contrast, sympathetic neuromuscular varicosities lack active zone-like morphological specializations. Using immunocytochemistry at the light and electron microscopic level we have now performed a systematic investigation of all five major classes of active zone proteins in peripheral neuromuscular junctions. Our results show that somatic neuromuscular endplates contain a full complement of all active zone proteins. In contrast, varicosities of the vas deferens contain a subset of active zone proteins including Bassoon and ELKS2, with the other four components being absent. We conclude that Bassoon and ELKS2 perform independent and specialized functions in synaptic transmission of autonomic synapses.

  20. Activity-based protein profiling identifies a host enzyme, carboxylesterase 1, which is differentially active during hepatitis C virus replication.

    PubMed

    Blais, David R; Lyn, Rodney K; Joyce, Michael A; Rouleau, Yanouchka; Steenbergen, Rineke; Barsby, Nicola; Zhu, Lin-Fu; Pegoraro, Adrian F; Stolow, Albert; Tyrrell, David L; Pezacki, John Paul

    2010-08-13

    Hepatitis C virus (HCV) relies on many interactions with host cell proteins for propagation. Successful HCV infection also requires enzymatic activity of host cell enzymes for key post-translational modifications. To identify such enzymes, we have applied activity-based protein profiling to examine the activity of serine hydrolases during HCV replication. Profiling of hydrolases in Huh7 cells replicating HCV identified CES1 (carboxylesterase 1) as a differentially active enzyme. CES1 is an endogenous liver protein involved in processing of triglycerides and cholesterol. We observe that CES1 expression and activity were altered in the presence of HCV. The knockdown of CES1 with siRNA resulted in lower levels of HCV replication, and up-regulation of CES1 was observed to favor HCV propagation, implying an important role for this host cell protein. Experiments in HCV JFH1-infected cells suggest that CES1 facilitates HCV release because less intracellular HCV core protein was observed, whereas HCV titers remained high. CES1 activity was observed to increase the size and density of lipid droplets, which are necessary for the maturation of very low density lipoproteins, one of the likely vehicles for HCV release. In transgenic mice containing human-mouse chimeric livers, HCV infection also correlates with higher levels of endogenous CES1, providing further evidence that CES1 has an important role in HCV propagation. PMID:20530478

  1. 3pK, a new mitogen-activated protein kinase-activated protein kinase located in the small cell lung cancer tumor suppressor gene region.

    PubMed Central

    Sithanandam, G; Latif, F; Duh, F M; Bernal, R; Smola, U; Li, H; Kuzmin, I; Wixler, V; Geil, L; Shrestha, S

    1996-01-01

    NotI linking clones, localized to the human chromosome 3p21.3 region and homozygously deleted in small cell lung cancer cell lines NCI-H740 and NCI-H1450, were used to search for a putative tumor suppressor gene(s). One of these clones, NL1G210, detected a 2.5-kb mRNA in all examined human tissues, expression being especially high in the heart and skeletal muscle. Two overlapping cDNA clones containing the entire open reading frame were isolated from a human heart cDNA library and fully characterized. Computer analysis and a search of the GenBank database to reveal high sequence identity of the product of this gene to serine-threonine kinases, especially to mitogen-activated protein kinase-activated protein kinase 2, a recently described substrate of mitogen-activated kinases. Sequence identitiy was 72% at the nucleotide level and 75% at the amino acid level, strongly suggesting that this protein is a serine-threonine kinase. Here we demonstrate that the new gene, referred to as 3pK (for chromosome 3p kinase), in fact encodes a mitogen-activated protein kinase-regulated protein serine-threonine kinase with a novel substrate specificity. PMID:8622688

  2. Differential regulation of protein subdomain activity with caged bivalent ligands.

    PubMed

    Mayer, Günter; Müller, Jens; Mack, Timo; Freitag, Daniel F; Höver, Thomas; Pötzsch, Bernd; Heckel, Alexander

    2009-03-01

    Subtle change: Spatiotemporal modulation of individual protein subdomains with light as the trigger signal becomes possible by using bivalent aptamers and introducing photolabile "caging groups" to switch individual aptamer modules ON or OFF differentially. To the best of our knowledge, this is the first study to show that it is possible to modulate individual domain activity in aptamers, and thus also domain activity in proteins, with light.

  3. Transcriptional bursting explains the noise–versus–mean relationship in mRNA and protein levels

    DOE PAGES

    Dar, Roy; Shaffer, Sydney M.; Singh, Abhyudai; Razooky, Brandon S.; Simpson, Michael L.; Raj, Arjun; Weinberger, Leor S.

    2016-07-28

    Recent analysis demonstrates that the HIV-1 Long Terminal Repeat (HIV LTR) promoter exhibits a range of possible transcriptional burst sizes and frequencies for any mean-expression level. However, these results have also been interpreted as demonstrating that cell-tocell expression variability (noise) and mean are uncorrelated, a significant deviation from previous results. Here, we re-examine the available mRNA and protein abundance data for the HIV LTR and find that noise in mRNA and protein expression scales inversely with the mean along analytically predicted transcriptional burst-size manifolds. We then experimentally perturb transcriptional activity to test a prediction of the multiple burst-size model: thatmore » increasing burst frequency will cause mRNA noise to decrease along given burst-size lines as mRNA levels increase. In conclusion, the data show that mRNA and protein noise decrease as mean expression increases, supporting the canonical inverse correlation between noise and mean.« less

  4. Transcriptional Bursting Explains the Noise–Versus–Mean Relationship in mRNA and Protein Levels

    PubMed Central

    Dar, Roy D.; Shaffer, Sydney M.; Singh, Abhyudai; Razooky, Brandon S.; Simpson, Michael L.; Raj, Arjun; Weinberger, Leor S.

    2016-01-01

    Recent analysis demonstrates that the HIV-1 Long Terminal Repeat (HIV LTR) promoter exhibits a range of possible transcriptional burst sizes and frequencies for any mean-expression level. However, these results have also been interpreted as demonstrating that cell-to-cell expression variability (noise) and mean are uncorrelated, a significant deviation from previous results. Here, we re-examine the available mRNA and protein abundance data for the HIV LTR and find that noise in mRNA and protein expression scales inversely with the mean along analytically predicted transcriptional burst-size manifolds. We then experimentally perturb transcriptional activity to test a prediction of the multiple burst-size model: that increasing burst frequency will cause mRNA noise to decrease along given burst-size lines as mRNA levels increase. The data show that mRNA and protein noise decrease as mean expression increases, supporting the canonical inverse correlation between noise and mean. PMID:27467384

  5. Serum levels of bone Gla-protein in inhabitants exposed to environmental cadmium

    SciTech Connect

    Kido, T.; Honda, R.; Tsuritani, I.; Ishizaki, M.; Yamada, Y.; Nakagawa, H.; Nogawa, K.; Dohi, Y. )

    1991-01-01

    Serum levels of bone Gla-protein (BGP)--the vitamin K-dependent CA2(+)-binding protein--were evaluated in 76 cadmium (Cd)-exposed subjects with renal tubular dysfunction (32 men, 44 women) and 133 nonexposed subjects (53 men, 80 women). Serum BGP levels were higher in the Cd-exposed subjects than in nonexposed subjects. Significant correlations between BGP and each index measured by bone microdensitometry (MD), serum alkaline phosphatase activity, and Cd in blood and urine were found. For all of the Cd-exposed and nonexposed men and women, BGP showed a significant standard partial regression coefficient (multiple regression analysis) with the metacarpal index (MCI), which was one of the MD indicators. Bone Gla-protein also correlated significantly with urinary beta 2-microglobulin in the men and with serum creatinine in the women. Serum BGP values strongly reflect the degree of bone damage and also reflect, although less strongly, the degree of renal damage induced by exposure to Cd.

  6. Bone GLA protein (BGP) levels and bone turnover in rheumatoid arthritis.

    PubMed

    Magaro, M; Altomonte, L; Mirone, L; Zoli, A; Corvino, G

    1989-06-01

    Bone GLA protein (BGP) and other biochemical indices of bone turnover were measured in 42 female patients with rheumatoid arthritis (RA) and in a group of normal subjects matched for sex and age. Mean serum BGP concentrations were significantly higher in patients with active arthritis than in patients with mild activity (p less than 0.01) and controls (p less than 0.01). No significant difference was found in serum BGP levels and in other parameters of bone turnover when the patients were stratified according to functional class or duration of disease. There was a correlation between BGP and alkaline phosphatase levels only in RA patients with high activity of disease. Our data suggest an accelerated bone turnover in patients with active RA. We infer that in such patients the impairment of bone metabolism is a determinant of RA-associated osteopenia. Disease activity rather than functional impairment or duration of arthritis should be regarded as a factor in the bone loss of RA.

  7. Cloning of three novel neuronal Cdk5 activator binding proteins.

    PubMed

    Ching, Y P; Qi, Z; Wang, J H

    2000-01-25

    Neuronal Cdc2-like kinase (Nclk) is involved in the regulation of neuronal differentiation and neuro-cytoskeleton dynamics. The active kinase consists of a catalytic subunit, Cdk5, and a 25 kDa activator protein (p25nck5a) derived from a 35 kDa neuronal-specific protein (p35nck5a). As an extension of our previous study (Qi, Z., Tang, D., Zhu, X., Fujita, D.J., Wang, J.H., 1998. Association of neurofilament proteins with neuronal Cdk5 activator. J. Biol. Chem. 270, 2329-2335), which showed that neurofilament is one of the p35nck5a-associated proteins, we now report the isolation of three other novel p35nck5a-associated proteins using the yeast two-hybrid screen. The full-length forms of these three novel proteins, designated C42, C48 and C53, have a molecular mass of 66, 24, and 57 kDa, respectively. Northern analysis indicates that these novel proteins are widely expressed in human tissues, including the heart, brain, skeletal muscle, placenta, lung, liver, kidney and pancreas. The bacterially expressed glutathione S-transferase (GST)-fusion forms of these three proteins were able to co-precipitate p35nck5a complexed with Cdk5 from insect cell lysate. Among these three proteins, only C48 and C53 can be phosphorylated by Nclk, suggesting that they may be the substrates of Nclk. Sequence homology searches have suggested that the C48 protein is marginally related to restin protein, whereas the C42 protein has homologues of unknown function in Caenorhabditis elegans and Arabidopsis thaliana. PMID:10721722

  8. Ferromagnetic interaction model of activity level in workplace communication

    NASA Astrophysics Data System (ADS)

    Akitomi, Tomoaki; Ara, Koji; Watanabe, Jun-ichiro; Yano, Kazuo

    2013-03-01

    The nature of human-human interaction, specifically, how people synchronize with each other in multiple-participant conversations, is described by a ferromagnetic interaction model of people’s activity levels. We found two microscopic human interaction characteristics from a real-environment face-to-face conversation. The first characteristic is that people quite regularly synchronize their activity level with that of the other participants in a conversation. The second characteristic is that the degree of synchronization increases as the number of participants increases. Based on these microscopic ferromagnetic characteristics, a “conversation activity level” was modeled according to the Ising model. The results of a simulation of activity level based on this model well reproduce macroscopic experimental measurements of activity level. This model will give a new insight into how people interact with each other in a conversation.

  9. Mass-tag labeling reveals site-specific and endogenous levels of protein S-fatty acylation.

    PubMed

    Percher, Avital; Ramakrishnan, Srinivasan; Thinon, Emmanuelle; Yuan, Xiaoqiu; Yount, Jacob S; Hang, Howard C

    2016-04-19

    Fatty acylation of cysteine residues provides spatial and temporal control of protein function in cells and regulates important biological pathways in eukaryotes. Although recent methods have improved the detection and proteomic analysis of cysteine fatty (S-fatty) acylated proteins, understanding how specific sites and quantitative levels of this posttranslational modification modulate cellular pathways are still challenging. To analyze the endogenous levels of protein S-fatty acylation in cells, we developed a mass-tag labeling method based on hydroxylamine-sensitivity of thioesters and selective maleimide-modification of cysteines, termed acyl-PEG exchange (APE). We demonstrate that APE enables sensitive detection of protein S-acylation levels and is broadly applicable to different classes of S-palmitoylated membrane proteins. Using APE, we show that endogenous interferon-induced transmembrane protein 3 is S-fatty acylated on three cysteine residues and site-specific modification of highly conserved cysteines are crucial for the antiviral activity of this IFN-stimulated immune effector. APE therefore provides a general and sensitive method for analyzing the endogenous levels of protein S-fatty acylation and should facilitate quantitative studies of this regulated and dynamic lipid modification in biological systems.

  10. Mass-tag labeling reveals site-specific and endogenous levels of protein S-fatty acylation.

    PubMed

    Percher, Avital; Ramakrishnan, Srinivasan; Thinon, Emmanuelle; Yuan, Xiaoqiu; Yount, Jacob S; Hang, Howard C

    2016-04-19

    Fatty acylation of cysteine residues provides spatial and temporal control of protein function in cells and regulates important biological pathways in eukaryotes. Although recent methods have improved the detection and proteomic analysis of cysteine fatty (S-fatty) acylated proteins, understanding how specific sites and quantitative levels of this posttranslational modification modulate cellular pathways are still challenging. To analyze the endogenous levels of protein S-fatty acylation in cells, we developed a mass-tag labeling method based on hydroxylamine-sensitivity of thioesters and selective maleimide-modification of cysteines, termed acyl-PEG exchange (APE). We demonstrate that APE enables sensitive detection of protein S-acylation levels and is broadly applicable to different classes of S-palmitoylated membrane proteins. Using APE, we show that endogenous interferon-induced transmembrane protein 3 is S-fatty acylated on three cysteine residues and site-specific modification of highly conserved cysteines are crucial for the antiviral activity of this IFN-stimulated immune effector. APE therefore provides a general and sensitive method for analyzing the endogenous levels of protein S-fatty acylation and should facilitate quantitative studies of this regulated and dynamic lipid modification in biological systems. PMID:27044110

  11. Activation of an Endoribonuclease by Non-intein Protein Splicing.

    PubMed

    Campbell, Stephen J; Stern, David B

    2016-07-29

    The Chlamydomonas reinhardtii chloroplast-localized poly(A)-binding protein RB47 is predicted to contain a non-conserved linker (NCL) sequence flanked by highly conserved N- and C-terminal sequences, based on the corresponding cDNA. RB47 was purified from chloroplasts in association with an endoribonuclease activity; however, protein sequencing failed to detect the NCL. Furthermore, while recombinant RB47 including the NCL did not display endoribonuclease activity in vitro, versions lacking the NCL displayed strong activity. Both full-length and shorter forms of RB47 could be detected in chloroplasts, with conversion to the shorter form occurring in chloroplasts isolated from cells grown in the light. This conversion could be replicated in vitro in chloroplast extracts in a light-dependent manner, where epitope tags and protein sequencing showed that the NCL was excised from a full-length recombinant substrate, together with splicing of the flanking sequences. The requirement for endogenous factors and light differentiates this protein splicing from autocatalytic inteins, and may allow the chloroplast to regulate the activation of RB47 endoribonuclease activity. We speculate that this protein splicing activity arose to post-translationally repair proteins that had been inactivated by deleterious insertions or extensions. PMID:27311716

  12. Recurrent Selection for Transgene Activity Levels in Maize Results in Proxy Selection for a Native Gene with the Same Promoter.

    PubMed

    Bodnar, Anastasia L; Schroder, Megan N; Scott, M Paul

    2016-01-01

    High activity levels of a transgene can be very useful, making a transgene easier to evaluate for safety and efficacy. High activity levels can also increase the economic benefit of the production of high value proteins in transgenic plants. The goal of this research is to determine if recurrent selection for activity of a transgene will result in higher activity, and if selection for activity of a transgene controlled by a native promoter will also increase protein levels of the native gene with the same promoter. To accomplish this goal we used transgenic maize containing a construct encoding green fluorescent protein controlled by the promoter for the maize endosperm-specific 27 kDa gamma zein seed storage protein. We carried out recurrent selection for fluorescence intensity in two breeding populations. After three generations of selection, both selected populations were significantly more fluorescent and had significantly higher levels of 27 kDa gamma zein than the unselected control populations. These higher levels of the 27 kDa gamma zein occurred independently of the presence of the transgene. The results show that recurrent selection can be used to increase activity of a transgene and that selection for a transgene controlled by a native promoter can increase protein levels of the native gene with the same promoter via proxy selection. Moreover, the increase in native gene protein level is maintained in the absence of the transgene, demonstrating that proxy selection can be used to produce non-transgenic plants with desired changes in gene expression.

  13. Protein metabolism in growing pigs fed corn or cassava peel based diets containing graded protein levels.

    PubMed

    Tewe, O O

    1985-05-01

    Sixty-four Large White cross Landrace weanling pigs were randomly allotted to eight treatments in a two by four factorial arrangement. The two dietary variables were cassava peel (0 and 40 per cent) and crude protein (20, 15, 10 and 5 per cent). Total serum protein concentration was significantly (P less than 0.01) reduced by protein deficiency and by its interaction with cassava peel. The multiple coefficient of determination (R2) showed that protein intake was the primary factor determining changes in serum protein. R2 values for cyanide intake (independent variable) on serum protein (dependent variable) increased from day 30 to 90 of the trial. Serum urea was increased on the 5 per cent protein diets on days 60 and 90 of the trial. The R2 values for cyanide and protein intake on serum urea concentration increased from day 30 to day 90 of the trial. Serum creatinine increased (P less than 0.05) on the 5 per cent protein diet on day 90 of the trial. The R2 value for the effects of protein intake on serum creatinine was higher than for cyanide intake on days 30 and 90. The results confirm the progressive and pronounced effects of long term cyanide intake on serum nitrogenous metabolites in pigs consuming between 110 and 120 ppm hydrocyanic acid, especially in diets containing 10 per cent or less protein. PMID:2989987

  14. Curcumin attenuates diet-induced hepatic steatosis by activating AMP-activated protein kinase.

    PubMed

    Um, Min Young; Hwang, Kwang Hyun; Ahn, Jiyun; Ha, Tae Youl

    2013-09-01

    Curcumin is a well-known component of traditional turmeric (Curcuma longa), which has been reported to prevent obesity and diabetes. However, the effect of curcumin on hepatic lipid metabolism remains unclear. The aim of this study was to examine the effects of curcumin on hepatic steatosis in high-fat/cholesterol diet (HFD)-induced obese mice. Male C57BL/6J mice were fed a normal diet (ND), HFD or HFD with 0.15% curcumin (HFD+C) for 11 weeks. We found that curcumin significantly lowered the body-weight and adipose tissue weight of mice in the HFD+C group compared with the findings for the HFD group (p < 0.05). The levels of total cholesterol, fasting glucose and insulin in serum were decreased, and HFD-induced impairment of insulin sensitivity was improved by curcumin supplementation (p < 0.05). Curcumin protected against the development of hepatic steatosis by reducing hepatic fat accumulation. Moreover, curcumin activated AMP-activated protein kinase (AMPK) and elevated the gene expression of peroxisome proliferator-activated receptor alpha. By contrast, curcumin suppressed the HFD-mediated increases in sterol regulatory element-binding protein-1, acetyl-CoA carboxylase 1, fatty acid synthase and cluster of differentiation 36 expression. Taken together, these findings indicate that curcumin attenuates HFD-induced hepatic steatosis by regulating hepatic lipid metabolism via AMPK activation, suggesting its use as a therapeutic for hepatic steatosis.

  15. Juvenile hormone-activated phospholipase C pathway enhances transcriptional activation by the methoprene-tolerant protein

    PubMed Central

    Liu, Pengcheng; Peng, Hong-Juan; Zhu, Jinsong

    2015-01-01

    Juvenile hormone (JH) is a key regulator of a wide diversity of developmental and physiological events in insects. Although the intracellular JH receptor methoprene-tolerant protein (MET) functions in the nucleus as a transcriptional activator for specific JH-regulated genes, some JH responses are mediated by signaling pathways that are initiated by proteins associated with plasma membrane. It is unknown whether the JH-regulated gene expression depends on the membrane-mediated signal transduction. In Aedes aegypti mosquitoes, we found that JH activated the phospholipase C (PLC) pathway and quickly increased the levels of inositol 1,4,5-trisphosphate, diacylglycerol, and intracellular calcium, leading to activation and autophosphorylation of calcium/calmodulin-dependent protein kinase II (CaMKII). When abdomens from newly emerged mosquitoes were cultured in vitro, the JH-activated gene expression was repressed substantially if specific inhibitors of PLC or CaMKII were added to the medium together with JH. In newly emerged female mosquitoes, RNAi-mediated depletion of PLC or CaMKII considerably reduced the expression of JH-responsive genes, including the Krüppel homolog 1 gene (AaKr-h1) and the early trypsin gene (AaET). JH-induced loading of MET to the promoters of AaKr-h1 and AaET was weakened drastically when either PLC or CaMKII was inactivated in the cultured tissues. Therefore, the results suggest that the membrane-initiated signaling pathway modifies the DNA-binding activity of MET via phosphorylation and thus facilitates the genomic responses to JH. In summary, this study reveals an interplay of genomic and nongenomic signaling mechanisms of JH. PMID:25825754

  16. Increased Levels of Antinutritional and/or Defense Proteins Reduced the Protein Quality of a Disease-Resistant Soybean Cultivar.

    PubMed

    Sousa, Daniele O B; Carvalho, Ana F U; Oliveira, José Tadeu A; Farias, Davi F; Castelar, Ivan; Oliveira, Henrique P; Vasconcelos, Ilka M

    2015-07-01

    The biochemical and nutritional attributes of two soybean (Glycine max (L.) Merr.) cultivars, one susceptible (Seridó) and the other resistant (Seridó-RCH) to stem canker, were examined to assess whether the resistance to pathogens was related to levels of antinutritional and/or defense proteins in the plant and subsequently affected the nutritional quality. Lectin, urease, trypsin inhibitor, peroxidase and chitinase activities were higher in the resistant cultivar. Growing rats were fed with isocaloric and isoproteic diets prepared with defatted raw soybean meals. Those on the Seridó-RCH diet showed the worst performance in terms of protein quality indicators. Based on regression analysis, lectin, trypsin inhibitor, peroxidase and chitinase appear to be involved in the resistance trait but also in the poorer nutritional quality of Seridó-RCH. Thus, the development of cultivars for disease resistance may lead to higher concentrations of antinutritional compounds, affecting the quality of soybean seeds. Further research that includes the assessment of more cultivars/genotypes is needed. PMID:26205163

  17. Increased Levels of Antinutritional and/or Defense Proteins Reduced the Protein Quality of a Disease-Resistant Soybean Cultivar

    PubMed Central

    Sousa, Daniele O. B.; Carvalho, Ana F. U.; Oliveira, José Tadeu A.; Farias, Davi F.; Castelar, Ivan; Oliveira, Henrique P.; Vasconcelos, Ilka M.

    2015-01-01

    The biochemical and nutritional attributes of two soybean (Glycine max (L.) Merr.) cultivars, one susceptible (Seridó) and the other resistant (Seridó-RCH) to stem canker, were examined to assess whether the resistance to pathogens was related to levels of antinutritional and/or defense proteins in the plant and subsequently affected the nutritional quality. Lectin, urease, trypsin inhibitor, peroxidase and chitinase activities were higher in the resistant cultivar. Growing rats were fed with isocaloric and isoproteic diets prepared with defatted raw soybean meals. Those on the Seridó-RCH diet showed the worst performance in terms of protein quality indicators. Based on regression analysis, lectin, trypsin inhibitor, peroxidase and chitinase appear to be involved in the resistance trait but also in the poorer nutritional quality of Seridó-RCH. Thus, the development of cultivars for disease resistance may lead to higher concentrations of antinutritional compounds, affecting the quality of soybean seeds. Further research that includes the assessment of more cultivars/genotypes is needed. PMID:26205163

  18. Immunotherapy for Prostate Cancer with Gc Protein-Derived Macrophage-Activating Factor, GcMAF.

    PubMed

    Yamamoto, Nobuto; Suyama, Hirofumi; Yamamoto, Nobuyuki

    2008-07-01

    Serum Gc protein (known as vitamin D(3)-binding protein) is the precursor for the principal macrophage-activating factor (MAF). The MAF precursor activity of serum Gc protein of prostate cancer patients was lost or reduced because Gc protein was deglycosylated by serum alpha-N-acetylgalactosaminidase (Nagalase) secreted from cancerous cells. Therefore, macrophages of prostate cancer patients having deglycosylated Gc protein cannot be activated, leading to immunosuppression. Stepwise treatment of purified Gc protein with immobilized beta-galactosidase and sialidase generated the most potent MAF (termed GcMAF) ever discovered, which produces no adverse effect in humans. Macrophages activated by GcMAF develop a considerable variation of receptors that recognize the abnormality in malignant cell surface and are highly tumoricidal. Sixteen nonanemic prostate cancer patients received weekly administration of 100 ng of GcMAF. As the MAF precursor activity increased, their serum Nagalase activity decreased. Because serum Nagalase activity is proportional to tumor burden, the entire time course analysis for GcMAF therapy was monitored by measuring the serum Nagalase activity. After 14 to 25 weekly administrations of GcMAF (100 ng/week), all 16 patients had very low serum Nagalase levels equivalent to those of healthy control values, indicating that these patients are tumor-free. No recurrence occurred for 7 years. PMID:18633461

  19. Structural mechanism of G protein activation by G protein-coupled receptor.

    PubMed

    Duc, Nguyen Minh; Kim, Hee Ryung; Chung, Ka Young

    2015-09-15

    G protein-coupled receptors (GPCRs) are a family of membrane receptors that regulate physiology and pathology of various organs. Consequently, about 40% of drugs in the market targets GPCRs. Heterotrimeric G proteins are composed of α, β, and γ subunits, and act as the key downstream signaling molecules of GPCRs. The structural mechanism of G protein activation by GPCRs has been of a great interest, and a number of biochemical and biophysical studies have been performed since the late 80's. These studies investigated the interface between GPCR and G proteins and the structural mechanism of GPCR-induced G protein activation. Recently, arrestins are also reported to be important molecular switches in GPCR-mediated signal transduction, and the physiological output of arrestin-mediated signal transduction is different from that of G protein-mediated signal transduction. Understanding the structural mechanism of the activation of G proteins and arrestins would provide fundamental information for the downstream signaling-selective GPCR-targeting drug development. This review will discuss the structural mechanism of GPCR-induced G protein activation by comparing previous biochemical and biophysical studies.

  20. Protein expression, characterization and activity comparisons of wild type and mutant DUSP5 proteins

    SciTech Connect

    Nayak, Jaladhi; Gastonguay, Adam J.; Talipov, Marat R.; Vakeel, Padmanabhan; Span, Elise A.; Kalous, Kelsey S.; Kutty, Raman G.; Jensen, Davin R.; Pokkuluri, Phani Raj; Sem, Daniel S.; Rathore, Rajendra; Ramchandran, Ramani

    2014-12-18

    Background: The mitogen-activated protein kinases (MAPKs) pathway is critical for cellular signaling, and proteins such as phosphatases that regulate this pathway are important for normal tissue development. Based on our previous work on dual specificity phosphatase-5 (DUSP5), and its role in embryonic vascular development and disease, we hypothesized that mutations in DUSP5 will affect its function. Results: In this study, we tested this hypothesis by generating full-length glutathione-S-transferase-tagged DUSP5 and serine 147 proline mutant (S147P) proteins from bacteria. Light scattering analysis, circular dichroism, enzymatic assays and molecular modeling approaches have been performed to extensively characterize the protein form and function. We demonstrate that both proteins are active and, interestingly, the S147P protein is hypoactive as compared to the DUSP5 WT protein in two distinct biochemical substrate assays. Furthermore, due to the novel positioning of the S147P mutation, we utilize computational modeling to reconstruct full-length DUSP5 and S147P to predict a possible mechanism for the reduced activity of S147P. Conclusion: Taken together, this is the first evidence of the generation and characterization of an active, full-length, mutant DUSP5 protein which will facilitate future structure-function and drug development-based studies.

  1. Protein expression, characterization and activity comparisons of wild type and mutant DUSP5 proteins

    DOE PAGES

    Nayak, Jaladhi; Gastonguay, Adam J.; Talipov, Marat R.; Vakeel, Padmanabhan; Span, Elise A.; Kalous, Kelsey S.; Kutty, Raman G.; Jensen, Davin R.; Pokkuluri, Phani Raj; Sem, Daniel S.; et al

    2014-12-18

    Background: The mitogen-activated protein kinases (MAPKs) pathway is critical for cellular signaling, and proteins such as phosphatases that regulate this pathway are important for normal tissue development. Based on our previous work on dual specificity phosphatase-5 (DUSP5), and its role in embryonic vascular development and disease, we hypothesized that mutations in DUSP5 will affect its function. Results: In this study, we tested this hypothesis by generating full-length glutathione-S-transferase-tagged DUSP5 and serine 147 proline mutant (S147P) proteins from bacteria. Light scattering analysis, circular dichroism, enzymatic assays and molecular modeling approaches have been performed to extensively characterize the protein form and function.more » We demonstrate that both proteins are active and, interestingly, the S147P protein is hypoactive as compared to the DUSP5 WT protein in two distinct biochemical substrate assays. Furthermore, due to the novel positioning of the S147P mutation, we utilize computational modeling to reconstruct full-length DUSP5 and S147P to predict a possible mechanism for the reduced activity of S147P. Conclusion: Taken together, this is the first evidence of the generation and characterization of an active, full-length, mutant DUSP5 protein which will facilitate future structure-function and drug development-based studies.« less

  2. Activated platelets rescue apoptotic cells via paracrine activation of EGFR and DNA-dependent protein kinase

    PubMed Central

    Au, A E-L; Sashindranath, M; Borg, R J; Kleifeld, O; Andrews, R K; Gardiner, E E; Medcalf, R L; Samson, A L

    2014-01-01

    Platelet activation is a frontline response to injury, not only essential for clot formation but also important for tissue repair. Indeed, the reparative influence of platelets has long been exploited therapeutically where application of platelet concentrates expedites wound recovery. Despite this, the mechanisms of platelet-triggered cytoprotection are poorly understood. Here, we show that activated platelets accumulate in the brain to exceptionally high levels following injury and release factors that potently protect neurons from apoptosis. Kinomic microarray and subsequent kinase inhibitor studies showed that platelet-based neuroprotection relies upon paracrine activation of the epidermal growth factor receptor (EGFR) and downstream DNA-dependent protein kinase (DNA-PK). This same anti-apoptotic cascade stimulated by activated platelets also provided chemo-resistance to several cancer cell types. Surprisingly, deep proteomic profiling of the platelet releasate failed to identify any known EGFR ligand, indicating that activated platelets release an atypical activator of the EGFR. This study is the first to formally associate platelet activation to EGFR/DNA-PK – an endogenous cytoprotective cascade. PMID:25210793

  3. The Saccharomyces cerevisiae PUT3 activator protein associates with proline-specific upstream activation sequences.

    PubMed Central

    Siddiqui, A H; Brandriss, M C

    1989-01-01

    The PUT1 and PUT2 genes encoding the enzymes of the proline utilization pathway of Saccharomyces cerevisiae are induced by proline and activated by the product of the PUT3 gene. Two upstream activation sequences (UASs) in the PUT1 promoter were identified by homology to the PUT2 UAS. Deletion analysis of the two PUT1 UASs showed that they were functionally independent and additive in producing maximal levels of gene expression. The consensus PUT UAS is a 21-base-pair partially palindromic sequence required in vivo for induction of both genes. The results of a gel mobility shift assay demonstrated that the proline-specific UAS is the binding site of a protein factor. In vitro complex formation was observed in crude extracts of yeast strains carrying either a single genomic copy of the PUT3 gene or the cloned PUT3 gene on a 2 microns plasmid, and the binding was dosage dependent. DNA-binding activity was not observed in extracts of strains carrying either a put3 mutation that caused a noninducible (Put-) phenotype or a deletion of the gene. Wild-type levels of complex formation were observed in an extract of a strain carrying an allele of PUT3 that resulted in a constitutive (Put+) phenotype. Extracts from a strain carrying a PUT3-lacZ gene fusion formed two complexes of slower mobility than the wild-type complex. We conclude that the PUT3 product is either a DNA-binding protein or part of a DNA-binding complex that recognizes the UASs of both PUT1 and PUT2. Binding was observed in extracts of a strain grown in the presence or absence of proline, demonstrating the constitutive nature of the DNA-protein interaction. Images PMID:2689862

  4. Telomere protein RAP1 levels are affected by cellular aging and oxidative stress

    PubMed Central

    Swanson, Mark J.; Baribault, Michelle E.; Israel, Joanna N.; Bae, Nancy S.

    2016-01-01

    Telomeres are important for maintaining the integrity of the genome through the action of the shelterin complex. Previous studies indicted that the length of the telomere did not have an effect on the amount of the shelterin subunits; however, those experiments were performed using immortalized cells with stable telomere lengths. The interest of the present study was to observe how decreasing telomere lengths over successive generations would affect the shelterin subunits. As neonatal human dermal fibroblasts aged and their telomeres became shorter, the levels of the telomere-binding protein telomeric repeat factor 2 (TRF2) decreased significantly. By contrast, the levels of one of its binding partners, repressor/activator protein 1 (RAP1), decreased to a lesser extent than would be expected from the decrease in TRF2. Other subunits, TERF1-interacting nuclear factor 2 and protection of telomeres protein 1, remained stable. The decrease in RAP1 in the older cells occurred in the nuclear and cytoplasmic fractions. Hydrogen peroxide (H2O2) stress was used as an artificial means of aging in the cells, and this resulted in RAP1 levels decreasing, but the effect was only observed in the nuclear portion. Similar results were obtained using U251 glioblastoma cells treated with H2O2 or grown in serum-depleted medium. The present findings indicate that TRF2 and RAP1 levels decrease as fibroblasts naturally age. RAP1 remains more stable compared to TRF2. RAP1 also responds to oxidative stress, but the response is different to that observed in aging. PMID:27446538

  5. Uncoupling protein 3 expression levels influence insulin sensitivity, fatty acid oxidation, and related signaling pathways.

    PubMed

    Senese, Rosalba; Valli, Vivien; Moreno, Maria; Lombardi, Assunta; Busiello, Rosa Anna; Cioffi, Federica; Silvestri, Elena; Goglia, Fernando; Lanni, Antonia; de Lange, Pieter

    2011-01-01

    Controversy exists on whether uncoupling protein 3 (UCP3) positively or negatively influences insulin sensitivity in vivo, and the underlying signaling pathways have been scarcely studied. We studied how a progressive reduction in UCP3 expression (using UCP3 +/+, UCP3 +/-, and UCP3 -/- mice) modulates insulin sensitivity and related metabolic parameters. In order to further validate our observations, we also studied animals in which insulin resistance was induced by administration of a high-fat diet (HFD). In UCP3 +/- and UCP3 -/- mice, gastrocnemius muscle Akt/protein kinase B (Akt/PKB) (serine 473) and AMP-activated protein kinase (AMPK) (threonine 171) phosphorylation, and glucose transporter 4 (GLUT4) membrane levels were reduced compared to UCP3 +/+ mice. The HOMA-IR index (insulin resistance parameter) was increased both in the UCP3 +/- and UCP3 -/- mice. In these mice, insulin administration normalized Akt/PKB phosphorylation between genotypes while AMPK phosphorylation was further reduced, and sarcolemmal GLUT4 levels were induced but did not reach control levels. Furthermore, non-insulin-stimulated muscle fatty acid oxidation and the expression of several involved genes both in muscle and in liver were reduced. HFD administration induced insulin resistance in UCP3 +/+ mice and the aforementioned parameters resulted similar to those of chow-fed UCP3 +/- and UCP3 -/- mice. In conclusion, high-fat-diet-induced insulin resistance in wild-type mice mimics that of chow-fed UCP3 +/- and UCP3 -/- mice showing that progressive reduction of UCP3 levels results in insulin resistance. This is accompanied by decreased fatty acid oxidation and a less intense Akt/PKB and AMPK signaling.

  6. Notum deacylates Wnt proteins to suppress signalling activity.

    PubMed

    Kakugawa, Satoshi; Langton, Paul F; Zebisch, Matthias; Howell, Steven A; Chang, Tao-Hsin; Liu, Yan; Feizi, Ten; Bineva, Ganka; O'Reilly, Nicola; Snijders, Ambrosius P; Jones, E Yvonne; Vincent, Jean-Paul

    2015-03-12

    Signalling by Wnt proteins is finely balanced to ensure normal development and tissue homeostasis while avoiding diseases such as cancer. This is achieved in part by Notum, a highly conserved secreted feedback antagonist. Notum has been thought to act as a phospholipase, shedding glypicans and associated Wnt proteins from the cell surface. However, this view fails to explain specificity, as glypicans bind many extracellular ligands. Here we provide genetic evidence in Drosophila that Notum requires glypicans to suppress Wnt signalling, but does not cleave their glycophosphatidylinositol anchor. Structural analyses reveal glycosaminoglycan binding sites on Notum, which probably help Notum to co-localize with Wnt proteins. They also identify, at the active site of human and Drosophila Notum, a large hydrophobic pocket that accommodates palmitoleate. Kinetic and mass spectrometric analyses of human proteins show that Notum is a carboxylesterase that removes an essential palmitoleate moiety from Wnt proteins and thus constitutes the first known extracellular protein deacylase. PMID:25731175

  7. Regulatory crosstalk by protein kinases on CFTR trafficking and activity

    NASA Astrophysics Data System (ADS)

    Farinha, Carlos Miguel; Swiatecka-Urban, Agnieszka; Brautigan, David; Jordan, Peter

    2016-01-01

    Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a member of the ATP binding cassette (ABC) transporter superfamily that functions as a cAMP-activated chloride ion channel in fluid-transporting epithelia. There is abundant evidence that CFTR activity (i.e. channel opening and closing) is regulated by protein kinases and phosphatases via phosphorylation and dephosphorylation. Here, we review recent evidence for the role of protein kinases in regulation of CFTR delivery to and retention in the plasma membrane. We review this information in a broader context of regulation of other transporters by protein kinases because the overall functional output of transporters involves the integrated control of both their number at the plasma membrane and their specific activity. While many details of the regulation of intracellular distribution of CFTR and other transporters remain to be elucidated, we hope that this review will motivate research providing new insights into how protein kinases control membrane transport to impact health and disease.

  8. Regulatory Crosstalk by Protein Kinases on CFTR Trafficking and Activity.

    PubMed

    Farinha, Carlos M; Swiatecka-Urban, Agnieszka; Brautigan, David L; Jordan, Peter

    2016-01-01

    Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a member of the ATP binding cassette (ABC) transporter superfamily that functions as a cAMP-activated chloride ion channel in fluid-transporting epithelia. There is abundant evidence that CFTR activity (i.e., channel opening and closing) is regulated by protein kinases and phosphatases via phosphorylation and dephosphorylation. Here, we review recent evidence for the role of protein kinases in regulation of CFTR delivery to and retention in the plasma membrane. We review this information in a broader context of regulation of other transporters by protein kinases because the overall functional output of transporters involves the integrated control of both their number at the plasma membrane and their specific activity. While many details of the regulation of intracellular distribution of CFTR and other transporters remain to be elucidated, we hope that this review will motivate research providing new insights into how protein kinases control membrane transport to impact health and disease.

  9. Regulatory Crosstalk by Protein Kinases on CFTR Trafficking and Activity

    PubMed Central

    Farinha, Carlos M.; Swiatecka-Urban, Agnieszka; Brautigan, David L.; Jordan, Peter

    2016-01-01

    Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a member of the ATP binding cassette (ABC) transporter superfamily that functions as a cAMP-activated chloride ion channel in fluid-transporting epithelia. There is abundant evidence that CFTR activity (i.e., channel opening and closing) is regulated by protein kinases and phosphatases via phosphorylation and dephosphorylation. Here, we review recent evidence for the role of protein kinases in regulation of CFTR delivery to and retention in the plasma membrane. We review this information in a broader context of regulation of other transporters by protein kinases because the overall functional output of transporters involves the integrated control of both their number at the plasma membrane and their specific activity. While many details of the regulation of intracellular distribution of CFTR and other transporters remain to be elucidated, we hope that this review will motivate research providing new insights into how protein kinases control membrane transport to impact health and disease. PMID:26835446

  10. JAB1 regulates unphosphorylated STAT3 DNA-binding activity through protein-protein interaction in human colon cancer cells.

    PubMed

    Nishimoto, Arata; Kugimiya, Naruji; Hosoyama, Toru; Enoki, Tadahiko; Li, Tao-Sheng; Hamano, Kimikazu

    2013-08-30

    Recent studies have revealed that unphosphorylated STAT3 forms a dimer, translocates to the nucleus, binds to the STAT3 binding site, and activates the transcription of STAT3 target genes, thereby playing an important role in oncogenesis in addition to phosphorylated STAT3. Among signaling steps of unphosphorylated STAT3, nuclear translocation and target DNA-binding are the critical steps for its activation. Therefore, elucidating the regulatory mechanism of these signaling steps of unphosphorylated STAT3 is a potential step in the discovery of a novel cancer drug. However, the mechanism of unphosphorylated STAT3 binding to the promoter of target genes remains unclear. In this study, we focused on Jun activation domain-binding protein 1 (JAB1) as a candidate protein that regulates unphosphorylated STAT3 DNA-binding activity. Initially, we observed that both unphosphorylated STAT3 and JAB1 existed in the nucleus of human colon cancer cell line COLO205 at the basal state (no cytokine stimulation). On the other hand, phosphorylated STAT3 did not exist in the nucleus of COLO205 cells at the basal state. Immunoprecipitation using nuclear extract of COLO205 cells revealed that JAB1 interacted with unphosphorylated STAT3. To investigate the effect of JAB1 on unphosphorylated STAT3 activity, RNAi studies were performed. Although JAB1 knockdown tended to increase nuclear STAT3 expression, it significantly decreased unphosphorylated STAT3 DNA-binding activity. Subsequently, JAB1 knockdown significantly decreased the expression levels of MDR1, NANOG, and VEGF, which are STAT3 target genes. Furthermore, the expression level of nuclear JAB1, but not nuclear STAT3, correlated with unphosphorylated STAT3 DNA-binding activity between COLO205 and LoVo cells. Taken together, these results suggest that nuclear JAB1 positively regulates unphosphorylated STAT3 DNA-binding activity through protein-protein interaction in human colon cancer cell line COLO205.

  11. Erythrocyte-derived microparticles supporting activated protein C-mediated regulation of blood coagulation.

    PubMed

    Koshiar, Ruzica Livaja; Somajo, Sofia; Norström, Eva; Dahlbäck, Björn

    2014-01-01

    Elevated levels of erythrocyte-derived microparticles are present in the circulation in medical conditions affecting the red blood cells. Erythrocyte-derived microparticles expose phosphatidylserine thus providing a suitable surface for procoagulant reactions leading to thrombin formation via the tenase and prothrombinase complexes. Patients with elevated levels of circulating erythrocyte-derived microparticles have increased thrombin generation in vivo. The aim of the present study was to investigate whether erythrocyte-derived microparticles are able to support the anticoagulant reactions of the protein C system. Erythrocyte-derived microparticles were isolated using ultracentrifugation after incubation of freshly prepared erythrocytes with the ionophore A23187 or from outdated erythrocyte concentrates, the different microparticles preparations yielding similar results. According to flow cytometry analysis, the microparticles exposed phoshatidylserine and bound lactadherin, annexin V, and protein S, which is a cofactor to activated protein C. The microparticles were able to assemble the tenase and prothrombinase complexes and to stimulate the formation of thrombin in plasma-based thrombin generation assay both in presence and absence of added tissue factor. The addition of activated protein C in the thrombin generation assay inhibited thrombin generation in a dose-dependent fashion. The anticoagulant effect of activated protein C in the thrombin generation assay was inhibited by a monoclonal antibody that prevents binding of protein S to microparticles and also attenuated by anti-TFPI antibodies. In the presence of erythrocyte-derived microparticles, activated protein C inhibited tenase and prothrombinase by degrading the cofactors FVIIIa and FVa, respectively. Protein S stimulated the Arg306-cleavage in FVa, whereas efficient inhibition of FVIIIa depended on the synergistic cofactor activity of protein S and FV. In summary, the erythrocyte-derived microparticle

  12. Pomegranate juice consumption increases GSH levels and reduces lipid and protein oxidation in human blood.

    PubMed

    Matthaiou, Chrysoula M; Goutzourelas, Nikolaos; Stagos, Dimitrios; Sarafoglou, Eleni; Jamurtas, Athanasios; Koulocheri, Sofia D; Haroutounian, Serkos A; Tsatsakis, Aristidis M; Kouretas, Dimitrios

    2014-11-01

    The aim of the present study was the assessment of the antioxidant effects of pomegranate juice (PJ) consumption in humans. Thus, 14 healthy volunteers consumed PJ daily for a period of 15days and the changes of oxidative stress markers in their blood were assessed at four different time points, immediately before the experiment (T1), after 15days of juice administration (T2), one (T3) and three weeks (T4) after the interruption of PJ administration. The markers studied were total antioxidant capacity (TAC), levels of malondialdehyde (MDA), and protein carbonyls (CARB) measured in plasma, as well as reduced glutathione (GSH), and catalase activity (CAT) measured in erythrocytes. The MDA was reduced by 24.4% at T3 and CARB were reduced by 19.6% and 17.7% at T2 and T3, respectively, supporting the evidence that PJ consumption enhances the antioxidant status in humans by decreasing lipid peroxidation and protein oxidation. Moreover, GSH levels were significantly increased (22.6%) at T2, indicating that PJ consumption improves the antioxidant mechanisms in erythrocytes by increasing GSH levels. Finally, it was shown that even a week after stopping PJ consumption some of its beneficial effects on antioxidant status still remained in the organism.

  13. A specific E3 ligase/deubiquitinase pair modulates TBP protein levels during muscle differentiation.

    PubMed

    Li, Li; Martinez, Silvia Sanchez; Hu, Wenxin; Liu, Zhe; Tjian, Robert

    2015-01-01

    TFIID-a complex of TATA-binding protein (TBP) and TBP-associated factors (TAFs)-is a central component of the Pol II promoter recognition apparatus. Recent studies have revealed significant downregulation of TFIID subunits in terminally differentiated myocytes, hepatocytes and adipocytes. Here, we report that TBP protein levels are tightly regulated by the ubiquitin-proteasome system. Using an in vitro ubiquitination assay coupled with biochemical fractionation, we identified Huwe1 as an E3 ligase targeting TBP for K48-linked ubiquitination and proteasome-mediated degradation. Upregulation of Huwe1 expression during myogenesis induces TBP degradation and myotube differentiation. We found that Huwe1 activity on TBP is antagonized by the deubiquitinase USP10, which protects TBP from degradation. Thus, modulating the levels of both Huwe1 and USP10 appears to fine-tune the requisite degradation of TBP during myogenesis. Together, our study unmasks a previously unknown interplay between an E3 ligase and a deubiquitinating enzyme regulating TBP levels during cellular differentiation.

  14. A specific E3 ligase/deubiquitinase pair modulates TBP protein levels during muscle differentiation

    PubMed Central

    Li, Li; Martinez, Silvia Sanchez; Hu, Wenxin; Liu, Zhe; Tjian, Robert

    2015-01-01

    TFIID—a complex of TATA-binding protein (TBP) and TBP-associated factors (TAFs)—is a central component of the Pol II promoter recognition apparatus. Recent studies have revealed significant downregulation of TFIID subunits in terminally differentiated myocytes, hepatocytes and adipocytes. Here, we report that TBP protein levels are tightly regulated by the ubiquitin-proteasome system. Using an in vitro ubiquitination assay coupled with biochemical fractionation, we identified Huwe1 as an E3 ligase targeting TBP for K48-linked ubiquitination and proteasome-mediated degradation. Upregulation of Huwe1 expression during myogenesis induces TBP degradation and myotube differentiation. We found that Huwe1 activity on TBP is antagonized by the deubiquitinase USP10, which protects TBP from degradation. Thus, modulating the levels of both Huwe1 and USP10 appears to fine-tune the requisite degradation of TBP during myogenesis. Together, our study unmasks a previously unknown interplay between an E3 ligase and a deubiquitinating enzyme regulating TBP levels during cellular differentiation. DOI: http://dx.doi.org/10.7554/eLife.08536.001 PMID:26393420

  15. Ku Protein Levels, Localization and Association to Replication Origins in Different Stages of Breast Tumor Progression

    PubMed Central

    Abdelbaqi, Khalil; Di Paola, Domenic; Rampakakis, Emmanouil; Zannis-Hadjopoulos, Maria

    2013-01-01

    Human origins of DNA replication are specific sequences within the genome whereby DNA replication is initiated. A select group of proteins, known as the pre-replication (pre-RC) complex, in whose formation the Ku protein (Ku70/Ku86) was shown to play a role, bind to replication origins to initiate DNA replication. In this study, we have examined the involvement of Ku in breast tumorigenesis and tumor progression and found that the Ku protein expression levels in human breast metastatic (MCF10AC1a) cells were higher in the chromatin fraction compared to hyperplastic (MCF10AT) and normal (MCF10A) human breast cells, but remained constant in both the nuclear and cytoplasmic fractions. In contrast, in human intestinal cells, the Ku expression level was relatively constant for all cell fractions. Nascent DNA abundance and chromatin association of Ku70/86 revealed that the c-myc origin activity in MCF10AC1a is 2.5 to 5-fold higher than in MCF10AT and MCF10A, respectively, and Ku was bound to the c-myc origin more abundantly in MCF10AC1a, by approximately 1.5 to 4.2-fold higher than in MCF10AT and MCF10A, respectively. In contrast, similar nascent DNA abundance and chromatin association was found for all cell lines for the lamin B2 origin, associated with the constitutively active housekeeping lamin B2 gene. Electrophoretic mobility shift assays (EMSAs) performed on the nuclear extracts (NEs) of the three cell types revealed the presence of protein-DNA replication complexes on both the c-myc and lamin B2 origins, but an increase in binding activity was observed from normal, to transformed, to cancer cells for the c-myc origin, whereas no such difference was seen for the lamin B2 origin. Overall, the results suggest that increased Ku chromatin association, beyond wild type levels, alters cellular processes, which have been implicated in tumorigenesis. PMID:23781282

  16. Ku protein levels, localization and association to replication origins in different stages of breast tumor progression.

    PubMed

    Abdelbaqi, Khalil; Di Paola, Domenic; Rampakakis, Emmanouil; Zannis-Hadjopoulos, Maria

    2013-01-01

    Human origins of DNA replication are specific sequences within the genome whereby DNA replication is initiated. A select group of proteins, known as the pre-replication (pre-RC) complex, in whose formation the Ku protein (Ku70/Ku86) was shown to play a role, bind to replication origins to initiate DNA replication. In this study, we have examined the involvement of Ku in breast tumorigenesis and tumor progression and found that the Ku protein expression levels in human breast metastatic (MCF10AC1a) cells were higher in the chromatin fraction compared to hyperplastic (MCF10AT) and normal (MCF10A) human breast cells, but remained constant in both the nuclear and cytoplasmic fractions. In contrast, in human intestinal cells, the Ku expression level was relatively constant for all cell fractions. Nascent DNA abundance and chromatin association of Ku70/86 revealed that the c-myc origin activity in MCF10AC1a is 2.5 to 5-fold higher than in MCF10AT and MCF10A, respectively, and Ku was bound to the c-myc origin more abundantly in MCF10AC1a, by approximately 1.5 to 4.2-fold higher than in MCF10AT and MCF10A, respectively. In contrast, similar nascent DNA abundance and chromatin association was found for all cell lines for the lamin B2 origin, associated with the constitutively active housekeeping lamin B2 gene. Electrophoretic mobility shift assays (EMSAs) performed on the nuclear extracts (NEs) of the three cell types revealed the presence of protein-DNA replication complexes on both the c-myc and lamin B2 origins, but an increase in binding activity was observed from normal, to transformed, to cancer cells for the c-myc origin, whereas no such difference was seen for the lamin B2 origin. Overall, the results suggest that increased Ku chromatin association, beyond wild type levels, alters cellular processes, which have been implicated in tumorigenesis.

  17. Ezrin Binds to DEAD-Box RNA Helicase DDX3 and Regulates Its Function and Protein Level

    PubMed Central

    Çelik, Haydar; Sajwan, Kamal P.; Selvanathan, Saravana P.; Marsh, Benjamin J.; Pai, Amrita V.; Kont, Yasemin Saygideger; Han, Jenny; Minas, Tsion Z.; Rahim, Said; Erkizan, Hayriye Verda; Toretsky, Jeffrey A.

    2015-01-01

    Ezrin is a key regulator of cancer metastasis that links the extracellular matrix to the actin cytoskeleton and regulates cell morphology and motility. We discovered a small-molecule inhibitor, NSC305787, that directly binds to ezrin and inhibits its function. In this study, we used a nano-liquid chromatography-tandem mass spectrometry (nano-LC–MS-MS)-based proteomic approach to identify ezrin-interacting proteins that are competed away by NSC305787. A large number of the proteins that interact with ezrin were implicated in protein translation and stress granule dynamics. We validated direct interaction between ezrin and the RNA helicase DDX3, and NSC305787 blocked this interaction. Downregulation or long-term pharmacological inhibition of ezrin led to reduced DDX3 protein levels without changes in DDX3 mRNA. Ectopic overexpression of ezrin in low-ezrin-expressing osteosarcoma cells caused a notable increase in DDX3 protein levels. Ezrin inhibited the RNA helicase activity of DDX3 but increased its ATPase activity. Our data suggest that ezrin controls the translation of mRNAs preferentially with a structured 5′ untranslated region, at least in part, by sustaining the protein level of DDX3 and/or regulating its function. Therefore, our findings suggest a novel function for ezrin in regulation of gene translation that is distinct from its canonical role as a cytoskeletal scaffold at the cell membrane. PMID:26149384

  18. Fused kinase is stabilized by Cdc37/Hsp90 and enhances Gli protein levels

    SciTech Connect

    Kise, Yoshiaki; Takenaka, Kei; Tezuka, Tohru; Yamamoto, Tadashi; Miki, Hiroaki . E-mail: miki@ims.u-tokyo.ac.jp

    2006-12-08

    Serine/threonine kinase Fused (Fu) is an essential component of Hedgehog (Hh) signaling in Drosophila, but the biochemical functions of Fu remain unclear. Here, we have investigated proteins co-precipitated with mammalian Fu and identified a kinase-specific chaperone complex, Cdc37/Hsp90, as a novel-binding partner of Fu. Inhibition of Hsp90 function by geldanamycin (GA) induces rapid degradation of Fu through a ubiquitin-proteasome pathway. We next show that co-expression of Fu with transcription factors Gli1 and Gli2 significantly increases their protein levels and luciferase reporter activities, which are blocked by GA. These increases can be ascribed to Fu-mediated stabilization of Gli because co-expression of Fu prolongs half-life of Gli1 and reduces polyubiquitination of Gli1. Finally, we show that GA inhibits proliferation of PC3, a Hh signaling-activated prostate cancer cell line. This growth inhibition is partially rescued by expression of ectopic Gli1, suggesting that Fu may contribute to enhance Hh signaling activity in cancer cells.

  19. Heritability and genetic basis of protein level variation in an outbred population

    PubMed Central

    Liu, Yi-Chun; Tekkedil, Manu M.; Steinmetz, Lars M.; Caudy, Amy A.; Fraser, Andrew G.

    2014-01-01

    The genetic basis of heritable traits has been studied for decades. Although recent mapping efforts have elucidated genetic determinants of transcript levels, mapping of protein abundance has lagged. Here, we analyze levels of 4084 GFP-tagged yeast proteins in the progeny of a cross between a laboratory and a wild strain using flow cytometry and high-content microscopy. The genotype of trans variants contributed little to protein level variation between individual cells but explained >50% of the variance in the population’s average protein abundance for half of the GFP fusions tested. To map trans-acting factors responsible, we performed flow sorting and bulk segregant analysis of 25 proteins, finding a median of five protein quantitative trait loci (pQTLs) per GFP fusion. Further, we find that cis-acting variants predominate; the genotype of a gene and its surrounding region had a large effect on protein level six times more frequently than the rest of the genome combined. We present evidence for both shared and independent genetic control of transcript and protein abundance: More than half of the expression QTLs (eQTLs) contribute to changes in protein levels of regulated genes, but several pQTLs do not affect their cognate transcript levels. Allele replacements of genes known to underlie trans eQTL hotspots confirmed the correlation of effects on mRNA and protein levels. This study represents the first genome-scale measurement of genetic contribution to protein levels in single cells and populations, identifies more than a hundred trans pQTLs, and validates the propagation of effects associated with transcript variation to protein abundance. PMID:24823668

  20. Effect of temperature on oxidative stress, antioxidant levels and uncoupling protein expression in striped hamsters.

    PubMed

    Zhou, Si-Si; Cao, Li-Li; Xu, Wei-Dong; Cao, Jing; Zhao, Zhi-Jun

    2015-11-01

    According to the rate of living-free radical hypothesis, higher metabolic rates should increase reactive oxygen species (ROS) production. However, the "uncoupling to survive" hypothesis postulates that uncoupling proteins (UCPs) can decrease ROS production by lowering the potential of the inner mitochondrial membrane, in which case the correlation between metabolic rate and ROS levels would be a negative rather than positive. In this study, we examined energy intake, oxidative stress levels, antioxidant activity and the expression of UCPs in brown adipose tissue (BAT), and in the liver, heart, skeletal muscle and brain, of striped hamsters (Cricetulus barabensis) acclimated to either 5 °C or 32.5 °C. The energy intake of hamsters acclimated to 5 °C increased by 70.7%, whereas the energy intake of hamsters acclimated to 32.5 °C decreased by 31.3%, relative to hamsters kept at room temperature (21 °C) (P<0.05). Malonadialdehyde (MDA) levels, total antioxidant capacity (T-AOC) and glutathione peroxidase (GSH-PX) activity in BAT significantly decreased in 5 °C group, but increased in 32.5 °C group, relative to the 21 °C group. Neither ROS levels (i.e. H2O2 levels), nor antioxidants in skeletal muscle, liver, heart or brain tissue, were affected by temperature. UCP1 expression in BAT was significantly up-regulated in 5 °C group, but down-regulated in 32.5 °C group, relative to the 21 °C group. UCP3 expression of skeletal muscle was also up-regulated significantly in hamsters acclimated to 5 °C. These results suggest that the relationship between ROS levels and metabolic rate was negative, rather than positive. UCP1 expression in BAT may have played a role in lowering ROS levels. PMID:26244518

  1. Effect of temperature on oxidative stress, antioxidant levels and uncoupling protein expression in striped hamsters.

    PubMed

    Zhou, Si-Si; Cao, Li-Li; Xu, Wei-Dong; Cao, Jing; Zhao, Zhi-Jun

    2015-11-01

    According to the rate of living-free radical hypothesis, higher metabolic rates should increase reactive oxygen species (ROS) production. However, the "uncoupling to survive" hypothesis postulates that uncoupling proteins (UCPs) can decrease ROS production by lowering the potential of the inner mitochondrial membrane, in which case the correlation between metabolic rate and ROS levels would be a negative rather than positive. In this study, we examined energy intake, oxidative stress levels, antioxidant activity and the expression of UCPs in brown adipose tissue (BAT), and in the liver, heart, skeletal muscle and brain, of striped hamsters (Cricetulus barabensis) acclimated to either 5 °C or 32.5 °C. The energy intake of hamsters acclimated to 5 °C increased by 70.7%, whereas the energy intake of hamsters acclimated to 32.5 °C decreased by 31.3%, relative to hamsters kept at room temperature (21 °C) (P<0.05). Malonadialdehyde (MDA) levels, total antioxidant capacity (T-AOC) and glutathione peroxidase (GSH-PX) activity in BAT significantly decreased in 5 °C group, but increased in 32.5 °C group, relative to the 21 °C group. Neither ROS levels (i.e. H2O2 levels), nor antioxidants in skeletal muscle, liver, heart or brain tissue, were affected by temperature. UCP1 expression in BAT was significantly up-regulated in 5 °C group, but down-regulated in 32.5 °C group, relative to the 21 °C group. UCP3 expression of skeletal muscle was also up-regulated significantly in hamsters acclimated to 5 °C. These results suggest that the relationship between ROS levels and metabolic rate was negative, rather than positive. UCP1 expression in BAT may have played a role in lowering ROS levels.

  2. Oxidative Stress Impairs the Stimulatory Effect of S100 Proteins on Protein Phosphatase 5 Activity.

    PubMed

    Yamaguchi, Fuminori; Tsuchiya, Mitsumasa; Shimamoto, Seiko; Fujimoto, Tomohito; Tokumitsu, Hiroshi; Tokuda, Masaaki; Kobayashi, Ryoji

    2016-01-01

    Oxidative stress is the consequence of an imbalance between the production of harmful reactive oxygen species and the cellular antioxidant system for neutralization, and it activates multiple intracellular signaling pathways, including apoptosis signal-regulating kinase 1 (ASK1). Protein phosphatase 5 (PP5) is a serine/threonine phosphatase involved in oxidative stress responses. Previously, we reported that S100 proteins activate PP5 in a calcium-dependent manner. S100 proteins belong to a family of small EF-hand calcium-binding proteins involved in many processes such as cell proliferation, differentiation, apoptosis, and inflammation. Therefore, we investigated the effects of oxidative stress on S100 proteins, their interaction with PP5, and PP5 enzyme activity. Recombinant S100A2 was easily air-oxidized or Cu-oxidized, and oxidized S100A2 formed cross-linked dimers and higher molecular-mass complexes. The binding of oxidized S100A2 to PP5 was reduced, resulting in decreased PP5 activation in vitro. Oxidation also impaired S100A1, S100A6, S100B, and S100P to activate PP5, although the low dose of oxidized S100 proteins still activated PP5. Hydrogen peroxide (H2O2) induced S100A2 oxidation in human keratinocytes (HaCaT) and human hepatocellular carcinoma (Huh-7) cells. Furthermore, H2O2 reduced the binding of S100A2 to PP5 and decreased PP5 activation in HaCaT and Huh-7 cells. Importantly, even the low dose of S100A2 achieved by knocking down increased dephosphorylation of ASK1 and reduced caspase 3/7 activity in Huh-7 cells treated with H2O2. These results indicate that oxidative stress impairs the ability of S100 proteins to bind and activate PP5, which in turn modulates the ASK1-mediated signaling cascades involved in apoptosis. PMID:27600583

  3. Physical activity as a determinant of fecal bile acid levels

    PubMed Central

    Wertheim, Betsy C.; Martínez, María Elena; Ashbeck, Erin L.; Roe, Denise J.; Jacobs, Elizabeth T.; Alberts, David S.; Thompson, Patricia A.

    2009-01-01

    Physical activity is protective against colon cancer, whereas colonic bile acid exposure is a suspected risk factor. While likely related, the association between physical activity and bile acid levels has not been well studied. Furthermore, the effect of triglycerides, which are known to modify bile acid levels, on this relationship has not been investigated. We conducted a cross-sectional analysis of baseline fecal bile acid levels for 735 colorectal adenoma formers obtained from participants in a phase III ursodeoxycholic acid chemoprevention trial. Compared to the lowest quartile of recreational physical activity duration, the highest quartile was associated with a 17% lower fecal bile acid concentration, adjusted for age, sex, dietary fiber intake, and body mass index (P = 0.042). Furthermore, consistent with a previously established relationship between serum triglyceride levels and bile acid metabolism, we stratified by triglyceride level and observed a 34% lower fecal bile acid concentration (highest versus lowest quartiles of physical activity) in individuals with low triglycerides (< 136 mg/dL; P = 0.002). In contrast, no association between physical activity and fecal bile acid concentration was observed for subjects with high triglycerides (≥ 136 mg/dL). Our results suggest that the biological mechanism responsible for the protective effect of physical activity on the incidence of colon cancer may be partially mediated by decreasing colonic bile acid exposure. However, this effect may be limited to individuals with lower triglyceride levels. PMID:19383885

  4. Light-dependent activation of G proteins by two isoforms of chicken melanopsins.

    PubMed

    Torii, Masaki; Kojima, Daisuke; Nishimura, Akiyuki; Itoh, Hiroshi; Fukada, Yoshitaka

    2015-11-01

    In the chicken pineal gland, light stimuli trigger signaling pathways mediated by two different subtypes, Gt and G11. These G proteins may be activated by any of the three major pineal opsins, pinopsin, OPN4-1 and OPN4-2, but biochemical evidence for the coupling has been missing except for functional coupling between pinopsin and Gt. Here we investigated the relative expression levels and the functional difference among the three pineal opsins. In the chicken pineal gland, the pinopsin mRNA level was significantly more abundant than the others, of which the OPN4-2 mRNA level was higher than that of OPN4-1. In G protein activation assays, Gt was strongly activated by pinopsin in a light-dependent manner, being consistent with previous studies, and weakly activated by OPN4-2. Unexpectedly, illuminated OPN4-2 more efficiently activated G protein(s) that was endogenously expressed in HEK293T cells in culture. On the other hand, Gq, the closest analogue of G11, was activated only by OPN4-1 although the activity was relatively weak under these conditions. These results suggest that OPN4-1 and OPN4-2 couple with Gq and Gt, respectively. Two melanopsins, OPN4-1 and OPN4-2, appear to have acquired mutually different functions through the evolution.

  5. A conserved patch of hydrophobic amino acids modulates Myb activity by mediating protein-protein interactions.

    PubMed

    Dukare, Sandeep; Klempnauer, Karl-Heinz

    2016-07-01

    The transcription factor c-Myb plays a key role in the control of proliferation and differentiation in hematopoietic progenitor cells and has been implicated in the development of leukemia and certain non-hematopoietic tumors. c-Myb activity is highly dependent on the interaction with the coactivator p300 which is mediated by the transactivation domain of c-Myb and the KIX domain of p300. We have previously observed that conservative valine-to-isoleucine amino acid substitutions in a conserved stretch of hydrophobic amino acids have a profound effect on Myb activity. Here, we have explored the function of the hydrophobic region as a mediator of protein-protein interactions. We show that the hydrophobic region facilitates Myb self-interaction and binding of the histone acetyl transferase Tip60, a previously identified Myb interacting protein. We show that these interactions are affected by the valine-to-isoleucine amino acid substitutions and suppress Myb activity by interfering with the interaction of Myb and the KIX domain of p300. Taken together, our work identifies the hydrophobic region in the Myb transactivation domain as a binding site for homo- and heteromeric protein interactions and leads to a picture of the c-Myb transactivation domain as a composite protein binding region that facilitates interdependent protein-protein interactions of Myb with regulatory proteins.

  6. Organization, structure and activity of proteins in monolayers.

    PubMed

    Boucher, Julie; Trudel, Eric; Méthot, Mario; Desmeules, Philippe; Salesse, Christian

    2007-08-01

    Many different processes take place at the cell membrane interface. Indeed, for instance, ligands bind membrane proteins which in turn activate peripheral membrane proteins, some of which are enzymes whose action is also located at the membrane interface. Native cell membranes are difficult to use to gain information on the activity of individual proteins at the membrane interface because of the large number of different proteins involved in membranous processes. Model membrane systems, such as monolayers at the air-water interface, have thus been extensively used during the last 50 years to reconstitute proteins and to gain information on their organization, structure and activity in membranes. In the present paper, we review the recent work we have performed with membrane and peripheral proteins as well as enzymes in monolayers at the air-water interface. We show that the structure and orientation of gramicidin has been determined by combining different methods. Furthermore, we demonstrate that the secondary structure of rhodopsin and bacteriorhodopsin is indistinguishable from that in native membranes when appropriate conditions are used. We also show that the kinetics and extent of monolayer binding of myristoylated recoverin is much faster than that of the nonmyristoylated form and that this binding is highly favored by the presence polyunsaturated phospholipids. Moreover, we show that the use of fragments of RPE65 allow determine which region of this protein is most likely involved in membrane binding. Monomolecular films were also used to further understand the hydrolysis of organized phospholipids by phospholipases A2 and C.

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

    PubMed

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

    2015-01-01

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

  8. Organization, Structure and Activity of Proteins in Monolayers

    SciTech Connect

    Boucher,J.; Trudel, E.; Methot, M.; Desmeules, P.; Salesse, C.

    2007-01-01

    Many different processes take place at the cell membrane interface. Indeed, for instance, ligands bind membrane proteins which in turn activate peripheral membrane proteins, some of which are enzymes whose action is also located at the membrane interface. Native cell membranes are difficult to use to gain information on the activity of individual proteins at the membrane interface because of the large number of different proteins involved in membranous processes. Model membrane systems, such as monolayers at the air-water interface, have thus been extensively used during the last 50 years to reconstitute proteins and to gain information on their organization, structure and activity in membranes. In the present paper, we review the recent work we have performed with membrane and peripheral proteins as well as enzymes in monolayers at the air-water interface. We show that the structure and orientation of gramicidin has been determined by combining different methods. Furthermore, we demonstrate that the secondary structure of rhodopsin and bacteriorhodopsin is indistinguishable from that in native membranes when appropriate conditions are used. We also show that the kinetics and extent of monolayer binding of myristoylated recoverin is much faster than that of the nonmyristoylated form and that this binding is highly favored by the presence polyunsaturated phospholipids. Moreover, we show that the use of fragments of RPE65 allow determine which region of this protein is most likely involved in membrane binding. Monomolecular films were also used to further understand the hydrolysis of organized phospholipids by phospholipases A2 and C.

  9. Severe acute respiratory syndrome coronavirus E protein transports calcium ions and activates the NLRP3 inflammasome.

    PubMed

    Nieto-Torres, Jose L; Verdiá-Báguena, Carmina; Jimenez-Guardeño, Jose M; Regla-Nava, Jose A; Castaño-Rodriguez, Carlos; Fernandez-Delgado, Raul; Torres, Jaume; Aguilella, Vicente M; Enjuanes, Luis

    2015-11-01

    Severe acute respiratory syndrome coronavirus (SARS-CoV) envelope (E) protein is a viroporin involved in virulence. E protein ion channel (IC) activity is specifically correlated with enhanced pulmonary damage, edema accumulation and death. IL-1β driven proinflammation is associated with those pathological signatures, however its link to IC activity remains unknown. In this report, we demonstrate that SARS-CoV E protein forms protein-lipid channels in ERGIC/Golgi membranes that are permeable to calcium ions, a highly relevant feature never reported before. Calcium ions together with pH modulated E protein pore charge and selectivity. Interestingly, E protein IC activity boosted the activation of the NLRP3 inflammasome, leading to IL-1β overproduction. Calcium transport through the E protein IC was the main trigger of this process. These findings strikingly link SARS-CoV E protein IC induced ionic disturbances at the cell level to immunopathological consequences and disease worsening in the infected organism.

  10. Control of Death-associated Protein Kinase (DAPK) Activity by Phosphorylation and Proteasomal Degradation*

    PubMed Central

    Jin, Yijun; Blue, Emily K.; Gallagher, Patricia J.

    2010-01-01

    Activation of death-associated protein kinase (DAPK) occurs via dephosphorylation of Ser-308 and subsequent association of calcium/calmodulin. In this study, we confirmed the existence of the alternatively spliced human DAPK-β, and we examined the levels of DAPK autophosphorylation and DAPK catalytic activity in response to tumor necrosis factor or ceramide. It was found that DAPK is rapidly dephosphorylated in response to tumor necrosis factor or ceramide and then subsequently degraded via proteasome activity. Dephosphorylation and activation of DAPK are shown to temporally precede its subsequent degradation. This results in an initial increase in kinase activity followed by a decrease in DAPK expression and activity. The decline in DAPK expression is paralleled with increased caspase activity and cell apoptosis. These results suggest that the apoptosis regulatory activities mediated by DAPK are controlled both by phosphorylation status and protein stability. PMID:17056602

  11. Biologically active protein fragments containing specific binding regions of serum albumin or related proteins

    NASA Technical Reports Server (NTRS)

    Carter, Daniel C. (Inventor)

    1998-01-01

    In accordance with the present invention, biologically active protein fragments can be constructed which contain only those specific portions of the serum albumin family of proteins such as regions known as subdomains IIA and IIIA which are primarily responsible for the binding properties of the serum albumins. The artificial serums that can be prepared from these biologically active protein fragments are advantageous in that they can be produced much more easily than serums containing the whole albumin, yet still retain all or most of the original binding potential of the full albumin proteins. In addition, since the protein fragment serums of the present invention can be made from non-natural sources using conventional recombinant DNA techniques, they are far safer than serums containing natural albumin because they do not carry the potentially harmful viruses and other contaminants that will be found in the natural substances.

  12. Latrepirdine is a potent activator of AMP-activated protein kinase and reduces neuronal excitability

    PubMed Central

    Weisová, P; Alvarez, S P; Kilbride, S M; Anilkumar, U; Baumann, B; Jordán, J; Bernas, T; Huber, H J; Düssmann, H; Prehn, J H M

    2013-01-01

    Latrepirdine/Dimebon is a small-molecule compound with attributed neurocognitive-enhancing activities, which has recently been tested in clinical trials for the treatment of Alzheimer's and Huntington's disease. Latrepirdine has been suggested to be a neuroprotective agent that increases mitochondrial function, however the molecular mechanisms underlying these activities have remained elusive. We here demonstrate that latrepirdine, at (sub)nanomolar concentrations (0.1 nM), activates the energy sensor AMP-activated protein kinase (AMPK). Treatment of primary neurons with latrepirdine increased intracellular ATP levels and glucose transporter 3 translocation to the plasma membrane. Latrepirdine also increased mitochondrial uptake of the voltage-sensitive probe TMRM. Gene silencing of AMPKα or its upstream kinases, LKB1 and CaMKKβ, inhibited this effect. However, studies using the plasma membrane potential indicator DisBAC2(3) demonstrated that the effects of latrepirdine on TMRM uptake were largely mediated by plasma membrane hyperpolarization, precluding a purely ‘mitochondrial' mechanism of action. In line with a stabilizing effect of latrepirdine on plasma membrane potential, pretreatment with latrepirdine reduced spontaneous Ca2+ oscillations as well as glutamate-induced Ca2+ increases in primary neurons, and protected neurons against glutamate toxicity. In conclusion, our experiments demonstrate that latrepirdine is a potent activator of AMPK, and suggest that one of the main pharmacological activities of latrepirdine is a reduction in neuronal excitability. PMID:24150226

  13. Blue Light Activates a Specific Protein Kinase in Higher Plants 1

    PubMed Central

    Reymond, Philippe; Short, Timothy W.; Briggs, Winslow R.

    1992-01-01

    Blue light mediates the phosphorylation of a membrane protein in seedlings from several plant species. When crude microsomal membrane proteins from dark-grown pea (Pisum sativum L.), sunflower (Helianthus annuus L.), zucchini (Cucurbita pepo L.), Arabidopsis (Arabidopsis thaliana L.), or tomato (Lycopersicon esculentum L.) stem segments, or from maize (Zea mays L.), barley (Hordeum vulgare L.), oat (Avena sativa L.), wheat (Triticum aestivum L.), or sorghum (Sorghum bicolor L.) coleoptiles are illuminated and incubated in vitro with [γ-32P]ATP, a protein of apparent molecular mass from 114 to 130 kD is rapidly phosphorylated. Hence, this system is probably ubiquitous in higher plants. Solubilized maize membranes exposed to blue light and added to unirradiated solubilized maize membranes show a higher level of phosphorylation of the light-affected protein than irradiated membrane proteins alone, suggesting that an unirradiated substrate is phosphorylated by a light-activated kinase. This finding is further demonstrated with membrane proteins from two different species, where the phosphorylated proteins are of different sizes and, hence, unambiguously distinguishable on gel electrophoresis. When solubilized membrane proteins from one species are irradiated and added to unirradiated membrane proteins from another species, the unirradiated protein becomes phosphorylated. These experiments indicate that the irradiated fraction can store the light signal for subsequent phosphorylation in the dark. They also support the hypothesis that light activates a specific kinase and that the systems share a close functional homology among different higher plants. Images Figure 1 Figure 2 Figure 5 PMID:16653043

  14. Physical Activity Levels in Portuguese High School Physical Education

    ERIC Educational Resources Information Center

    Marmeleira, Jose Francisco Filipe; Aldeias, Nuno Micael Carrasqueira; da Graca, Pedro Miguel dos Santos Medeira

    2012-01-01

    The main aim of this study was to evaluate the physical activity (PA) levels of high school Portuguese students during physical education (PE) and investigate the association of PA levels with students' goal orientation and intrinsic motivation. Forty-six students from three high schools participated. Heart rate telemetry and pedometry were used…

  15. Movement Activity Levels on Traditional and Contemporary Playground Structures.

    ERIC Educational Resources Information Center

    Gabbard, Carl P.; LeBlanc, Elizabeth

    This study investigated playground activity levels of children in grades K-4 and compared levels of use of traditional and creative playground apparatus. The traditional playground area consisted of climbing bars, slides, ladders, chin bars, swings, see saws, and a merry-go-round. The creative playground contained tire hurdles, tire walk, tire…

  16. Factor H-related proteins determine complement-activating surfaces.

    PubMed

    Józsi, Mihály; Tortajada, Agustin; Uzonyi, Barbara; Goicoechea de Jorge, Elena; Rodríguez de Córdoba, Santiago

    2015-06-01

    Complement factor H-related proteins (FHRs) are strongly associated with different diseases involving complement dysregulation, which suggests a major role for these proteins regulating complement activation. Because FHRs are evolutionarily and structurally related to complement inhibitor factor H (FH), the initial assumption was that the FHRs are also negative complement regulators. Whereas weak complement inhibiting activities were originally reported for these molecules, recent developments indicate that FHRs may enhance complement activation, with important implications for the role of these proteins in health and disease. We review these findings here, and propose that FHRs represent a complex set of surface recognition molecules that, by competing with FH, provide improved discrimination of self and non-self surfaces and play a central role in determining appropriate activation of the complement pathway.

  17. Solvating atomic level fine-grained proteins in supra-molecular level coarse-grained water for molecular dynamics simulations.

    PubMed

    Riniker, Sereina; Eichenberger, Andreas P; van Gunsteren, Wilfred F

    2012-08-01

    Simulation of the dynamics of a protein in aqueous solution using an atomic model for both the protein and the many water molecules is still computationally extremely demanding considering the time scale of protein motions. The use of supra-atomic or supra-molecular coarse-grained (CG) models may enhance the computational efficiency, but inevitably at the cost of reduced accuracy. Coarse-graining solvent degrees of freedom is likely to yield a favourable balance between reduced accuracy and enhanced computational speed. Here, the use of a supra-molecular coarse-grained water model that largely preserves the thermodynamic and dielectric properties of atomic level fine-grained (FG) water in molecular dynamics simulations of an atomic model for four proteins is investigated. The results of using an FG, a CG, an implicit, or a vacuum solvent environment of the four proteins are compared, and for hen egg-white lysozyme a comparison to NMR data is made. The mixed-grained simulations do not show large differences compared to the FG atomic level simulations, apart from an increased tendency to form hydrogen bonds between long side chains, which is due to the reduced ability of the supra-molecular CG beads that represent five FG water molecules to make solvent-protein hydrogen bonds. But, the mixed-grained simulations are at least an order of magnitude faster than the atomic level ones.

  18. Influence of particle size on the binding activity of proteins adsorbed onto gold nanoparticles.

    PubMed

    Kaur, Kanwarjeet; Forrest, James A

    2012-02-01

    We used optical extinction spectroscopy to study the structure of proteins adsorbed onto gold nanoparticles of sizes 5-60 nm and their resulting biological binding activity. For these studies, proteins differing in size and shape, with well-characterized and specific interactions-rabbit immunoglobulin G (IgG), goat anti-rabbit IgG (anti-IgG), Staphylococcal protein A, streptavidin, and biotin-were used as model systems. Protein interaction with gold nanoparticles was probed by optical extinction measurements of localized surface plasmon resonance (LSPR) of the gold nanoparticles. Binding of the ligands in solution to protein molecules already immobilized on the surface of gold causes a small but detectable shift in the LSPR peak of the gold nanoparticles. This shift can be used to probe the binding activity of the adsorbed protein. Within the context of Mie theory calculations, the thickness of the adsorbed protein layer as well as its apparent refractive index is shown to depend on the size of the gold nanoparticle. The results suggest that proteins can adopt different orientations that depend on the size of the gold nanospheres. These different orientations, in turn, can result in different levels of biological activity. For example, we find that IgG adsorbed on spheres with diameter ≥20 nm does not bind to protein A. This study illustrates the principle that the size of nanoparticles can strongly influence the binding activity of adsorbed proteins. In addition to the importance of this in cases of direct exposure of proteins to nanoparticles, the results have implications for proteins adsorbed to materials with nanometer scale surface roughness.

  19. Energy transfer at the active sites of heme proteins

    SciTech Connect

    Dlott, D.D.; Hill, J.R.

    1995-12-31

    Experiments using a picosecond pump-probe apparatus at the Picosecond Free-electron Laser Center at Stanford University, were performed to investigate the relaxation of carbon monoxide bound to the active sites of heme proteins. The significance of these experiments is two-fold: (1) they provide detailed information about molecular dynamics occurring at the active sites of proteins; and (2) they provide insight into the nature of vibrational relaxation processes in condensed matter. Molecular engineering is used to construct various molecular systems which are studied with the FEL. We have studied native proteins, mainly myoglobin obtained from different species, mutant proteins produced by genetic engineering using recombinant DNA techniques, and a variety of model systems which mimic the structures of the active sites of native proteins, which are produced using molecular synthesis. Use of these different systems permits us to investigate how specific molecular structural changes affect dynamical processes occurring at the active sites. This research provides insight into the problems of how different species needs are fulfilled by heme proteins which have greatly different functionality, which is induced by rather small structural changes.

  20. The Role of Various Curriculum Models on Physical Activity Levels

    ERIC Educational Resources Information Center

    Culpepper, Dean O.; Tarr, Susan J.; Killion, Lorraine E.

    2011-01-01

    Researchers have suggested that physical education curricula can be highly effective in increasing physical activity levels at school (Sallis & Owen, 1999). The purpose of this study was to investigate the impact of various curriculum models on physical activity. Total steps were measured on 1,111 subjects and three curriculum models were studied…

  1. African American Preschool Children's Physical Activity Levels in Head Start

    ERIC Educational Resources Information Center

    Shen, Bo; Reinhart-Lee, Tamara; Janisse, Heather; Brogan, Kathryn; Danford, Cynthia; Jen, K-L. C.

    2012-01-01

    The purpose of this study was to describe the physical activity levels of urban inner city preschoolers while attending Head Start, the federally funded preschool program for children from low-income families. Participants were 158 African American children. Their physical activity during Head Start days was measured using programmed RT-3…

  2. Active Ageing Level of Older Persons: Regional Comparison in Thailand.

    PubMed

    Haque, Md Nuruzzaman

    2016-01-01

    Active ageing level and its discrepancy in different regions (Bangkok, Central, North, Northeast, and South) of Thailand have been examined for prioritizing the policy agenda to be implemented. Attempt has been made to test preliminary active ageing models for Thai older persons and hence active ageing index (AAI, ranges from 0 to 1) has been estimated. Using nationally representative data and confirmatory factor analysis approach, this study justified active ageing models for female and male older persons in Thailand. Results revealed that active ageing level of Thai older persons is not high (mean AAIs for female and male older persons are 0.64 and 0.61, resp., and those are significantly different (p < 0.001)). Mean AAI in Central region is lower than North, Northeast, and South regions but there is no significant difference in the latter three regions of Thailand. Special emphasis should be given to Central region and policy should be undertaken for increasing active ageing level. Implementation of an Integrated Active Ageing Package (IAAP), containing policies for older persons to improve their health and economic security, to promote participation in social groups and longer working lives, and to arrange learning programs, would be helpful for increasing older persons' active ageing level in Thailand. PMID:27375903

  3. Active Ageing Level of Older Persons: Regional Comparison in Thailand.

    PubMed

    Haque, Md Nuruzzaman

    2016-01-01

    Active ageing level and its discrepancy in different regions (Bangkok, Central, North, Northeast, and South) of Thailand have been examined for prioritizing the policy agenda to be implemented. Attempt has been made to test preliminary active ageing models for Thai older persons and hence active ageing index (AAI, ranges from 0 to 1) has been estimated. Using nationally representative data and confirmatory factor analysis approach, this study justified active ageing models for female and male older persons in Thailand. Results revealed that active ageing level of Thai older persons is not high (mean AAIs for female and male older persons are 0.64 and 0.61, resp., and those are significantly different (p < 0.001)). Mean AAI in Central region is lower than North, Northeast, and South regions but there is no significant difference in the latter three regions of Thailand. Special emphasis should be given to Central region and policy should be undertaken for increasing active ageing level. Implementation of an Integrated Active Ageing Package (IAAP), containing policies for older persons to improve their health and economic security, to promote participation in social groups and longer working lives, and to arrange learning programs, would be helpful for increasing older persons' active ageing level in Thailand.

  4. Seasonality in Children's Pedometer-Measured Physical Activity Levels

    ERIC Educational Resources Information Center

    Beighle, Aaron; Alderman, Brandon; Morgan, Charles F.; Le Masurier, Guy

    2008-01-01

    Seasonality appears to have an impact on children's physical activity levels, but equivocal findings demand more study in this area. With the increased use of pedometers in both research and practice, collecting descriptive data in various seasons to examine the impact of seasonality on pedometer-measured physical activity among children is…

  5. Porins from Salmonella enterica Serovar Typhimurium Activate the Transcription Factors Activating Protein 1 and NF-κB through the Raf-1-Mitogen-Activated Protein Kinase Cascade

    PubMed Central

    Galdiero, Massimiliano; Vitiello, Mariateresa; Sanzari, Emma; D’Isanto, Marina; Tortora, Annalisa; Longanella, Anna; Galdiero, Stefania

    2002-01-01

    In this study we examined the ability of Salmonella enterica serovar Typhimurium porins to activate activating protein 1 (AP-1) and nuclear factor κB (NF-κB) through the mitogen-activated protein kinase (MAPK) cascade, and we identified the AP-1-induced protein subunits. Our results demonstrate that these enzymes may participate in cell signaling pathways leading to AP-1 and NF-κB activation following porin stimulation of cells. Raf-1 was phosphorylated in response to the treatment of U937 cells with porins; moreover, the porin-mediated increase in Raf-1 phosphorylation is accompanied by the phosphorylation of MAPK kinase 1/2 (MEK1/2), p38, extracellular-signal-regulated kinase 1/2, and c-Jun N-terminal kinase. We used three different inhibitors of phosphorylation pathways: 2′-amino-3′-methoxyflavone (PD-098059), a selective inhibitor of MEK1 activator and the MAPK cascade; 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580), a specific inhibitor of the p38 pathway; and 7β-acetoxy-1α,6β,9α-trihydroxy-8,13-epoxy-labd-14-en-11-one (forskolin), an inhibitor at the level of Raf-1 kinase. PD-098059 pretreatment of cells decreases AP-1 and NF-κB activation by lipopolysaccharide (LPS) but not by porins, and SB203580 pretreatment of cells decreases mainly AP-1 and NF-κB activation by porins; in contrast, forskolin pretreatment of cells does not affect AP-1 and NF-κB activation following either porin or LPS stimulation. Our data suggest that the p38 signaling pathway mainly regulates AP-1 and NF-κB activation in cells treated with S. enterica serovar Typhimurium porins. Antibody electrophoretic mobility shift assays showed that JunD and c-Fos binding is found in cells treated with porins, in cells treated with LPS, and in unstimulated cells. However, by 30 to 60 min of stimulation, a different complex including c-Jun appears in cells treated with porins or LPS, while the Fra-2 subunit is present only after porin stimulation

  6. Utilizing avidity to improve antifreeze protein activity: a type III antifreeze protein trimer exhibits increased thermal hysteresis activity.

    PubMed

    Can, Özge; Holland, Nolan B

    2013-12-01

    Antifreeze proteins (AFPs) are ice growth inhibitors that allow the survival of several species living at temperatures colder than the freezing point of their bodily fluids. AFP activity is commonly defined in terms of thermal hysteresis, which is the difference observed for the solution freezing and melting temperatures. Increasing the thermal hysteresis activity of these proteins, particularly at low concentrations, is of great interest because of their wide range of potential applications. In this study, we have designed and expressed one-, two-, and three-domain antifreeze proteins to improve thermal hysteresis activity through increased binding avidity. The three-domain type III AFP yielded significantly greater activity than the one- and two-domain proteins, reaching a thermal hysteresis of >1.6 °C at a concentration of <1 mM. To elucidate the basis of this increase, the data were fit to a multidomain protein adsorption model based on the classical Langmuir isotherm. Fits of the data to the modified isotherms yield values for the equilibrium binding constants for the adsorption of AFP to ice and indicate that protein surface coverage is proportional to thermal hysteresis activity.

  7. Gc protein (vitamin D-binding protein): Gc genotyping and GcMAF precursor activity.

    PubMed

    Nagasawa, Hideko; Uto, Yoshihiro; Sasaki, Hideyuki; Okamura, Natsuko; Murakami, Aya; Kubo, Shinichi; Kirk, Kenneth L; Hori, Hitoshi

    2005-01-01

    The Gc protein (human group-specific component (Gc), a vitamin D-binding protein or Gc globulin), has important physiological functions that include involvement in vitamin D transport and storage, scavenging of extracellular G-actin, enhancement of the chemotactic activity of C5a for neutrophils in inflammation and macrophage activation (mediated by a GalNAc-modified Gc protein (GcMAF)). In this review, the structure and function of the Gc protein is focused on especially with regard to Gc genotyping and GcMAF precursor activity. A discussion of the research strategy "GcMAF as a target for drug discovery" is included, based on our own research. PMID:16302727

  8. Interactive Effects of Indigestible Carbohydrates, Protein Type, and Protein Level on Biomarkers of Large Intestine Health in Rats.

    PubMed

    Taciak, Marcin; Barszcz, Marcin; Tuśnio, Anna; Pastuszewska, Barbara

    2015-01-01

    The effects of indigestible carbohydrates, protein type, and protein level on large intestine health were examined in rats. For 21 days, 12 groups of six 12-week-old male Wistar rats were fed diets with casein (CAS), or potato protein concentrate (PPC), providing 14% (lower protein level; LP), or 20% (higher protein level; HP) protein, and containing cellulose, resistant potato starch, or pectin. Fermentation end-products, pH, and β-glucuronidase levels in cecal digesta, and ammonia levels in colonic digesta were determined. Cecal digesta, tissue weights, cecal and colon morphology, and colonocyte DNA damage were also analyzed. Digesta pH was lower, whereas relative mass of cecal tissue and digesta were higher in rats fed pectin diets than in those fed cellulose. Cecal parameters were greater in rats fed PPC and HP diets than in those fed CAS and LP diets, respectively. Short-chain fatty acid (SCFA) concentrations were unaffected by protein or carbohydrate type. Total SCFA, acetic acid, and propionic acid concentrations were greater in rats fed LP diets than in those fed HP. Cecal pool of isobutyric and isovaleric acids was greater in rats fed PPC than in those fed CAS diets. PPC diets decreased phenol concentration and increased ammonia concentration in cecal and colonic digesta, respectively. Cecal crypt depth was greater in rats fed PPC and HP diets, and was unaffected by carbohydrates; whereas colonic crypt depth was greater in rats fed cellulose. Myenteron thickness in the cecum was unaffected by nutrition, but was greater in the colon of rats fed cellulose. Colonocyte DNA damage was greater in rats fed LP diets than in those fed HP diets, and was unaffected by carbohydrate or protein type. It was found that nutritional factors decreasing cecal digesta weight contribute to greater phenol production, increased DNA damage, and reduced ammonia concentration in the colon. PMID:26536028

  9. Interactive Effects of Indigestible Carbohydrates, Protein Type, and Protein Level on Biomarkers of Large Intestine Health in Rats

    PubMed Central

    Taciak, Marcin; Barszcz, Marcin; Tuśnio, Anna; Pastuszewska, Barbara

    2015-01-01

    The effects of indigestible carbohydrates, protein type, and protein level on large intestine health were examined in rats. For 21 days, 12 groups of six 12-week-old male Wistar rats were fed diets with casein (CAS), or potato protein concentrate (PPC), providing 14% (lower protein level; LP), or 20% (higher protein level; HP) protein, and containing cellulose, resistant potato starch, or pectin. Fermentation end-products, pH, and β-glucuronidase levels in cecal digesta, and ammonia levels in colonic digesta were determined. Cecal digesta, tissue weights, cecal and colon morphology, and colonocyte DNA damage were also analyzed. Digesta pH was lower, whereas relative mass of cecal tissue and digesta were higher in rats fed pectin diets than in those fed cellulose. Cecal parameters were greater in rats fed PPC and HP diets than in those fed CAS and LP diets, respectively. Short-chain fatty acid (SCFA) concentrations were unaffected by protein or carbohydrate type. Total SCFA, acetic acid, and propionic acid concentrations were greater in rats fed LP diets than in those fed HP. Cecal pool of isobutyric and isovaleric acids was greater in rats fed PPC than in those fed CAS diets. PPC diets decreased phenol concentration and increased ammonia concentration in cecal and colonic digesta, respectively. Cecal crypt depth was greater in rats fed PPC and HP diets, and was unaffected by carbohydrates; whereas colonic crypt depth was greater in rats fed cellulose. Myenteron thickness in the cecum was unaffected by nutrition, but was greater in the colon of rats fed cellulose. Colonocyte DNA damage was greater in rats fed LP diets than in those fed HP diets, and was unaffected by carbohydrate or protein type. It was found that nutritional factors decreasing cecal digesta weight contribute to greater phenol production, increased DNA damage, and reduced ammonia concentration in the colon. PMID:26536028

  10. Fluorogen-Activating-Proteins as Universal Affinity Biosensors for Immunodetection

    PubMed Central

    Gallo, Eugenio; Vasilev, Kalin V.; Jarvik, Jonathan

    2014-01-01

    Fluorogen-activating-proteins (FAPs) are a novel platform of fluorescence biosensors utilized for protein discovery. The technology currently demands molecular manipulation methods that limit its application and adaptability. Here, we highlight an alternative approach based on universal affinity reagents for protein detection. The affinity reagents were engineered as bi-partite fusion proteins, where the specificity moiety is derived from IgG-binding proteinsProtein-A or Protein-G – and the signaling element is a FAP. In this manner, primary antibodies provide the antigenic selectivity against a desired protein in biological samples, while FAP affinity reagents target the constant region (Fc) of antibodies and provide the biosensor component of detection. Fluorescence results using various techniques indicate minimal background and high target specificity for exogenous and endogenous proteins in mammalian cells. Additionally, FAP-based affinity reagents provide enhanced properties of detection previously absent using conventional affinity systems. Distinct features explored in this report include: (1) unfixed signal wavelengths (excitation and emission) determined by the particular fluorogen chosen, (2) real-time user controlled fluorescence on-set and off-set, (3) signal wavelength substitution while performing live analysis, and (4) enhanced resistance to photobleaching. PMID:24122476

  11. Nature's "silver bullet" for anticoagulation: mechanism of Zymogen Protein C to Activated Protein C.

    PubMed

    Bruley, Duane F; Streiff, Michael B

    2013-01-01

    We have defined the Zymogen Protein C (ZPC) to Activated Protein C (APC) process as the "silver bullet" of blood anticoagulation. This definition suggests that the anticoagulation activity occurs when and where it is needed, resulting in local anticoagulation without enhanced bleeding. It is important for man to be able to manufacture an inexpensive ZPC product or to find a substitute drug to duplicate one of God's natural anticoagulant/antithrombotic processes, in vivo, in human blood. After intense research and at great expense scientists have not been able to produce a safe anticoagulant. All products that are now being used can cause bleeding even if dosing is carefully monitored. In fact many professionals in the health care and the pharmaceutical industries define an anticoagulant as a drug that "does" cause bleeding. This results in a large financial burden that has been placed on the health care industry because of necessary emergency treatments for dangerous occurrences. In addition, many patients are dying annually due to internal and external bleeds created or enhanced by presently administered anticoagulants. Since there are no safe drugs available it is necessary to use the existing products when a medical condition calls for an anticoagulant. This paper will discuss the ZPC process and why its mechanistic design is one of nature's unique defenses against unwanted blood clotting. The prevention and lysis of clots allows normal blood flow and therefore results in the required tissue oxygenation for cell function and survival. If clinical research is carried out with great care it could uncover other uses of ZPC that will allow safer medical procedures, in addition to its use with standard PC deficiency cases. An important example might be for some brain surgeries where the use of existing anticoagulants is unsafe because of potential bleeds. Clinical research could reveal an efficacious ZPC level (for instance, 125, 150, or 200% of normal) that would

  12. Muscle contractile activity regulates Sirt3 protein expression in rat skeletal muscles.

    PubMed

    Hokari, Fumi; Kawasaki, Emi; Sakai, Atsushi; Koshinaka, Keiichi; Sakuma, Kunihiro; Kawanaka, Kentaro

    2010-08-01

    Sirt3, a member of the sirtuin family, is known to control cellular mitochondrial function. Furthermore, because sirtuins require NAD for their deacetylase activity, nicotinamide phosphoribosyltransferase (Nampt), which is a rate-limiting enzyme in the intracellular NAD biosynthetic pathway, influences their activity. We examined the effects of exercise training and normal postural contractile activity on Sirt3 and Nampt protein expression in rat skeletal muscles. Male rats were trained by treadmill running at 20 m/min, 60 min/day, 7 days/wk for 4 wk. This treadmill training program increased the Sirt3 protein expression in the soleus and plantaris muscles by 49% and 41%, respectively (P < 0.05). Moreover, a 4-wk voluntary wheel-running program also induced 66% and 95% increases in Sirt3 protein in the plantaris and triceps muscles of rats, respectively (P < 0.05). Treadmill-running and voluntary running training induced no significant changes in Nampt protein expression in skeletal muscles. In resting rats, the soleus muscle, which is recruited during normal postural activity, possessed the greatest expression levels of the Sirt3 and Nampt proteins, followed by the plantaris and triceps muscles. Furthermore, the Sirt3, but not Nampt, protein level was reduced in the soleus muscles from immobilized hindlimbs compared with that shown in the contralateral control muscle. These results demonstrated that 1) Sirt3 protein expression is upregulated by exercise training in skeletal muscles and 2) local postural contractile activity plays an important role in maintaining a high level of Sirt3 protein expression in postural muscle.

  13. Peptides and proteins with antimicrobial activity.

    PubMed

    Coutinho, Henrique Douglas Melo; Lôbo, Katiuscia Menezes; Bezerra, Denise Aline Casimiro; Lôbo, Inalzuir

    2008-01-01

    The increase of microbial resistance to antibiotics has led to a continuing search for newer and more effective drugs. Antimicrobial peptides are generally found in animals, plants, and microorganisms and are of great interest to medicine, pharmacology, and the food industry. These peptides are capable of inhibiting pathogenic microorganisms. They can attack parasites, while causing little or no harm to the host cells. The defensins are peptides found in granules in the polymorphonuclear neutrophils (PMNs) and are responsible for the defense of the organism. Several animal defensins, like dermaseptin, antileukoprotease, protegrin, and others, have had their activities and efficacy tested and been shown to be effective against bacteria, fungi, and protists; there are also specific defensins from invertebrates, e.g., drosomycin and heliomicin; from plants, e.g., the types A and B; and the bacteriocins, e.g., acrocin, marcescin, etc. The aim of the present work was to compile a comprehensive bibliographic review of the diverse potentially antimicrobial peptides in an effort to systematize the current knowledge on these substances as a contribution for further researches. The currently available bibliography does not give a holistic approach on this subject. The present work intends to show that the mechanism of defense represented by defensins is promising from the perspective of its application in the treatment of infectious diseases in human, animals and plants.

  14. Heated Proteins are Still Active in a Functionalized Nanoporous Support

    SciTech Connect

    Chen, Baowei; Qi, Wen N.; Li, Xiaolin; Lei, Chenghong; Liu, Jun

    2013-07-08

    We report that even under the heated condition, the conformation and activity of a protein can be hoarded in a functionalized nanoporous support via non-covalent interaction, although the hoarded protein was not exhibiting the full protein activity, the protein released subsequently still maintained its native conformation and activity. Glucose oxidase (GOX) was spontaneously and largely entrapped in aminopropyl-functionalized mesoporous silica (NH2-FMS) at 20 oC via a dominant electrostatic interaction. Although FMS-GOX displayed 45% activity of the free enzyme in solution, the GOX released from FMS exhibited its 100% activity prior to the entrapment. Surprisingly, the released GOX from FMS still maintained 89% of its initial activity prior to the entrapment after FMS-GOX was incubated at 60 oC for 1 h prior to release, while the free GOX in solution lost nearly all activity under the same incubation. Intrinsic fluorescence emission of GOX and native electrophoresis demonstrated that the heating resulted in significant conformational changes and oligomeric structures of the free GOX, but FMS efficiently maintained the thermal stability of GOX therein and resisted the thermal denaturation and oligomeric aggregation.

  15. Staphylokinase as a Plasminogen Activator Component in Recombinant Fusion Proteins

    PubMed Central

    Szarka, S. J.; Sihota, E. G.; Habibi, H. R.; Wong, S.-L.

    1999-01-01

    The plasminogen activator staphylokinase (SAK) is a promising thrombolytic agent for treatment of myocardial infarction. It can specifically stimulate the thrombolysis of both erythrocyte-rich and platelet-rich clots. However, SAK lacks fibrin-binding and thrombin inhibitor activities, two functions which would supplement and potentially improve its thrombolytic potency. Creating a recombinant fusion protein is one approach for combining protein domains with complementary functions. To evaluate SAK for use in a translational fusion protein, both N- and C-terminal fusions to SAK were constructed by using hirudin as a fusion partner. Recombinant fusion proteins were secreted from Bacillus subtilis and purified from culture supernatants. The rate of plasminogen activation by SAK was not altered by the presence of an additional N- or C-terminal protein sequence. However, cleavage at N-terminal lysines within SAK rendered the N-terminal fusion unstable in the presence of plasmin. The results of site-directed mutagenesis of lysine 10 and lysine 11 in SAK suggested that a plasmin-resistant variant cannot be created without interfering with the plasmin processing necessary for activation of SAK. Although putative plasmin cleavage sites are located at the C-terminal end of SAK at lysine 135 and lysine 136, these sites were resistant to plasmin cleavage in vitro. Therefore, C-terminal fusions represent stable configurations for developing improved thrombolytic agents based on SAK as the plasminogen activator component. PMID:9925575

  16. Ca2+ activates human homologous recombination protein Rad51 by modulating its ATPase activity

    PubMed Central

    Bugreev, Dmitry V.; Mazin, Alexander V.

    2004-01-01

    Human Rad51 (hRad51) protein plays a key role in homologous recombination and DNA repair. hRad51 protein forms a helical filament on single-stranded DNA (ssDNA), which performs the basic steps of homologous recombination: a search for homologous double-stranded DNA (dsDNA) and DNA strand exchange. hRad51 protein possesses DNA-dependent ATPase activity; however, the role of this activity has not been understood. Our current results show that Ca2+ greatly stimulates DNA strand exchange activity of hRad51 protein. We found that Ca2+ exerts its stimulatory effect by modulating the ATPase activity of hRad51 protein. Our data demonstrate that, in the presence of Mg2+, the hRad51-ATP-ssDNA filament is quickly converted to an inactive hRad51-ADP-ssDNA form, due to relatively rapid ATP hydrolysis and slow dissociation of ADP. Ca2+ maintains the active hRad51-ATP-ssDNA filament by reducing the ATP hydrolysis rate. These findings demonstrate a crucial role of the ATPase activity in regulation of DNA strand exchange activity of hRad51 protein. This mechanism of Rad51 protein regulation by modulating its ATPase activity is evolutionarily recent; we found no such mechanism for yeast Rad51 (yRad51) protein. PMID:15226506

  17. Identification of lysosomal Npc1-binding proteins: Cathepsin D activity is regulated by NPC1.

    PubMed

    Macías-Vidal, Judit; Guerrero-Hernández, Martina; Estanyol, Josep Maria; Aguado, Carmen; Knecht, Erwin; Coll, Maria Josep; Bachs, Oriol

    2016-01-01

    Niemann-Pick type C (NPC) disease is an inherited lysosomal storage disorder, characterized by severe neurodegeneration. It is mostly produced by mutations in the NPC1 gene, encoding for a protein of the late endosomes/lysosomes membrane, involved in cholesterol metabolism. However, the specific role of this protein in NPC disease still remains unknown. We aimed to identify Npc1-binding proteins in order to define new putative NPC1 lysosomal functions. By affinity chromatography using an Npc1 peptide (amino acids 1032-1066 of loop I), as bait, we fished 31 lysosomal proteins subsequently identified by LC-MS/MS. Most of them were involved in proteolysis and lipid catabolism and included the protease cathepsin D. Cathepsin D and NPC1 interaction was validated by immunoprecipitation and the functional relevance of this interaction was studied. We found that fibroblasts from NPC patients with low levels of NPC1 protein have high amounts of procathepsin D but reduced quantities of the mature protein, thus showing a diminished cathepsin D activity. The increase of NPC1 protein levels in NPC cells by treatment with the proteasome inhibitor bortezomib, induced an elevation of cathepsin D activity. All these results suggest a new lysosomal function of NPC1 as a regulator of cathepsin D processing and activity.

  18. Effect of process conditions on recovery of protein activity after freezing and freeze-drying.

    PubMed

    Jiang, S; Nail, S L

    1998-05-01

    The objective of this research was to gain a better understanding of the degree to which recovery of activity of model proteins after freeze-drying can be maximized by manipulation of freeze-dry process conditions in the absence of protective solutes. Catalase, beta-galactosidase and lactate dehydrogenase (LDH) were used as model proteins. All of the three proteins exhibited a concentration-dependent loss of activity after freezing, with significantly higher recovery at higher concentration. The freezing method and the type of buffer were also important, with sodium phosphate buffer and freezing by immersion of vials in liquid nitrogen associated with the lowest recovery of activity. Differential scanning calorimetry was predictive of the onset of collapse during freeze-drying only for beta-galactosidase. For the other proteins, either no Tg' transition was observed, or the apparent glass transition did not correlate with the microscopically-observed collapse temperature. The time course of activity loss for beta-galactosidase and LDH was compared during freeze-drying under conditions which produced collapse of the dried matrix and conditions which produced retention of microstructure in the dried solid. Recovery of activity decreased continuously during primary drying, with no sharp drop in recovery of activity associated with the onset of collapse. The most important drying process variable affecting recovery of activity was residual moisture level, with a dramatic drop in activity recovery associated with residual moisture levels less than about 10%. PMID:9653629

  19. Calcium, phosphorus and protein levels as factors in the distribution of the pheasant

    USGS Publications Warehouse

    Dale, F.H.; DeWitt, J.B.

    1958-01-01

    Summary of work on pheasant nutrition conducted since 1949 at the Patuxent Research Refuge. Pheasant chicks fed experimental diets failed to develop normally on protein levels of 15 and 18%. With 22% protein they grew at a reduced rate as compared to those on 28%. Protein level of the reproductive diet was shown to be important; low production of eggs and young resulted from levels below 25%. Calcium was found to be even more critical than protein level for reproduction; birds on a winter diet that furnished 145 mg./kg. per day had poor reproductive success the following spring. About 600 mg./kg. of Ca per day was necessary in the reproduction diet. Birds on an intermediate level of Ca (about 0.5% of diet) showed evidence of cumulative deficiency. It was concluded that pheasants receiving levels of Ca no higher than 0.5% in nature might display 'straggling failure' such as has been observed in several midwestern areas.

  20. Low Serum Level α-Synuclein and Tau Protein in Autism Spectrum Disorder Compared to Controls.

    PubMed

    Kadak, Muhammed Tayyib; Cetin, Ihsan; Tarakçıoğlu, Mahmut Cem; Özer, Ömer Faruk; Kaçar, Selma; Çimen, Behzat

    2015-12-01

    α-Synuclein (α-syn) and tau proteins are thought to be related with the synaptic loss and cell death underlying several important neurodegenerative diseases. The aim of our study was to investigate serum α-syn and tau levels in autism. Serum levels of α-syn and tau were measured, and autism spectrum disorder (ASD) severity was assessed at admission using the Childhood Autism Rating Scale (CARS) total score. The mean CARS score of the autism group on admission was 47.91 points (SD: 5.97). The results indicated that the mean serum α-syn and serum tau levels were significantly (p < 0.001) lower in children with ASD as compared with normal cases (33.01 ± 20.78 and 55.19 ± 15.34 ng/mL and 241.23 ± 290.5 and 509.78 ± 269.25 ng/mL, respectively). There was a significant positive correlation between serum α-syn levels and serum levels of tau identified by Pearson correlation analysis (r = 0.922, n = 28, p < 0.001). Synaptic abnormality in autism may result from microglial activity. Furthermore, α-syn and tau aggregation may lead to synaptic dysfunction, and this may contribute to either neuronal or synaptic dysfunction or neurodegeneration. Our preliminary study suggests that low levels of serum α-syn and tau may be implicated in the relationship between synaptic activity and autism.

  1. Effect of the level of dietary protein on the utilization of alpha-ketoisocaproate for protein synthesis

    SciTech Connect

    Kang, C.W.; Tungsanga, K.; Walser, M.

    1986-04-01

    The efficiency of alpha-ketoisocaproate (KIC) as a dietary substitute for leucine in rats on varying protein intake was estimated by an isotopic method, previously shown to yield the same results as comparative growth experiments. /sup 14/C-KIC and /sup 3/H-leucine are injected orally. Six hours later the ratio, R, of /sup 14/C//sup 3/H in isolated proteins, divided by the same ratio in the injectate is measured. This ratio has been shown to be approximately equal to nutritional efficiency of KIC relative to leucine. As dietary protein increased from 6.3% to 48.3%, whole body protein R decreased from 0.515 +/- 0.045 to 0.299 +/- 0.016. Variations with protein intake were noted in R of protein isolated from individual organs. The magnitude of R in these organs varied two-fold, in the following sequence: brain greater than heart greater than or equal to skeletal muscle greater than or equal to salivary gland greater than or equal to kidney greater than liver. Whole body protein R could be confidently predicted (r2 = 0.992) from R in the protein of kidney and muscle. Thus, the nutritional efficiency of KIC as a dietary substitute for leucine in individual organs as well as in the whole animal is strongly dependent on the level of protein intake.

  2. Plasma levels of the chemokines monocyte chemotactic proteins-1 and -2 are elevated in human sepsis.

    PubMed

    Bossink, A W; Paemen, L; Jansen, P M; Hack, C E; Thijs, L G; Van Damme, J

    1995-11-15

    Because of their effects on monocytes, monocyte chemotactic proteins-1 and -2 (MCP-1 and MCP-2) may participate in the pathophysiology of sepsis. We measured circulating MCP-1 and MCP-2 levels in 42 septic patients having positive local or blood cultures. MCP-1 and MCP-2 levels were elevated in 24 (57%) and 25 (59%) of 42 septic patients, respectively, compared with healthy volunteers. Both patients with gram-positive and gram-negative infections had elevated MCP-1 plasma levels (P = .0001) and P < .0001), respectively; Mann-Whitney-U test), whereas patients with gram-positive infection, but not those with gram-negative infection, had increased MCP-2 plasma levels (P= .0182). No relative differences in MCP-1 and MCP-2 plasma levels were observed between several subgroups of patients (sepsis v septic shock; survivors v nonsurvivors), although levels of MCP-1 were the highest in patients with the more severe forms of sepsis, ie, those with shock or a lethal outcome. Serial observations showed that MCP-1 and MCP-2 plasma levels remained elevated for at least 48 hours. MCP-1 correlated weakly with interleukin-8 and MCP-2, the correlations for which were most pronounced in patients with septic shock. MCP-2 correlated with interleukin-8, and surprisingly, with the complement activation product C3a; these correlations further improved when analyzing patients with septic shock or when applying gram-positive infections. Thus, our results not only show increased MCP-1 and MCP-2 levels in patients with sepsis, but also suggest that the synthesis and release of MCP-1 and MCP-2 in sepsis are differently regulated in part.

  3. Copper uptake is required for pyrrolidine dithiocarbamate-mediated oxidation and protein level increase of p53 in cells.

    PubMed Central

    Furuta, Saori; Ortiz, Fausto; Zhu Sun, Xiu; Wu, Hsiao-Huei; Mason, Andrew; Momand, Jamil

    2002-01-01

    The p53 tumour-suppressor protein is a transcription factor that activates the expression of genes involved in cell cycle arrest, apoptosis and DNA repair. The p53 protein is vulnerable to oxidation at cysteine thiol groups. The metal-chelating dithiocarbamates, pyrrolidine dithiocarbamate (PDTC), diethyldithiocarbamate, ethylene(bis)dithiocarbamate and H(2)O(2) were tested for their oxidative effects on p53 in cultured human breast cancer cells. Only PDTC oxidized p53, although all oxidants tested increased the p53 level. Inductively coupled plasma MS analysis indicated that the addition of 60 microM PDTC increased the cellular copper concentration by 4-fold, which was the highest level of copper accumulated amongst all the oxidants tested. Bathocuproinedisulphonic acid, a membrane-impermeable Cu(I) chelator inhibited the PDTC-mediated copper accumulation. Bathocuproinedisulphonic acid as well as the hydroxyl radical scavenger d-mannitol inhibited the PDTC-dependent increase in p53 protein and oxidation. Our results show that a low level of copper accumulation in the range of 25-40 microg/g of cellular protein increases the steady-state levels of p53. At copper accumulation levels higher than 60 microg/g of cellular protein, p53 is oxidized. These results suggest that p53 is vulnerable to free radical-mediated oxidation at cysteine residues. PMID:11964141

  4. Circulating IGF-axis Protein Levels and Their Relation with Levels of Plasma Adipocytokines and Macronutrient Consumption in Women

    PubMed Central

    Beasley, Jeannette M.; Wedick, Nicole M.; Rajpathak, Swapnil N.; Xue, Xiaonan; Holmes, Michelle D.; Gunter, Marc J.; Wylie-Rosett, Judith; Rohan, Thomas E.; Pollak, Michael; Kaplan, Robert C.; Hu, Frank B.; Sun, Qi; Strickler, Howard D.

    2014-01-01

    Objective Circulating free insulin-like growth factor (IGF)-I and its binding proteins, most notably, IGFBP-1 and IGFBP-2, have been prospectively associated with incident type 2 diabetes in women. However, little is known regarding the factors that may influence these IGF-axis protein levels. To study the relation of IGF-axis protein levels with adipocytokines, macronutrient consumption, and other factors related to diabetes. Design Fasting plasma from 558 controls enrolled in a nested case-control study within the Nurses’ Health Study of incident type 2 diabetes in women were tested for: IGF-axis proteins (free and total IGF-I, IGFBP-1, IGFBP-2, IGFBP-3), adipocytokines (leptin, adiponectin, resistin), soluble leptin receptor (sOB-R), inflammatory factors (IL-18 and C-reactive protein (CRP)), insulin, and glycated hemoglobin (HbA1C). Results In multivariate models, each 1% increase in sOB-R (mean 34.9 ng/mL, standard deviation (SD) ±11.3) was associated with −0.20% total IGF-I (P=0.0003) and −0.42% free IGF-I (P=0.002), as well as 0.73% higher IGFBP-1 (P<0.0001) and 0.27% IGFBP-2 (P=0.003). For example, a one SD change from the mean sOB-R level was associated with 11% lower free IGF-I. Insulin levels (mean 6.8 μU/mL ±5.3) were inversely and adiponectin (mean 18.3 μg/mL ±7.4) positively associated with IGFBP-1 and IGFBP-2 (all P<0.01). Consumption of dairy protein, monounsaturated fats, and saturated fats, was also correlated with IGF-axis protein levels (all P<0.05). Conclusions Several molecular factors and macronutrients were independently associated with plasma IGF-axis protein levels. Which of these, if any, reflect biologic relationships that can be intervened upon to influence IGF-axis protein concentrations warrants further investigation. PMID:24888819

  5. A local average connectivity-based method for identifying essential proteins from the network level.

    PubMed

    Li, Min; Wang, Jianxin; Chen, Xiang; Wang, Huan; Pan, Yi

    2011-06-01

    Identifying essential proteins is very important for understanding the minimal requirements of cellular survival and development. Fast growth in the amount of available protein-protein interactions has produced unprecedented opportunities for detecting protein essentiality from the network level. Essential proteins have been found to be more abundant among those highly connected proteins. However, there exist a number of highly connected proteins which are not essential. By analyzing these proteins, we find that few of their neighbors interact with each other. Thus, we propose a new local method, named LAC, to determine a protein's essentiality by evaluating the relationship between a protein and its neighbors. The performance of LAC is validated based on the yeast protein interaction networks obtained from two different databases: DIP and BioGRID. The experimental results of the two networks show that the number of essential proteins predicted by LAC clearly exceeds that explored by Degree Centrality (DC). More over, LAC is also compared with other seven measures of protein centrality (Neighborhood Component (DMNC), Betweenness Centrality (BC), Closeness Centrality (CC), Bottle Neck (BN), Information Centrality (IC), Eigenvector Centrality (EC), and Subgraph Centrality (SC)) in identifying essential proteins. The comparison results based on the validations of sensitivity, specificity, F-measure, positive predictive value, negative predictive value, and accuracy consistently show that LAC outweighs these seven previous methods. PMID:21704260

  6. Endocytosis of Seven-Transmembrane RGS Protein Activates G- protein Coupled Signaling in Arabidopsis

    PubMed Central

    Urano, Daisuke; Phan, Nguyen; Jones, Janice C.; Yang, Jing; Huang, Jirong; Grigston, Jeffrey; Taylor, J. Philip; Jones, Alan M.

    2012-01-01

    Signal transduction typically begins by ligand-dependent activation of a concomitant partner which is otherwise in its resting state. However, in cases where signal activation is constitutive by default, the mechanism of regulation is unknown. The Arabidopsis thaliana heterotrimeric Gα protein self-activates without accessory proteins, and is kept in its resting state by the negative regulator, AtRGS1 (Regulator of G protein Signaling 1), which is the prototype of a seven transmembrane receptor fused with an RGS domain. Endocytosis of AtRGS1 by ligand-dependent endocytosis physically uncouples the GTPase accelerating activity of AtRGS1 from the Gα protein, permitting sustained activation. Phosphorylation of AtRGS1 by AtWNK8 kinase causes AtRGS1 endocytosis, required both for G protein-mediated sugar signaling and cell proliferation. In animals, receptor endocytosis results in signal desensitization, whereas in plants, endocytosis results in signal activation. These findings reveal how different organisms rearrange a regulatory system to result in opposite outcomes using similar phosphorylation-dependent endocytosis. PMID:22940907

  7. Gain and kinetics of activation in the G-protein cascade of phototransduction.

    PubMed Central

    Lamb, T D

    1996-01-01

    The guanine nucleotide binding protein (G protein) cascade underlying phototransduction is one of the best understood of all signaling pathways. The diffusional interactions of the proteins underlying the cascade have been analyzed, both at a macroscopic level and also in terms of the stochastic nature of the molecular contacts. In response to a single activated rhodopsin (R*) formed as a result of a single photon hit, it can be shown that molecules of the G-protein transducin will be activated approximately linearly with time. This, in turn, will cause the number of activated molecules of the effector protein (the phosphodiesterase) also to increase linearly with time. These kinetics of protein activation provide an accurate description of the time course of the rising phase of the photoreceptor's electrical response over a wide range of flash intensities. Recent estimates indicate that at room temperature each R* triggers activation of the phosphodiesterase at a rate of 1000-2000 subunits.s-1. Now that a quantitative description of the activation steps in transduction has been obtained, perhaps the greatest challenge for the future is to provide a comprehensive description of the shutoff reactions, so that a complete account of the photoreceptor's response to light can be achieved. Images Fig. 1 Fig. 2 PMID:8570596

  8. Competition between members of the tribbles pseudokinase protein family shapes their interactions with mitogen activated protein kinase pathways.

    PubMed

    Guan, Hongtao; Shuaib, Aban; Leon, David Davila De; Angyal, Adrienn; Salazar, Maria; Velasco, Guillermo; Holcombe, Mike; Dower, Steven K; Kiss-Toth, Endre

    2016-01-01

    Spatio-temporal regulation of intracellular signalling networks is key to normal cellular physiology; dysregulation of which leads to disease. The family of three mammalian tribbles proteins has emerged as an important controller of signalling via regulating the activity of mitogen activated protein kinases (MAPK), the PI3-kinase induced signalling network and E3 ubiquitin ligases. However, the importance of potential redundancy in the action of tribbles and how the differences in affinities for the various binding partners may influence signalling control is currently unclear. We report that tribbles proteins can bind to an overlapping set of MAPK-kinases (MAPKK) in live cells and dictate the localisation of the complexes. Binding studies in transfected cells reveal common regulatory mechanisms and suggest that tribbles and MAPKs may interact with MAPKKs in a competitive manner. Computational modelling of the impact of tribbles on MAPK activation suggests a high sensitivity of this system to changes in tribbles levels, highlighting that these proteins are ideally placed to control the dynamics and balance of activation of concurrent signalling pathways. PMID:27600771

  9. Competition between members of the tribbles pseudokinase protein family shapes their interactions with mitogen activated protein kinase pathways

    PubMed Central

    Guan, Hongtao; Shuaib, Aban; Leon, David Davila De; Angyal, Adrienn; Salazar, Maria; Velasco, Guillermo; Holcombe, Mike; Dower, Steven K.; Kiss-Toth, Endre

    2016-01-01

    Spatio-temporal regulation of intracellular signalling networks is key to normal cellular physiology; dysregulation of which leads to disease. The family of three mammalian tribbles proteins has emerged as an important controller of signalling via regulating the activity of mitogen activated protein kinases (MAPK), the PI3-kinase induced signalling network and E3 ubiquitin ligases. However, the importance of potential redundancy in the action of tribbles and how the differences in affinities for the various binding partners may influence signalling control is currently unclear. We report that tribbles proteins can bind to an overlapping set of MAPK-kinases (MAPKK) in live cells and dictate the localisation of the complexes. Binding studies in transfected cells reveal common regulatory mechanisms and suggest that tribbles and MAPKs may interact with MAPKKs in a competitive manner. Computational modelling of the impact of tribbles on MAPK activation suggests a high sensitivity of this system to changes in tribbles levels, highlighting that these proteins are ideally placed to control the dynamics and balance of activation of concurrent signalling pathways. PMID:27600771

  10. NEU3 activity enhances EGFR activation without affecting EGFR expression and acts on its sialylation levels.

    PubMed

    Mozzi, Alessandra; Forcella, Matilde; Riva, Alice; Difrancesco, Carlotta; Molinari, Francesca; Martin, Vittoria; Papini, Nadia; Bernasconi, Barbara; Nonnis, Simona; Tedeschi, Gabriella; Mazzucchelli, Luca; Monti, Eugenio; Fusi, Paola; Frattini, Milo

    2015-08-01

    Several studies performed over the last decade have focused on the role of sialylation in the progression of cancer and, in particular, on the association between deregulation of sialidases and tumorigenic transformation. The plasma membrane-associated sialidase NEU3 is often deregulated in colorectal cancer (CRC), and it was shown that this enzyme co-immunoprecipitates in HeLa cells with epidermal growth factor receptor (EGFR), the molecular target of most recent monoclonal antibody-based therapies against CRC. To investigate the role of NEU3 sialidase on EGFR deregulation in CRC, we first collected data on NEU3 gene expression levels from a library of commercial colon cell lines, demonstrating that NEU3 transcription is upregulated in these cell lines. We also found EGFR to be hyperphosphorylated in all cell lines, with the exception of SW620 cells and the CCD841 normal intestinal cell line. By comparing the effects induced by overexpression of either the wild-type or the inactive mutant form of NEU3 on EGFR, we demonstrated that the active form of NEU3 enhanced receptor activation without affecting EGFR mRNA or protein expression. Moreover, through western blots and mass spectrometry analysis, we found that EGFR immunoprecipitated from cells overexpressing active NEU3, unlike the receptor from mock cells and cells overexpressing inactive NEU3, is desialylated. On the whole, our data demonstrate that, besides the already reported indirect EGFR activation through GM3, sialidase NEU3 could also play a role on EGFR activation through its desialylation. PMID:25922362

  11. Beyond the zone: protein needs of active individuals.

    PubMed

    Lemon, P W

    2000-10-01

    There has been debate among athletes and nutritionists regarding dietary protein needs for centuries. Although contrary to traditional belief, recent scientific information collected on physically active individuals tends to indicate that regular exercise increases daily protein requirements; however, the precise details remain to be worked out. Based on laboratory measures, daily protein requirements are increased by perhaps as much as 100% vs. recommendations for sedentary individuals (1.6-1.8 vs. 0.8 g/kg). Yet even these intakes are much less than those reported by most athletes. This may mean that actual requirements are below what is needed to optimize athletic performance, and so the debate continues. Numerous interacting factors including energy intake, carbohydrate availability, exercise intensity, duration and type, dietary protein quality, training history, gender, age, timing of nutrient intake and the like make this topic extremely complex. Many questions remain to be resolved. At the present time, substantial data indicate that the current recommended protein intake should be adjusted upward for those who are physically active, especially in populations whose needs are elevated for other reasons, e.g., growing individuals, dieters, vegetarians, individuals with muscle disease-induced weakness and the elderly. For these latter groups, specific supplementation may be appropriate, but for most North Americans who consume a varied diet, including complete protein foods (meat, eggs, fish and dairy products), and sufficient energy the increased protein needs induced by a regular exercise program can be met in one's diet.

  12. A theoretical approach to spot active regions in antimicrobial proteins

    PubMed Central

    2009-01-01

    Background Much effort goes into identifying new antimicrobial compounds able to evade the increasing resistance of microorganisms to antibiotics. One strategy relies on antimicrobial peptides, either derived from fragments released by proteolytic cleavage of proteins or designed from known antimicrobial protein regions. Results To identify these antimicrobial determinants, we developed a theoretical approach that predicts antimicrobial proteins from their amino acid sequence in addition to determining their antimicrobial regions. A bactericidal propensity index has been calculated for each amino acid, using the experimental data reported from a high-throughput screening assay as reference. Scanning profiles were performed for protein sequences and potentially active stretches were identified by the best selected threshold parameters. The method was corroborated against positive and negative datasets. This successful approach means that we can spot active sequences previously reported in the literature from experimental data for most of the antimicrobial proteins examined. Conclusion The method presented can correctly identify antimicrobial proteins with an accuracy of 85% and a sensitivity of 90%. The method can also predict their key active regions, making this a tool for the design of new antimicrobial drugs. PMID:19906288

  13. Angiogenic activity of an Onchocerca volvulus Ancylostoma secreted protein homologue.

    PubMed

    Higazi, Tarig B; Pearlman, Eric; Whikehart, David R; Unnasch, Thomas R

    2003-06-01

    Angiogenesis is an important step in the development of ocular onchocercaisis. In previous studies, it has been demonstrated that Onchocerca volvulus homologues of the Ancylostoma secreted protein family have pronounced angiogenic activity. The overall goal of the current study was to determine if this angiogenic effect is exerted through a direct or indirect mechanism. These studies focused on one member of this family, OvASP-2, as this protein is expressed in microfilaria, the stage of the parasite that causes ocular onchocercaisis. Clones encoding truncated and full length open reading frames were expressed as fusion proteins with Escherichia coli maltose binding protein (MBP), and angiogenic activity was compared in vitro and in vivo with MBP alone. Truncated constructs expressing only the first 105 amino acids of OvASP-2 were as active as the full length protein in inducing new blood vessel formation. The full length fusion protein did not stimulate proliferation or production of vascular endothelial growth factor in vascular endothelial cells in vitro, indicating that OvASP-2 does not directly stimulate angiogenesis. Sequence analysis demonstrated that the gene encoding OvASP-2 contained five introns. Sequence comparisons of the genomic loci from West African blinding and non-blinding strains of O. volvulus revealed that some polymorphism existed among the various isolates tested. However, none of these polymorphisms could be used to differentiate the parasite strains, suggesting that qualitative variation in OvASP-2 could not explain the difference in ocular pathogenic potential of the two parasite strains.

  14. Cyclic AMP-dependent protein kinase activity in Trypanosoma cruzi.

    PubMed Central

    Ulloa, R M; Mesri, E; Esteva, M; Torres, H N; Téllez-Iñón, M T

    1988-01-01

    A cyclic AMP-dependent protein kinase activity from epimastigote forms of Trypanosoma cruzi was characterized. Cytosolic extracts were chromatographed on DEAE-cellulose columns, giving two peaks of kinase activity, which were eluted at 0.15 M- and 0.32 M-NaCl respectively. The second activity peak was stimulated by nanomolar concentrations of cyclic AMP. In addition, a cyclic AMP-binding protein co-eluted with the second kinase activity peak. Cyclic AMP-dependent protein kinase activity was further purified by gel filtration, affinity chromatography on histone-agarose and cyclic AMP-agarose, as well as by chromatography on CM-Sephadex. The enzyme ('holoenzyme') could be partially dissociated into two different components: 'catalytic' and 'regulatory'. The 'regulatory' component had specific binding for cyclic AMP, and it inhibited phosphotransferase activity of the homologous 'catalytic component' or of the 'catalytic subunit' from bovine heart. Cyclic AMP reversed these inhibitions. A 'holoenzyme preparation' was phosphorylated in the absence of exogenous phosphate acceptor and analysed by polyacrylamide-gel electrophoresis. A 56 kDa band was phosphorylated. The same preparation was analysed by Western blotting, by using polyclonal antibodies to the regulatory subunits of protein kinases type I or II. Both antibodies reacted with the 56 kDa band. Images Fig. 7. Fig. 8. PMID:2848508

  15. Hydrodynamic collective effects of active proteins in biological membranes.

    PubMed

    Koyano, Yuki; Kitahata, Hiroyuki; Mikhailov, Alexander S

    2016-08-01

    Lipid bilayers forming biological membranes are known to behave as viscous two-dimensional fluids on submicrometer scales; usually they contain a large number of active protein inclusions. Recently, it was shown [A. S. Mikhailov and R. Kapral, Proc. Natl. Acad. Sci. USA 112, E3639 (2015)PNASA60027-842410.1073/pnas.1506825112] that such active proteins should induce nonthermal fluctuating lipid flows leading to diffusion enhancement and chemotaxislike drift for passive inclusions in biomembranes. Here, a detailed analytical and numerical investigation of such effects is performed. The attention is focused on the situations when proteins are concentrated within lipid rafts. We demonstrate that passive particles tend to become attracted by active rafts and are accumulated inside them. PMID:27627343

  16. MAPK-Activated Protein Kinases (MKs): Novel Insights and Challenges.

    PubMed

    Gaestel, Matthias

    2015-01-01

    Downstream of MAPKs, such as classical/atypical ERKs and p38 MAPKs, but not of JNKs, signaling is often mediated by protein kinases which are phosphorylated and activated by MAPKs and, therefore, designated MAPK-activated protein kinases (MAPKAPKs). Recently, novel insights into the specificity of the assembly of MAPK/MAPKAPK hetero-dimeric protein kinase signaling complexes have been gained. In addition, new functional aspects of MKs have been described and established functions have been challenged. This short review will summarize recent developments including the linear motif (LM) in MKs, the ERK-independent activation of RSK, the RSK-independent effects of some RSK-inhibitors and the challenged role of MK5/PRAK in tumor suppression. PMID:26779481

  17. Hydrodynamic collective effects of active proteins in biological membranes

    NASA Astrophysics Data System (ADS)

    Koyano, Yuki; Kitahata, Hiroyuki; Mikhailov, Alexander S.

    2016-08-01

    Lipid bilayers forming biological membranes are known to behave as viscous two-dimensional fluids on submicrometer scales; usually they contain a large number of active protein inclusions. Recently, it was shown [A. S. Mikhailov and R. Kapral, Proc. Natl. Acad. Sci. USA 112, E3639 (2015), 10.1073/pnas.1506825112] that such active proteins should induce nonthermal fluctuating lipid flows leading to diffusion enhancement and chemotaxislike drift for passive inclusions in biomembranes. Here, a detailed analytical and numerical investigation of such effects is performed. The attention is focused on the situations when proteins are concentrated within lipid rafts. We demonstrate that passive particles tend to become attracted by active rafts and are accumulated inside them.

  18. Effects of dietary protein level on growth and utilization of protein and energy by juvenile mangrove red snapper (Lutjanus argentimaculatus)

    NASA Astrophysics Data System (ADS)

    Ghulam, Abbas; Khalid, Jamil; Rukhsana, Akhtar; Lin, Hong

    2005-01-01

    A feeding trial was conducted in a recirculating water system to investigate the effects of dietary protein levels on growth, feed utilization, hepatosomatic index and liver lipid deposition of juvenile red snapper, Lutjanus argentimaculatus (average initial wet weight 8.0 ± 0.39 g and total length 3.14 ± 0.3 cm). In the experiment, six fishmeal-based diets were formulated to contain various protein levels (20% to 45% in 5% increments), with dietary energy ranging from 2210.7kJ lOOg to 2250.2kJlOOg dry matter. The protein to energy ratios of diets ranged from 8.58 mg protein kJ-1 to 20.03 mg protein kJ-1. Diets were fed for 90d to triplicate groups of fish stocked in 0.128m3 seawater tanks, 25 individuals each. The daily ration of 2% wet body weight was offered to the fish thrice a day. The fish at the end of the study had more than ten-fold (77.0g) increase in weight compared to the initial (8.0g). Fish fed diets of 40% and 45% protein produced significantly (P<0.05) higher weight gain of 77.2g and 76.5g, and specific growth rate (SGR) of 2.65% and 2.62% than those of 67.0 g and 68.3g, and 2.49% and 2.51% of the other diets. The broken-line regression of SGR against dietary protein level yielded an optimum dietary protein requirement of 42.6% (Y=-1.6295 + 0.1114 X 2,P<0.05). Survival remained 100% among groups. Feed conversion ratio decreased from 0.45 for fish fed 20% dietary protein to 0.35 for fish fed 45% dietary protein. Nitrogen intake increased with an increase in dietary protein, which in turn resulted in an increase in nitrogen gain of fish whole body. Fish fed 40% and 45% protein diets showed higher (P<0.05) nitrogen gain (0.27g and 0.26g) than those (0.23g and 025g) fed all other diets. Gross energy intake (GEI) in fish fed 45% protein was lower (600.67kJ) than that (607.97 kJ) of 40% protein diet, though the differences were not statistically significant (P>0.05); GEI ranging from 677.31 kJ to 663.20 kJ at remaining four diets (20% to 35% protein

  19. Integrated Stability and Activity Control of the Drosophila Rbf1 Retinoblastoma Protein*

    PubMed Central

    Zhang, Liang; Wei, Yiliang; Pushel, Irina; Heinze, Karolin; Elenbaas, Jared; Henry, R. William; Arnosti, David N.

    2014-01-01

    The retinoblastoma (RB) family transcriptional corepressors regulate diverse cellular events including cell cycle, senescence, and differentiation. The activity and stability of these proteins are mediated by post-translational modifications; however, we lack a general understanding of how distinct modifications coordinately impact both of these properties. Previously, we showed that protein turnover and activity are tightly linked through an evolutionarily conserved C-terminal instability element (IE) in the Drosophila RB-related protein Rbf1; surprisingly, mutant proteins with enhanced stability were less, not more active. To better understand how activity and turnover are controlled in this model RB protein, we assessed the impact of Cyclin-Cdk kinase regulation on Rbf1. An evolutionarily conserved N-terminal threonine residue is required for Cyclin-Cdk response and showed a dominant impact on turnover and activity; however, specific residues in the C-terminal IE differentially impacted Rbf1 activity and turnover, indicating an additional level of regulation. Strikingly, specific IE mutations that impaired turnover but not activity induced dramatic developmental phenotypes in the Drosophila eye. Mutation of the highly conserved Lys-774 residue induced hypermorphic phenotypes that mimicked the loss of phosphorylation control; mutation of the corresponding codon of the human RBL2 gene has been reported in lung tumors. Our data support a model in which closely intermingled residues within the conserved IE govern protein turnover, presumably through interactions with E3 ligases, and protein activity via contacts with E2F transcription partners. Such functional relationships are likely to similarly impact mammalian RB family proteins, with important implications for development and disease. PMID:25049232

  20. Changes in Protein Kinase A Activity Accompany Sclerotial Development in Sclerotinia sclerotiorum.

    PubMed

    Harel, A; Gorovits, R; Yarden, O

    2005-04-01

    ABSTRACT Sclerotia of Sclerotinia sclerotiorum are pigmented, multihyphal structures that play a central role in the life and infection cycles of this pathogen. Sclerotial formation has been shown to be affected by increased intracellular cAMP levels. Cyclic AMP (cAMP) is a key modulator of cAMP-dependent protein kinase A (PKA) and the latter may prove to play a significant role in sclerotial development. Therefore, we monitored changes in relative PKA activity levels during sclerotial development. To do so, we first developed conditions for near-synchronous sclerotial development in culture, based on hyphal maceration and filtering. Relative PKA activity levels increased during the white-sclerotium stage in the wild-type strain, while low levels were maintained in nonsclerotium-producing mutants. Furthermore, applying caffeine, an inducer of PKA activity, resulted in increased relative PKA activity levels and was correlated with the formation of sclerotial initial-like aggregates in cultures of the non-sclerotium-producing mutants. In addition, low PKA activities were found in an antisense smk1 strain, which exhibits low extracellular-signal-regulated kinase (ERK)-type mitogen-activated protein kinase (MAPK) activity, and does not produce sclerotia. The changes in PKA activity, as well as the abundance of phosphorylated MAPKs (ERK-like as well as p38-like) that accompany sclerotial development in a distinct developmental phase manner represent a potential target for antifungal intervention. PMID:18943042

  1. Influence of protein level and supplemental methionine in practical rations for young endangered masked bobwhite quail

    USGS Publications Warehouse

    Serafin, J.A.

    1982-01-01

    A study was conducted to examine the protein requirement of young endangered masked Bobwhite quail (Colinus virginianus ridgwayi). Five practical starting rations containing 24 to 32% protein were fed alone and supplemented with methionine for 5 weeks. Supplemental methionine significantly improved growth of quail fed diets containing 24 and 26% protein. Increasing the protein level improved growth of quail fed unsupplemented diets but did not do so when diets contained supplemental methionine. A methionine-supplemented ration containing 24% protein appeared adequate for supporting rapid growth of masked Bobwhite quail.

  2. Signal peptides are allosteric activators of the protein translocase

    PubMed Central

    Gouridis, Giorgos; Karamanou, Spyridoula; Gelis, Ioannis; Kalodimos, Charalampos G.; Economou, Anastassios

    2010-01-01

    Extra-cytoplasmic polypeptides are usually synthesized as “preproteins” carrying aminoterminal, cleavable signal peptides1 and secreted across membranes by translocases. The main bacterial translocase comprises the SecYEG protein-conducting channel and the peripheral ATPase motor SecA2,3. Most proteins destined for the periplasm and beyond are exported post-translationally by SecA2,3. Preprotein targeting to SecA is thought to involve signal peptides4 and chaperones like SecB5,6. Here we reveal that signal peptides have a novel role beyond targeting: they are essential allosteric activators of the translocase. Upon docking on their binding groove on SecA, signal peptides act in trans to drive three successive states: first, “triggering” that drives the translocase to a lower activation energy state; then “trapping” that engages non-native preprotein mature domains docked with high affinity on the secretion apparatus and, finally, “secretion” during which trapped mature domains undergo multiple turnovers of translocation in segments7. A significant contribution by mature domains renders signal peptides less critical in bacterial secretory protein targeting than currently assumed. Rather, it is their function as allosteric activators of the translocase that renders signal peptides essential for protein secretion. A role for signal peptides and targeting sequences as allosteric activators may be universal in protein translocases. PMID:19924216

  3. Suppression of Myc oncogenic activity by ribosomal protein haploinsufficiency.

    PubMed

    Barna, Maria; Pusic, Aya; Zollo, Ornella; Costa, Maria; Kondrashov, Nadya; Rego, Eduardo; Rao, Pulivarthi H; Ruggero, Davide

    2008-12-18

    The Myc oncogene regulates the expression of several components of the protein synthetic machinery, including ribosomal proteins, initiation factors of translation, RNA polymerase III and ribosomal DNA. Whether and how increasing the cellular protein synthesis capacity affects the multistep process leading to cancer remains to be addressed. Here we use ribosomal protein heterozygote mice as a genetic tool to restore increased protein synthesis in Emu-Myc/+ transgenic mice to normal levels, and show that the oncogenic potential of Myc in this context is suppressed. Our findings demonstrate that the ability of Myc to increase protein synthesis directly augments cell size and is sufficient to accelerate cell cycle progression independently of known cell cycle targets transcriptionally regulated by Myc. In addition, when protein synthesis is restored to normal levels, Myc-overexpressing precancerous cells are more efficiently eliminated by programmed cell death. Our findings reveal a new mechanism that links increases in general protein synthesis rates downstream of an oncogenic signal to a specific molecular impairment in the modality of translation initiation used to regulate the expression of selective messenger RNAs. We show that an aberrant increase in cap-dependent translation downstream of Myc hyperactivation specifically impairs the translational switch to internal ribosomal entry site (IRES)-dependent translation that is required for accurate mitotic progression. Failure of this translational switch results in reduced mitotic-specific expression of the endogenous IRES-dependent form of Cdk11 (also known as Cdc2l and PITSLRE), which leads to cytokinesis defects and is associated with increased centrosome numbers and genome instability in Emu-Myc/+ mice. When accurate translational control is re-established in Emu-Myc/+ mice, genome instability is suppressed. Our findings demonstrate how perturbations in translational control provide a highly specific outcome

  4. Cannabinoid receptor 2 expression modulates Gβ(1)γ(2) protein interaction with the activator of G protein signalling 2/dynein light chain protein Tctex-1.

    PubMed

    Nagler, Marina; Palkowitsch, Lysann; Rading, Sebastian; Moepps, Barbara; Karsak, Meliha

    2016-01-01

    The activator of G protein signalling AGS2 (Tctex-1) forms protein complexes with Gβγ, and controls cell proliferation by regulating cell cycle progression. A direct interaction of Tctex-1 with various G protein-coupled receptors has been reported. Since the carboxyl terminal portion of CB2 carries a putative Tctex-1 binding motif, we investigated the potential interplay of CB2 and Tctex-1 in the absence and presence of Gβγ. The supposed interaction of cannabinoid receptor CB2 with Tctex-1 and the influence of CB2 on the formation of Tctex-1-Gβγ-complexes were studied by co- and/or immunoprecipitation experiments in transiently transfected HEK293 cells. The analysis on Tctex-1 protein was performed in the absence and presence of the ligands JWH 133, 2-AG, and AM 630, the protein biosynthesis inhibitor cycloheximide or the protein degradation blockers MG132, NH4Cl/leupeptin or bafilomycin. Our results show that CB2 neither directly nor indirectly via Gβγ interacts with Tctex-1, but competes with Tctex-1 in binding to Gβγ. The Tctex-1-Gβγ protein interaction was disrupted by CB2 receptor expression resulting in a release of Tctex-1 from the complex, and its degradation by the proteasome and partly by lysosomes. The decrease in Tctex-1 protein levels is induced by CB2 expression "dose-dependently" and is independent of stimulation by agonist or blocking by an inverse agonist treatment. The results suggest that CB2 receptor expression independent of its activation by agonists is sufficient to competitively disrupt Gβγ-Tctex-1 complexes, and to initiate Tctex-1 degradation. These findings implicate that CB2 receptor expression modifies the stability of intracellular protein complexes by a non-canonical pathway.

  5. RNA helicase: a novel activity associated with a protein encoded by a positive strand RNA virus.

    PubMed Central

    Laín, S; Riechmann, J L; García, J A

    1990-01-01

    Most positive strand RNA viruses infecting plants and animals encode proteins containing the so-called nucleotide binding motif (NTBM) (1) in their amino acid sequences (2). As suggested from the high level of sequence similarity of these viral proteins with the recently described superfamilies of helicase-like proteins (3-5), the NTBM-containing cylindrical inclusion (CI) protein from plum pox virus (PPV), which belongs to the potyvirus group of positive strand RNA viruses, is shown to be able to unwind RNA duplexes. This activity was found to be dependent on the hydrolysis of NTP to NDP and Pi, and thus it can be considered as an RNA helicase activity. In the in vitro assay used, the PPV CI protein was only able to unwind double strand RNA substrates with 3' single strand overhangs. This result indicates that the helicase activity of the PPV CI protein functions in the 3' to 5' direction (6). To our knowledge, this is the first report on a helicase activity associated with a protein encoded by an RNA virus. Images PMID:2263459

  6. Protein levels and colony development of Africanized and European honey bees fed natural and artificial diets.

    PubMed

    Morais, M M; Turcatto, A P; Pereira, R A; Francoy, T M; Guidugli-Lazzarini, K R; Gonçalves, L S; de Almeida, J M V; Ellis, J D; De Jong, D

    2013-12-19

    Pollen substitute diets are a valuable resource for maintaining strong and health honey bee colonies. Specific diets may be useful in one region or country and inadequate or economically unviable in others. We compared two artificial protein diets that had been formulated from locally-available ingredients in Brazil with bee bread and a non-protein sucrose diet. Groups of 100 newly-emerged, adult workers of Africanized honey bees in Brazil and European honey bees in the USA were confined in small cages and fed on one of four diets for seven days. The artificial diets included a high protein diet made of soy milk powder and albumin, and a lower protein level diet consisting of soy milk powder, brewer's yeast and rice bran. The initial protein levels in newly emerged bees were approximately 18-21 µg/µL hemolymph. After feeding on the diets for seven days, the protein levels in the hemolymph were similar among the protein diet groups (~37-49 µg/µL after seven days), although Africanized bees acquired higher protein levels, increasing 145 and 100% on diets D1 and D2, respectively, versus 83 and 60% in the European bees. All the protein diets resulted in significantly higher levels of protein than sucrose solution alone. In the field, the two pollen substitute diets were tested during periods of low pollen availability in the field in two regions of Brazil. Food consumption, population development, colony weight, and honey production were evaluated to determine the impact of the diets on colony strength parameters. The colonies fed artificial diets had a significant improvement in all parameters, while control colonies dwindled during the dearth period. We conclude that these two artificial protein diets have good potential as pollen substitutes during dearth periods and that Africanized bees more efficiently utilize artificial protein diets than do European honey bees.

  7. ELEVATED LEVELS OF SOLUBLE ST2 PROTEIN IN DENGUE VIRUS INFECTED PATIENTS

    PubMed Central

    Becerra, Aniuska; Warke, Rajas V.; de Bosch, Norma; Rothman, Alan L.; Bosch, Irene

    2008-01-01

    Levels of the soluble form of the interleukin-1 receptor like 1 protein (IL-1RL-1 / ST2) are elevated in the serum of patients with diseases characterized by an inflammatory response. The objective of this study was to determine the concentration of soluble ST2 (sST2) in dengue infected patients during the course of the disease. Twenty four patients with confirmed dengue infection, classified as dengue fever, and eleven patients with other febrile illness (OFI) were evaluated. Levels of sST2 in serum and laboratory variables usually altered during dengue infections were measured. Dengue infected patients had higher serum sST2 levels than OFI at the end of the febrile stage and at defervescence (p=0.0088 and p=0.0004 respectively). Patients with secondary dengue infections had higher serum sST2 levels compared with patients with primary dengue infections (p=0.047 at last day of fever and p=0.030 at defervescence). Furthermore, in dengue infected patients, we found a significant negative correlation of sST2 with platelet and WBC counts, and positive correlation with thrombin time and transaminases activity. We suggest that sST2 could be a potential marker of dengue infection, could be associated with severity or could play a role in the immune response in secondary dengue virus infection. PMID:18226917

  8. Overexpression of hydroxynitrile lyase in cassava roots elevates protein and free amino acids while reducing residual cyanogen levels.

    PubMed

    Narayanan, Narayanan N; Ihemere, Uzoma; Ellery, Claire; Sayre, Richard T

    2011-01-01

    Cassava is the major source of calories for more than 250 million Sub-Saharan Africans, however, it has the lowest protein-to-energy ratio of any major staple food crop in the world. A cassava-based diet provides less than 30% of the minimum daily requirement for protein. Moreover, both leaves and roots contain potentially toxic levels of cyanogenic glucosides. The major cyanogen in cassava is linamarin which is stored in the vacuole. Upon tissue disruption linamarin is deglycosylated by the apolplastic enzyme, linamarase, producing acetone cyanohydrin. Acetone cyanohydrin can spontaneously decompose at pHs >5.0 or temperatures >35°C, or is enzymatically broken down by hydroxynitrile lyase (HNL) to produce acetone and free cyanide which is then volatilized. Unlike leaves, cassava roots have little HNL activity. The lack of HNL activity in roots is associated with the accumulation of potentially toxic levels of acetone cyanohydrin in poorly processed roots. We hypothesized that the over-expression of HNL in cassava roots under the control of a root-specific, patatin promoter would not only accelerate cyanogenesis during food processing, resulting in a safer food product, but lead to increased root protein levels since HNL is sequestered in the cell wall. Transgenic lines expressing a patatin-driven HNL gene construct exhibited a 2-20 fold increase in relative HNL mRNA levels in roots when compared with wild type resulting in a threefold increase in total root protein in 7 month old plants. After food processing, HNL overexpressing lines had substantially reduced acetone cyanohydrin and cyanide levels in roots relative to wild-type roots. Furthermore, steady state linamarin levels in intact tissues were reduced by 80% in transgenic cassava roots. These results suggest that enhanced linamarin metabolism contributed to the elevated root protein levels.

  9. Synergistic Control of Kinetochore Protein Levels by Psh1 and Ubr2

    PubMed Central

    Herrero, Eva; Thorpe, Peter H.

    2016-01-01

    The accurate segregation of chromosomes during cell division is achieved by attachment of chromosomes to the mitotic spindle via the kinetochore, a large multi-protein complex that assembles on centromeres. The budding yeast kinetochore comprises more than 60 different proteins. Although the structure and function of many of these proteins has been investigated, we have little understanding of the steady state regulation of kinetochores. The primary model of kinetochore homeostasis suggests that kinetochores assemble hierarchically from the centromeric DNA via the inclusion of a centromere-specific histone into chromatin. We tested this model by trying to perturb kinetochore protein levels by overexpressing an outer kinetochore gene, MTW1. This increase in protein failed to change protein recruitment, consistent with the hierarchical assembly model. However, we find that deletion of Psh1, a key ubiquitin ligase that is known to restrict inner kinetochore protein loading, does not increase levels of outer kinetochore proteins, thus breaking the normal kinetochore stoichiometry. This perturbation leads to chromosome segregation defects, which can be partially suppressed by mutation of Ubr2, a second ubiquitin ligase that normally restricts protein levels at the outer kinetochore. Together these data show that Psh1 and Ubr2 synergistically control the amount of proteins at the kinetochore. PMID:26891228

  10. Structure-activity relationship of synthetic branched-chain distearoylglycerol (distearin) as protein kinase C activators

    SciTech Connect

    Zhou, Qingzhong; Raynor, R.L.; Wood, M.G. Jr.; Menger, F.M.; Kuo, J.F. )

    1988-09-20

    Several representative branched-chain analogues of distearin (DS) were synthesized and tested for their abilities to activate protein kinase C (PKC) and to compete for the binding of ({sup 3}H)phorbol 12,13-dibutyrate (PDBu) to the enzyme. Substitutions of stearoyl moieties at sn-1 and sn-2 with 8-methylstearate decreased activities on these parameters, relative to those of the parental diacylglycerol DS, a weak PKC activator. Substitutions with 8-butyl, 4-butyl, or 8-phenyl derivatives, on the other hand, increased activities of the resulting analogues to levels comparable to those seen for diolein (DO), a diacylglycerol prototype shown to be a potent PKC activator. Kinetic analysis indicated that 8-methyldistearin (8-MeDS) acted by decreasing, whereas 8-butyldistearin (8-BuDS) and 8-phenyldistearin (8-PhDS) acted by increasing, the affinities of PKC for phosphatidylserine (PS, a phospholipid cofactor) and Ca{sup 2+} compared to the values seen in the absence or presence of DS. The stimulatory effect of 8-BuDS and 8-PhDS on PKC, as DO, was additive to that of 1,2-(8-butyl)distearoylphosphatidylcholine (1,2(8-Bu)DSPC) and, moreover, they abolished the marked inhibition of the enzyme activity caused by high concentrations of 1,2(8-Bu)DSPC. The present findings demonstrated a structure-activity relationship of the branched-chain DS analogues in the regulation of PKC, perhaps related to their abilities to specifically modify interactions of PKC with PS and/or Ca{sup 2+} critically involved in enzyme activation/inactivation.

  11. Physicochemical effects of the lipid phase and protein level on meat emulsion stability, texture, and microstructure.

    PubMed

    Youssef, M K; Barbut, S

    2010-03-01

    The effects of beef fat (25%) substitution with rendered beef fat, canola oil, palm oil, or hydrogenated palm oil at varying meat protein levels (8%, 11%, and 14%) were studied in emulsified beef meat batters. There was no significant difference in fat loss among meat batters made with beef fat, rendered beef fat, or palm oil. Hydrogenated palm oil provided the most stable batters at all protein levels. Increasing meat protein to 14% resulted in high fat loss in batters prepared with canola oil, which did not occur in the other formulations. This indicates that the physicochemical characteristics of fat/oil affect emulsion stability. Cooked batter hardness was higher (P < 0.05) when protein level was raised; highest in hydrogenated palm oil batters when compared at similar protein levels. As protein level was raised springiness values were increased in all the meat treatments. Springiness was higher in the canola oil treatments. Light microscopy revealed fat globule coalescence in canola oil meat batters prepared with 14% protein, as well as the development of fat channels and more protein aggregation; both seem to result in lower emulsion stability. Hydrogenated palm oil batters showed fat particles with sharp edges as opposed to the round ones seen in all other treatments.

  12. Contextual interactions determine whether the Drosophila homeodomain protein, Vnd, acts as a repressor or activator

    PubMed Central

    Yu, Zhongxin; Syu, Li-Jyun; Mellerick, Dervla M.

    2005-01-01

    At the molecular level, members of the NKx2.2 family of transcription factors establish neural compartment boundaries by repressing the expression of homeobox genes specific for adjacent domains [Muhr et al. (2001) Cell, 104, 861–873; Weiss et al. (1998) Genes Dev., 12, 3591–3602]. The Drosophila homologue, vnd, interacts genetically with the high-mobility group protein, Dichaete, in a manner suggesting co-operative activation [Zhao and Skeath (2002) Development, 129, 1165–1174]. However, evidence for direct interactions and transcriptional activation is lacking. Here, we present molecular evidence for the interaction of Vnd and Dichaete that leads to the activation of target gene expression. Two-hybrid interaction assays indicate that Dichaete binds the Vnd homeodomain, and additional Vnd sequences stabilize this interaction. In addition, Vnd has two activation domains that are typically masked in the intact protein. Whether vnd can activate or repress transcription is context-dependent. Full-length Vnd, when expressed as a Gal4 fusion protein, acts as a repressor containing multiple repression domains. A divergent domain in the N-terminus, not found in vertebrate Vnd-like proteins, causes the strongest repression. The co-repressor, Groucho, enhances Vnd repression, and these two proteins physically interact. The data presented indicate that the activation and repression domains of Vnd are complex, and whether Vnd functions as a transcriptional repressor or activator depends on both intra- and inter-molecular interactions. PMID:15640442

  13. Efficient monitoring of protein ubiquitylation levels using TUBEs-based microarrays.

    PubMed

    Serna, Sonia; Xolalpa, Wendy; Lang, Valérie; Aillet, Fabienne; England, Patrick; Reichardt, Niels; Rodriguez, Manuel S

    2016-08-01

    Analyzing protein ubiquitylation changes during physiological or pathological processes is challenging due to its high reversibility and dynamic turnover of modified targets. We have developed a protein microarray to assess endogenous ubiquitylation levels from cell cultures, employing tandem ubiquitin-binding entities (TUBEs) with three or four ubiquitin-associated (UBA) domains as capture probes. Adriamycin (ADR)-stimulated MCF7 cells were used to differentiate protein ubiquitylation levels between cells that are sensitive or resistant to ADR treatment. We show that TUBEs-based microarrays can be used for the analysis of cellular processes regulated by ubiquitylation and for the detection of pathologies with aberrant ubiquitylation levels. PMID:27410252

  14. Efficient monitoring of protein ubiquitylation levels using TUBEs-based microarrays.

    PubMed

    Serna, Sonia; Xolalpa, Wendy; Lang, Valérie; Aillet, Fabienne; England, Patrick; Reichardt, Niels; Rodriguez, Manuel S

    2016-08-01

    Analyzing protein ubiquitylation changes during physiological or pathological processes is challenging due to its high reversibility and dynamic turnover of modified targets. We have developed a protein microarray to assess endogenous ubiquitylation levels from cell cultures, employing tandem ubiquitin-binding entities (TUBEs) with three or four ubiquitin-associated (UBA) domains as capture probes. Adriamycin (ADR)-stimulated MCF7 cells were used to differentiate protein ubiquitylation levels between cells that are sensitive or resistant to ADR treatment. We show that TUBEs-based microarrays can be used for the analysis of cellular processes regulated by ubiquitylation and for the detection of pathologies with aberrant ubiquitylation levels.

  15. Structural basis for chemokine recognition and activation of a viral G protein-coupled receptor

    SciTech Connect

    Burg, John S.; Ingram, Jessica R.; Venkatakrishnan, A.J.; Jude, Kevin M.; Dukkipati, Abhiram; Feinberg, Evan N.; Angelini, Alessandro; Waghray, Deepa; Dror, Ron O.; Ploegh, Hidde L.; Garcia, K. Christopher

    2015-03-05

    Chemokines are small proteins that function as immune modulators through activation of chemokine G protein-coupled receptors (GPCRs). Several viruses also encode chemokines and chemokine receptors to subvert the host immune response. How protein ligands activate GPCRs remains unknown. We report the crystal structure at 2.9 angstrom resolution of the human cytomegalovirus GPCR US28 in complex with the chemokine domain of human CX3CL1 (fractalkine). The globular body of CX3CL1 is perched on top of the US28 extracellular vestibule, whereas its amino terminus projects into the central core of US28. The transmembrane helices of US28 adopt an active-state-like conformation. Atomic-level simulations suggest that the agonist-independent activity of US28 may be due to an amino acid network evolved in the viral GPCR to destabilize the receptor’s inactive state.

  16. Autophosphorylation Activity of a Soluble Hexameric Histidine Kinase Correlates with the Shift in Protein Conformational Equilibrium

    PubMed Central

    Wojnowska, Marta; Yan, Jun; Sivalingam, Ganesh N.; Cryar, Adam; Gor, Jayesh; Thalassinos, Konstantinos; Djordjevic, Snezana

    2013-01-01

    Summary In a commonly accepted model, in response to stimuli, bacterial histidine kinases undergo a conformational transition between an active and inactive form. Structural information on histidine kinases is limited. By using ion mobility-mass spectrometry (IM-MS), we demonstrate an exchange between two conformational populations of histidine kinase ExsG that are linked to different levels of kinase activity. ExsG is an atypical signaling protein that incorporates an uncommon histidine kinase catalytic core at the C terminus preceded by an N-terminal “receiver domain” that is normally associated with the response regulator proteins in two-component signal transduction systems. IM-MS analysis and enzymatic assays indicate that phosphorylation of the ExsG receiver domain stabilizes the “compact” form of the protein and inhibits kinase core activity; in contrast, nucleotide binding required for kinase activity is associated with the more open conformation of ExsG. PMID:24210218

  17. Maternal folic acid supplementation to dams on marginal protein level alters brain fatty acid levels of their adult offspring.

    PubMed

    Rao, Shobha; Joshi, Sadhana; Kale, Anvita; Hegde, Mahabaleshwar; Mahadik, Sahebarao

    2006-05-01

    Studies on fetal programming of adult diseases have highlighted the importance of maternal nutrition during pregnancy. Folic acid and long-chain essential polyunsaturated fatty acids (LC-PUFAs) have independent effects on fetal growth. However, folic acid effects may also involve alteration of LC-PUFA metabolism. Because marginal deficiency of LC-PUFAs during critical periods of brain growth and development is associated with risks for adult diseases, it is highly relevant to investigate how maternal supplementation of such nutrients can alter brain fatty acid levels. We examined the impact of folic acid supplementation, conventionally used in maternal intervention, on brain essential fatty acid levels and plasma corticosterone concentrations in adult offspring at 11 months of age. Pregnant female rats from 4 groups (6 in each) were fed with casein diets either with 18 g protein/100 g diet (control diet) or treatment diets that were marginal in protein (MP), such as 12 g protein/100 g diet supplemented with 8 mg folic acid (FAS/MP), 12 g protein/100 g diet without folic acid (FAD/MP), or 12 g protein/100 g diet (MP) with 2 mg folic acid. Pups were weaned to a standard laboratory diet with 18 g protein/100 g diet. All male adult offspring in the FAS/MP group showed lower docosahexaenoic acid (P<.05) as compared with control adult offspring (6.04+/-2.28 vs 10.33+/-0.86 g/100 g fatty acids) and higher n-6/n-3 ratio (P<.05). Docosahexaenoic acid levels in FAS/MP adult offspring were also lower (P<.05) when compared with the MP group. Plasma corticosterone concentrations were higher (P<.05) in male adult offspring from the FAS/MP group compared with control as well as the MP adult offspring. Results suggest that maternal folic acid supplementation at MP intake decreased brain docosahexaenoic acid levels probably involving corticosterone increase. PMID:16631439

  18. Maternal folic acid supplementation to dams on marginal protein level alters brain fatty acid levels of their adult offspring.

    PubMed

    Rao, Shobha; Joshi, Sadhana; Kale, Anvita; Hegde, Mahabaleshwar; Mahadik, Sahebarao

    2006-05-01

    Studies on fetal programming of adult diseases have highlighted the importance of maternal nutrition during pregnancy. Folic acid and long-chain essential polyunsaturated fatty acids (LC-PUFAs) have independent effects on fetal growth. However, folic acid effects may also involve alteration of LC-PUFA metabolism. Because marginal deficiency of LC-PUFAs during critical periods of brain growth and development is associated with risks for adult diseases, it is highly relevant to investigate how maternal supplementation of such nutrients can alter brain fatty acid levels. We examined the impact of folic acid supplementation, conventionally used in maternal intervention, on brain essential fatty acid levels and plasma corticosterone concentrations in adult offspring at 11 months of age. Pregnant female rats from 4 groups (6 in each) were fed with casein diets either with 18 g protein/100 g diet (control diet) or treatment diets that were marginal in protein (MP), such as 12 g protein/100 g diet supplemented with 8 mg folic acid (FAS/MP), 12 g protein/100 g diet without folic acid (FAD/MP), or 12 g protein/100 g diet (MP) with 2 mg folic acid. Pups were weaned to a standard laboratory diet with 18 g protein/100 g diet. All male adult offspring in the FAS/MP group showed lower docosahexaenoic acid (P<.05) as compared with control adult offspring (6.04+/-2.28 vs 10.33+/-0.86 g/100 g fatty acids) and higher n-6/n-3 ratio (P<.05). Docosahexaenoic acid levels in FAS/MP adult offspring were also lower (P<.05) when compared with the MP group. Plasma corticosterone concentrations were higher (P<.05) in male adult offspring from the FAS/MP group compared with control as well as the MP adult offspring. Results suggest that maternal folic acid supplementation at MP intake decreased brain docosahexaenoic acid levels probably involving corticosterone increase.

  19. Extraction of Children's Friendship Relation from Activity Level

    NASA Astrophysics Data System (ADS)

    Kono, Aki; Shintani, Kimio; Katsuki, Takuya; Kihara, Shin'ya; Ueda, Mari; Kaneda, Shigeo; Haga, Hirohide

    Children learn to fit into society through living in a group, and it's greatly influenced by their friend relations. Although preschool teachers need to observe them to assist in the growth of children's social progress and support the development each child's personality, only experienced teachers can watch over children while providing high-quality guidance. To resolve the problem, this paper proposes a mathematical and objective method that assists teachers with observation. It uses numerical data of activity level recorded by pedometers, and we make tree diagram called dendrogram based on hierarchical clustering with recorded activity level. Also, we calculate children's ``breadth'' and ``depth'' of friend relations by using more than one dendrogram. When we record children's activity level in a certain kindergarten for two months and evaluated the proposed method, the results usually coincide with remarks of teachers about the children.

  20. AMP-activated protein kinase regulates nicotinamide phosphoribosyl transferase expression in skeletal muscle

    PubMed Central

    Brandauer, Josef; Vienberg, Sara G; Andersen, Marianne A; Ringholm, Stine; Risis, Steve; Larsen, Per S; Kristensen, Jonas M; Frøsig, Christian; Leick, Lotte; Fentz, Joachim; Jørgensen, Sebastian; Kiens, Bente; Wojtaszewski, Jørgen F P; Richter, Erik A; Zierath, Juleen R; Goodyear, Laurie J; Pilegaard, Henriette; Treebak, Jonas T

    2013-01-01

    Deacetylases such as sirtuins (SIRTs) convert NAD to nicotinamide (NAM). Nicotinamide phosphoribosyl transferase (Nampt) is the rate-limiting enzyme in the NAD salvage pathway responsible for converting NAM to NAD to maintain cellular redox state. Activation of AMP-activated protein kinase (AMPK) increases SIRT activity by elevating NAD levels. As NAM directly inhibits SIRTs, increased Nampt activation or expression could be a metabolic stress response. Evidence suggests that AMPK regulates Nampt mRNA content, but whether repeated AMPK activation is necessary for increasing Nampt protein levels is unknown. To this end, we assessed whether exercise training- or 5-amino-1-β-d-ribofuranosyl-imidazole-4-carboxamide (AICAR)-mediated increases in skeletal muscle Nampt abundance are AMPK dependent. One-legged knee-extensor exercise training in humans increased Nampt protein by 16% (P < 0.05) in the trained, but not the untrained leg. Moreover, increases in Nampt mRNA following acute exercise or AICAR treatment (P < 0.05 for both) were maintained in mouse skeletal muscle lacking a functional AMPK α2 subunit. Nampt protein was reduced in skeletal muscle of sedentary AMPK α2 kinase dead (KD), but 6.5 weeks of endurance exercise training increased skeletal muscle Nampt protein to a similar extent in both wild-type (WT) (24%) and AMPK α2 KD (18%) mice. In contrast, 4 weeks of daily AICAR treatment increased Nampt protein in skeletal muscle in WT mice (27%), but this effect did not occur in AMPK α2 KD mice. In conclusion, functional α2-containing AMPK heterotrimers are required for elevation of skeletal muscle Nampt protein, but not mRNA induction. These findings suggest AMPK plays a post-translational role in the regulation of skeletal muscle Nampt protein abundance, and further indicate that the regulation of cellular energy charge and nutrient sensing is mechanistically related. PMID:23918774

  1. Complement activation and protein adsorption by carbon nanotubes.

    PubMed

    Salvador-Morales, Carolina; Flahaut, Emmanuel; Sim, Edith; Sloan, Jeremy; Green, Malcolm L H; Sim, Robert B

    2006-02-01

    As a first step to validate the use of carbon nanotubes as novel vaccine or drug delivery devices, their interaction with a part of the human immune system, complement, has been explored. Haemolytic assays were conducted to investigate the activation of the human serum complement system via the classical and alternative pathways. Western blot and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) techniques were used to elucidate the mechanism of activation of complement via the classical pathway, and to analyse the interaction of complement and other plasma proteins with carbon nanotubes. We report for the first time that carbon nanotubes activate human complement via both classical and alternative pathways. We conclude that complement activation by nanotubes is consistent with reported adjuvant effects, and might also in various circumstances promote damaging effects of excessive complement activation, such as inflammation and granuloma formation. C1q binds directly to carbon nanotubes. Protein binding to carbon nanotubes is highly selective, since out of the many different proteins in plasma, very few bind to the carbon nanotubes. Fibrinogen and apolipoproteins (AI, AIV and CIII) were the proteins that bound to carbon nanotubes in greatest quantity.

  2. Protein-tyrosine-phosphatase 2C is phosphorylated and inhibited by 44-kDa mitogen-activated protein kinase.

    PubMed Central

    Peraldi, P; Zhao, Z; Filloux, C; Fischer, E H; Van Obberghen, E

    1994-01-01

    Protein-tyrosine-phosphatase 2C (PTP2C, also named SHPTP2, SHPTP3, or PTP1D) is a cytosolic enzyme with two Src homology 2 domains. We have investigated its regulation by phosphorylation in PC12 rat pheochromocytoma cells. In untreated cells, PTP2C was phosphorylated predominantly on serine residues. A 5-min treatment with epidermal growth factor (EGF) induced an increase in phosphorylation on threonine and, to a lesser degree, on serine. After 45 min of exposure to EGF, PTP2C phosphorylation returned to basal levels. Using an in vitro kinase assay, we found that the 44-kDa mitogen-activated protein kinase, p44mapk, phosphorylated PTP2C on serine and threonine residues. This phosphorylation resulted in a pronounced inhibition of PTP2C enzyme activity measured with phosphorylated EGF receptors as substrate. Moreover, in intact PC12 cells, PTP2C was also inhibited following a short EGF treatment, but its activity returned to normal when the exposure to EGF was maintained for 45 min. The profile of this response to EGF can be inversely correlated to that of the stimulatory action of EGF on p44mapk. These data suggest that the EGF-induced regulation of PTP2C activity is mediated by p44mapk. These findings provide evidence for an additional role of the mitogen-activated protein kinase cascade--namely, the regulation of a PTP. Images PMID:8197172

  3. Dual level inhibition of E2F-1 activity by adeno-associated virus Rep78.

    PubMed

    Batchu, R B; Shammas, M A; Wang, J Y; Munshi, N C

    2001-06-29

    E2F-1, a major cellular transcription factor, plays a pivotal role in regulating the cell cycle. The activity of E2F-1 is negatively regulated by its interaction with retinoblastoma protein (pRB), and disruption of the pRB-E2F-1 complex, a hallmark of cellular transformation by DNA tumor viruses, leads to cell proliferation. Adeno-associated virus-2 (AAV) is known to have onco-suppressive properties against DNA tumor viruses. Here we provide, for the first time, the molecular basis for antioncogenic activity of AAV. Rep78, a major regulatory protein of AAV, interacts at the protein level with E2F-1 and stabilizes the pRB-E2F-1 complex. At the DNA level, Rep78 binds to a putative site on the E2F-1 promoter and down-regulates the adenovirus-induced E2F-1 transcription. This dual level of Rep78 activity leads to decreased cellular levels of free E2F-1, leading to its onco-suppressive properties.

  4. Dietary thiamin level influences levels of its diphosphate form and thiamin-dependent enzymic activities of rat liver.

    PubMed

    Blair, P V; Kobayashi, R; Edwards, H M; Shay, N F; Baker, D H; Harris, R A

    1999-03-01

    This study was prompted by our incomplete understanding of the mechanism responsible for the clinical benefits of pharmacological doses of thiamin in some patients with maple syrup urine disease (MSUD) and the question of whether thiamin diphosphate (TDP), a potent inhibitor of the activity of the protein kinase that phosphorylates and inactivates the isolated branched-chain alpha-ketoacid dehydrogenase (BCKDH) complex, affects the activity state of the complex. Rats were fed a chemically-defined diet containing graded levels of thiamin (0, 0.275, 0.55, 5.5, and 55 mg thiamin/kg diet). Maximal weight gain was attained over a 3-wk period only in rats fed diets with 5.5 and 55 mg thiamin/kg. Feeding rats the thiamin-free diet for just 2 d caused loss of nearly half of the TDP from liver mitochondria. Three more days caused over 70% loss, an additional 3 wk, over 90%. Starvation for 2 d had no effect, suggesting a mechanism for conservation of TDP in this nutritional state. Mitochondrial TDP was higher in rats fed pharmacological amounts of thiamin (55 mg thiamin/kg diet) than in rats fed adequate thiamin for maximal growth. Varying dietary thiamin had marked but opposite effects on the activities of alpha-ketoglutarate dehydrogenase (alpha-KGDH) and BCKDH. Thiamin deficiency decreased alpha-KGDH activity, increased BCKDH activity, and increased the proportion of BCKDH in the active, dephosphorylated, state. Excess dietary thiamin had the opposite effects. TDP appears to be more tightly associated with alpha-KGDH than BCKDH in thiamin-deficient rats, perhaps denoting retention of alpha-KGDH activity at the expense of BCKDH activity. Thus, thiamin deficiency and excess cause large changes in mitochondrial TDP levels that have a major influence on the activities of the keto acid dehydrogenase complexes.

  5. Noise exposure immediately activates cochlear mitogen-activated protein kinase signaling.

    PubMed

    Alagramam, Kumar N; Stepanyan, Ruben; Jamesdaniel, Samson; Chen, Daniel H-C; Davis, Rickie R

    2014-01-01

    Noise-induced hearing loss (NIHL) is a major public health issue worldwide. Uncovering the early molecular events associated with NIHL would reveal mechanisms leading to the hearing loss. Our aim is to investigate the immediate molecular responses after different levels of noise exposure and identify the common and distinct pathways that mediate NIHL. Previous work showed mice exposed to 116 decibels sound pressure level (dB SPL) broadband noise for 1 h had greater threshold shifts than the mice exposed to 110 dB SPL broadband noise, hence we used these two noise levels in this study. Groups of 4-8-week-old CBA/CaJ mice were exposed to no noise (control) or to broadband noise for 1 h, followed by transcriptome analysis of total cochlear RNA isolated immediately after noise exposure. Previously identified and novel genes were found in all data sets. Following exposure to noise at 116 dB SPL, the earliest responses included up-regulation of 243 genes and down-regulation of 61 genes, while a similar exposure at 110 dB SPL up-regulated 155 genes and down-regulated 221 genes. Bioinformatics analysis indicated that mitogen-activated protein kinase (MAPK) signaling was the major pathway in both levels of noise exposure. Nevertheless, both qualitative and quantitative differences were noticed in some MAPK signaling genes, after exposure to different noise levels. Cacna1b , Cacna1g , and Pla2g6 , related to calcium signaling were down-regulated after 110 dB SPL exposure, while the fold increase in the expression of Fos was relatively lower than what was observed after 116 dB SPL exposure. These subtle variations provide insight on the factors that may contribute to the differences in NIHL despite the activation of a common pathway.

  6. The product of a novel growth factor activated gene, fos B, interacts with JUN proteins enhancing their DNA binding activity.

    PubMed Central

    Zerial, M; Toschi, L; Ryseck, R P; Schuermann, M; Müller, R; Bravo, R

    1989-01-01

    We have identified a gene, fos B, encoding a nuclear protein of 338 amino acids presenting a 70% homology with c-fos, whose expression is activated during G0/G1 transition. Growth factor stimulation of quiescent cells leads to a rapid and transient accumulation of fos B mRNA, with kinetics similar to those of c-fos. The induction of fos B mRNA levels is in part due to a dramatic increase in the transcription of the gene. The half-life of fos B mRNA is in the order of 10-15 min. Both transcriptional activation and mRNA stability are substantially increased in the presence of protein synthesis inhibitors. Immunoprecipitation studies showed that fos B as c-fos protein, forms a complex in vitro with c-jun and jun B proteins in the absence of a target binding sequence. Gel retardation assays demonstrated that fos B protein positively influences the binding of c-jun and jun B proteins to an AP-1 binding consensus sequence, suggesting that fos B protein plays a role in control of gene expression. Images PMID:2498083

  7. Thymic Stromal Lymphopoietin Promotes Fibrosis and Activates Mitogen-Activated Protein Kinases in MRC-5 Cells

    PubMed Central

    Li, Li; Tang, Su; Tang, Xiaodong

    2016-01-01

    Background Acute lung injury (ALI) is a life-threatening hypoxemic respiratory disorder with high incidence and mortality. ALI usually manifests as widespread inflammation and lung fibrosis with the accumulation of pro-inflammatory and pro-fibrotic factors and collagen. Thymic stromal lymphopoietin (TSLP) has a significant role in regulation of inflammation but little is known about its roles in lung fibrosis or ALI. This study aimed to define the role and possible regulatory mechanism of TSLP in lung fibrosis. Material/Methods We cultured human lung fibroblast MRC-5 cells and overexpressed or inhibited TSLP by the vector or small interfering RNA transfection. Then, the pro-fibrotic factors skeletal muscle actin alpha (α-SMA) and collagen I, and the 4 mitogen-activated protein kinases (MAPKs) – MAPK7, p38, extracellular signal-regulated kinase 1 (ERK1), and c-Jun N-terminal kinase 1 (JNK1) – were detected by Western blot. Results Results showed that TSLP promoted the production of α-SMA and collagen I (P<0.001), suggesting that it can accelerate MRC-5 cell fibrosis. It also activated the expression of MAPK7, p-p38, p-ERK1, and p-JNK1, but the total MAPK7, p-38, ERK1, and JNK1 protein levels were mostly unchanged, indicating the activated MAPK pathways that might contribute to the promotion of cell fibrosis. Conclusions This study shows the pro-fibrotic role of TSLP in MRC-5 cells, suggesting TSLP is a potential therapeutic target for treating lung fibrosis in ALI. It possibly functions via activating MAPKs. These findings add to our understanding of the mechanism of fibrosis. PMID:27385084

  8. Methods to distinguish various types of protein phosphatase activity

    SciTech Connect

    Brautigan, D.L.; Shriner, C.L.

    1988-01-01

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

  9. Resveratrol up-regulates AMPA receptor expression via AMP-activated protein kinase-mediated protein translation.

    PubMed

    Wang, Guan; Amato, Stephen; Gilbert, James; Man, Heng-Ye

    2015-08-01

    Resveratrol is a phytoalexin that confers overall health benefits including positive regulation in brain function such as learning and cognition. However, whether and how resveratrol affects synaptic activity remains largely unknown. α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are glutamatergic receptors that mediate the majority of fast excitatory transmission and synaptic plasticity, and thus play a critical role in higher brain functions, including learning and memory. We find that in rat primary neurons, resveratrol can rapidly increase AMPAR protein level, AMPAR synaptic accumulation and the strength of excitatory synaptic transmission. The resveratrol effect on AMPAR protein expression is independent of sirtuin 1 (SIRT1), the conventional downstream target of resveratrol, but rather is mediated by AMP-activated protein kinase (AMPK) and subsequent downstream phosphoinositide 3-kinase (PI3K)/Akt signaling. Application of the AMPK specific activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) mimics the effects of resveratrol on both signaling and AMPAR expression. The resveratrol-induced increase in AMPAR expression results from elevated protein synthesis via regulation of the eukaryotic initiation factor (eIF) 4E/4G complex. Disruption of the translation initiation complex completely blocks resveratrol-dependent AMPAR up-regulation. These findings indicate that resveratrol may regulate brain function through facilitation of AMPAR biogenesis and synaptic transmission.

  10. Actions of Rho family small G proteins and p21-activated protein kinases on mitogen-activated protein kinase family members.

    PubMed Central

    Frost, J A; Xu, S; Hutchison, M R; Marcus, S; Cobb, M H

    1996-01-01

    The mitogen-activated protein (MAP) kinases are a family of serine/threonine kinases that are regulated by distinct extracellular stimuli. The currently known members include extracellular signal-regulated protein kinase 1 (ERK1), ERK2, the c-Jun N-terminal kinase/stress-activated protein kinases (JNK/SAPKs), and p38 MAP kinases. We find that overexpression of the Ste20-related enzymes p21-activated kinase 1 (PAK1) and PAK2 in 293 cells is sufficient to activate JNK/SAPK and to a lesser extent p38 MAP kinase but not ERK2. Rat MAP/ERK kinase kinase 1 can stimulate the activity of each of these MAP kinases. Although neither activated Rac nor the PAKs stimulate ERK2 activity, overexpression of either dominant negative Rac2 or the N-terminal regulatory domain of PAK1 inhibits Ras-mediated activation of ERK2, suggesting a permissive role for Rac in the control of the ERK pathway. Furthermore, constitutively active Rac2, Cdc42hs, and RhoA synergize with an activated form of Raf to increase ERK2 activity. These findings reveal a previously unrecognized connection between Rho family small G proteins and the ERK pathway. PMID:8668187

  11. Sasa borealis extract exerts an antidiabetic effect via activation of the AMP-activated protein kinase.

    PubMed

    Nam, Jung Soo; Chung, Hee Jin; Jang, Min Kyung; Jung, In Ah; Park, Seong Ha; Cho, Su In; Jung, Myeong Ho

    2013-02-01

    Leaf of Sasa borealis, a species of bamboo, has been reported to exhibit anti-hyperglycemic effect. However, its antidiabetic mechanism is not fully understood. In this study, we examined whether an extract of S. borealis activates AMP-activated protein kinase (AMPK) and exerts anti-hyperglycemic effects. Treatment with the S. borealis extract increased insulin signaling and phosphorylation of AMPK and stimulated the expression of its downstream targets, including PPARα, ACO, and CPT-1 in C2C12 cells and PPARα in HepG2 cells. However, inhibition of AMPK activation attenuated insulin signaling and prevented the stimulation of AMPK target genes. The S. borealis extract increased glucose uptake in C2C12 cells and suppressed expression of the gluconeogenic gene, PEPCK in HepG2 cells. The extract significantly reduced blood glucose and triglyceride levels in STZ-induced diabetic mice. The extract enhanced AMPK phosphorylation and increased Glut-4 expression in the skeletal muscle of the mice. These findings demonstrated that the S. borealis extract exerts its anti-hyperglycemic effect through activation of AMPK and enhancement of insulin signaling. PMID:23423690

  12. Sasa borealis extract exerts an antidiabetic effect via activation of the AMP-activated protein kinase

    PubMed Central

    Nam, Jung Soo; Chung, Hee Jin; Jang, Min Kyung; Jung, In Ah; Park, Seong Ha; Cho, Su In

    2013-01-01

    Leaf of Sasa borealis, a species of bamboo, has been reported to exhibit anti-hyperglycemic effect. However, its antidiabetic mechanism is not fully understood. In this study, we examined whether an extract of S. borealis activates AMP-activated protein kinase (AMPK) and exerts anti-hyperglycemic effects. Treatment with the S. borealis extract increased insulin signaling and phosphorylation of AMPK and stimulated the expression of its downstream targets, including PPARα, ACO, and CPT-1 in C2C12 cells and PPARα in HepG2 cells. However, inhibition of AMPK activation attenuated insulin signaling and prevented the stimulation of AMPK target genes. The S. borealis extract increased glucose uptake in C2C12 cells and suppressed expression of the gluconeogenic gene, PEPCK in HepG2 cells. The extract significantly reduced blood glucose and triglyceride levels in STZ-induced diabetic mice. The extract enhanced AMPK phosphorylation and increased Glut-4 expression in the skeletal muscle of the mice. These findings demonstrated that the S. borealis extract exerts its anti-hyperglycemic effect through activation of AMPK and enhancement of insulin signaling. PMID:23423690

  13. AMP-activated protein kinase and metabolic control

    PubMed Central

    Viollet, Benoit; Andreelli, Fabrizio

    2011-01-01

    AMP-activated protein kinase (AMPK), a phylogenetically conserved serine/threonine protein kinase, is a major regulator of cellular and whole-body energy homeostasis that coordinates metabolic pathways in order to balance nutrient supply with energy demand. It is now recognized that pharmacological activation of AMPK improves blood glucose homeostasis, lipid profile and blood pressure in insulin-resistant rodents. Indeed, AMPK activation mimics the beneficial effects of physical activity or those of calorie restriction by acting on multiple cellular targets. In addition it is now demonstrated that AMPK is one of the probable (albeit indirect) targets of major antidiabetic drugs including, the biguanides (metformin) and thiazolidinediones, as well as of insulin sensitizing adipokines (e.g., adiponectin). Taken together, such findings highlight the logic underlying the concept of targeting the AMPK pathway for the treatment of metabolic syndrome and type 2 diabetes. PMID:21484577

  14. Detergent activation of the binding protein in the folate radioassay

    SciTech Connect

    Hansen, S.I.; Holm, J.; Lyngbye, J.

    1982-01-01

    A minor cow's whey protein associated with ..beta..-lactoglobulin is used as binding protein in the competitive radioassay for serum and erythrocyte folate. Seeking to optimize the assay, we tested the performance of binder solutions of increasing purity. The folate binding protein was isolated from cow's whey by means of CM-Sepharose CL-6B cation-exchange chromatography, and further purified on a methotrexate-AH-Sepharose 4B affinity matrix. In contrast to ..beta..-lactoglobulin, the purified protein did not bind folate unless the detergents cetyltrimethylammonium (10 mmol/Ll) or Triton X-100 (1 g/L) were present. Such detergent activation was not needed in the presence of serum. There seems to be a striking analogy between these phenomena and the well-known reactivation of certain purified membrane-derived enzymes by surfactants (lipids/detergents).

  15. Controlling Protein Activity and Degradation Using Blue Light.

    PubMed

    Lutz, Anne P; Renicke, Christian; Taxis, Christof

    2016-01-01

    Regulation of protein stability is a fundamental process in eukaryotic cells and pivotal to, e.g., cell cycle progression, faithful chromosome segregation, or protein quality control. Synthetic regulation of protein stability requires conditional degradation sequences (degrons) that induce a stability switch upon a specific signal. Fusion to a selected target protein permits to influence virtually every process in a cell. Light as signal is advantageous due to its precise applicability in time, space, quality, and quantity. Light control of protein stability was achieved by fusing the LOV2 photoreceptor domain of Arabidopsis thaliana phototropin1 with a synthetic degron (cODC1) derived from the carboxy-terminal degron of ornithine decarboxylase to obtain the photosensitive degron (psd) module. The psd module can be attached to the carboxy terminus of target proteins that are localized to the cytosol or nucleus to obtain light control over their stability. Blue light induces structural changes in the LOV2 domain, which in turn lead to activation of the degron and thus proteasomal degradation of the whole fusion protein. Variants of the psd module with diverse characteristics are useful to fine-tune the stability of a selected target at permissive (darkness) and restrictive conditions (blue light). PMID:26965116

  16. Altered Activation of Protein Kinase PKR and Enhanced Apoptosis in Dystonia Cells Carrying a Mutation in PKR Activator Protein PACT*

    PubMed Central

    Vaughn, Lauren S; Bragg, D. Cristopher; Sharma, Nutan; Camargos, Sarah; Cardoso, Francisco; Patel, Rekha C

    2015-01-01

    PACT is a stress-modulated activator of the interferon-induced double-stranded RNA-activated protein kinase (PKR). Stress-induced phosphorylation of PACT is essential for PACT's association with PKR leading to PKR activation. PKR activation leads to phosphorylation of translation initiation factor eIF2α inhibition of protein synthesis and apoptosis. A recessively inherited form of early-onset dystonia DYT16 has been recently identified to arise due to a homozygous missense mutation P222L in PACT. To examine if the mutant P222L protein alters the stress-response pathway, we examined the ability of mutant P222L to interact with and activate PKR. Our results indicate that the substitution mutant P222L activates PKR more robustly and for longer duration albeit with slower kinetics in response to the endoplasmic reticulum stress. In addition, the affinity of PACT-PACT and PACT-PKR interactions is enhanced in dystonia patient lymphoblasts, thereby leading to intensified PKR activation and enhanced cellular death. P222L mutation also changes the affinity of PACT-TRBP interaction after cellular stress, thereby offering a mechanism for the delayed PKR activation in response to stress. Our results demonstrate the impact of a dystonia-causing substitution mutation on stress-induced cellular apoptosis. PMID:26231208

  17. Steroidogenic Acute Regulatory Protein Overexpression Correlates with Protein Kinase A Activation in Adrenocortical Adenoma

    PubMed Central

    Xie, Jing; Su, Tingwei; Jiang, Lei; Jiang, Yiran; Cao, Yanan; Liu, Jianmin; Ning, Guang; Wang, Weiqing

    2016-01-01

    The association of pathological features of cortisol-producing adrenocortical adenomas (ACAs) with somatic driver mutations and their molecular classification remain unclear. In this study, we explored the association between steroidogenic acute regulatory protein (StAR) expression and the driver mutations activating cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling to identify the pathological markers of ACAs. Immunohistochemical staining for StAR and mutations in the protein kinase cAMP-activated catalytic subunit alpha (PRKACA), protein kinase cAMP-dependent type I regulatory subunit alpha (PRKAR1A) and guanine nucleotide binding protein, alpha stimulating (GNAS) genes were examined in 97 ACAs. The association of StAR expression with the clinical and mutational features of the ACAs was analyzed. ACAs with mutations in PRKACA, GNAS, and PRKAR1A showed strong immunopositive staining for StAR. The concordance between high StAR expression and mutations activating cAMP/PKA signaling in the ACAs was 99.0%. ACAs with high expression of StAR had significantly smaller tumor volume (P < 0.001) and higher urinary cortisol per tumor volume (P = 0.032) than those with low expression of StAR. Our findings suggest that immunohistochemical staining for StAR is a reliable pathological approach for the diagnosis and classification of ACAs with cAMP/PKA signaling-activating mutations. PMID:27606678

  18. Steroidogenic Acute Regulatory Protein Overexpression Correlates with Protein Kinase A Activation in Adrenocortical Adenoma.

    PubMed

    Zhou, Weiwei; Wu, Luming; Xie, Jing; Su, Tingwei; Jiang, Lei; Jiang, Yiran; Cao, Yanan; Liu, Jianmin; Ning, Guang; Wang, Weiqing

    2016-01-01

    The association of pathological features of cortisol-producing adrenocortical adenomas (ACAs) with somatic driver mutations and their molecular classification remain unclear. In this study, we explored the association between steroidogenic acute regulatory protein (StAR) expression and the driver mutations activating cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling to identify the pathological markers of ACAs. Immunohistochemical staining for StAR and mutations in the protein kinase cAMP-activated catalytic subunit alpha (PRKACA), protein kinase cAMP-dependent type I regulatory subunit alpha (PRKAR1A) and guanine nucleotide binding protein, alpha stimulating (GNAS) genes were examined in 97 ACAs. The association of StAR expression with the clinical and mutational features of the ACAs was analyzed. ACAs with mutations in PRKACA, GNAS, and PRKAR1A showed strong immunopositive staining for StAR. The concordance between high StAR expression and mutations activating cAMP/PKA signaling in the ACAs was 99.0%. ACAs with high expression of StAR had significantly smaller tumor volume (P < 0.001) and higher urinary cortisol per tumor volume (P = 0.032) than those with low expression of StAR. Our findings suggest that immunohistochemical staining for StAR is a reliable pathological approach for the diagnosis and classification of ACAs with cAMP/PKA signaling-activating mutations.

  19. Steroidogenic Acute Regulatory Protein Overexpression Correlates with Protein Kinase A Activation in Adrenocortical Adenoma.

    PubMed

    Zhou, Weiwei; Wu, Luming; Xie, Jing; Su, Tingwei; Jiang, Lei; Jiang, Yiran; Cao, Yanan; Liu, Jianmin; Ning, Guang; Wang, Weiqing

    2016-01-01

    The association of pathological features of cortisol-producing adrenocortical adenomas (ACAs) with somatic driver mutations and their molecular classification remain unclear. In this study, we explored the association between steroidogenic acute regulatory protein (StAR) expression and the driver mutations activating cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling to identify the pathological markers of ACAs. Immunohistochemical staining for StAR and mutations in the protein kinase cAMP-activated catalytic subunit alpha (PRKACA), protein kinase cAMP-dependent type I regulatory subunit alpha (PRKAR1A) and guanine nucleotide binding protein, alpha stimulating (GNAS) genes were examined in 97 ACAs. The association of StAR expression with the clinical and mutational features of the ACAs was analyzed. ACAs with mutations in PRKACA, GNAS, and PRKAR1A showed strong immunopositive staining for StAR. The concordance between high StAR expression and mutations activating cAMP/PKA signaling in the ACAs was 99.0%. ACAs with high expression of StAR had significantly smaller tumor volume (P < 0.001) and higher urinary cortisol per tumor volume (P = 0.032) than those with low expression of StAR. Our findings suggest that immunohistochemical staining for StAR is a reliable pathological approach for the diagnosis and classification of ACAs with cAMP/PKA signaling-activating mutations. PMID:27606678

  20. Astragaloside II triggers T cell activation through regulation of CD45 protein tyrosine phosphatase activity

    PubMed Central

    Wan, Chun-ping; Gao, Li-xin; Hou, Li-fei; Yang, Xiao-qian; He, Pei-lan; Yang, Yi-fu; Tang, Wei; Yue, Jian-min; Li, Jia; Zuo, Jian-ping

    2013-01-01

    Aim: To investigate the immunomodulating activity of astragalosides, the active compounds from a traditional tonic herb Astragalus membranaceus Bge, and to explore the molecular mechanisms underlying the actions, focusing on CD45 protein tyrosine phosphatase (CD45 PTPase), which plays a critical role in T lymphocyte activation. Methods: Primary splenocytes and T cells were prepared from mice. CD45 PTPase activity was assessed using a colorimetric assay. Cell proliferation was measured using a [3H]-thymidine incorporation assay. Cytokine proteins and mRNAs were examined with ELISA and RT-PCR, respectively. Activation markers, including CD25 and CD69, were analyzed using flow cytometry. Activation of LCK (Tyr505) was detected using Western blot analysis. Mice were injected with the immunosuppressant cyclophosphamide (CTX, 80 mg/kg), and administered astragaloside II (50 mg/kg). Results: Astragaloside I, II, III, and IV concentration-dependently increased the CD45-mediated of pNPP/OMFP hydrolysis with the EC50 values ranged from 3.33 to 10.42 μg/mL. Astragaloside II (10 and 30 nmol/L) significantly enhanced the proliferation of primary splenocytes induced by ConA, alloantigen or anti-CD3. Astragaloside II (30 nmol/L) significantly increased IL-2 and IFN-γ secretion, upregulated the mRNA levels of IFN-γ and T-bet in primary splenocytes, and promoted CD25 and CD69 expression on primary CD4+ T cells upon TCR stimulation. Furthermore, astragaloside II (100 nmol/L) promoted CD45-mediated dephosphorylation of LCK (Tyr505) in primary T cells, which could be blocked by a specific CD45 PTPase inhibitor. In CTX-induced immunosuppressed mice, oral administration of astragaloside II restored the proliferation of splenic T cells and the production of IFN-γ and IL-2. However, astragaloside II had no apparent effects on B cell proliferation. Conclusion: Astragaloside II enhances T cell activation by regulating the activity of CD45 PTPase, which may explain why Astragalus

  1. MAP17 and SGLT1 Protein Expression Levels as Prognostic Markers for Cervical Tumor Patient Survival

    PubMed Central

    Perez, Marco; Praena-Fernandez, Juan M.; Felipe-Abrio, Blanca; Lopez-Garcia, Maria A.; Lucena-Cacace, Antonio; Garcia, Angel; Lleonart, Matilde; Roncador, Guiovanna; Marin, Juan J.; Carnero, Amancio

    2013-01-01

    MAP17 is a membrane-associated protein that is overexpressed in human tumors. Because the expression of MAP17 increases reactive oxygen species (ROS) generation through SGLT1 in cancer cells, in the present work, we investigated whether MAP17 and/or SGLT1 might be markers for the activity of treatments involving oxidative stress, such as cisplatin or radiotherapy. First, we confirmed transcriptional alterations in genes involved in the oxidative stress induced by MAP17 expression in HeLa cervical tumor cells and found that Hela cells expressing MAP17 were more sensitive to therapies that induce ROS than were parental cells. Furthermore, MAP17 increased glucose uptake through SGLT receptors. We then analyzed MAP17 and SGLT1 expression levels in cervical tumors treated with cisplatin plus radiotherapy and correlated the expression levels with patient survival. MAP17 and SGLT1 were expressed in approximately 70% and 50% of cervical tumors of different types, respectively, but they were not expressed in adenoma tumors. Furthermore, there was a significant correlation between MAP17 and SGLT1 expression levels. High levels of either MAP17 or SGLT1 correlated with improved patient survival after treatment. However, the patients with high levels of both MAP17 and SGLT1 survived through the end of this study. Therefore, the combination of high MAP17 and SGLT1 levels is a marker for good prognosis in patients with cervical tumors after cisplatin plus radiotherapy treatment. These results also suggest that the use of MAP17 and SGLT1 markers may identify patients who are likely to exhibit a better response to treatments that boost oxidative stress in other cancer types. PMID:23418532

  2. Installing hydrolytic activity into a completely de novo protein framework

    NASA Astrophysics Data System (ADS)

    Burton, Antony J.; Thomson, Andrew R.; Dawson, William M.; Brady, R. Leo; Woolfson, Derek N.

    2016-09-01

    The design of enzyme-like catalysts tests our understanding of sequence-to-structure/function relationships in proteins. Here we install hydrolytic activity predictably into a completely de novo and thermostable α-helical barrel, which comprises seven helices arranged around an accessible channel. We show that the lumen of the barrel accepts 21 mutations to functional polar residues. The resulting variant, which has cysteine-histidine-glutamic acid triads on each helix, hydrolyses p-nitrophenyl acetate with catalytic efficiencies that match the most-efficient redesigned hydrolases based on natural protein scaffolds. This is the first report of a functional catalytic triad engineered into a de novo protein framework. The flexibility of our system also allows the facile incorporation of unnatural side chains to improve activity and probe the catalytic mechanism. Such a predictable and robust construction of truly de novo biocatalysts holds promise for applications in chemical and biochemical synthesis.

  3. Installing hydrolytic activity into a completely de novo protein framework

    NASA Astrophysics Data System (ADS)

    Burton, Antony J.; Thomson, Andrew R.; Dawson, William M.; Brady, R. Leo; Woolfson, Derek N.

    2016-09-01

    The design of enzyme-like catalysts tests our understanding of sequence-to-structure/function relationships in proteins. Here we install hydrolytic activity predictably into a completely de novo and thermostable α-helical barrel, which comprises seven helices arranged around an accessible channel. We show that the lumen of the barrel accepts 21 mutations to functional polar residues. The resulting variant, which has cysteine–histidine–glutamic acid triads on each helix, hydrolyses p-nitrophenyl acetate with catalytic efficiencies that match the most-efficient redesigned hydrolases based on natural protein scaffolds. This is the first report of a functional catalytic triad engineered into a de novo protein framework. The flexibility of our system also allows the facile incorporation of unnatural side chains to improve activity and probe the catalytic mechanism. Such a predictable and robust construction of truly de novo biocatalysts holds promise for applications in chemical and biochemical synthesis.

  4. Installing hydrolytic activity into a completely de novo protein framework.

    PubMed

    Burton, Antony J; Thomson, Andrew R; Dawson, William M; Brady, R Leo; Woolfson, Derek N

    2016-09-01

    The design of enzyme-like catalysts tests our understanding of sequence-to-structure/function relationships in proteins. Here we install hydrolytic activity predictably into a completely de novo and thermostable α-helical barrel, which comprises seven helices arranged around an accessible channel. We show that the lumen of the barrel accepts 21 mutations to functional polar residues. The resulting variant, which has cysteine-histidine-glutamic acid triads on each helix, hydrolyses p-nitrophenyl acetate with catalytic efficiencies that match the most-efficient redesigned hydrolases based on natural protein scaffolds. This is the first report of a functional catalytic triad engineered into a de novo protein framework. The flexibility of our system also allows the facile incorporation of unnatural side chains to improve activity and probe the catalytic mechanism. Such a predictable and robust construction of truly de novo biocatalysts holds promise for applications in chemical and biochemical synthesis. PMID:27554410

  5. The impact of carbohydrate and protein level and sources on swine manure foaming properties

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study explored the impact of swine diet on the composition, methane production potential, and foaming properties of manure. Samples of swine manure were collected from controlled feeding trials with diets varying in protein and carbohydrate levels and sources. Protein sources consisted of corn ...

  6. Degradation of Keap1 activates BH3-only proteins Bim and PUMA during hepatocyte lipoapoptosis.

    PubMed

    Cazanave, S C; Wang, X; Zhou, H; Rahmani, M; Grant, S; Durrant, D E; Klaassen, C D; Yamamoto, M; Sanyal, A J

    2014-08-01

    Non-alcoholic steatohepatitis is characterized by hepatic steatosis, elevated levels of circulating free fatty acids (FFA) and hepatocyte lipoapoptosis. This lipoapoptosis requires increased JNK phosphorylation and activation of the pro-apoptotic BH3-only proteins Bim and PUMA. Kelch-like ECH-associated protein (Keap)-1 is a BTB/Kelch protein that can regulate the expression of Bcl-2 protein and control apoptotic cell death. Yet, the role of Keap1 in hepatocyte lipotoxicity is unclear. Here we demonstrate that Keap1 protein was rapidly degraded in hepatocytes, through autophagy in a p62-dependent manner, in response to the toxic saturated FFA palmitate, but not following incubation with the non-toxic FFA oleic acid. Stable knockdown of Keap1 expression, using shRNA technology, in hepatocarcinoma cell lines induced spontaneous cell toxicity that was associated with JNK1-dependent upregulation of Bim and PUMA protein levels. Also, Keap1 knockdown further sensitized hepatocytes to lipoapoptosis by palmitate. Likewise, primary hepatocytes isolated from liver-specific Keap1(-/-) mice displayed higher Bim and PUMA protein levels and demonstrated increased sensitivity to palmitate-induced apoptosis than wild-type mouse hepatocytes. Finally, stable knockdown of Bim or PUMA expression prevented cell toxicity induced by loss of Keap1. These results implicate p62-dependent autophagic degradation of Keap1 by palmitate as a mechanism contributing to hepatocyte lipoapoptosis.

  7. Phytochrome regulates GTP-binding protein activity in the envelope of pea nuclei

    NASA Technical Reports Server (NTRS)

    Clark, G. B.; Memon, A. R.; Thompson, G. A. Jr; Roux, S. J.

    1993-01-01

    Three GTP-binding proteins with apparent molecular masses of 27, 28 and 30 kDa have been detected in isolated nuclei of etiolated pea plumules. After LDS-PAGE and transfer to nitrocellulose these proteins bind [32P]GTP in the presence of excess ATP, suggesting that they are monomeric G proteins. When nuclei are disrupted, three proteins co-purify with the nuclear envelope fraction and are highly enriched in this fraction. The level of [32P]GTP-binding for all three protein bands is significantly increased when harvested pea plumules are irradiated by red light, and this effect is reversed by far-red light. The results indicate that GTP-binding activity associated with the nuclear envelope of plant cells is photoreversibly regulated by the pigment phytochrome.

  8. Elevated serum interleukin-23 levels in ankylosing spondylitis patients and the relationship with disease activity.

    PubMed

    Ugur, Mahir; Baygutalp, Nurcan Kilic; Melikoglu, Meltem Alkan; Baygutalp, Fatih; Altas, Elif Umay; Seferoglu, Buminhan

    2015-11-01

    This study was aimed to evaluate the relationship between serum interleukin-23 (IL-23) levels and ankylosing spondylitis (AS).Twenty male patients diagnosed with ankylosing spondylitis according to the 1984 modified New York criteria for AS and twenty male healthy controls were included in this study.The demographic characteristics, clinical and laboratory findings of the patients were recorded. Serum IL-23 levels, C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) were measured in both the AS and control groups. The Bath ankylosing spondylitis disease activity ındex (BASDAI), the Bath ankylosing spondylitis functional index (BASFI), and the Bath ankylosing spondylitis metrology index (BASMI) were evaluated as disease activity parameters. The AS patients were divided into two subgroups as active and inactive in respect of CRP, ESR levels and BASDAI scores. The mean serum IL-23 levels of the AS and control groups were 334.45±176.54 pg/ml and 166.49±177.50 pg/ml respectively, and there was a significant difference between the groups. Correlation analysis of serum IL-23 levels with clinical and laboratory parameters showed that there were positive correlations between serum IL-23 levels and the BASDAI, BASFI scores in total, active and inactive patients and the BASMI scores in total and inactive patients and negative correlations between serum IL-23 levels and ESR in inactive patients. It was shown that altered serum IL-23 levels were related to AS disease activity. Further studies in large patient series are necessary to investigate the role of IL-23 protein in etiopathogenesis of AS.

  9. Monitoring Brain Activity with Protein Voltage and Calcium Sensors

    PubMed Central

    Storace, Douglas A.; Braubach, Oliver R.; Jin, Lei; Cohen, Lawrence B.; Sung, Uhna

    2015-01-01

    Understanding the roles of different cell types in the behaviors generated by neural circuits requires protein indicators that report neural activity with high spatio-temporal resolution. Genetically encoded fluorescent protein (FP) voltage sensors, which optically report the electrical activity in distinct cell populations, are, in principle, ideal candidates. Here we demonstrate that the FP voltage sensor ArcLight reports odor-evoked electrical activity in the in vivo mammalian olfactory bulb in single trials using both wide-field and 2-photon imaging. ArcLight resolved fast odorant-responses in individual glomeruli, and distributed odorant responses across a population of glomeruli. Comparisons between ArcLight and the protein calcium sensors GCaMP3 and GCaMP6f revealed that ArcLight had faster temporal kinetics that more clearly distinguished activity elicited by individual odorant inspirations. In contrast, the signals from both GCaMPs were a saturating integral of activity that returned relatively slowly to the baseline. ArcLight enables optical electrophysiology of mammalian neuronal population activity in vivo. PMID:25970202

  10. Monitoring brain activity with protein voltage and calcium sensors.

    PubMed

    Storace, Douglas A; Braubach, Oliver R; Jin, Lei; Cohen, Lawrence B; Sung, Uhna

    2015-05-13

    Understanding the roles of different cell types in the behaviors generated by neural circuits requires protein indicators that report neural activity with high spatio-temporal resolution. Genetically encoded fluorescent protein (FP) voltage sensors, which optically report the electrical activity in distinct cell populations, are, in principle, ideal candidates. Here we demonstrate that the FP voltage sensor ArcLight reports odor-evoked electrical activity in the in vivo mammalian olfactory bulb in single trials using both wide-field and 2-photon imaging. ArcLight resolved fast odorant-responses in individual glomeruli, and distributed odorant responses across a population of glomeruli. Comparisons between ArcLight and the protein calcium sensors GCaMP3 and GCaMP6f revealed that ArcLight had faster temporal kinetics that more clearly distinguished activity elicited by individual odorant inspirations. In contrast, the signals from both GCaMPs were a saturating integral of activity that returned relatively slowly to the baseline. ArcLight enables optical electrophysiology of mammalian neuronal population activity in vivo.

  11. Low Levels of p53 Protein and Chromatin Silencing of p53 Target Genes Repress Apoptosis in Drosophila Endocycling Cells

    PubMed Central

    Zhang, Bingqing; Mehrotra, Sonam; Ng, Wei Lun; Calvi, Brian R.

    2014-01-01

    Apoptotic cell death is an important response to genotoxic stress that prevents oncogenesis. It is known that tissues can differ in their apoptotic response, but molecular mechanisms are little understood. Here, we show that Drosophila polyploid endocycling cells (G/S cycle) repress the apoptotic response to DNA damage through at least two mechanisms. First, the expression of all the Drosophila p53 protein isoforms is strongly repressed at a post-transcriptional step. Second, p53-regulated pro-apoptotic genes are epigenetically silenced in endocycling cells, preventing activation of a paused RNA Pol II by p53-dependent or p53-independent pathways. Over-expression of the p53A isoform did not activate this paused RNA Pol II complex in endocycling cells, but over-expression of the p53B isoform with a longer transactivation domain did, suggesting that dampened p53B protein levels are crucial for apoptotic repression. We also find that the p53A protein isoform is ubiquitinated and degraded by the proteasome in endocycling cells. In mitotic cycling cells, p53A was the only isoform expressed to detectable levels, and its mRNA and protein levels increased after irradiation, but there was no evidence for an increase in protein stability. However, our data suggest that p53A protein stability is regulated in unirradiated cells, which likely ensures that apoptosis does not occur in the absence of stress. Without irradiation, both p53A protein and a paused RNA pol II were pre-bound to the promoters of pro-apoptotic genes, preparing mitotic cycling cells for a rapid apoptotic response to genotoxic stress. Together, our results define molecular mechanisms by which different cells in development modulate their apoptotic response, with broader significance for the survival of normal and cancer polyploid cells in mammals. PMID:25211335

  12. Accumulation and degradation of thiamin-binding protein and level of thiamin in wheat seeds during seed maturation and germination.

    PubMed

    Watanabe, Katsumi; Nishida, Naoko; Adachi, Takashi; Ueda, Motoko; Mitsunaga, Toshio; Kawamura, Yukio

    2004-06-01

    Changes in the levels of thiamin-binding globulin and thiamin in wheat seeds during maturation and germination were studied. The thiamin-binding activity of the seed proteins increased with seed development after flowering. The thiamin content of the seeds also increased with development. Thiamin-binding activity decreased during seed germination. On the other hand, immunological analysis using an antibody directed against the thiamin-binding protein isolated from wheat seeds showed that the thiamin-binding globulin accumulated in the aleurone layer of the seeds during maturation, and then the protein was degraded and disappeared during seed germination. These results suggested that the thiamin-binding globulin of wheat seeds was synthesized and accumulated in the aleurone layer of the seeds with seed development, similar to the thiamin-binding albumin in sesame seeds, and that thiamin bound to the thiamin-binding globulin in the dormant wheat seeds for germ growth during germination.

  13. Adaptor protein containing PH domain, PTB domain and leucine zipper (APPL1) regulates the protein level of EGFR by modulating its trafficking

    SciTech Connect

    Lee, Jae-Rin; Hahn, Hwa-Sun; Kim, Young-Hoon; Nguyen, Hong-Hoa; Yang, Jun-Mo; Kang, Jong-Sun; Hahn, Myong-Joon

    2011-11-11

    Highlights: Black-Right-Pointing-Pointer APPL1 regulates the protein level of EGFR in response to EGF stimulation. Black-Right-Pointing-Pointer Depletion of APPL1 accelerates the movement of EGF/EGFR from the cell surface to the perinuclear region in response to EGF. Black-Right-Pointing-Pointer Knockdown of APPL1 enhances the activity of Rab5. -- Abstract: The EGFR-mediated signaling pathway regulates multiple biological processes such as cell proliferation, survival and differentiation. Previously APPL1 (adaptor protein containing PH domain, PTB domain and leucine zipper 1) has been reported to function as a downstream effector of EGF-initiated signaling. Here we demonstrate that APPL1 regulates EGFR protein levels in response to EGF stimulation. Overexpression of APPL1 enhances EGFR stabilization while APPL1 depletion by siRNA reduces EGFR protein levels. APPL1 depletion accelerates EGFR internalization and movement of EGF/EGFR from cell surface to the perinuclear region in response to EGF treatment. Conversely, overexpression of APPL1 decelerates EGFR internalization and translocation of EGF/EGFR to the perinuclear region. Furthermore, APPL1 depletion enhances the activity of Rab5 which is involved in internalization and trafficking of EGFR and inhibition of Rab5 in APPL1-depleted cells restored EGFR levels. Consistently, APPL1 depletion reduced activation of Akt, the downstream signaling effector of EGFR and this is restored by inhibition of Rab5. These findings suggest that APPL1 is required for EGFR signaling by regulation of EGFR stabilities through inhibition of Rab5.

  14. Protein synthesis inhibitors reveal differential regulation of mitogen-activated protein kinase and stress-activated protein kinase pathways that converge on Elk-1.

    PubMed Central

    Zinck, R; Cahill, M A; Kracht, M; Sachsenmaier, C; Hipskind, R A; Nordheim, A

    1995-01-01

    Inhibitors of protein synthesis, such as anisomycin and cycloheximide, lead to superinduction of immediate-early genes. We demonstrate that these two drugs activate intracellular signaling pathways involving both the mitogen-activated protein kinase (MAPK) and stress-activated protein kinase (SAPK) cascades. The activation of either pathway correlates with phosphorylation of the c-fos regulatory transcription factor Elk-1. In HeLa cells, anisomycin stabilizes c-fos mRNA when protein synthesis is inhibited to only 50%. Under these conditions, anisomycin, in contrast to cycloheximide, rapidly induces kinase activation and efficient Elk-1 phosphorylation. However, full inhibition of translation by either drug leads to prolonged activation of SAPK activity, while MAPK induction is transient. This correlates with prolonged Elk-1 phosphorylation and c-fos transcription. Elk-1 induction and c-fos activation are also observed in KB cells, in which anisomycin strongly induces SAPKs but not MAPKs. Purified p54 SAPK alpha efficiently phosphorylates the Elk-1 C-terminal domain in vitro and comigrates with anisomycin-activated kinases in in-gel kinase assays. Thus, Elk-1 provides a potential convergence point for the MAPK and SAPK signaling pathways. The activation of signal cascades and control of transcription factor function therefore represent prominent processes in immediate-early gene superinduction. PMID:7651411

  15. Differential Requirement for Pten Lipid and Protein Phosphatase Activity during Zebrafish Embryonic Development

    PubMed Central

    Stumpf, Miriam; den Hertog, Jeroen

    2016-01-01

    The lipid- and protein phosphatase PTEN is one of the most frequently mutated tumor suppressor genes in human cancers and many mutations found in tumor samples directly affect PTEN phosphatase activity. In order to understand the functional consequences of these mutations in vivo, the aim of our study was to dissect the role of Pten phosphatase activities during zebrafish embryonic development. As in other model organisms, zebrafish mutants lacking functional Pten are embryonically lethal. Zebrafish have two pten genes and pten double homozygous zebrafish embryos develop a severe pleiotropic phenotype around 4 days post fertilization, which can be largely rescued by re-introduction of pten mRNA at the one-cell stage. We used this assay to characterize the rescue-capacity of Pten and variants with mutations that disrupt lipid, protein or both phosphatase activities. The pleiotropic phenotype at 4dpf could only be rescued by wild type Pten, indicating that both phosphatase activities are required for normal zebrafish embryonic development. An earlier aspect of the phenotype, hyperbranching of intersegmental vessels, however, was rescued by Pten that retained lipid phosphatase activity, independent of protein phosphatase activity. Lipid phosphatase activity was also required for moderating pAkt levels at 4 dpf. We propose that the role of Pten during angiogenesis mainly consists of suppressing PI3K signaling via its lipid phosphatase activity, whereas the complex process of embryonic development requires lipid and protein phosphatase of Pten. PMID:26848951

  16. Immersion freezing of ice nucleating active protein complexes

    NASA Astrophysics Data System (ADS)

    Hartmann, S.; Augustin, S.; Clauss, T.; Voigtländer, J.; Niedermeier, D.; Wex, H.; Stratmann, F.

    2012-08-01

    Biological particles, e.g. bacteria and their Ice Nucleating Active (INA) protein complexes, might play an important role for the ice formation in atmospheric mixed-phase clouds. Therefore, the immersion freezing behavior of INA protein complexes generated from a SnomaxTM solution/suspension was investigated as function of temperature in a range of -5 °C to -38 °C at the Leipzig Aerosol Cloud Interaction Simulator (LACIS). The immersion freezing of droplets containing small numbers of INA protein complexes occurs in a temperature range of -7 °C and -10 °C. The experiments performed in the lower temperature range, where all droplets freeze which contain at least one INA protein complex, are used to determine the average number of INA protein complexes present, assuming that the INA protein complexes are Poisson distributed over the droplet ensemble. Knowing the average number of INA protein complexes, the heterogeneous ice nucleation rate and rate coefficient of a single INA protein complex is determined by using the newly-developed CHESS model (stoCHastic model of idEntical poiSSon distributed ice nuclei). Therefore, we assume the ice nucleation process to be of stochastic nature, and a parameterization of the INA protein complex's nucleation rate. Analyzing the results of immersion freezing experiments from literature (SnomaxTM and Pseudomonas syringae bacteria), to results gained in this study, demonstrates that first, a similar temperature dependence of the heterogeneous ice nucleation rate for a single INA protein complex was found in all experiments, second, the shift of the ice fraction curves to higher temperatures can be explained consistently by a higher average number of INA protein complexes being present in the droplet ensemble, and finally the heterogeneous ice nucleation rate of one single INA protein complex might be also applicable for intact Pseudomonas syringae bacteria cells. The results obtained in this study allow a new perspective on the

  17. Identification and validation of genetic variants that influence transcription factor and cell signaling protein levels.

    PubMed

    Hause, Ronald J; Stark, Amy L; Antao, Nirav N; Gorsic, Lidija K; Chung, Sophie H; Brown, Christopher D; Wong, Shan S; Gill, Daniel F; Myers, Jamie L; To, Lida Anita; White, Kevin P; Dolan, M Eileen; Jones, Richard Baker

    2014-08-01

    Many genetic variants associated with human disease have been found to be associated with alterations in mRNA expression. Although it is commonly assumed that mRNA expression changes will lead to consequent changes in protein levels, methodological challenges have limited our ability to test the degree to which this assumption holds true. Here, we further developed the micro-western array approach and globally examined relationships between human genetic variation and cellular protein levels. We collected more than 250,000 protein level measurements comprising 441 transcription factor and signaling protein isoforms across 68 Yoruba (YRI) HapMap lymphoblastoid cell lines (LCLs) and identified 12 cis and 160 trans protein level QTLs (pQTLs) at a false discovery rate (FDR) of 20%. Whereas up to two thirds of cis mRNA expression QTLs (eQTLs) were also pQTLs, many pQTLs were not associated with mRNA expression. Notably, we replicated and functionally validated a trans pQTL relationship between the KARS lysyl-tRNA synthetase locus and levels of the DIDO1 protein. This study demonstrates proof of concept in applying an antibody-based microarray approach to iteratively measure the levels of human proteins and relate these levels to human genome variation and other genomic data sets. Our results suggest that protein-based mechanisms might functionally buffer genetic alterations that influence mRNA expression levels and that pQTLs might contribute phenotypic diversity to a human population independently of influences on mRNA expression.

  18. Education Finance Legislative Activity and Trends at the State Level.

    ERIC Educational Resources Information Center

    Crampton, Faith E.

    1999-01-01

    Reviews 1997 school finance legislation, comparing legislative activity levels from 1994 to 1997. In 1997, 32 states passed legislation pertaining to capital-outlay funding, tax bases, and taxation for education funding. Half passed legislation for state aid, technology, special-purpose education, budgeting/fiscal management, and school personnel…

  19. Pedometer-Assessed Physical Activity Levels of Rural Appalachian Youth

    ERIC Educational Resources Information Center

    Oh, Hyun-Ju; Rana, Sharon

    2014-01-01

    The purposes of this investigation were to examine whether pedometer-assessed physical activity (PA) in Appalachian Ohio students differed by body mass index (BMI), school level (middle school vs. high school), and gender during school days and nonschool days and whether students met the recommended PA guidelines. Participants (N = 149) were…

  20. Genetic Influences on Mechanically-Assessed Activity Level in Children

    ERIC Educational Resources Information Center

    Wood, Alexis C.; Saudino, Kimberly J.; Rogers, Hannah; Asherson, Philip; Kuntsi, Jonna

    2007-01-01

    Background: Activity level is an important component of children's temperament, as well as being part of the core symptom domain of hyperactivity-impulsivity within attention deficit hyperactivity disorder (ADHD). Yet it is poorly understood, due partly to limitations on parent and teacher ratings, which are typically used as measurements of these…

  1. Cardiovascular effects of variations in habitual levels of physical activity

    NASA Technical Reports Server (NTRS)

    Blomqvist, C. G.; Mitchell, J. H.

    1975-01-01

    Mechanisms involved in human cardiovascular adaption to stress, particularly adaption to different levels of physical activity are determined along with quantitative noninvasive methods for evaluation of cardiovascular function during stess in normal subjects and in individuals with latent or manifest cardiovascular disease. Results are summarized.

  2. Increased Myeloperoxidase Activity and Protein Nitration Are Indicators of Inflammation in Patients with Chagas' Disease▿

    PubMed Central

    Dhiman, Monisha; Estrada-Franco, Jose Guillermo; Pando, Jasmine M.; Ramirez-Aguilar, Francisco J.; Spratt, Heidi; Vazquez-Corzo, Sara; Perez-Molina, Gladys; Gallegos-Sandoval, Rosa; Moreno, Roberto; Garg, Nisha Jain

    2009-01-01

    In this study, we investigated whether inflammatory responses contribute to oxidative/nitrosative stress in patients with Chagas' disease. We used three tests (enzyme-linked immunosorbent assay, immuno-flow cytometry, and STAT-PAK immunochromatography) to screen human serum samples (n = 1,481) originating from Chiapas, Mexico, for Trypanosoma cruzi-specific antibodies. We identified 121 subjects who were seropositive for T. cruzi-specific antibodies, a finding indicative of an 8.5% seroprevalence in the rural population from Chiapas. Seropositive and seronegative subjects were examined for plasma levels of biomarkers of inflammation, i.e., myeloperoxidase (MPO), inducible nitric oxide synthase (iNOS), and xanthine oxidase (XOD), as well as for oxidative (advanced oxidation protein products [AOPPs]) and nitrosative (3-nitrotyrosine [3NT]) biomarkers. The seropositive subjects exhibited a significant increase in MPO activity and protein level, the indicator of neutrophil activation. Subsequently, a corresponding increase in AOPP contents, formed by MPO-dependent hypochlorous acid and chloramine formation, was noted in seropositive subjects. The plasma level of 3NT was significantly increased in seropositive subjects, yet we observed no change in XOD activity (O2− source) and nitrate/nitrite contents (denotes iNOS activation and NO production), which implied that direct peroxynitrite formation does not contribute to increased nitrosative damage in chagasic subjects. Instead, a positive correlation between increased MPO activity and protein 3NT formation was observed, which suggested to us that MPO-dependent formation of nitrylchloride that occurs in the presence of physiological NO and O2− concentrations contributes to protein nitration. Overall, our data demonstrate that T. cruzi-induced neutrophil activation is pathological and contributes to MPO-mediated collateral protein oxidative and nitrosative damage in human patients with Chagas' disease. Therapies

  3. Activation of immobilized, biotinylated choleragen AI protein by a 19-kilodalton guanine nucleotide-binding protein.

    PubMed

    Noda, M; Tsai, S C; Adamik, R; Bobak, D A; Moss, J; Vaughan, M

    1989-09-19

    Cholera toxin catalyzes the ADP-ribosylation that results in activation of the stimulatory guanine nucleotide-binding protein of the adenylyl cyclase system, known as Gs. The toxin also ADP-ribosylates other proteins and simple guanidino compounds and auto-ADP-ribosylates its AI protein (CTA1). All of the ADP-ribosyltransferase activities of CTAI are enhanced by 19-21-kDa guanine nucleotide-binding proteins known as ADP-ribosylation factors, or ARFs. CTAI contains a single cysteine located near the carboxy terminus. CTAI was immobilized through this cysteine by reaction with iodoacetyl-N-biotinyl-hexylenediamine and binding of the resulting biotinylated protein to avidin-agarose. Immobilized CTAI catalyzed the ARF-stimulated ADP-ribosylation of agmatine. The reaction was enhanced by detergents and phospholipid, but the fold stimulation by purified sARF-II from bovine brain was considerably less than that observed with free CTA. ADP-ribosylation of Gsa by immobilized CTAI, which was somewhat enhanced by sARF-II, was much less than predicted on the basis of the NAD:agmatine ADP-ribosyltransferase activity. Immobilized CTAI catalyzed its own auto-ADP-ribosylation as well as the ADP-ribosylation of the immobilized avidin and CTA2, with relatively little stimulation by sARF-II. ADP-ribosylation of CTA2 by free CTAI is minimal. These observations are consistent with the conclusion that the cysteine near the carboxy terminus of the toxin is not critical for ADP-ribosyltransferase activity or for its regulation by sARF-II. Biotinylation and immobilization of the toxin through this cysteine may, however, limit accessibility to Gsa or SARF-II, or perhaps otherwise reduce interaction with these proteins whether as substrates or activator.

  4. Regulation of the activity of the dual-function DnaA protein in Caulobacter crescentus.

    PubMed

    Fernandez-Fernandez, Carmen; Gonzalez, Diego; Collier, Justine

    2011-01-01

    DnaA is a conserved essential bacterial protein that acts as the initiator of chromosomal replication as well as a master transcriptional regulator in Caulobacter crescentus. Thus, the intracellular levels of active DnaA need to be tightly regulated during the cell cycle. Our previous work suggested that DnaA may be regulated at the level of its activity by the replisome-associated protein HdaA. Here, we describe the construction of a mutant DnaA protein [DnaA(R357A)]. The R357 residue in the AAA+ domain of the C. crescentus DnaA protein is equivalent to the R334 residue of the E. coli DnaA protein, which is required for the Regulatory Inactivation of DnaA (RIDA). We found that the expression of the DnaA(R357A) mutant protein in C. crescentus, but not the expression of the wild-type DnaA protein at similar levels, causes a severe phenotype of over-initiation of chromosomal replication and that it blocks cell division. Thus, the mutant DnaA(R357A) protein is hyper-active to promote the initiation of DNA replication, compared to the wild-type DnaA protein. DnaA(R357A) could not replace DnaA in vivo, indicating that the switch in DnaA activity once chromosomal replication has started may be an essential process in C. crescentus. We propose that the inactivation of DnaA is the main mechanism ensuring that chromosomal replication starts only once per cell cycle. We further observed that the R357A substitution in DnaA does not promote the activity of DnaA as a direct transcriptional activator of four important genes, encoding HdaA, the GcrA master cell cycle regulator, the FtsZ cell division protein and the MipZ spatial regulator of cell division. Thus, the AAA+ domain of DnaA may play a role in temporally regulating the bifunctionality of DnaA by reallocating DnaA molecules from initiating DNA replication to transcribing genes within the unique DnaA regulon of C. crescentus.

  5. Reassessing the Potential Activities of Plant CGI-58 Protein

    PubMed Central

    Khatib, Abdallah; Arhab, Yani; Bentebibel, Assia; Abousalham, Abdelkarim; Noiriel, Alexandre

    2016-01-01

    Comparative Gene Identification-58 (CGI-58) is a widespread protein found in animals and plants. This protein has been shown to participate in lipolysis in mice and humans by activating Adipose triglyceride lipase (ATGL), the initial enzyme responsible for the triacylglycerol (TAG) catabolism cascade. Human mutation of CGI-58 is the cause of Chanarin-Dorfman syndrome, an orphan disease characterized by a systemic accumulation of TAG which engenders tissue disorders. The CGI-58 protein has also been shown to participate in neutral lipid metabolism in plants and, in this case, a mutation again provokes TAG accumulation. Although its roles as an ATGL coactivator and in lipid metabolism are quite clear, the catalytic activity of CGI-58 is still in question. The acyltransferase activities of CGI-58 have been speculated about, reported or even dismissed and experimental evidence that CGI-58 expressed in E. coli possesses an unambiguous catalytic activity is still lacking. To address this problem, we developed a new set of plasmids and site-directed mutants to elucidate the in vivo effects of CGI-58 expression on lipid metabolism in E. coli. By analyzing the lipid composition in selected E. coli strains expressing CGI-58 proteins, and by reinvestigating enzymatic tests with adequate controls, we show here that recombinant plant CGI-58 has none of the proposed activities previously described. Recombinant plant and mouse CGI-58 both lack acyltransferase activity towards either lysophosphatidylglycerol or lysophosphatidic acid to form phosphatidylglycerol or phosphatidic acid and recombinant plant CGI-58 does not catalyze TAG or phospholipid hydrolysis. However, expression of recombinant plant CGI-58, but not mouse CGI-58, led to a decrease in phosphatidylglycerol in all strains of E. coli tested, and a mutation of the putative catalytic residues restored a wild-type phenotype. The potential activities of plant CGI-58 are subsequently discussed. PMID:26745266

  6. Reassessing the Potential Activities of Plant CGI-58 Protein.

    PubMed

    Khatib, Abdallah; Arhab, Yani; Bentebibel, Assia; Abousalham, Abdelkarim; Noiriel, Alexandre

    2016-01-01

    Comparative Gene Identification-58 (CGI-58) is a widespread protein found in animals and plants. This protein has been shown to participate in lipolysis in mice and humans by activating Adipose triglyceride lipase (ATGL), the initial enzyme responsible for the triacylglycerol (TAG) catabolism cascade. Human mutation of CGI-58 is the cause of Chanarin-Dorfman syndrome, an orphan disease characterized by a systemic accumulation of TAG which engenders tissue disorders. The CGI-58 protein has also been shown to participate in neutral lipid metabolism in plants and, in this case, a mutation again provokes TAG accumulation. Although its roles as an ATGL coactivator and in lipid metabolism are quite clear, the catalytic activity of CGI-58 is still in question. The acyltransferase activities of CGI-58 have been speculated about, reported or even dismissed and experimental evidence that CGI-58 expressed in E. coli possesses an unambiguous catalytic activity is still lacking. To address this problem, we developed a new set of plasmids and site-directed mutants to elucidate the in vivo effects of CGI-58 expression on lipid metabolism in E. coli. By analyzing the lipid composition in selected E. coli strains expressing CGI-58 proteins, and by reinvestigating enzymatic tests with adequate controls, we show here that recombinant plant CGI-58 has none of the propos