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Sample records for affect protein activity

  1. Rice proteins, extracted by alkali and α-amylase, differently affect in vitro antioxidant activity.

    PubMed

    Wang, Zhengxuan; Liu, Ye; Li, Hui; Yang, Lin

    2016-09-01

    Alkali treatment and α-amylase degradation are different processes for rice protein (RP) isolation. The major aim of this study was to determine the influence of two different extraction methods on the antioxidant capacities of RPA, extracted by alkaline (0.2% NaOH), and RPE, extracted by α-amylase, during in vitro digestion for 2h with pepsin and for 3h with pancreatin. Upon pepsin-pancreatin digestion, the protein hydrolysates (RPA-S, RPE-S), which were the supernatants in the absence of undigested residue, and the whole protein digests (RPA, RPE), in which undigested residue remained, were measured. RPE exhibited the stronger antioxidant responses to free radical scavenging activity, metal chelating activity, and reducing power, whereas the weakest antioxidant capacities were produced by RPE-S. In contrast, no significant differences in antioxidant activity were observed between RPA and RPA-S. The present study demonstrated that the in vitro antioxidant responses induced by the hydrolysates and the protein digests of RPs could be affected differently by alkali treatment and α-amylase degradation, suggesting that the extraction is a vital processing step to modify the antioxidant capacities of RPs. The results of the current study indicated that the protein digests, in which undigested residues remained, could exhibit more efficacious antioxidant activity compared to the hydrolysates.

  2. Opaque7 Encodes an Acyl-Activating Enzyme-Like Protein That Affects Storage Protein Synthesis in Maize Endosperm

    PubMed Central

    Wang, Gang; Sun, Xiaoliang; Wang, Guifeng; Wang, Fei; Gao, Qiang; Sun, Xin; Tang, Yuanping; Chang, Chong; Lai, Jinsheng; Zhu, Lihuang; Xu, Zhengkai; Song, Rentao

    2011-01-01

    In maize, a series of seed mutants with starchy endosperm could increase the lysine content by decreased amount of zeins, the main storage proteins in endosperm. Cloning and characterization of these mutants could reveal regulatory mechanisms for zeins accumulation in maize endosperm. Opaque7 (o7) is a classic maize starchy endosperm mutant with large effects on zeins accumulation and high lysine content. In this study, the O7 gene was cloned by map-based cloning and confirmed by transgenic functional complementation and RNAi. The o7-ref allele has a 12-bp in-frame deletion. The four-amino-acid deletion caused low accumulation of o7 protein in vivo. The O7 gene encodes an acyl-activating enzyme with high similarity to AAE3. The opaque phenotype of the o7 mutant was produced by the reduction of protein body size and number caused by a decrease in the α-zeins concentrations. Analysis of amino acids and metabolites suggested that the O7 gene might affect amino acid biosynthesis by affecting α-ketoglutaric acid and oxaloacetic acid. Transgenic rice seeds containing RNAi constructs targeting the rice ortholog of maize O7 also produced lower amounts of seed proteins and displayed an opaque endosperm phenotype, indicating a conserved biological function of O7 in cereal crops. The cloning of O7 revealed a novel regulatory mechanism for storage protein synthesis and highlighted an effective target for the genetic manipulation of storage protein contents in cereal seeds. PMID:21954158

  3. Catalytic activities of Werner protein are affected by adduction with 4-hydroxy-2-nonenal.

    PubMed

    Czerwińska, Jolanta; Poznański, Jarosław; Dębski, Janusz; Bukowy, Zuzanna; Bohr, Vilhelm A; Tudek, Barbara; Speina, Elżbieta

    2014-01-01

    4-Hydroxy-2-nonenal (HNE) is a reactive α,β-unsaturated aldehyde generated during oxidative stress and subsequent peroxidation of polyunsaturated fatty acids. Here, Werner protein (WRN) was identified as a novel target for modification by HNE. Werner syndrome arises through mutations in the WRN gene that encodes the RecQ DNA helicase which is critical for maintaining genomic stability. This hereditary disease is associated with chromosomal instability, premature aging and cancer predisposition. WRN appears to participate in the cellular response to oxidative stress and cells devoid of WRN display elevated levels of oxidative DNA damage. We demonstrated that helicase/ATPase and exonuclease activities of HNE-modified WRN protein were inhibited both in vitro and in immunocomplexes purified from the cell extracts. Sites of HNE adduction in human WRN were identified at Lys577, Cys727, His1290, Cys1367, Lys1371 and Lys1389. We applied in silico modeling of the helicase and RQC domains of WRN protein with HNE adducted to Lys577 and Cys727 and provided a potential mechanism of the observed deregulation of the protein catalytic activities. In light of the obtained results, we postulate that HNE adduction to WRN is a post-translational modification, which may affect WRN conformational stability and function, contributing to features and diseases associated with premature senescence.

  4. Catalytic activities of Werner protein are affected by adduction with 4-hydroxy-2-nonenal

    PubMed Central

    Czerwińska, Jolanta; Poznański, Jarosław; Dębski, Janusz; Bukowy, Zuzanna; Bohr, Vilhelm A.; Tudek, Barbara; Speina, Elżbieta

    2014-01-01

    4-Hydroxy-2-nonenal (HNE) is a reactive α,β-unsaturated aldehyde generated during oxidative stress and subsequent peroxidation of polyunsaturated fatty acids. Here, Werner protein (WRN) was identified as a novel target for modification by HNE. Werner syndrome arises through mutations in the WRN gene that encodes the RecQ DNA helicase which is critical for maintaining genomic stability. This hereditary disease is associated with chromosomal instability, premature aging and cancer predisposition. WRN appears to participate in the cellular response to oxidative stress and cells devoid of WRN display elevated levels of oxidative DNA damage. We demonstrated that helicase/ATPase and exonuclease activities of HNE-modified WRN protein were inhibited both in vitro and in immunocomplexes purified from the cell extracts. Sites of HNE adduction in human WRN were identified at Lys577, Cys727, His1290, Cys1367, Lys1371 and Lys1389. We applied in silico modeling of the helicase and RQC domains of WRN protein with HNE adducted to Lys577 and Cys727 and provided a potential mechanism of the observed deregulation of the protein catalytic activities. In light of the obtained results, we postulate that HNE adduction to WRN is a post-translational modification, which may affect WRN conformational stability and function, contributing to features and diseases associated with premature senescence. PMID:25170083

  5. The microtubule-associated protein MAP18 affects ROP2 GTPase activity during root hair growth.

    PubMed

    Kang, Erfang; Zheng, Mingzhi; Zhang, Yan; Yuan, Ming; Yalovsky, Shaul; Zhu, Lei; Fu, Ying

    2017-03-17

    Establishment and maintenance of the polar site are important for root hair tip growth. We previously reported that Arabidopsis (Arabidopsis thaliana) MICROTUBULE-ASSOCIATED PROTEIN18 (MAP18) functions in controlling the direction of pollen tube growth and root hair elongation. Additionally, the Rop GTPase ROP2 was reported as a positive regulator of both root hair initiation and tip growth in Arabidopsis. Both loss-of-function of ROP2 or knock-down of MAP18 leads to a decrease in root hair length, whereas overexpression of either MAP18 or ROP2 causes multiple tips or a branching hair phenotype. However, it is unclear whether MAP18 and ROP2 coordinately regulate root hair growth. In the present study, we demonstrate that MAP18 and ROP2 interact genetically and functionally. MAP18 physically interacts with ROP2 in vitro and in vivo and preferentially binds to the inactive form of the ROP2 protein. MAP18 promotes ROP2 activity during root hair tip growth. Further investigation revealed that MAP18 competes with RhoGTPase GDP dissociation inhibitor 1 (AtRhoGDI1)/SUPERCENTIPEDE1 (SCN1) for binding to ROP2, in turn affecting localization of active ROP2 in the plasma membrane of the root hair tip. These results reveal a novel function of MAP18 in the regulation of ROP2 activation during root hair growth.

  6. Balancing protein stability and activity in cancer: a new approach for identifying driver mutations affecting CBL ubiquitin ligase activation

    PubMed Central

    Li, Minghui; Kales, Stephen C.; Ma, Ke; Shoemaker, Benjamin A.; Crespo-Barreto, Juan; Cangelosi, Andrew L.; Lipkowitz, Stanley; Panchenko, Anna R.

    2015-01-01

    Oncogenic mutations in the monomeric Casitas B-lineage lymphoma (Cbl) gene have been found in many tumors, but their significance remains largely unknown. Several human c-Cbl (CBL) structures have recently been solved depicting the protein at different stages of its activation cycle and thus provide mechanistic insight underlying how stability-activity tradeoffs in cancer-related proteins may influence disease onset and progression. In this study, we computationally modeled the effects of missense cancer mutations on structures representing four stages of the CBL activation cycle to identify driver mutations that affect CBL stability, binding, and activity. We found that recurrent, homozygous, and leukemia-specific mutations had greater destabilizing effects on CBL states than did random non-cancer mutations. We further tested the ability of these computational models assessing the changes in CBL stability and its binding to ubiquitin conjugating enzyme E2, by performing blind CBL-mediated EGFR ubiquitination assays in cells. Experimental CBL ubiquitin ligase activity was in agreement with the predicted changes in CBL stability and, to a lesser extent, with CBL-E2 binding affinity. Two-thirds of all experimentally tested mutations affected the ubiquitin ligase activity by either destabilizing CBL or disrupting CBL-E2 binding, whereas about one-third of tested mutations were found to be neutral. Collectively, our findings demonstrate that computational methods incorporating multiple protein conformations and stability and binding affinity evaluations can successfully predict the functional consequences of cancer mutations on protein activity, and provide a proof of concept for mutations in CBL. PMID:26676746

  7. 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.

  8. The Interaction of the Gammaherpesvirus 68 orf73 Protein with Cellular BET Proteins Affects the Activation of Cell Cycle Promoters▿

    PubMed Central

    Ottinger, Matthias; Pliquet, Daniel; Christalla, Thomas; Frank, Ronald; Stewart, James P.; Schulz, Thomas F.

    2009-01-01

    Infection of mice with murine gammaherpesvirus 68 (MHV-68) provides a valuable animal model for gamma-2 herpesvirus (rhadinovirus) infection and pathogenesis. The MHV-68 orf73 protein has been shown to be required for the establishment of viral latency in vivo. This study describes a novel transcriptional activation function of the MHV-68 orf73 protein and identifies the cellular bromodomain containing BET proteins Brd2/RING3, Brd3/ORFX, and BRD4 as interaction partners for the MHV-68 orf73 protein. BET protein members are known to interact with acetylated histones, and Brd2 and Brd4 have been implicated in fundamental cellular processes, including cell cycle regulation and transcriptional regulation. Using MHV-68 orf73 peptide array assays, we identified Brd2 and Brd4 interaction sites in the orf73 protein. Mutation of one binding site led to a loss of the interaction with Brd2/4 but not the retinoblastoma protein Rb, to impaired chromatin association, and to a decreased ability to activate the BET-responsive cyclin D1, D2, and E promoters. The results therefore pinpoint the binding site for Brd2/4 in a rhadinoviral orf73 protein and suggest that the recruitment of a member of the BET protein family allows the MHV-68 orf73 protein to activate the promoters of G1/S cyclins. These findings point to parallels between the transcriptional activator functions of rhadinoviral orf73 proteins and papillomavirus E2 proteins. PMID:19244327

  9. Inhibition of Protein Farnesylation Arrests Adipogenesis and Affects PPARγ Expression and Activation in Differentiating Mesenchymal Stem Cells

    PubMed Central

    Rivas, Daniel; Akter, Rahima; Duque, Gustavo

    2007-01-01

    Protein farnesylation is required for the activation of multiple proteins involved in cell differentiation and function. In white adipose tissue protein, farnesylation has shown to be essential for the successful differentiation of preadipocytes into adipocytes. We hypothesize that protein farnesylation is required for PPARγ2 expression and activation, and therefore for the differentiation of human mesenchymal stem cells (MSCs) into adipocytes. MSCs were plated and induced to differentiate into adipocytes for three weeks. Differentiating cells were treated with either an inhibitor of farnesylation (FTI-277) or vehicle alone. The effect of inhibition of farnesylation in differentiating adipocytes was determined by oil red O staining. Cell survival was quantified using MTS Formazan. Additionally, nuclear extracts were obtained and prelamin A, chaperon protein HDJ-2, PPARγ, and SREBP-1 were determined by western blot. Finally, DNA binding PPARγ activity was determined using an ELISA-based PPARγ activation quantification method. Treatment with an inhibitor of farnesylation (FTI-277) arrests adipogenesis without affecting cell survival. This effect was concomitant with lower levels of PPARγ expression and activity. Finally, accumulation of prelamin A induced an increased proportion of mature SREBP-1 which is known to affect PPARγ activity. In summary, inhibition of protein farnesylation arrests the adipogenic differentiation of MSCs and affects PPARγ expression and activity. PMID:18274630

  10. Inhibition of Protein Farnesylation Arrests Adipogenesis and Affects PPARgamma Expression and Activation in Differentiating Mesenchymal Stem Cells.

    PubMed

    Rivas, Daniel; Akter, Rahima; Duque, Gustavo

    2007-01-01

    Protein farnesylation is required for the activation of multiple proteins involved in cell differentiation and function. In white adipose tissue protein, farnesylation has shown to be essential for the successful differentiation of preadipocytes into adipocytes. We hypothesize that protein farnesylation is required for PPARgamma2 expression and activation, and therefore for the differentiation of human mesenchymal stem cells (MSCs) into adipocytes. MSCs were plated and induced to differentiate into adipocytes for three weeks. Differentiating cells were treated with either an inhibitor of farnesylation (FTI-277) or vehicle alone. The effect of inhibition of farnesylation in differentiating adipocytes was determined by oil red O staining. Cell survival was quantified using MTS Formazan. Additionally, nuclear extracts were obtained and prelamin A, chaperon protein HDJ-2, PPARgamma, and SREBP-1 were determined by western blot. Finally, DNA binding PPARgamma activity was determined using an ELISA-based PPARgamma activation quantification method. Treatment with an inhibitor of farnesylation (FTI-277) arrests adipogenesis without affecting cell survival. This effect was concomitant with lower levels of PPARgamma expression and activity. Finally, accumulation of prelamin A induced an increased proportion of mature SREBP-1 which is known to affect PPARgamma activity. In summary, inhibition of protein farnesylation arrests the adipogenic differentiation of MSCs and affects PPARgamma expression and activity.

  11. Prion Protein M129V Polymorphism Affects Retrieval-Related Brain Activity

    ERIC Educational Resources Information Center

    Buchmann, Andreas; Mondadori, Christian R. A.; Hanggi, Jurgen; Aerni, Amanda; Vrticka, Pascal; Luechinger, Roger; Boesiger, Peter; Hock, Christoph; Nitsch, Roger M.; de Quervain, Dominique J.-F.; Papassotiropoulos, Andreas; Henke, Katharina

    2008-01-01

    The prion protein Met129Val polymorphism has recently been related to human long-term memory with carriers of either the 129[superscript MM] or the 129[superscript MV] genotype recalling 17% more words than 129[superscript VV] carriers at 24 h following learning. Here, we sampled genotype differences in retrieval-related brain activity at 30 min…

  12. Interaction of Silver Nanoparticles with Serum Proteins Affects Their Antimicrobial Activity In Vivo

    PubMed Central

    Gnanadhas, Divya Prakash; Ben Thomas, Midhun; Thomas, Rony; Raichur, Ashok M.

    2013-01-01

    The emergence of multidrug-resistant bacteria is a global threat for human society. There exist recorded data that silver was used as an antimicrobial agent by the ancient Greeks and Romans during the 8th century. Silver nanoparticles (AgNPs) are of potential interest because of their effective antibacterial and antiviral activities, with minimal cytotoxic effects on the cells. However, very few reports have shown the usage of AgNPs for antibacterial therapy in vivo. In this study, we deciphered the importance of the chosen methods for synthesis and capping of AgNPs for their improved activity in vivo. The interaction of AgNPs with serum albumin has a significant effect on their antibacterial activity. It was observed that uncapped AgNPs exhibited no antibacterial activity in the presence of serum proteins, due to the interaction with bovine serum albumin (BSA), which was confirmed by UV-Vis spectroscopy. However, capped AgNPs [with citrate or poly(vinylpyrrolidone)] exhibited antibacterial properties due to minimized interactions with serum proteins. The damage in the bacterial membrane was assessed by flow cytometry, which also showed that only capped AgNPs exhibited antibacterial properties, even in the presence of BSA. In order to understand the in vivo relevance of the antibacterial activities of different AgNPs, a murine salmonellosis model was used. It was conclusively proved that AgNPs capped with citrate or PVP exhibited significant antibacterial activities in vivo against Salmonella infection compared to uncapped AgNPs. These results clearly demonstrate the importance of capping agents and the synthesis method for AgNPs in their use as antimicrobial agents for therapeutic purposes. PMID:23877702

  13. In Absence of the Cellular Prion Protein, Alterations in Copper Metabolism and Copper-Dependent Oxidase Activity Affect Iron Distribution.

    PubMed

    Gasperini, Lisa; Meneghetti, Elisa; Legname, Giuseppe; Benetti, Federico

    2016-01-01

    Essential elements as copper and iron modulate a wide range of physiological functions. Their metabolism is strictly regulated by cellular pathways, since dysregulation of metal homeostasis is responsible for many detrimental effects. Neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and prion diseases are characterized by alterations of metal ions. These neurodegenerative maladies involve proteins that bind metals and mediate their metabolism through not well-defined mechanisms. Prion protein, for instance, interacts with divalent cations via multiple metal-binding sites and it modulates several metal-dependent physiological functions, such as S-nitrosylation of NMDA receptors. In this work we focused on the effect of prion protein absence on copper and iron metabolism during development and adulthood. In particular, we investigated copper and iron functional values in serum and several organs such as liver, spleen, total brain and isolated hippocampus. Our results show that iron content is diminished in prion protein-null mouse serum, while it accumulates in liver and spleen. Our data suggest that these alterations can be due to impairments in copper-dependent cerulopalsmin activity which is known to affect iron mobilization. In prion protein-null mouse total brain and hippocampus, metal ion content shows a fluctuating trend, suggesting the presence of homeostatic compensatory mechanisms. However, copper and iron functional values are likely altered also in these two organs, as indicated by the modulation of metal-binding protein expression levels. Altogether, these results reveal that the absence of the cellular prion protein impairs copper metabolism and copper-dependent oxidase activity, with ensuing alteration of iron mobilization from cellular storage compartments.

  14. In Absence of the Cellular Prion Protein, Alterations in Copper Metabolism and Copper-Dependent Oxidase Activity Affect Iron Distribution

    PubMed Central

    Gasperini, Lisa; Meneghetti, Elisa; Legname, Giuseppe; Benetti, Federico

    2016-01-01

    Essential elements as copper and iron modulate a wide range of physiological functions. Their metabolism is strictly regulated by cellular pathways, since dysregulation of metal homeostasis is responsible for many detrimental effects. Neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and prion diseases are characterized by alterations of metal ions. These neurodegenerative maladies involve proteins that bind metals and mediate their metabolism through not well-defined mechanisms. Prion protein, for instance, interacts with divalent cations via multiple metal-binding sites and it modulates several metal-dependent physiological functions, such as S-nitrosylation of NMDA receptors. In this work we focused on the effect of prion protein absence on copper and iron metabolism during development and adulthood. In particular, we investigated copper and iron functional values in serum and several organs such as liver, spleen, total brain and isolated hippocampus. Our results show that iron content is diminished in prion protein-null mouse serum, while it accumulates in liver and spleen. Our data suggest that these alterations can be due to impairments in copper-dependent cerulopalsmin activity which is known to affect iron mobilization. In prion protein-null mouse total brain and hippocampus, metal ion content shows a fluctuating trend, suggesting the presence of homeostatic compensatory mechanisms. However, copper and iron functional values are likely altered also in these two organs, as indicated by the modulation of metal-binding protein expression levels. Altogether, these results reveal that the absence of the cellular prion protein impairs copper metabolism and copper-dependent oxidase activity, with ensuing alteration of iron mobilization from cellular storage compartments. PMID:27729845

  15. Jasmonic acid affects plant morphology and calcium-dependent protein kinase expression and activity in Solanum tuberosum.

    PubMed

    Ulloa, Rita M; Raíces, Marcela; MacIntosh, Gustavo C; Maldonado, Sara; Téllez-Iñón, María T

    2002-07-01

    The effect of jasmonic acid (JA) on plant growth and on calcium-dependent protein kinase (CDPK) activity and expression was studied in non-photoperiodic potato plants, Solanum tuberosum L. var. Spunta, grown in vitro. Stem cuttings were grown for 45 days (long treatment, LT) in MS medium with increasing concentrations of JA. For short treatments (ST) adult plants grown in MS were transferred for 1, 4 and 20 h to JA containing media. During the LT, low concentrations of JA promoted cell expansion and shoot elongation while higher concentrations caused growth inhibition. Under these conditions, treated plants showed root shortening and tuber formation was not induced. Morphological and histochemical studies using light microscopy and TEM analysis of leaves from treated plants revealed that JA also affected subcellular organelles of mesophyll cells. Peroxisomes increased in size and number, and an autophagic process was triggered in response to high concentrations of the hormone. CDPK activity, determined in crude extracts of treated plants (LT), was inhibited (up to 80%). Plant growth and CDPK inhibition were reverted upon transfer of the plants to hormone-free medium. Soluble CDPK activity decreased in response to JA short treatment. Concomitantly, a decline in the steady state levels of StCDPK2 mRNA, a potato CDPK isoform that is expressed in leaves, was observed. These data suggest that the phytohormone down-regulated the expression and activity of the kinase.

  16. The absence of myristic acid decreases membrane binding of p60src but does not affect tyrosine protein kinase activity.

    PubMed Central

    Buss, J E; Kamps, M P; Gould, K; Sefton, B M

    1986-01-01

    We have constructed two point mutants of Rous sarcoma virus in which the amino-terminal glycine residue of the transforming protein, p60src, was changed to an alanine or a glutamic acid residue. Both mutant proteins failed to become myristylated and, more importantly, no longer transformed cells. The lack of transformation could not be attributed to defects in the catalytic activity of the mutant p60src proteins. In vitro phosphorylation of the peptide angiotensin or of the cellular substrate proteins enolase and p36 revealed no significant differences in the Km or specific activity of the mutant and wild-type p60src proteins. However, when cellular fractions were prepared, less than 12% of the nonmyristylated p60src proteins was bound to membranes. In contrast, more than 82% of the wild-type protein was associated with membranes. Wild-type p60src was phosphorylated by protein kinase C, a protein kinase which associates with membranes when activated. The mutant proteins were not. This finding supports the idea that within the intact cell the nonmyristylated p60src proteins are cytoplasmic and suggests that this apparent solubility is not an artifact of the cell fractionation procedure. The myristyl groups of p60src apparently encourages a tight association between protein and membranes and, by determining the cellular location of the enzyme, allows transformation to occur. Images PMID:3009860

  17. Metformin revisited: Does this regulator of AMP-activated protein kinase secondarily affect bone metabolism and prevent diabetic osteopathy

    PubMed Central

    McCarthy, Antonio Desmond; Cortizo, Ana María; Sedlinsky, Claudia

    2016-01-01

    Patients with long-term type 1 and type 2 diabetes mellitus (DM) can develop skeletal complications or “diabetic osteopathy”. These include osteopenia, osteoporosis and an increased incidence of low-stress fractures. In this context, it is important to evaluate whether current anti-diabetic treatments can secondarily affect bone metabolism. Adenosine monophosphate-activated protein kinase (AMPK) modulates multiple metabolic pathways and acts as a sensor of the cellular energy status; recent evidence suggests a critical role for AMPK in bone homeostasis. In addition, AMPK activation is believed to mediate most clinical effects of the insulin-sensitizer metformin. Over the past decade, several research groups have investigated the effects of metformin on bone, providing a considerable body of pre-clinical (in vitro, ex vivo and in vivo) as well as clinical evidence for an anabolic action of metformin on bone. However, two caveats should be kept in mind when considering metformin treatment for a patient with type 2 DM at risk for diabetic osteopathy. In the first place, metformin should probably not be considered an anti-osteoporotic drug; it is an insulin sensitizer with proven macrovascular benefits that can secondarily improve bone metabolism in the context of DM. Secondly, we are still awaiting the results of randomized placebo-controlled studies in humans that evaluate the effects of metformin on bone metabolism as a primary endpoint. PMID:27022443

  18. Activity of cGMP-Dependent Protein Kinase (PKG) Affects Sucrose Responsiveness and Habituation in "Drosophila melanogaster"

    ERIC Educational Resources Information Center

    Scheiner, Ricarda; Sokolowski, Marla B.; Erber, Joachim

    2004-01-01

    The cGMP-dependent protein kinase (PKG) has many cellular functions in vertebrates and insects that affect complex behaviors such as locomotion and foraging. The "foraging" ("for") gene encodes a PKG in "Drosophila melanogaster." Here, we demonstrate a function for the "for" gene in sensory responsiveness and nonassociative learning. Larvae of the…

  19. Aging and exercise affect the level of protein acetylation and SIRT1 activity in cerebellum of male rats.

    PubMed

    Marton, Orsolya; Koltai, Erika; Nyakas, Csaba; Bakonyi, Tibor; Zenteno-Savin, Tania; Kumagai, Shuzo; Goto, Sataro; Radak, Zsolt

    2010-12-01

    Aging is associated with a gradual decline in cognitive and motor functions, the result of complex biochemical processes including pre- and posttranslational modifications of proteins. Sirtuins are NAD(+) dependent protein deacetylases. These enzymes modulate the aging process by lysine deacetylation, which alters the activity and stability of proteins. Exercise can increase mean life-span and improve quality of life. Data from our laboratories revealed that 4 weeks of treadmill running improves performance in the Morris Maze test for young (4 months, old) but not old (30 months, old) male rats, and the exercise could not prevent the age-associated loss in muscle strength assessed by a gripping test. The positive correlation between protein acetylation and the gripping test suggests that the age-dependent decrease in relative activity of SIRT1 in the cerebellum impairs motor function. Similarly to the acetylation level of total proteins, the acetylation of ά -tubulin is also increased with aging, while the effect of exercise training was not found to be significant. Moreover, the protein content of nicotinamide phosphoribosyltransferase, one of the key enzymes of NAD biosynthesis, decreased in the young exercise group. These data suggest that aging results in decreased specific activity of SIRT1 in cerebellum, which could lead to increased acetylation of protein residues, including ά-tubulin, that interfere with motor function.

  20. Sequences near the Active Site in Chimeric Penicillin Binding Proteins 5 and 6 Affect Uniform Morphology of Escherichia coli

    PubMed Central

    Ghosh, Anindya S.; Young, Kevin D.

    2003-01-01

    Penicillin binding protein (PBP) 5, a dd-carboxypeptidase that removes the terminal d-alanine from peptide side chains of peptidoglycan, plays an important role in creating and maintaining the uniform cell shape of Escherichia coli. PBP 6, a highly similar homologue, cannot substitute for PBP 5 in this respect. Previously, we localized the shape-maintaining characteristics of PBP 5 to the globular domain that contains the active site (domain I), where PBPs 5 and 6 share substantial identity. To identify the specific segment of domain I responsible for shape control, we created a set of hybrids and determined which ones complemented the aberrant morphology of a misshapen PBP mutant, E. coli CS703-1. Fusion proteins were constructed in which 47, 199 and 228 amino-terminal amino acids of one PBP were fused to the corresponding carboxy-terminal amino acids of the other. The morphological phenotype was reversed only by hybrid proteins containing PBP 5 residues 200 to 228, which are located next to the KTG motif of the active site. Because residues 220 to 228 were identical in these proteins, the morphological effect was determined by alterations in amino acids 200 to 219. To confirm the importance of this segment, we constructed mosaic proteins in which these 20 amino acids were grafted from PBP 5 into PBP 6 and vice versa. The PBP 6/5/6 mosaic complemented the aberrant morphology of CS703-1, whereas PBP 5/6/5 did not. Site-directed mutagenesis demonstrated that the Asp218 and Lys219 residues were important for shape maintenance by these mosaic PBPs, but the same mutations in wild-type PBP 5 did not eliminate its shape-promoting activity. Homologous enzymes from five other bacteria also complemented the phenotype of CS703-1. The overall conclusion is that creation of a bacterial cell of regular diameter and uniform contour apparently depends primarily on a slight alteration of the enzymatic activity or substrate accessibility at the active site of E. coli PBP 5. PMID

  1. The unfolded protein response is activated in disease-affected brain regions in progressive supranuclear palsy and Alzheimer’s disease

    PubMed Central

    2013-01-01

    Background Progressive supranuclear palsy (PSP) is a neurodegenerative disorder pathologically characterized by intracellular tangles of hyperphosphorylated tau protein distributed throughout the neocortex, basal ganglia, and brainstem. A genome-wide association study identified EIF2AK3 as a risk factor for PSP. EIF2AK3 encodes PERK, part of the endoplasmic reticulum’s (ER) unfolded protein response (UPR). PERK is an ER membrane protein that senses unfolded protein accumulation within the ER lumen. Recently, several groups noted UPR activation in Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis, multiple system atrophy, and in the hippocampus and substantia nigra of PSP subjects. Here, we evaluate UPR PERK activation in the pons, medulla, midbrain, hippocampus, frontal cortex and cerebellum in subjects with PSP, AD, and in normal controls. Results We found UPR activation primarily in disease-affected brain regions in both disorders. In PSP, the UPR was primarily activated in the pons and medulla and to a much lesser extent in the hippocampus. In AD, the UPR was extensively activated in the hippocampus. We also observed UPR activation in the hippocampus of some elderly normal controls, severity of which positively correlated with both age and tau pathology but not with Aβ plaque burden. Finally, we evaluated EIF2AK3 coding variants that influence PERK activation. We show that a haplotype associated with increased PERK activation is genetically associated with increased PSP risk. Conclusions The UPR is activated in disease affected regions in PSP and the genetic evidence shows that this activation increases risk for PSP and is not a protective response. PMID:24252572

  2. Na,K-ATPase reconstituted in ternary liposome: the presence of cholesterol affects protein activity and thermal stability.

    PubMed

    Yoneda, Juliana Sakamoto; Rigos, Carolina Fortes; de Lourenço, Thaís Fernanda Aranda; Sebinelli, Heitor Gobbi; Ciancaglini, Pietro

    2014-12-15

    Differential scanning calorimetry (DSC) was applied to investigate the effect of cholesterol on the thermotropic properties of the lipid membrane (DPPC and DPPE). The thermostability and unfolding of solubilized and reconstituted Na,K-ATPase in DPPC:DPPE:cholesterol-liposomes was also studied to gain insight into the role of cholesterol in the Na,K-ATPase modulation of enzyme function and activity. The tertiary system (DPPC:DPPE:cholesterol) (molar ratio DPPC:DPPE equal 1:1) when cholesterol content was increased from 0% up to 40% results in a slight decrease in the temperature of transition and enthalpy, and an increase in width. We observed that, without heating treatment, at 37°C, the activity was higher for 20mol% cholesterol. However, thermal inactivation experiments showed that the enzyme activity loss time depends on the cholesterol membrane content. The unfolding of the enzyme incorporated to liposomes of DPPC:DPPE (1:1mol) with different cholesterol contents, ranging from 0% to 40% mol was also studied by DSC. Some differences between the thermograms indicate that the presence of lipids promotes a conformational change in protein structure and this change is enough to change the way Na,K-ATPase thermally unfolds.

  3. Alpha7-nicotinic acetylcholine receptors affect growth regulation of human mesothelioma cells: role of mitogen-activated protein kinase pathway.

    PubMed

    Trombino, Sonya; Cesario, Alfredo; Margaritora, Stefano; Granone, PierLuigi; Motta, Giovanni; Falugi, Carla; Russo, Patrizia

    2004-01-01

    This study presents data suggesting that both human mesothelioma (cell lines and human mesothelioma biopsies) and human normal mesothelial cells express receptors for acetylcholine and that stimulation of these receptors by nicotine prompted cell growth via activation of nicotinic cholinergic receptors. Thus, these data demonstrate that: (a) human mesothelioma cells and human biopsies of mesothelioma as well as of normal pleural mesothelial cells express functionally alpha-7 nicotinic acethlycholine receptors, evaluated by alpha-bungarotoxin-FITC binding, receptor binding assay, Western blot, and reverse transcription-PCR; (b) choline acetyltransferase immunostaining is present in mesothelioma cells; (c) mesothelioma cell growth is modulated by the cholinergic system in which agonists (i.e., nicotine) has a proliferative effect, and antagonists (i.e., curare) has an inhibitory effect, evaluated by cell cloning, DNA synthesis and cell cycle; (d) nicotine induces Ca(+2) influx, evaluated by [(45)Ca(2+)] uptake, and consequently activation of mitogen-activated protein kinase pathway (extracellular signal-regulated kinase and p90(RSK) phosphorylation), evaluated by Western blot; and (e) apoptosis mechanisms in mesothelioma cells are under the control of the cholinergic system (nicotine antiapoptotic via induction of nuclear factor-kappaB complexes and phosphorylation of Bad at Ser(112); curare proapoptotic via G(0)-G(1) arrest p21(waf-1) dependent but p53 independent). The involvement of the nonneuronal cholinergic system in mesothelioma appears reasonable and open up new therapeutic strategies.

  4. The SNF5 protein of Saccharomyces cerevisiae is a glutamine- and proline-rich transcriptional activator that affects expression of a broad spectrum of genes.

    PubMed Central

    Laurent, B C; Treitel, M A; Carlson, M

    1990-01-01

    The Saccharomyces cerevisiae SNF5 gene affects expression of both glucose- and phosphate-regulated genes and appears to function in transcription. We report the nucleotide sequence, which predicts that SNF5 encodes a 102,536-dalton protein. The N-terminal third of the protein is extremely rich in glutamine and proline. Mutants carrying a deletion of the coding sequence were viable but grew slowly, indicating that the SNF5 gene is important but not essential. Evidence that SNF5 affects expression of the cell type-specific genes MF alpha 1 and BAR1 at the RNA level extends the known range of SNF5 function. SNF5 is apparently required for expression of a wide variety of differently regulated genes. A bifunctional SNF5-beta-galactosidase fusion protein was localized in the nucleus by immunofluorescence. No DNA-binding activity was detected for SNF5. A LexA-SNF5 fusion protein, when bound to a lexA operator, functioned as a transcriptional activator. Images PMID:2233708

  5. AMP-Activated Protein Kinase α2 in Neutrophils Regulates Vascular Repair via Hypoxia-Inducible Factor-1α and a Network of Proteins Affecting Metabolism and Apoptosis

    PubMed Central

    Abdel Malik, Randa; Zippel, Nina; Frömel, Timo; Heidler, Juliana; Zukunft, Sven; Walzog, Barbara; Ansari, Nariman; Pampaloni, Francesco; Wingert, Susanne; Rieger, Michael A.; Wittig, Ilka; Fisslthaler, Beate

    2017-01-01

    Rationale: The AMP-activated protein kinase (AMPK) is stimulated by hypoxia, and although the AMPKα1 catalytic subunit has been implicated in angiogenesis, little is known about the role played by the AMPKα2 subunit in vascular repair. Objective: To determine the role of the AMPKα2 subunit in vascular repair. Methods and Results: Recovery of blood flow after femoral artery ligation was impaired (>80%) in AMPKα2−/− versus wild-type mice, a phenotype reproduced in mice lacking AMPKα2 in myeloid cells (AMPKα2ΔMC). Three days after ligation, neutrophil infiltration into ischemic limbs of AMPKα2ΔMC mice was lower than that in wild-type mice despite being higher after 24 hours. Neutrophil survival in ischemic tissue is required to attract monocytes that contribute to the angiogenic response. Indeed, apoptosis was increased in hypoxic neutrophils from AMPKα2ΔMC mice, fewer monocytes were recruited, and gene array analysis revealed attenuated expression of proangiogenic proteins in ischemic AMPKα2ΔMC hindlimbs. Many angiogenic growth factors are regulated by hypoxia-inducible factor, and hypoxia-inducible factor-1α induction was attenuated in AMPKα2-deficient cells and accompanied by its enhanced hydroxylation. Also, fewer proteins were regulated by hypoxia in neutrophils from AMPKα2ΔMC mice. Mechanistically, isocitrate dehydrogenase expression and the production of α-ketoglutarate, which negatively regulate hypoxia-inducible factor-1α stability, were attenuated in neutrophils from wild-type mice but remained elevated in cells from AMPKα2ΔMC mice. Conclusions: AMPKα2 regulates α-ketoglutarate generation, hypoxia-inducible factor-1α stability, and neutrophil survival, which in turn determine further myeloid cell recruitment and repair potential. The activation of AMPKα2 in neutrophils is a decisive event in the initiation of vascular repair after ischemia. PMID:27777247

  6. Protein Molecular Structures, Protein SubFractions, and Protein Availability Affected by Heat Processing: A Review

    SciTech Connect

    Yu,P.

    2007-01-01

    The utilization and availability of protein depended on the types of protein and their specific susceptibility to enzymatic hydrolysis (inhibitory activities) in the gastrointestine and was highly associated with protein molecular structures. Studying internal protein structure and protein subfraction profiles leaded to an understanding of the components that make up a whole protein. An understanding of the molecular structure of the whole protein was often vital to understanding its digestive behavior and nutritive value in animals. In this review, recently obtained information on protein molecular structural effects of heat processing was reviewed, in relation to protein characteristics affecting digestive behavior and nutrient utilization and availability. The emphasis of this review was on (1) using the newly advanced synchrotron technology (S-FTIR) as a novel approach to reveal protein molecular chemistry affected by heat processing within intact plant tissues; (2) revealing the effects of heat processing on the profile changes of protein subfractions associated with digestive behaviors and kinetics manipulated by heat processing; (3) prediction of the changes of protein availability and supply after heat processing, using the advanced DVE/OEB and NRC-2001 models, and (4) obtaining information on optimal processing conditions of protein as intestinal protein source to achieve target values for potential high net absorbable protein in the small intestine. The information described in this article may give better insight in the mechanisms involved and the intrinsic protein molecular structural changes occurring upon processing.

  7. Inhibition of Mitogen-activated Protein Kinase (MAPK)-interacting Kinase (MNK) Preferentially Affects Translation of mRNAs Containing Both a 5'-Terminal Cap and Hairpin.

    PubMed

    Korneeva, Nadejda L; Song, Anren; Gram, Hermann; Edens, Mary Ann; Rhoads, Robert E

    2016-02-12

    The MAPK-interacting kinases 1 and 2 (MNK1 and MNK2) are activated by extracellular signal-regulated kinases 1 and 2 (ERK1/2) or p38 in response to cellular stress and extracellular stimuli that include growth factors, cytokines, and hormones. Modulation of MNK activity affects translation of mRNAs involved in the cell cycle, cancer progression, and cell survival. However, the mechanism by which MNK selectively affects translation of these mRNAs is not understood. MNK binds eukaryotic translation initiation factor 4G (eIF4G) and phosphorylates the cap-binding protein eIF4E. Using a cell-free translation system from rabbit reticulocytes programmed with mRNAs containing different 5'-ends, we show that an MNK inhibitor, CGP57380, affects translation of only those mRNAs that contain both a cap and a hairpin in the 5'-UTR. Similarly, a C-terminal fragment of human eIF4G-1, eIF4G(1357-1600), which prevents binding of MNK to intact eIF4G, reduces eIF4E phosphorylation and inhibits translation of only capped and hairpin-containing mRNAs. Analysis of proteins bound to m(7)GTP-Sepharose reveals that both CGP and eIF4G(1357-1600) decrease binding of eIF4E to eIF4G. These data suggest that MNK stimulates translation only of mRNAs containing both a cap and 5'-terminal RNA duplex via eIF4E phosphorylation, thereby enhancing the coupled cap-binding and RNA-unwinding activities of eIF4F.

  8. N-linked glycans do not affect plasma membrane localization of multidrug resistance protein 4 (MRP4) but selectively alter its prostaglandin E2 transport activity.

    PubMed

    Miah, M Fahad; Conseil, Gwenaëlle; Cole, Susan P C

    2016-01-22

    Multidrug resistance protein 4 (MRP4) is a member of subfamily C of the ATP-binding cassette superfamily of membrane transport proteins. MRP4 mediates the ATP-dependent efflux of many endogenous and exogenous solutes across the plasma membrane, and in polarized cells, it localizes to the apical or basolateral plasma membrane depending on the tissue type. MRP4 is a 170 kDa glycoprotein and here we show that MRP4 is simultaneously N-glycosylated at Asn746 and Asn754. Furthermore, confocal immunofluorescence studies showed that N-glycans do not affect MRP4's apical membrane localization in polarized LLC-PK1 cells or basolateral membrane localization in polarized MDCKI cells. However, vesicular transport assays showed that N-glycans differentially affect MRP4's ability to transport prostaglandin E2, but not estradiol glucuronide. Together these data indicate that N-glycosylation at Asn746 and Asn754 is not essential for plasma membrane localization of MRP4 but cause substrate-selective effects on its transport activity.

  9. Ketogenic diet delays the phase of circadian rhythms and does not affect AMP-activated protein kinase (AMPK) in mouse liver.

    PubMed

    Genzer, Yoni; Dadon, Maayan; Burg, Chen; Chapnik, Nava; Froy, Oren

    2015-12-05

    Ketogenic diet (KD) is used for weight loss or to treat epilepsy. KD leads to liver AMP-activated protein kinase (AMPK) activation, which would be expected to inhibit gluconeogenesis. However, KD leads to increased hepatic glucose output. As AMPK and its active phosphorylated form (pAMPK) show circadian oscillation, this discrepancy could stem from wrong-time-of-day sampling. The effect of KD was tested on mouse clock gene expression, AMPK, mTOR, SIRT1 and locomotor activity for 2 months and compared to low-fat diet (LFD). KD led to 1.5-fold increased levels of blood glucose and insulin. Brain pAMPK/AMPK ratio was 40% higher under KD, whereas that in liver was not affected. KD led to 40% and 20% down-regulation of the ratio of pP70S6K/P70S6K, the downstream target of mTOR, in the brain and liver, respectively. SIRT1 levels were 40% higher in the brain, but 40% lower in the liver of KD-fed mice. Clock genes showed delayed rhythms under KD. In the brain of KD-fed mice, amplitudes of clock genes were down-regulated, whereas 6-fold up-regulation was found in the liver. The metabolic state under KD indicates reduced satiety in the brain and reduced anabolism alongside increased gluconeogenesis in the liver.

  10. Can Supersaturation Affect Protein Crystal Quality?

    NASA Technical Reports Server (NTRS)

    Gorti, Sridhar

    2013-01-01

    In quiescent environments (microgravity, capillary tubes, gels) formation of a depletion zone is to be expected, due either to limited sedimentation, density driven convection or a combination of both. The formation of a depletion zone can: Modify solution supersaturation near crystal; Give rise to impurity partitioning. It is conjectured that both supersaturation and impurity partitioning affect protein crystal quality and size. Further detailed investigations on various proteins are needed to assess above hypothesis.

  11. A mutation in TRPC6 channels abolishes their activation by hypoosmotic stretch but does not affect activation by diacylglycerol or G protein signaling cascades.

    PubMed

    Wilson, Cory; Dryer, Stuart E

    2014-05-01

    Canonical transient receptor potential-6 (TRPC6) channels have been implicated in the pathogenesis of kidney disease and in the regulation of vascular smooth muscle tone, podocyte function, and a variety of processes in other cell types. The question of whether their gating is intrinsically mechanosensitive has been controversial. In this study we have examined activation of two alleles of TRPC6 transiently expressed in CHO-K1 cells: the wild-type human TRPC6 channel, and TRPC6-N143S, an allele originally identified in a family with autosomal dominant familial focal and segmental glomerulosclerosis (FSGS). We observed that both channel variants carried robust cationic currents that could be evoked by application of membrane-permeable analogs of diacylglycerol (DAG) or by the P2Y receptor agonist ATP. The amplitudes and characteristics of currents evoked by the DAG analog or ATP were indistinguishable in cells expressing the two TRPC6 alleles. By contrast, hypoosmotic stretch evoked robust currents in wild-type TRPC6 channels but had no discernible effect on currents in cells expressing TRPC6-N143S, indicating that the mutant form lacks mechanosensitivity. Coexpression of TRPC6-N143S with wild-type TRPC6 or TRPC3 channels did not alter stretch-evoked responses compared with when TRPC3 channels were expressed by themselves, indicating that TRPC6-N143S does not function as a dominant-negative. These data indicate that mechanical activation and activation evoked by DAG or ATP occur through fundamentally distinct biophysical mechanisms, and they provide support for the hypothesis that protein complexes containing wild-type TRPC6 subunits can be intrinsically mechanosensitive.

  12. Can Solution Supersaturation Affect Protein Crystal Quality?

    NASA Technical Reports Server (NTRS)

    Gorti, Sridhar

    2013-01-01

    The formation of large protein crystals of "high quality" is considered a characteristic manifestation of microgravity. The physical processes that predict the formation of large, high quality protein crystals in the microgravity environment of space are considered rooted in the existence of a "depletion zone" in the vicinity of crystal. Namely, it is considered reasonable that crystal quality suffers in earth-grown crystals as a result of the incorporation of large aggregates, micro-crystals and/or large molecular weight "impurities", processes which are aided by density driven convective flow or mixing at the crystal-liquid interface. Sedimentation and density driven convection produce unfavorable solution conditions in the vicinity of the crystal surface, which promotes rapid crystal growth to the detriment of crystal size and quality. In this effort, we shall further present the hypothesis that the solution supersaturatoin at the crystal surface determines the growth mechanism, or mode, by which protein crystals grow. It is further hypothesized that protein crystal quality is affected by the mechanism or mode of crystal growth. Hence the formation of a depletion zone in microgravity environment is beneficial due to inhibition of impurity incorporatoin as well as preventing a kinetic roughening transition. It should be noted that for many proteins the magnitude of neither protein crystal growth rates nor solution supersaturation are predictors of a kinetic roughening transition. That is, the kinetic roughening transition supersaturation must be dtermined for each individual protein.

  13. Variants in Complement Factor H and Complement Factor H-Related Protein Genes, CFHR3 and CFHR1, Affect Complement Activation in IgA Nephropathy

    PubMed Central

    Zhu, Li; Zhai, Ya-Ling; Wang, Feng-Mei; Hou, Ping; Lv, Ji-Cheng; Xu, Da-Min; Shi, Su-Fang; Liu, Li-Jun; Yu, Feng; Zhao, Ming-Hui; Novak, Jan; Gharavi, Ali G.

    2015-01-01

    Complement activation is common in patients with IgA nephropathy (IgAN) and associated with disease severity. Our recent genome-wide association study of IgAN identified susceptibility loci on 1q32 containing the complement regulatory protein-encoding genes CFH and CFHR1–5, with rs6677604 in CFH as the top single-nucleotide polymorphism and CFHR3–1 deletion (CFHR3–1∆) as the top signal for copy number variation. In this study, to explore the clinical effects of variation in CFH, CFHR3, and CFHR1 on IgAN susceptibility and progression, we enrolled two populations. Group 1 included 1178 subjects with IgAN and available genome-wide association study data. Group 2 included 365 subjects with IgAN and available clinical follow-up data. In group 1, rs6677604 was associated with mesangial C3 deposition by genotype–phenotype correlation analysis. In group 2, we detected a linkage between the rs6677604-A allele and CFHR3–1∆ and found that the rs6677604-A allele was associated with higher serum levels of CFH and lower levels of the complement activation split product C3a. Furthermore, CFH levels were positively associated with circulating C3 levels and negatively associated with mesangial C3 deposition. Moreover, serum levels of the pathogenic galactose-deficient glycoform of IgA1 were also associated with the degree of mesangial C3 deposition in patients with IgAN. Our findings suggest that genetic variants in CFH, CFHR3, and CFHR1 affect complement activation and thereby, predispose patients to develop IgAN. PMID:25205734

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

    PubMed

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

    2009-01-01

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

  15. Regulation of mRNA abundance in activated T lymphocytes: identification of mRNA species affected by the inhibition of protein synthesis.

    PubMed Central

    Coleclough, C; Kuhn, L; Lefkovits, I

    1990-01-01

    Inhibition of protein synthesis has often been observed to increase the concentration of mRNAs that encode proteins associated with the regulation of cell division. As two-dimensional gel electrophoresis permits the simultaneous monitoring of individual elements in large populations of gene products, we have used this technique to assess the effect of cycloheximide treatment on the mRNA complement of activated mouse T cells in an objective fashion. Two-dimensional gels of proteins generated by cell-free translation of mRNA from T-cell blasts display about 400 spots; only 5 of these are reproducibly enhanced by cycloheximide treatment and about 4 are diminished. The cDNA cloning vector lambda jac allows analysis of large arrays of molecular clones by cell-free expression, and we have used it in a sibling selection scheme to isolate a clone of one of the prominently induced mRNA species, which we refer to as chx1. chx1 mRNA concentration is increased by cycloheximide treatment of activated B cells, as well as T cells, and it is rapidly and transiently induced, in a cycloheximide-enhanced manner, upon serum stimulation of resting 3T3 fibroblastoid cells. The chx1 protein is hydrophilic, is slightly basic, and has patches of homology with the Jun-D gene product. The chx1 gene is remarkable in its lack of detectable introns and of strong bias against CpG dinucleotides. Images PMID:2308934

  16. Leucine does not affect mechanistic target of rapamycin complex 1 assembly but is required for maximal ribosomal protein s6 kinase 1 activity in human skeletal muscle following resistance exercise.

    PubMed

    Apró, William; Moberg, Marcus; Hamilton, D Lee; Ekblom, Björn; Rooyackers, Olav; Holmberg, Hans-Christer; Blomstrand, Eva

    2015-10-01

    We examined how the stimulatory effect of leucine on the mechanistic target of rapamycin complex 1 (mTORC1) pathway is affected by the presence of the remaining essential amino acids (EAAs). Nine male subjects performed resistance exercise on 4 occasions and were randomly supplied EAAs with leucine, EAAs without leucine (EAA-Leu), leucine alone, or flavored water (placebo; control). Muscle biopsies were taken from the vastus lateralis before and 60 and 90 min after exercise. Biopsies were analyzed for protein phosphorylation, kinase activity, protein-protein interactions, amino acid concentrations, and tracer incorporation. Leucine alone stimulated ribosomal protein s6 kinase 1 (S6K1) phosphorylation ∼280% more than placebo and EAA-Leu after exercise. Moreover, this response was enhanced by 60-75% after intake of EAAs compared with that of leucine alone (P < 0.05). Kinase activity of S6K1 reflected that of S6K1 phosphorylation; 60 min after exercise, the activity was elevated 3.3- and 4.2-fold with intake of leucine alone and with EAAs, respectively (P < 0.05). The interaction between mammalian target of rapamycin and regulatory-associated protein of mammalian target of rapamycin was unaltered in response to both resistance exercise and amino acid provision. Leucine alone stimulates mTORC1 signaling, although this response is enhanced by other EAAs and does not appear to be caused by alterations in mTORC1 assembly.

  17. The Hydroxyl at Position C1 of Genipin Is the Active Inhibitory Group that Affects Mitochondrial Uncoupling Protein 2 in Panc-1 Cells

    PubMed Central

    Hou, Jianwei; Ding, Yue; Zhang, Tong; Zhang, Yong; Wang, Jianying; Shi, Chenchen; Fu, Wenwei; Cai, Zhenzhen

    2016-01-01

    Genipin (GNP) effectively inhibits uncoupling protein 2 (UCP2), which regulates the leakage of protons across the inner mitochondrial membrane. UCP2 inhibition may induce pancreatic adenocarcinoma cell death by increasing reactive oxygen species (ROS) levels. In this study, the hydroxyls at positions C10 (10-OH) and C1 (1-OH) of GNP were hypothesized to be the active groups that cause these inhibitory effects. Four GNP derivatives in which the hydroxyl at position C10 or C1 was replaced with other chemical groups were synthesized and isolated. Differences in the inhibitory effects of GNP and its four derivatives on pancreatic carcinoma cell (Panc-1) proliferation were assessed. The effects of GNP and its derivatives on apoptosis, UCP2 inhibition and ROS production were also studied to explore the relationship between GNP’s activity and its structure. The derivatives with 1-OH substitutions, geniposide (1-GNP1) and 1-ethyl-genipin (1-GNP2) lacked cytotoxic effects, while the other derivatives that retained 1-OH, 10-piv-genipin (10-GNP1) and 10-acetic acid-genipin (10-GNP2) exerted biological effects similar to those of GNP, even in the absence of 10-OH. Thus, 1-OH is the key functional group in the structure of GNP that is responsible for GNP’s apoptotic effects. These cytotoxic effects involve the induction of Panc-1 cell apoptosis through UCP2 inhibition and subsequent ROS production. PMID:26771380

  18. 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.

  19. Pharmacological inhibition of p38 mitogen-activated protein kinases affects KC/CXCL1-induced intraluminal crawling, transendothelial migration, and chemotaxis of neutrophils in vivo.

    PubMed

    Xu, Najia; Hossain, Mokarram; Liu, Lixin

    2013-01-01

    p38 mitogen-activated protein kinase (MAPK) signalling is critical in the pathophysiology of a variety of inflammatory processes. Leukocyte recruitment to the site of inflammation is a multistep process governed by specific signalling cascades. After adhesion in the lumen, many leukocytes crawl to optimal sites at endothelial junctions and transmigrate to extravascular tissue in a Mac-1-dependent manner. The signalling mechanisms that regulate postadhesion steps of intraluminal crawling, transmigration, and chemotaxis in tissue remain incompletely understood. The present study explored the effect of p38 MAPK inhibitor SB203580 on various parameters of neutrophil recruitment triggered by chemokine KC (CXCL1) gradient. Neutrophil-endothelial interactions in microvasculature of murine cremaster muscle were determined using intravital microscopy and time-lapsed video analysis. SB203580 (100 nM) did not change leukocyte rolling but significantly attenuated neutrophil adhesion, emigration, and transmigration and impaired the initiation of neutrophil crawling and transmigration. In response to KC chemotactic gradient, SB203580 significantly reduced the velocity of migration and chemotaxis index of neutrophils in tissue. The upregulation of Mac-1 expression in neutrophils stimulated by KC was significantly blunted by SB203580 in vitro. Collectively, our findings demonstrate that pharmacological suppression of p38 MAPK significantly impairs multiple steps of neutrophil recruitment in vivo.

  20. [Protein nutrition and physical activity].

    PubMed

    Navarro, M P

    1992-09-01

    The relationship between physical exercise and diet in order to optimize performance is getting growing interest. This review examines protein needs and protein intakes as well as the role of protein in the body and the metabolic changes occurring at the synthesis and catabolic levels during exercise. Protein synthesis in muscle or liver, amino acids oxidation, glucose production via gluconeogenesis from amino acids, etc., are modified, and consequently plasma and urinary nitrogen metabolites are affected. A brief comment on the advantages, disadvantages and forms of different protein supplements for sportsmen is given.

  1. 28 CFR 55.15 - Affected activities.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... RIGHTS ACT REGARDING LANGUAGE MINORITY GROUPS Minority Language Materials and Assistance § 55.15 Affected... of applicable language minority groups to be effectively informed of and participate effectively in voting-connected activities. Accordingly, the quoted language should be broadly construed to apply to...

  2. 28 CFR 55.15 - Affected activities.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... RIGHTS ACT REGARDING LANGUAGE MINORITY GROUPS Minority Language Materials and Assistance § 55.15 Affected... of applicable language minority groups to be effectively informed of and participate effectively in voting-connected activities. Accordingly, the quoted language should be broadly construed to apply to...

  3. 28 CFR 55.15 - Affected activities.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... RIGHTS ACT REGARDING LANGUAGE MINORITY GROUPS Minority Language Materials and Assistance § 55.15 Affected... of applicable language minority groups to be effectively informed of and participate effectively in voting-connected activities. Accordingly, the quoted language should be broadly construed to apply to...

  4. 28 CFR 55.15 - Affected activities.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... RIGHTS ACT REGARDING LANGUAGE MINORITY GROUPS Minority Language Materials and Assistance § 55.15 Affected... of applicable language minority groups to be effectively informed of and participate effectively in voting-connected activities. Accordingly, the quoted language should be broadly construed to apply to...

  5. 28 CFR 55.15 - Affected activities.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... RIGHTS ACT REGARDING LANGUAGE MINORITY GROUPS Minority Language Materials and Assistance § 55.15 Affected... of applicable language minority groups to be effectively informed of and participate effectively in voting-connected activities. Accordingly, the quoted language should be broadly construed to apply to...

  6. 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.

  7. Do recreational activities affect coastal biodiversity?

    NASA Astrophysics Data System (ADS)

    Riera, Rodrigo; Menci, Cristiano; Sanabria-Fernández, José Antonio; Becerro, Mikel A.

    2016-09-01

    Human activities are largely affecting coastal communities worldwide. Recreational perturbations have been overlooked in comparison to other perturbations, yet they are potential threats to marine biodiversity. They affect coastal communities in different ways, underpinning consistent shifts in fish and invertebrates assemblages. Several sites were sampled subjected to varying effects by recreational fishermen (low and high pressure) and scuba divers (low and high) in an overpopulated Atlantic island. Non-consistent differences in ecological, trophic and functional diversity were found in coastal communities, considering both factors ("diving" and "fishing"). Multivariate analyses only showed significant differences in benthic invertebrates between intensively-dived and non-dived sites. The lack of clear trends may be explained by the depletion of coastal resources in the study area, an extensively-affected island by overfishing.

  8. Solar activity affects avian timing of reproduction

    PubMed Central

    Visser, Marcel E.; Sanz, Juan José

    2009-01-01

    Avian timing of reproduction is strongly affected by ambient temperature. Here we show that there is an additional effect of sunspots on laying date, from five long-term population studies of great and blue tits (Parus major and Cyanistes caeruleus), demonstrating for the first time that solar activity not only has an effect on population numbers but that it also affects the timing of animal behaviour. This effect is statistically independent of ambient temperature. In years with few sunspots, birds initiate laying late while they are often early in years with many sunspots. The sunspot effect may be owing to a crucial difference between the method of temperature measurements by meteorological stations (in the shade) and the temperatures experienced by the birds. A better understanding of the impact of all the thermal components of weather on the phenology of ecosystems is essential when predicting their responses to climate change. PMID:19574283

  9. Mutations affecting enzymatic activity in liver arginase

    SciTech Connect

    Vockley, J.G.; Tabor, D.E.; Goodman, B.K.

    1994-09-01

    The hydrolysis of arginine to ornithine and urea is catalyzed by arginase in the last step of the urea cycle. We examined a group of arginase deficient patients by PCR-SSCP analysis to characterize the molecular basis of this disorder. A heterogeneous population of nonsense mutations, microdeletions, and missense mutations has been identified in our cohort. Microdeletions which introduce premature stop codons downstream of the deletion and nonsense mutations result in no arginase activity. These mutations occur randomly along the gene. The majority of missense mutations identified appear to occur in regions of high cross-species homology. To test the effect of these missense mutations on arginase activity, site-directed mutagenesis was used to re-create the patient mutations for in vivo expression studies in a prokaryotic fusion-protein expression system. Of 4 different missense mutations identified in 6 individuals, only one was located outside of a conserved region. The three substitution mutations within the conserved regions had a significant effect on enzymatic activity (0-3.1 nmole/30min, normal is 1300-1400 nmoles/30min, as determined by in vitro arginase assay), while the fourth mutation, a T to S substitution, did not. In addition, site-directed mutagenesis was utilized to create mutations not in residues postulated to play a significant role in the enzymatic function or active site formation in manganese-binding proteins such as arginase. We have determined that the substitution of glycine for a histidine residue, located in a very highly conserved region of exon 3, and the substitution of a histidine and an aspartic acid residue within a similarly conserved region in exon 4, totally abolishes enzymatic activity. Mutations substituting glycine for an additional histidine and aspartic acid residue in exon 4 and two aspartic acid residues in exon 7 have also been created. We are currently in the process of characterizing these mutations.

  10. Role of AMP-activated protein kinase activators in antiproliferative multi-drug pituitary tumour therapies: effects of combined treatments with compounds affecting the mTOR-p70S6 kinase axis in cultured pituitary tumour cells.

    PubMed

    Tulipano, G; Faggi, L; Cacciamali, A; Spinello, M; Cocchi, D; Giustina, A

    2015-01-01

    AMP-activated protein kinase (AMPK) is activated under conditions that deplete cellular ATP levels and elevate AMP levels. We have recently shown that AMPK can represent a valid target for improving the medical treatment of growth hormone (GH)-secreting pituitary adenomas and the effects of its activation or inhibition in pituitary tumour cells are worthy of further characterisation. We aimed to determine whether AMPK may have a role in combined antiproliferative therapies based on multiple drugs targeting cell anabolic functions at different levels in pituitary tumour cells to overcome the risk of cell growth escape phenomena. Accordingly, we tried to determine whether a rationale exists in combining compounds activating AMPK with compounds targeting the phosphatidylinositol-3-kinase (PI3K)/Akt/mTOR/p70S6K signalling pathway. AMPK down-regulation by specific small-interfering RNAs confirmed that activated AMPK had a role in restraining growth of GH3 cells. Hence, we compared the effects of compounds directly targeting the mTOR-p70S6K axis, namely the mTOR inhibitor rapamycin and the p70S6K inhibitor PF-4708671, with the effects of the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) on cell signalling and cell growth, in rat pituitary GH3 cells. AICAR was able to reduce growth factor-induced p70S6K activity, as shown by the decrease of phospho-p70S6K levels. However, it was far less effective than rapamycin and PF-4708671. We observed significant differences between the growth inhibitory effects of the three compounds in GH3 and GH1 cells. Interestingly, PF-4708671 was devoid of any effect. AICAR was at least as effective as rapamycin and the co-treatment was more effective than single treatments. AICAR induced apoptosis of GH3 cells, whereas rapamycin caused preferentially a decrease of cell proliferation. Finally, AICAR and rapamycin differed in their actions on growth factor-induced extracellular signal regulated kinase 1/2 phosphorylation

  11. Oxalomalate affects the inducible nitric oxide synthase expression and activity.

    PubMed

    Irace, Carlo; Esposito, Giuseppe; Maffettone, Carmen; Rossi, Antonietta; Festa, Michela; Iuvone, Teresa; Santamaria, Rita; Sautebin, Lidia; Carnuccio, Rosa; Colonna, Alfredo

    2007-03-13

    Inducible nitric oxide synthase (iNOS) is an homodimeric enzyme which produces large amounts of nitric oxide (NO) in response to inflammatory stimuli. Several factors affect the synthesis and catalytic activity of iNOS. Particularly, dimerization of NOS monomers is promoted by heme, whereas an intracellular depletion of heme and/or L-arginine considerably decreases NOS resistance to proteolysis. In this study, we found that oxalomalate (OMA, oxalomalic acid, alpha-hydroxy-beta-oxalosuccinic acid), an inhibitor of both aconitase and NADP-dependent isocitrate dehydrogenase, inhibited nitrite production and iNOS protein expression in lipopolysaccharide (LPS)-activated J774 macrophages, without affecting iNOS mRNA content. Furthermore, injection of OMA precursors to LPS-stimulated rats also decreased nitrite production and iNOS expression in isolated peritoneal macrophages. Interestingly, alpha-ketoglutarate or succinyl-CoA administration reversed OMA effect on NO production, thus correlating NO biosynthesis with the anabolic capacity of Krebs cycle. When protein synthesis was blocked by cycloheximide in LPS-activated J774 cells treated with OMA, iNOS protein levels, evaluated by Western blot analysis and (35)S-metabolic labelling, were decreased, suggesting that OMA reduces iNOS biosynthesis and induces an increase in the degradation rate of iNOS protein. Moreover, we showed that OMA inhibits the activity of the iNOS from lung of LPS-treated rats by enzymatic assay. Our results, demonstrating that OMA acts regulating synthesis, catalytic activity and degradation of iNOS, suggest that this compound might have a potential role in reducing the NO overproduction occurring in some pathological conditions.

  12. Temperature and dietary starch level affected protein but not starch digestibility in gilthead sea bream juveniles.

    PubMed

    Couto, A; Enes, P; Peres, H; Oliva-Teles, A

    2012-06-01

    A study was carried out with gilthead sea bream juveniles to assess the effect of water temperature (18 and 25°C) and dietary pregelatinized starch level (10, 20 and 30%) on digestibility of protein and starch and on the activity of proteolytic and amylolytic enzymes. ADC of pregelatinized starch was very high (>99%) irrespectively of dietary inclusion level, and it was not affected by water temperature. ADC of protein was also high (>90%) but improved at the higher water temperature. Dietary starch interacted with protein digestibility, which decreased as dietary starch level increased. Temperature affected both acid and basic protease activities, with acid protease activity being higher at 25°C and basic protease activity being higher at 18°C. However, total proteolytic activity and amylase activities were not affected by water temperature. Dietary carbohydrate exerted no effect on proteolytic or amylolitic activities. It is concluded that gilthead sea bream juveniles digest pregelatinized starch very efficiently irrespective of water temperature, due to adjustments of amylase activity to cope with temperature differences. Pregelatinized starch interacts negatively with protein digestibility, with the ADC of protein decreasing as dietary starch levels increase.

  13. Nitrogen Assimilation and Protein Synthesis in Wheat Seedlings as Affected by Mineral Nutrition. II. Micronutrients 1

    PubMed Central

    Harper, James E.; Paulsen, Gary M.

    1969-01-01

    Activity of nitrate reductase from Triticum aestivum L. seedlings was decreased by deficiencies of molybdenum, zinc, and chlorine. Nitrate accumulated in molybdenum-deficient seedlings, declined in zinc-deficient seedlings, and was unaffected by the other micronutrient treatments. Glutamic acid dehydrogenase activity was decreased by deficiency of molybdenum, the only nutrient that affected the enzyme. Glutamine synthetase activity was decreased only by copper deficiency, and glutamic-oxaloacetic transaminase was not affected by any micronutrient deficiencies. Incorporation of 14C-leucine into protein by wheat seedlings was increased by molybdenum deficiency, apparently because of decreased inhibition from endogenous amino acids, and was decreased by copper deficiency. Protein content was not affected significantly by the micronutrient treatments. PMID:16657114

  14. Degradation of Activated Protein Kinases by Ubiquitination

    PubMed Central

    Lu, Zhimin; Hunter, Tony

    2009-01-01

    Protein kinases are important regulators of intracellular signal transduction pathways and play critical roles in diverse cellular functions. Once a protein kinase is activated, its activity is subsequently downregulated through a variety of mechanisms. Accumulating evidence indicates that the activation of protein kinases commonly initiates their downregulation via the ubiquitin/proteasome pathway. Failure to regulate protein kinase activity or expression levels can cause human diseases. PMID:19489726

  15. Plant Protein and Animal Proteins: Do They Differentially Affect Cardiovascular Disease Risk?12

    PubMed Central

    Richter, Chesney K; Skulas-Ray, Ann C; Champagne, Catherine M; Kris-Etherton, Penny M

    2015-01-01

    Proteins from plant-based compared with animal-based food sources may have different effects on cardiovascular disease (CVD) risk factors. Numerous epidemiologic and intervention studies have evaluated their respective health benefits; however, it is difficult to isolate the role of plant or animal protein on CVD risk. This review evaluates the current evidence from observational and intervention studies, focusing on the specific protein-providing foods and populations studied. Dietary protein is derived from many food sources, and each provides a different composite of nonprotein compounds that can also affect CVD risk factors. Increasing the consumption of protein-rich foods also typically results in lower intakes of other nutrients, which may simultaneously influence outcomes. Given these complexities, blanket statements about plant or animal protein may be too general, and greater consideration of the specific protein food sources and the background diet is required. The potential mechanisms responsible for any specific effects of plant and animal protein are similarly multifaceted and include the amino acid content of particular foods, contributions from other nonprotein compounds provided concomitantly by the whole food, and interactions with the gut microbiome. Evidence to date is inconclusive, and additional studies are needed to further advance our understanding of the complexity of plant protein vs. animal protein comparisons. Nonetheless, current evidence supports the idea that CVD risk can be reduced by a dietary pattern that provides more plant sources of protein compared with the typical American diet and also includes animal-based protein foods that are unprocessed and low in saturated fat. PMID:26567196

  16. Plant protein and animal proteins: do they differentially affect cardiovascular disease risk?

    PubMed

    Richter, Chesney K; Skulas-Ray, Ann C; Champagne, Catherine M; Kris-Etherton, Penny M

    2015-11-01

    Proteins from plant-based compared with animal-based food sources may have different effects on cardiovascular disease (CVD) risk factors. Numerous epidemiologic and intervention studies have evaluated their respective health benefits; however, it is difficult to isolate the role of plant or animal protein on CVD risk. This review evaluates the current evidence from observational and intervention studies, focusing on the specific protein-providing foods and populations studied. Dietary protein is derived from many food sources, and each provides a different composite of nonprotein compounds that can also affect CVD risk factors. Increasing the consumption of protein-rich foods also typically results in lower intakes of other nutrients, which may simultaneously influence outcomes. Given these complexities, blanket statements about plant or animal protein may be too general, and greater consideration of the specific protein food sources and the background diet is required. The potential mechanisms responsible for any specific effects of plant and animal protein are similarly multifaceted and include the amino acid content of particular foods, contributions from other nonprotein compounds provided concomitantly by the whole food, and interactions with the gut microbiome. Evidence to date is inconclusive, and additional studies are needed to further advance our understanding of the complexity of plant protein vs. animal protein comparisons. Nonetheless, current evidence supports the idea that CVD risk can be reduced by a dietary pattern that provides more plant sources of protein compared with the typical American diet and also includes animal-based protein foods that are unprocessed and low in saturated fat.

  17. New functional assays to selectively quantify the activated protein C- and tissue factor pathway inhibitor-cofactor activities of protein S in plasma.

    PubMed

    Alshaikh, N A; Rosing, J; Thomassen, M C L G D; Castoldi, E; Simioni, P; Hackeng, T M

    2017-02-17

    Essentials Protein S is a cofactor of activated protein C (APC) and tissue factor pathway inhibitor (TFPI). There are no assays to quantify separate APC and TFPI cofactor activities of protein S in plasma. We developed assays to measure the APC- and TFPI-cofactor activities of protein S in plasma. The assays were sensitive to protein S deficiency, and not affected by the Factor V Leiden mutation.

  18. Do Non-Collagenous Proteins Affect Skeletal Mechanical Properties?

    PubMed Central

    Morgan, Stacyann; Poundarik, Atharva A.; Vashishth, Deepak

    2015-01-01

    The remarkable mechanical behavior of bone is attributed to its complex nanocomposite structure that, in addition to mineral and collagen, comprises a variety of non-collagenous matrix proteins or NCPs. Traditionally, NCPs have been studied as signaling molecules in biological processes including bone formation, resorption and turnover. Limited attention has been given to their role in determining the mechanical properties of bone. Recent studies have highlighted that NCPs can indeed be lost or modified with aging, diseases and drug therapies. Homozygous and heterozygous mice models of key NCP provide a useful approach to determine the impact of NCPs on bone morphology as well as matrix quality, and to carry out detailed mechanical analysis for elucidating the pathway by which NCPs can affect the mechanical properties of bone. In this article, we present a systematic analysis of a large cohort of NCPs on bone’s structural and material hierarchy, and identify three principal pathways by which they determine bone’s mechanical properties. These pathways include alterations of bone morphological parameters crucial for bone’s structural competency, bone quality changes in key matrix parameters (mineral and collagen), and a direct role as load bearing structural proteins. PMID:26048282

  19. Lengths of Orthologous Prokaryotic Proteins Are Affected by Evolutionary Factors

    PubMed Central

    Tatarinova, Tatiana; Dien Bard, Jennifer; Cohen, Irit

    2015-01-01

    Proteins of the same functional family (for example, kinases) may have significantly different lengths. It is an open question whether such variation in length is random or it appears as a response to some unknown evolutionary driving factors. The main purpose of this paper is to demonstrate existence of factors affecting prokaryotic gene lengths. We believe that the ranking of genomes according to lengths of their genes, followed by the calculation of coefficients of association between genome rank and genome property, is a reasonable approach in revealing such evolutionary driving factors. As we demonstrated earlier, our chosen approach, Bubble-sort, combines stability, accuracy, and computational efficiency as compared to other ranking methods. Application of Bubble Sort to the set of 1390 prokaryotic genomes confirmed that genes of Archaeal species are generally shorter than Bacterial ones. We observed that gene lengths are affected by various factors: within each domain, different phyla have preferences for short or long genes; thermophiles tend to have shorter genes than the soil-dwellers; halophiles tend to have longer genes. We also found that species with overrepresentation of cytosines and guanines in the third position of the codon (GC3 content) tend to have longer genes than species with low GC3 content. PMID:26114113

  20. Lengths of Orthologous Prokaryotic Proteins Are Affected by Evolutionary Factors.

    PubMed

    Tatarinova, Tatiana; Salih, Bilal; Dien Bard, Jennifer; Cohen, Irit; Bolshoy, Alexander

    2015-01-01

    Proteins of the same functional family (for example, kinases) may have significantly different lengths. It is an open question whether such variation in length is random or it appears as a response to some unknown evolutionary driving factors. The main purpose of this paper is to demonstrate existence of factors affecting prokaryotic gene lengths. We believe that the ranking of genomes according to lengths of their genes, followed by the calculation of coefficients of association between genome rank and genome property, is a reasonable approach in revealing such evolutionary driving factors. As we demonstrated earlier, our chosen approach, Bubble-sort, combines stability, accuracy, and computational efficiency as compared to other ranking methods. Application of Bubble Sort to the set of 1390 prokaryotic genomes confirmed that genes of Archaeal species are generally shorter than Bacterial ones. We observed that gene lengths are affected by various factors: within each domain, different phyla have preferences for short or long genes; thermophiles tend to have shorter genes than the soil-dwellers; halophiles tend to have longer genes. We also found that species with overrepresentation of cytosines and guanines in the third position of the codon (GC3 content) tend to have longer genes than species with low GC3 content.

  1. DNA affects the composition of lipoplex protein corona: a proteomics approach.

    PubMed

    Capriotti, Anna L; Caracciolo, Giulio; Caruso, Giuseppe; Foglia, Patrizia; Pozzi, Daniela; Samperi, Roberto; Laganà, Aldo

    2011-08-01

    The distribution of drug delivery systems into the body is affected by plasma proteins adsorbed onto their surface. Furthermore, an exact understanding of the structure and morphology of drug carriers is fundamental to understand their role as gene delivery systems. In this work, the adsorption of human plasma proteins bound to cationic liposomes and to their relative DNA lipoplexes was compared. A shotgun proteomics approach based on HPLC coupled to high resolution MS was used for an efficient identification of proteins adsorbed onto liposome and lipoplex surfaces. The distinct pattern of proteins adsorbed helps to better understand the DNA compaction process. The experimental evidence leads us to hypothesize that polyanionic DNA is associated to the lipoplex surface and can interact with basic plasma proteins. Such a finding is in agreement with recent results showing that lipoplexes are multilamellar DNA/lipid domains partially decorated with DNA at their surface. Proteomics experiments showed that the lipoplex corona is rich of biologically relevant proteins such as fibronectin, histones and complement proteins. Our results provide novel insights to understand how lipoplexes activate the immune system and why they are rapidly cleared from the blood stream. The differences in the protein adsorption data detected in the presented experiments could be the basis for the establishment of a correlation between protein adsorption pattern and in vivo fate of intravenously administered nanoparticles and will require some consideration in the future.

  2. Biologically active extracts with kidney affections applications

    NASA Astrophysics Data System (ADS)

    Pascu (Neagu), Mihaela; Pascu, Daniela-Elena; Cozea, Andreea; Bunaciu, Andrei A.; Miron, Alexandra Raluca; Nechifor, Cristina Aurelia

    2015-12-01

    This paper is aimed to select plant materials rich in bioflavonoid compounds, made from herbs known for their application performances in the prevention and therapy of renal diseases, namely kidney stones and urinary infections (renal lithiasis, nephritis, urethritis, cystitis, etc.). This paper presents a comparative study of the medicinal plant extracts composition belonging to Ericaceae-Cranberry (fruit and leaves) - Vaccinium vitis-idaea L. and Bilberry (fruit) - Vaccinium myrtillus L. Concentrated extracts obtained from medicinal plants used in this work were analyzed from structural, morphological and compositional points of view using different techniques: chromatographic methods (HPLC), scanning electronic microscopy, infrared, and UV spectrophotometry, also by using kinetic model. Liquid chromatography was able to identify the specific compounds of the Ericaceae family, present in all three extracts, arbutosid, as well as specific components of each species, mostly from the class of polyphenols. The identification and quantitative determination of the active ingredients from these extracts can give information related to their therapeutic effects.

  3. Human carotid atherosclerotic plaque protein(s) change HDL protein(s) composition and impair HDL anti-oxidant activity.

    PubMed

    Cohen, Elad; Aviram, Michael; Khatib, Soliman; Volkova, Nina; Vaya, Jacob

    2016-01-01

    High density lipoprotein (HDL) anti-atherogenic functions are closely associated with cardiovascular disease risk factor, and are dictated by its composition, which is often affected by environmental factors. The present study investigates the effects of the human carotid plaque constituents on HDL composition and biological functions. To this end, human carotid plaques were homogenized and incubated with HDL. Results showed that after incubation, most of the apolipoprotein A1 (Apo A1) protein was released from the HDL, and HDL diameter increased by an average of approximately 2 nm. In parallel, HDL antioxidant activity was impaired. In response to homogenate treatment HDL could not prevent the accelerated oxidation of LDL caused by the homogenate. Boiling of the homogenate prior to its incubation with HDL abolished its effects on HDL composition changes. Moreover, tryptophan fluorescence quenching assay revealed an interaction between plaque component(s) and HDL, an interaction that was reduced by 50% upon using pre-boiled homogenate. These results led to hypothesize that plaque protein(s) interacted with HDL-associated Apo A1 and altered the HDL composition. Immuno-precipitation of Apo A1 that was released from the HDL after its incubation with the homogenate revealed a co-precipitation of three isomers of actin. However, beta-actin alone did not significantly affect the HDL composition, and yet the active protein within the plaque was elusive. In conclusion then, protein(s) in the homogenate interact with HDL protein(s), leading to release of Apo A1 from the HDL particle, a process that was associated with an increase in HDL diameter and with impaired HDL anti-oxidant activity.

  4. Single site mutations in the hetero-oligomeric Mrp antiporter from alkaliphilic Bacillus pseudofirmus OF4 that affect Na+/H+ antiport activity, sodium exclusion, individual Mrp protein levels, or Mrp complex formation.

    PubMed

    Morino, Masato; Natsui, Shinsuke; Ono, Tomohiro; Swartz, Talia H; Krulwich, Terry A; Ito, Masahiro

    2010-10-01

    Mrp systems are widely distributed and structurally complex cation/proton antiporters. Antiport activity requires hetero-oligomeric complexes of all six or seven hydrophobic Mrp proteins (MrpA-MrpG). Here, a panel of site-directed mutants in conserved or proposed motif residues was made in the Mrp Na(+)(Li(+))/H(+) antiporter from an alkaliphilic Bacillus. The mutant operons were expressed in antiporter-deficient Escherichia coli KNabc and assessed for antiport properties, support of sodium resistance, membrane levels of each Mrp protein, and presence of monomeric and dimeric Mrp complexes. Antiport did not depend on a VFF motif or a conserved tyrosine pair, but a role for a conserved histidine in a potential quinone binding site of MrpA was supported. The importance of several acidic residues for antiport was confirmed, and the importance of additional residues was demonstrated (e.g. three lysine residues conserved across MrpA, MrpD, and membrane-bound respiratory Complex I subunits (NuoL/M/N)). The results extended indications that MrpE is required for normal membrane levels of other Mrp proteins and for complex formation. Moreover, mutations in several other Mrp proteins lead to greatly reduced membrane levels of MrpE. Thus, changes in either of the two Mrp modules, MrpA-MrpD and MrpE-MrpG, influence the other. Two mutants, MrpB-P37G and MrpC-Q70A, showed a normal phenotype but lacked the MrpA-MrpG monomeric complex while retaining the dimeric hetero-oligomeric complex. Finally, MrpG-P81A and MrpG-P81G mutants exhibited no antiport activity but supported sodium resistance and a low [Na(+)](in). Such mutants could be used to screen hypothesized but uncharacterized sodium efflux functions of Mrp apart from Na(+) (Li(+))/H(+) antiport.

  5. Oligomeric state affects oxygen dissociation and diguanylate cyclase activity of globin coupled sensors.

    PubMed

    Burns, Justin L; Deer, D Douglas; Weinert, Emily E

    2014-11-01

    Bacterial biofilm formation is regulated by enzymes, such as diguanylate cyclases, that respond to environmental signals and alter c-di-GMP levels. Diguanylate cyclase activity of two globin coupled sensors is shown to be regulated by gaseous ligands, with cyclase activity and O2 dissociation affected by protein oligomeric state.

  6. Axonal localization of Ca2+-dependent activator protein for secretion 2 is critical for subcellular locality of brain-derived neurotrophic factor and neurotrophin-3 release affecting proper development of postnatal mouse cerebellum.

    PubMed

    Sadakata, Tetsushi; Kakegawa, Wataru; Shinoda, Yo; Hosono, Mayu; Katoh-Semba, Ritsuko; Sekine, Yukiko; Sato, Yumi; Saruta, Chihiro; Ishizaki, Yasuki; Yuzaki, Michisuke; Kojima, Masami; Furuichi, Teiichi

    2014-01-01

    Ca2+-dependent activator protein for secretion 2 (CAPS2) is a protein that is essential for enhanced release of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) from cerebellar granule cells. We previously identified dex3, a rare alternative splice variant of CAPS2, which is overrepresented in patients with autism and is missing an exon 3 critical for axonal localization. We recently reported that a mouse model CAPS2Δex3/Δex3 expressing dex3 showed autistic-like behavioral phenotypes including impaired social interaction and cognition and increased anxiety in an unfamiliar environment. Here, we verified impairment in axonal, but not somato-dendritic, localization of dex3 protein in cerebellar granule cells and demonstrated cellular and physiological phenotypes in postnatal cerebellum of CAPS2Δex3/Δex3 mice. Interestingly, both BDNF and NT-3 were markedly reduced in axons of cerebellar granule cells, resulting in a significant decrease in their release. As a result, dex3 mice showed developmental deficits in dendritic arborization of Purkinje cells, vermian lobulation and fissurization, and granule cell precursor proliferation. Paired-pulse facilitation at parallel fiber-Purkinje cell synapses was also impaired. Together, our results indicate that CAPS2 plays an important role in subcellular locality (axonal vs. somato-dendritic) of enhanced BDNF and NT-3 release, which is indispensable for proper development of postnatal cerebellum.

  7. An Arabidopsis Stomatin-Like Protein Affects Mitochondrial Respiratory Supercomplex Organization1[C][W][OPEN

    PubMed Central

    Gehl, Bernadette; Lee, Chun Pong; Bota, Pedro; Blatt, Michael R.; Sweetlove, Lee J.

    2014-01-01

    Stomatins belong to the band-7 protein family, a diverse group of conserved eukaryotic and prokaryotic membrane proteins involved in the formation of large protein complexes as protein-lipid scaffolds. The Arabidopsis (Arabidopsis thaliana) genome contains two paralogous genes encoding stomatin-like proteins (SLPs; AtSLP1 and AtSLP2) that are phylogenetically related to human SLP2, a protein involved in mitochondrial fusion and protein complex formation in the mitochondrial inner membrane. We used reverse genetics in combination with biochemical methods to investigate the function of AtSLPs. We demonstrate that both SLPs localize to mitochondrial membranes. SLP1 migrates as a large (approximately 3 MDa) complex in blue-native gel electrophoresis. Remarkably, slp1 knockout mutants have reduced protein and activity levels of complex I and supercomplexes, indicating that SLP affects the assembly and/or stability of these complexes. These findings point to a role for SLP1 in the organization of respiratory supercomplexes in Arabidopsis. PMID:24424325

  8. Lumenal protein within secretory granules affects fusion pore expansion.

    PubMed

    Weiss, Annita Ngatchou; Anantharam, Arun; Bittner, Mary A; Axelrod, Daniel; Holz, Ronald W

    2014-07-01

    It is often assumed that upon fusion of the secretory granule membrane with the plasma membrane, lumenal contents are rapidly discharged and dispersed into the extracellular medium. Although this is the case for low-molecular-weight neurotransmitters and some proteins, there are numerous examples of the dispersal of a protein being delayed for many seconds after fusion. We have investigated the role of fusion-pore expansion in determining the contrasting discharge rates of fluorescent-tagged neuropeptide-Y (NPY) (within 200 ms) and tissue plasminogen activator (tPA) (over many seconds) in adrenal chromaffin cells. The endogenous proteins are expressed in separate chromaffin cell subpopulations. Fusion pore expansion was measured by two independent methods, orientation of a fluorescent probe within the plasma membrane using polarized total internal reflection fluorescence microscopy and amperometry of released catecholamine. Together, they probe the continuum of the fusion-pore duration, from milliseconds to many seconds after fusion. Polarized total internal reflection fluorescence microscopy revealed that 71% of the fusion events of tPA-cer-containing granules maintained curvature for >10 s, with approximately half of the structures likely connected to the plasma membrane by a short narrow neck. Such events were not commonly observed upon fusion of NPY-cer-containing granules. Amperometry revealed that the expression of tPA-green fluorescent protein (GFP) prolonged the duration of the prespike foot ∼2.5-fold compared to NPY-GFP-expressing cells and nontransfected cells, indicating that expansion of the initial fusion pore in tPA granules was delayed. The t1/2 of the main catecholamine spike was also increased, consistent with a prolonged delay of fusion-pore expansion. tPA added extracellularly bound to the lumenal surface of fused granules. We propose that tPA within the granule lumen controls its own discharge. Its intrinsic biochemistry determines not only

  9. Biologically active proteins from natural product extracts.

    PubMed

    O'Keefe, B R

    2001-10-01

    The term "biologically active proteins" is almost redundant. All proteins produced by living creatures are, by their very nature, biologically active to some extent in their homologous species. In this review, a subset of these proteins will be discussed that are biologically active in heterologous systems. The isolation and characterization of novel proteins from natural product extracts including those derived from microorganisms, plants, insects, terrestrial vertebrates, and marine organisms will be reviewed and grouped into several distinct classes based on their biological activity and their structure.

  10. Rational Design of Protein C Activators

    PubMed Central

    Barranco-Medina, Sergio; Murphy, Mary; Pelc, Leslie; Chen, Zhiwei; Di Cera, Enrico; Pozzi, Nicola

    2017-01-01

    In addition to its procoagulant and proinflammatory functions mediated by cleavage of fibrinogen and PAR1, the trypsin-like protease thrombin activates the anticoagulant protein C in a reaction that requires the cofactor thrombomodulin and the endothelial protein C receptor. Once in the circulation, activated protein C functions as an anticoagulant, anti-inflammatory and regenerative factor. Hence, availability of a protein C activator would afford a therapeutic for patients suffering from thrombotic disorders and a diagnostic tool for monitoring the level of protein C in plasma. Here, we present a fusion protein where thrombin and the EGF456 domain of thrombomodulin are connected through a peptide linker. The fusion protein recapitulates the functional and structural properties of the thrombin-thrombomodulin complex, prolongs the clotting time by generating pharmacological quantities of activated protein C and effectively diagnoses protein C deficiency in human plasma. Notably, these functions do not require exogenous thrombomodulin, unlike other anticoagulant thrombin derivatives engineered to date. These features make the fusion protein an innovative step toward the development of protein C activators of clinical and diagnostic relevance. PMID:28294177

  11. Dietary protein source affects lipid metabolism in the European seabass (Dicentrarchus labrax).

    PubMed

    Dias, J; Alvarez, M J; Arzel, J; Corraze, G; Diez, A; Bautista, J M; Kaushik, S J

    2005-09-01

    The study was undertaken to evaluate the effects of dietary protein sources on lipogenesis and fat deposition in a marine teleost, the European seabass (Dicentrarchus labrax). Four isonitrogenous (crude protein (CP, Nx6.25), 44% DM) and isoenergetic (22-23 kJ/g DM) diets were formulated to contain one of the following as the major protein source: fish meal (FM), one of two soy protein concentrates (SPC) and corn gluten meal (CGM). Apparent digestibility coefficients of the diets and raw ingredients, as well as soluble nitrogen (ammonia and urea) and phosphorus excretion were measured. Growth rates of seabass fed plant protein-based diets were significantly lower than those fed fish meal based diet. The protein utilisation was strongly correlated to the dietary essential amino acids index. Measurements of N excretion (ammonia and urea nitrogen) confirmed these data. Daily fat gain at the whole body level ranged between 1.1 to 1.7 g/kg BW, with the highest values being recorded in fish fed the fish meal based diet. Levels of plasma triglycerides and cholesterol were lower in fish fed soy protein diets than in those fed the diet solely based on fish meal. Soy protein rich diets decreased the activities of selected hepatic lipogenic enzymes (glucose 6-phosphate dehydrogenase, malic enzyme, ATP-citrate lysase, acetylcoenzyme A carboxylase and fatty acid synthetase). Highest lipogenic enzyme activities where found in fish fed the fish meal diet, except for fatty acid synthetase which was increased in seabass fed the corn-gluten meal based diets. Overall data suggest that dietary protein sources affects fat deposition and the lipogenic potential in European seabass.

  12. Cocaine affects the dynamics of cytoskeletal proteins via sigma(1) receptors.

    PubMed

    Su, T P; Hayashi, T

    2001-09-01

    Cytoskeletal proteins are important in protein trafficking, membrane protein clustering, dendrite growth and the morphological maintenance of neurons. Sigma(1) receptors are unique endoplasmic reticular (ER) proteins that bind (+)benzomorphans, neurosteroids and psychotropic drugs such as cocaine. Cocaine, via sigma(1) receptors, can cause the dissociation of a cytoskeletal adaptor protein ankyrin from inositol (1,4,5)-trisphosphate [Ins(1,4,5)P(3)] receptors on the ER as a sigma(1)-receptor-ankyrin complex, which then translocates to the plasma membrane and nucleus. The dissociation of sigma(1)-receptor-ankyrin from Ins(1,4,5)P(3) receptors also increases the intracellular Ca(2+) concentration [[Ca(2+)](i)], which affects the activity of cytoskeletal proteins. Furthermore, cocaine might increase [Ca(2+)](i) via phospholipase C (PLC)-linked dopamine D1 receptors. We hypothesize that cocaine might cause life-long changes in neurons via cytoskeletal proteins by interacting with both D1 receptors and sigma(1) receptors.

  13. Amyloid precursor protein (APP) affects global protein synthesis in dividing human cells.

    PubMed

    Sobol, Anna; Galluzzo, Paola; Liang, Shuang; Rambo, Brittany; Skucha, Sylvia; Weber, Megan J; Alani, Sara; Bocchetta, Maurizio

    2015-05-01

    Hypoxic non-small cell lung cancer (NSCLC) is dependent on Notch-1 signaling for survival. Targeting Notch-1 by means of γ-secretase inhibitors (GSI) proved effective in killing hypoxic NSCLC. Post-mortem analysis of GSI-treated, NSCLC-burdened mice suggested enhanced phosphorylation of 4E-BP1 at threonines 37/46 in hypoxic tumor tissues. In vitro dissection of this phenomenon revealed that Amyloid Precursor Protein (APP) inhibition was responsible for a non-canonical 4E-BP1 phosphorylation pattern rearrangement-a process, in part, mediated by APP regulation of the pseudophosphatase Styx. Upon APP depletion we observed modifications of eIF-4F composition indicating increased recruitment of eIF-4A to the mRNA cap. This phenomenon was supported by the observation that cells with depleted APP were partially resistant to silvestrol, an antibiotic that interferes with eIF-4A assembly into eIF-4F complexes. APP downregulation in dividing human cells increased the rate of global protein synthesis, both cap- and IRES-dependent. Such an increase seemed independent of mTOR inhibition. After administration of Torin-1, APP downregulation and Mechanistic Target of Rapamycin Complex 1 (mTORC-1) inhibition affected 4E-BP1 phosphorylation and global protein synthesis in opposite fashions. Additional investigations indicated that APP operates independently of mTORC-1. Key phenomena described in this study were reversed by overexpression of the APP C-terminal domain. The presented data suggest that APP may be a novel regulator of protein synthesis in dividing human cells, both cancerous and primary. Furthermore, APP appears to affect translation initiation using mechanisms seemingly dissimilar to mTORC-1 regulation of cap-dependent protein synthesis.

  14. BIMOLECULAR FLUORESCENCE COMPLEMENTATION ANALYSIS OF INDUCIBLE PROTEIN INTERACTIONS: EFFECTS OF FACTORS AFFECTING PROTEIN FOLDING ON FLUORESCENT PROTEIN FRAGMENT ASSOCIATION

    PubMed Central

    Robida, Aaron M; Kerppola, Tom K

    2009-01-01

    adaptation to protein folding stress. In summary, BiFC analysis enables detection of protein interactions within minutes after complex formation in living cells, but does not allow detection of complex dissociation. Conditional BiFC complex formation depends on the folding efficiencies of fluorescent protein fragments and can be affected by the cellular protein folding environment. PMID:19733184

  15. Expression of proteins and protein kinase activity during germination of aerial spores of Streptomyces granaticolor.

    PubMed

    Mikulík, Karel; Bobek, Jan; Bezousková, Silvia; Benada, Oldrich; Kofronová, Olga

    2002-11-29

    Dormant aerial spores of Streptomyces granaticolor contain pre-existing pool of mRNA and active ribosomes for rapid translation of proteins required for earlier steps of germination. Activated spores were labeled for 30 min with [35S]methionine/cysteine in the presence or absence of rifamycin (400 microg/ml) and resolved by two-dimensional electrophoresis. About 320 proteins were synthesized during the first 30 min of cultivation at the beginning of swelling, before the first DNA replication. Results from nine different experiments performed in the presence of rifamycin revealed 15 protein spots. Transition from dormant spores to swollen spores is not affected by the presence of rifamycin but further development of spores is stopped. To support existence of pre-existing pool of mRNA in spores, cell-free extract of spores (S30 fraction) was used for in vitro protein synthesis. These results indicate that RNA of spores possesses mRNA functionally competent and provides templates for protein synthesis. Cell-free extracts isolated from spores, activated spores, and during spore germination were further examined for in vitro protein phosphorylation. The analyses show that preparation from dormant spores catalyzes phosphorylation of only seven proteins. In the absence of phosphatase inhibitors, several proteins were partially dephosphorylated. The activation of spores leads to a reduction in phosphorylation activity. Results from in vitro phosphorylation reaction indicate that during germination phosphorylation/dephosphorylation of proteins is a complex function of developmental changes.

  16. A Protein Aggregation Based Test for Screening of the Agents Affecting Thermostability of Proteins

    PubMed Central

    Eronina, Tatyana; Borzova, Vera; Maloletkina, Olga; Kleymenov, Sergey; Asryants, Regina; Markossian, Kira; Kurganov, Boris

    2011-01-01

    To search for agents affecting thermal stability of proteins, a test based on the registration of protein aggregation in the regime of heating with a constant rate was used. The initial parts of the dependences of the light scattering intensity (I) on temperature (T) were analyzed using the following empiric equation: I = Kagg(T−T0)2, where Kagg is the parameter characterizing the initial rate of aggregation and T0 is a temperature at which the initial increase in the light scattering intensity is registered. The aggregation data are interpreted in the frame of the model assuming the formation of the start aggregates at the initial stages of the aggregation process. Parameter T0 corresponds to the moment of the origination of the start aggregates. The applicability of the proposed approach was demonstrated on the examples of thermal aggregation of glycogen phosphorylase b from rabbit skeletal muscles and bovine liver glutamate dehydrogenase studied in the presence of agents of different chemical nature. The elaborated approach to the study of protein aggregation may be used for rapid identification of small molecules that interact with protein targets. PMID:21760963

  17. Cbl proteins in platelet activation.

    PubMed

    Buitrago, Lorena; Tsygankov, Alexander; Sanjay, Archana; Kunapuli, Satya P

    2013-01-01

    Platelets play a fundamental role in hemostasis. Their functional responses have to be tightly controlled as any disturbance may lead to bleeding disorders or thrombosis. It is thus important to clearly identify and understand the signaling mechanisms involved in platelet function. An important role of c-Cbl and Cbl-b ubiquitin ligases in platelet functional responses and in hematological malignancies has been recently described. Cbl proteins perform negative and positive regulation of several signaling pathways in platelets. In this review, we explore the role of Cbl proteins in platelet functional responses.

  18. 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

  19. 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.

  20. Remotely activated protein-producing nanoparticles.

    PubMed

    Schroeder, Avi; Goldberg, Michael S; Kastrup, Christian; Wang, Yingxia; Jiang, Shan; Joseph, Brian J; Levins, Christopher G; Kannan, Sneha T; Langer, Robert; Anderson, Daniel G

    2012-06-13

    The development of responsive nanomaterials, nanoscale systems that actively respond to stimuli, is one general goal of nanotechnology. Here we develop nanoparticles that can be controllably triggered to synthesize proteins. The nanoparticles consist of lipid vesicles filled with the cellular machinery responsible for transcription and translation, including amino acids, ribosomes, and DNA caged with a photolabile protecting group. These particles served as nanofactories capable of producing proteins including green fluorescent protein (GFP) and enzymatically active luciferase. In vitro and in vivo, protein synthesis was spatially and temporally controllable, and could be initiated by irradiating micrometer-scale regions on the time scale of milliseconds. The ability to control protein synthesis inside nanomaterials may enable new strategies to facilitate the study of orthogonal proteins in a confined environment and for remotely activated drug delivery.

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

    SciTech Connect

    Van De Walle, Jacqueline; Sergent, Therese; Piront, Neil; Toussaint, Olivier; Schneider, Yves-Jacques; Larondelle, Yvan

    2010-06-15

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

  2. Antibody Array Revealed PRL-3 Affects Protein Phosphorylation and Cytokine Secretion.

    PubMed

    Yang, Yongyong; Lian, Shenyi; Meng, Lin; Qu, Like; Shou, Chengchao

    2017-01-01

    Phosphatase of regenerating liver 3 (PRL-3) promotes cancer metastasis and progression via increasing cell motility and invasiveness, however the mechanism is still not fully understood. Previous reports showed that PRL-3 increases the phosphorylation of many important proteins and suspected that PRL-3-enhanced protein phosphorylation may be due to its regulation on cytokines. To investigate PRL-3's impact on protein phosphorylation and cytokine secretion, we performed antibody arrays against protein phosphorylation and cytokines separately. The data showed that PRL-3 could enhance tyrosine phosphorylation and serine/threonine phosphorylation of diverse signaling proteins. Meanwhile, PRL-3 could affect the secretion of a subset of cytokines. Furthermore, we discovered the PRL-3-increased IL-1α secretion was regulated by NF-κB and Jak2-Stat3 pathways and inhibiting IL-1α could reduce PRL-3-enhanced cell migration. Therefore, our result indicated that PRL-3 promotes protein phosphorylation by acting as an 'activator kinase' and consequently regulates cytokine secretion.

  3. Antibody Array Revealed PRL-3 Affects Protein Phosphorylation and Cytokine Secretion

    PubMed Central

    Meng, Lin; Qu, Like; Shou, Chengchao

    2017-01-01

    Phosphatase of regenerating liver 3 (PRL-3) promotes cancer metastasis and progression via increasing cell motility and invasiveness, however the mechanism is still not fully understood. Previous reports showed that PRL-3 increases the phosphorylation of many important proteins and suspected that PRL-3-enhanced protein phosphorylation may be due to its regulation on cytokines. To investigate PRL-3’s impact on protein phosphorylation and cytokine secretion, we performed antibody arrays against protein phosphorylation and cytokines separately. The data showed that PRL-3 could enhance tyrosine phosphorylation and serine/threonine phosphorylation of diverse signaling proteins. Meanwhile, PRL-3 could affect the secretion of a subset of cytokines. Furthermore, we discovered the PRL-3-increased IL-1α secretion was regulated by NF-κB and Jak2-Stat3 pathways and inhibiting IL-1α could reduce PRL-3-enhanced cell migration. Therefore, our result indicated that PRL-3 promotes protein phosphorylation by acting as an ‘activator kinase’ and consequently regulates cytokine secretion. PMID:28068414

  4. HB-EGF affects astrocyte morphology, proliferation, differentiation, and the expression of intermediate filament proteins.

    PubMed

    Puschmann, Till B; Zandén, Carl; Lebkuechner, Isabell; Philippot, Camille; de Pablo, Yolanda; Liu, Johan; Pekny, Milos

    2014-03-01

    Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a vascular-derived trophic factor, belongs to the epidermal growth factor (EGF) family of neuroprotective, hypoxia-inducible proteins released by astrocytes in CNS injuries. It was suggested that HB-EGF can replace fetal calf serum (FCS) in astrocyte cultures. We previously demonstrated that in contrast to standard 2D cell culture systems, Bioactive3D culture system, when used with FCS, minimizes the baseline activation of astrocytes and preserves their complex morphology. Here, we show that HB-EGF induced EGF receptor (EGFR) activation by Y1068 phosphorylation, Mapk/Erk pathway activation, and led to an increase in cell proliferation, more prominent in Bioactive3D than in 2D cultures. HB-EGF changed morphology of 2D and Bioactive3D cultured astrocytes toward a radial glia-like phenotype and induced the expression of intermediate filament and progenitor cell marker protein nestin. Glial fibrillary acidic protein (GFAP) and vimentin protein expression was unaffected. RT-qPCR analysis demonstrated that HB-EGF affected the expression of Notch signaling pathway genes, implying a role for the Notch signaling in HB-EGF-mediated astrocyte response. HB-EGF can be used as a FCS replacement for astrocyte expansion and in vitro experimentation both in 2D and Bioactive3D culture systems; however, caution should be exercised since it appears to induce partial de-differentiation of astrocytes.

  5. How optimization of potential functions affects protein folding.

    PubMed Central

    Hao, M H; Scheraga, H A

    1996-01-01

    The relationship between the optimization of the potential function and the foldability of theoretical protein models is studied based on investigations of a 27-mer cubic-lattice protein model and a more realistic lattice model for the protein crambin. In both the simple and the more complicated systems, optimization of the energy parameters achieves significant improvements in the statistical-mechanical characteristics of the systems and leads to foldable protein models in simulation experiments. The foldability of the protein models is characterized by their statistical-mechanical properties--e.g., by the density of states and by Monte Carlo folding simulations of the models. With optimized energy parameters, a high level of consistency exists among different interactions in the native structures of the protein models, as revealed by a correlation function between the optimized energy parameters and the native structure of the model proteins. The results of this work are relevant to the design of a general potential function for folding proteins by theoretical simulations. PMID:8643516

  6. A novel family of small proteins that affect plant development

    SciTech Connect

    John Charles Walker

    2011-04-29

    The DVL genes represent a new group of plant proteins that influence plant growth and development. Overexpression of DVL1, and other members of the DVL family, causes striking phenotypic changes. The DVL proteins share sequence homology in their C-terminal half. Point mutations in the C-terminal domain show it is necessary and deletion studies demonstrate the C-terminal domain is sufficient to confer the overexpression phenotypes. The phenotypes observed, and the conservation of the protein sequence in the plant kingdom, does suggest the DVL proteins have a role in modulating plant growth and development. Our working hypothesis is the DVL proteins function as regulators of cellular signaling pathways that control growth and development.

  7. Dietary protein considerations to support active aging.

    PubMed

    Wall, Benjamin T; Cermak, Naomi M; van Loon, Luc J C

    2014-11-01

    Given our rapidly aging world-wide population, the loss of skeletal muscle mass with healthy aging (sarcopenia) represents an important societal and public health concern. Maintaining or adopting an active lifestyle alleviates age-related muscle loss to a certain extent. Over time, even small losses of muscle tissue can hinder the ability to maintain an active lifestyle and, as such, contribute to the development of frailty and metabolic disease. Considerable research focus has addressed the application of dietary protein supplementation to support exercise-induced gains in muscle mass in younger individuals. In contrast, the role of dietary protein in supporting the maintenance (or gain) of skeletal muscle mass in active older persons has received less attention. Older individuals display a blunted muscle protein synthetic response to dietary protein ingestion. However, this reduced anabolic response can largely be overcome when physical activity is performed in close temporal proximity to protein consumption. Moreover, recent evidence has helped elucidate the optimal type and amount of dietary protein that should be ingested by the older adult throughout the day in order to maximize the skeletal muscle adaptive response to physical activity. Evidence demonstrates that when these principles are adhered to, muscle maintenance or hypertrophy over prolonged periods can be further augmented in active older persons. The present review outlines the current understanding of the role that dietary protein occupies in the lifestyle of active older adults as a means to increase skeletal muscle mass, strength and function, and thus support healthier aging.

  8. Copper(I) and nickel(II) complexes with 1:1 vs. 1:2 coordination of ferrocenyl hydrazone ligands: do the geometry and composition of complexes affect DNA binding/cleavage, protein binding, antioxidant and cytotoxic activities?

    PubMed

    Krishnamoorthy, Paramasivam; Sathyadevi, Palanisamy; Butorac, Rachel R; Cowley, Alan H; Bhuvanesh, Nattamai S P; Dharmaraj, Nallasamy

    2012-04-21

    A new series of geometrically different complexes containing ferrocenyl hydrazone ligands were synthesised by reacting suitable precursor complex [MCl(2)(PPh(3))(2)] with the ligands HL(1) or HL(2) (where M = Cu(II) or Ni(II); HL(1) = [Cp(2)Fe(CH=N-NH-CO-C(6)H(5))] (1) and HL(2) = [Cp(2)Fe(CH=N-NH-CO-C(5)H(4)N)]) (2). The new complexes of the composition [Cu(L(1))(PPh(3))(2)], (3) [Cu(L(2))(PPh(3))(2)] (4), [Ni(L(1))(2)] (5) and [Ni(L(2))(2)] (6) were characterised by various spectral studies. Among them, complexes 3 and 5 characterised by single crystal X-ray diffraction showed a distorted tetrahedral structure for the former with 1:1 metal-ligand stoichiometry, but a distorted square planar geometry with 1:2 metal-ligand stoichiometry in the case of the latter. Systematic biological investigations like DNA binding, DNA cleavage, protein binding, free radical scavenging and cytotoxicity activities were carried out using all the synthesised compounds and the results obtained were explained on the basis of structure-activity relationships. The binding constant (K(b)) values of the synthesised compounds are found to be in the order of magnitude 10(3)-10(5) M(-1) and also they exhibit significant cleavage of supercoiled (SC) pUC19 DNA in the presence of H(2)O(2) as co-oxidant. The conformational changes of bovine serum albumin (BSA) upon binding with the above complexes were also studied. In addition, concentration dependent free radical scavenging potential of all the synthesised compounds (1-6) was also carried out under in vitro conditions. Assays on the cytotoxicity of the above complexes against HeLa and A431 tumor cells and NIH 3T3 normal cells were also carried out.

  9. Corrugator activity confirms immediate negative affect in surprise

    PubMed Central

    Topolinski, Sascha; Strack, Fritz

    2015-01-01

    The emotion of surprise entails a complex of immediate responses, such as cognitive interruption, attention allocation to, and more systematic processing of the surprising stimulus. All these processes serve the ultimate function to increase processing depth and thus cognitively master the surprising stimulus. The present account introduces phasic negative affect as the underlying mechanism responsible for this switch in operating mode. Surprising stimuli are schema-discrepant and thus entail cognitive disfluency, which elicits immediate negative affect. This affect in turn works like a phasic cognitive tuning switching the current processing mode from more automatic and heuristic to more systematic and reflective processing. Directly testing the initial elicitation of negative affect by surprising events, the present experiment presented high and low surprising neutral trivia statements to N = 28 participants while assessing their spontaneous facial expressions via facial electromyography. High compared to low surprising trivia elicited higher corrugator activity, indicative of negative affect and mental effort, while leaving zygomaticus (positive affect) and frontalis (cultural surprise expression) activity unaffected. Future research shall investigate the mediating role of negative affect in eliciting surprise-related outcomes. PMID:25762956

  10. Does the DFT Self-Interaction Error Affect Energies Calculated in Proteins with Large QM Systems?

    PubMed

    Fouda, Adam; Ryde, Ulf

    2016-11-08

    We have examined how the self-interaction error in density-functional theory (DFT) calculations affects energies calculated on large systems (600-1000 atoms) involving several charged groups. We employ 18 different quantum mechanical (QM) methods, including Hartree-Fock, as well as pure, hybrid, and range-separated DFT methods. They are used to calculate reaction and activation energies for three different protein models in vacuum, in a point-charge surrounding, or with a continuum-solvent model. We show that pure DFT functionals give rise to a significant delocalization of the charges in charged groups in the protein, typically by ∼0.1 e, as evidenced from the Mulliken charges. This has a clear effect on how the surroundings affect calculated reaction and activation energies, indicating that these methods should be avoided for DFT calculations on large systems. Fortunately, methods such as CAM-B3LYP, BHLYP, and M06-2X give results that agree within a few kilojoules per mole, especially when the calculations are performed in a point-charge surrounding. Therefore, we recommend these methods to estimate the effect of the surroundings with large QM systems (but other QM methods may be used to study the intrinsic reaction and activation energies).

  11. 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.

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

    DOE PAGES

    Nayak, Jaladhi; Gastonguay, Adam J.; Talipov, Marat R.; ...

    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

  13. Draxin, an axon guidance protein, affects chick trunk neural crest migration.

    PubMed

    Su, Yuhong; Naser, Iftekhar B; Islam, Shahidul M; Zhang, Sanbing; Ahmed, Giasuddin; Chen, Sandy; Shinmyo, Yohei; Kawakami, Minoru; Yamamura, Ken-ichi; Tanaka, Hideaki

    2009-12-01

    The neural crest is a multipotent population of migratory cells that arises in the central nervous system and subsequently migrates along defined stereotypic pathways. In the present work, we analyzed the role of a repulsive axon guidance protein, draxin, in the migration of neural crest cells. Draxin is expressed in the roof plate of the chick trunk spinal cord and around the early migration pathway of neural crest cells. Draxin modulates chick neural crest cell migration in vitro by reducing the polarization of these cells. When exposed to draxin, the velocity of migrating neural crest cells was reduced, and the cells changed direction so frequently that the net migration distance was also reduced. Overexpression of draxin also caused some early migrating neural crest cells to change direction to the dorsolateral pathway in the chick trunk region, presumably due to draxin's inhibitory activity. These results demonstrate that draxin, an axon guidance protein, can also affect trunk neural crest migration in the chick embryo.

  14. Mutations of the Wiskott-Aldrich Syndrome Protein affect protein expression and dictate the clinical phenotypes.

    PubMed

    Ochs, Hans D

    2009-01-01

    Mutations of the Wiskott-Aldrich Syndrome Protein (WASP) are responsible for classic Wiskott-Aldrich Syndrome (WAS), X-linked thrombocytopenia (XLT), and in rare instances congenital X-linked neutropenia (XLN). WASP is a regulator of actin polymerization in hematopoietic cells with well-defined functional domains that are involved in cell signaling and cell locomotion, immune synapse formation, and apoptosis. Mutations of WASP are located throughout the gene and either inhibit or disregulate normal WASP function. Analysis of a large patient population demonstrates a strong phenotype-genotype correlation. Classic WAS occurs when WASP is absent, XLT when mutated WASP is expressed and XLN when missense mutations occur in the Cdc42-binding site. However, because there are exceptions to this rule it is difficult to predict the long-term prognosis of a given affected boy solely based on the analysis of WASP expression.

  15. Serum heat inactivation affects protein corona composition and nanoparticle uptake.

    PubMed

    Lesniak, Anna; Campbell, Abigail; Monopoli, Marco P; Lynch, Iseult; Salvati, Anna; Dawson, Kenneth A

    2010-12-01

    Nanoparticles are of an appropriate size to interact with cells, and are likely to use a range of cellular machinery for internalisation and trafficking to various sub-cellular compartments. It is now understood that once in contact with biological fluids, the nanoparticle surface gets covered by a highly specific layer of proteins, forming the nanoparticle protein corona. This protein layer is stable for times longer than the typical time scale of nanoparticle import, and thus can impact on particle uptake and trafficking inside the cells. In this work, the effect of the corona composition on nanoparticle uptake has been investigated, by studying the impact of serum heat inactivation and complement depletion on the load of nanoparticles accumulated inside the cell. For the same material and nanoparticle size, cellular uptake was found to be significantly different when the nanoparticles were dispersed in medium where the serum was heat inactivated or not heat inactivated, even for non-specialized cells, suggesting that different sera can lead to different nanoparticle doses. The fact that uptake was correlated with the amount of protein bound into the nanoparticle corona suggests the need for commonly agreed dispersion protocols for in vitro nanoparticle-cell studies.

  16. Hexavalent chromium affects sperm motility by influencing protein tyrosine phosphorylation in the midpiece of boar spermatozoa.

    PubMed

    Zhen, Linqing; Wang, Lirui; Fu, Jieli; Li, Yuhua; Zhao, Na; Li, Xinhong

    2016-01-01

    Hexavalent chromium reportedly induces reproductive toxicity and further inhibits male fertility in mammals. In this study, we investigated the molecular mechanism by which hexavalent chromium affects motility signaling in boar spermatozoa in vitro. The results indicated that Cr(VI) decreased sperm motility, protein phosphorylation, mitochondrial membrane potential (ΔΨm) and metabolic enzyme activity starting at 4μmol/mL following incubation for 1.5h. Notably, all parameters were potently inhibited by 10μmol/mL Cr, while supplementation with the dibutyryl-cAMP (dbcAMP) and the 3-isobutyl-1-methylxanthine (IBMX) prevented the inhibition of protein phosphorylation. Interestingly, high concentrations of Cr (>10μmol/mL) increased the tyrosine phosphorylation of some high-molecular-weight proteins in the principle piece but decreased that in the middle piece associated with an extreme reduction of sperm motility. These results suggest that chromium affects boar sperm motility by impairing tyrosine phosphorylation in the midpiece of sperm by blocking the cAMP/PKA pathway in boar sperm in vitro.

  17. Golgi Anti-apoptotic Proteins Are Highly Conserved Ion Channels That Affect Apoptosis and Cell Migration*

    PubMed Central

    Carrara, Guia; Saraiva, Nuno; Parsons, Maddy; Byrne, Bernadette; Prole, David L.; Taylor, Colin W.; Smith, Geoffrey L.

    2015-01-01

    Golgi anti-apoptotic proteins (GAAPs) are multitransmembrane proteins that are expressed in the Golgi apparatus and are able to homo-oligomerize. They are highly conserved throughout eukaryotes and are present in some prokaryotes and orthopoxviruses. Within eukaryotes, GAAPs regulate the Ca2+ content of intracellular stores, inhibit apoptosis, and promote cell adhesion and migration. Data presented here demonstrate that purified viral GAAPs (vGAAPs) and human Bax inhibitor 1 form ion channels and that vGAAP from camelpox virus is selective for cations. Mutagenesis of vGAAP, including some residues conserved in the recently solved structure of a related bacterial protein, BsYetJ, altered the conductance (E207Q and D219N) and ion selectivity (E207Q) of the channel. Mutation of residue Glu-207 or -178 reduced the effects of GAAP on cell migration and adhesion without affecting protection from apoptosis. In contrast, mutation of Asp-219 abrogated the anti-apoptotic activity of GAAP but not its effects on cell migration and adhesion. These results demonstrate that GAAPs are ion channels and define residues that contribute to the ion-conducting pore and affect apoptosis, cell adhesion, and migration independently. PMID:25713081

  18. 16 CFR 801.3 - Activities in or affecting commerce.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 16 Commercial Practices 1 2014-01-01 2014-01-01 false Activities in or affecting commerce. 801.3 Section 801.3 Commercial Practices FEDERAL TRADE COMMISSION RULES, REGULATIONS, STATEMENTS AND INTERPRETATIONS UNDER THE HART-SCOTT-RODINO ANTITRUST IMPROVEMENTS ACT OF 1976 COVERAGE RULES § 801.3...

  19. 16 CFR 801.3 - Activities in or affecting commerce.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 16 Commercial Practices 1 2012-01-01 2012-01-01 false Activities in or affecting commerce. 801.3 Section 801.3 Commercial Practices FEDERAL TRADE COMMISSION RULES, REGULATIONS, STATEMENTS AND INTERPRETATIONS UNDER THE HART-SCOTT-RODINO ANTITRUST IMPROVEMENTS ACT OF 1976 COVERAGE RULES § 801.3...

  20. 16 CFR 801.3 - Activities in or affecting commerce.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 16 Commercial Practices 1 2010-01-01 2010-01-01 false Activities in or affecting commerce. 801.3 Section 801.3 Commercial Practices FEDERAL TRADE COMMISSION RULES, REGULATIONS, STATEMENTS AND INTERPRETATIONS UNDER THE HART-SCOTT-RODINO ANTITRUST IMPROVEMENTS ACT OF 1976 COVERAGE RULES § 801.3...

  1. 16 CFR 801.3 - Activities in or affecting commerce.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 16 Commercial Practices 1 2013-01-01 2013-01-01 false Activities in or affecting commerce. 801.3 Section 801.3 Commercial Practices FEDERAL TRADE COMMISSION RULES, REGULATIONS, STATEMENTS AND INTERPRETATIONS UNDER THE HART-SCOTT-RODINO ANTITRUST IMPROVEMENTS ACT OF 1976 COVERAGE RULES § 801.3...

  2. 16 CFR 801.3 - Activities in or affecting commerce.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 16 Commercial Practices 1 2011-01-01 2011-01-01 false Activities in or affecting commerce. 801.3 Section 801.3 Commercial Practices FEDERAL TRADE COMMISSION RULES, REGULATIONS, STATEMENTS AND INTERPRETATIONS UNDER THE HART-SCOTT-RODINO ANTITRUST IMPROVEMENTS ACT OF 1976 COVERAGE RULES § 801.3...

  3. Monitoring Affect States during Effortful Problem Solving Activities

    ERIC Educational Resources Information Center

    D'Mello, Sidney K.; Lehman, Blair; Person, Natalie

    2010-01-01

    We explored the affective states that students experienced during effortful problem solving activities. We conducted a study where 41 students solved difficult analytical reasoning problems from the Law School Admission Test. Students viewed videos of their faces and screen captures and judged their emotions from a set of 14 states (basic…

  4. Microparticulation of whey protein: related factors affecting the solubility.

    PubMed

    Lieske, B; Konrad, G

    1994-10-01

    Solubility of Simplesse 100, the only whey-based fat substitute, was found to be good, considering the fact that technology for preparation of Simplesse 100 is a sequence of thermal steps. To characterize this phenomen, gel chromatography on Sephadex G-100, Sephacryl S-1000 and SDS-PAGE were used, supported by high-speed separation, UV studies and analytical procedures. Results show that the unusual solubility characteristic of microparticulated whey protein is related to two molecular effects: (1) optimal defolding of protein molecules and (2) stabilization of the defolded status by carbohydrate. Both effects were considered to favour non-covalent bonds, which contribute to the outstanding physico-functional and nutritive properties of microparticles.

  5. Yeast mutants affecting possible quality control of plasma membrane proteins.

    PubMed

    Li, Y; Kane, T; Tipper, C; Spatrick, P; Jenness, D D

    1999-05-01

    Mutations gef1, stp22, STP26, and STP27 in Saccharomyces cerevisiae were identified as suppressors of the temperature-sensitive alpha-factor receptor (mutation ste2-3) and arginine permease (mutation can1(ts)). These suppressors inhibited the elimination of misfolded receptors (synthesized at 34 degrees C) as well as damaged surface receptors (shifted from 22 to 34 degrees C). The stp22 mutation (allelic to vps23 [M. Babst and S. Emr, personal communication] and the STP26 mutation also caused missorting of carboxypeptidase Y, and ste2-3 was suppressed by mutations vps1, vps8, vps10, and vps28 but not by mutation vps3. In the stp22 mutant, both the mutant and the wild-type receptors (tagged with green fluorescent protein [GFP]) accumulated within an endosome-like compartment and were excluded from the vacuole. GFP-tagged Stp22p also accumulated in this compartment. Upon reaching the vacuole, cytoplasmic domains of both mutant and wild-type receptors appeared within the vacuolar lumen. Stp22p and Gef1p are similar to tumor susceptibility protein TSG101 and voltage-gated chloride channel, respectively. These results identify potential elements of plasma membrane quality control and indicate that cytoplasmic domains of membrane proteins are translocated into the vacuolar lumen.

  6. 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.

  7. MYST protein acetyltransferase activity requires active site lysine autoacetylation.

    PubMed

    Yuan, Hua; Rossetto, Dorine; Mellert, Hestia; Dang, Weiwei; Srinivasan, Madhusudan; Johnson, Jamel; Hodawadekar, Santosh; Ding, Emily C; Speicher, Kaye; Abshiru, Nebiyu; Perry, Rocco; Wu, Jiang; Yang, Chao; Zheng, Y George; Speicher, David W; Thibault, Pierre; Verreault, Alain; Johnson, F Bradley; Berger, Shelley L; Sternglanz, Rolf; McMahon, Steven B; Côté, Jacques; Marmorstein, Ronen

    2012-01-04

    The MYST protein lysine acetyltransferases are evolutionarily conserved throughout eukaryotes and acetylate proteins to regulate diverse biological processes including gene regulation, DNA repair, cell-cycle regulation, stem cell homeostasis and development. Here, we demonstrate that MYST protein acetyltransferase activity requires active site lysine autoacetylation. The X-ray crystal structures of yeast Esa1 (yEsa1/KAT5) bound to a bisubstrate H4K16CoA inhibitor and human MOF (hMOF/KAT8/MYST1) reveal that they are autoacetylated at a strictly conserved lysine residue in MYST proteins (yEsa1-K262 and hMOF-K274) in the enzyme active site. The structure of hMOF also shows partial occupancy of K274 in the unacetylated form, revealing that the side chain reorients to a position that engages the catalytic glutamate residue and would block cognate protein substrate binding. Consistent with the structural findings, we present mass spectrometry data and biochemical experiments to demonstrate that this lysine autoacetylation on yEsa1, hMOF and its yeast orthologue, ySas2 (KAT8) occurs in solution and is required for acetylation and protein substrate binding in vitro. We also show that this autoacetylation occurs in vivo and is required for the cellular functions of these MYST proteins. These findings provide an avenue for the autoposttranslational regulation of MYST proteins that is distinct from other acetyltransferases but draws similarities to the phosphoregulation of protein kinases.

  8. MYST protein acetyltransferase activity requires active site lysine autoacetylation

    PubMed Central

    Yuan, Hua; Rossetto, Dorine; Mellert, Hestia; Dang, Weiwei; Srinivasan, Madhusudan; Johnson, Jamel; Hodawadekar, Santosh; Ding, Emily C; Speicher, Kaye; Abshiru, Nebiyu; Perry, Rocco; Wu, Jiang; Yang, Chao; Zheng, Y George; Speicher, David W; Thibault, Pierre; Verreault, Alain; Johnson, F Bradley; Berger, Shelley L; Sternglanz, Rolf; McMahon, Steven B; Côté, Jacques; Marmorstein, Ronen

    2012-01-01

    The MYST protein lysine acetyltransferases are evolutionarily conserved throughout eukaryotes and acetylate proteins to regulate diverse biological processes including gene regulation, DNA repair, cell-cycle regulation, stem cell homeostasis and development. Here, we demonstrate that MYST protein acetyltransferase activity requires active site lysine autoacetylation. The X-ray crystal structures of yeast Esa1 (yEsa1/KAT5) bound to a bisubstrate H4K16CoA inhibitor and human MOF (hMOF/KAT8/MYST1) reveal that they are autoacetylated at a strictly conserved lysine residue in MYST proteins (yEsa1-K262 and hMOF-K274) in the enzyme active site. The structure of hMOF also shows partial occupancy of K274 in the unacetylated form, revealing that the side chain reorients to a position that engages the catalytic glutamate residue and would block cognate protein substrate binding. Consistent with the structural findings, we present mass spectrometry data and biochemical experiments to demonstrate that this lysine autoacetylation on yEsa1, hMOF and its yeast orthologue, ySas2 (KAT8) occurs in solution and is required for acetylation and protein substrate binding in vitro. We also show that this autoacetylation occurs in vivo and is required for the cellular functions of these MYST proteins. These findings provide an avenue for the autoposttranslational regulation of MYST proteins that is distinct from other acetyltransferases but draws similarities to the phosphoregulation of protein kinases. PMID:22020126

  9. 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

  10. Water molecules inside protein structure affect binding of monosaccharides with HIV-1 antibody 2G12.

    PubMed

    Ueno-Noto, Kaori; Takano, Keiko

    2016-10-05

    Water molecules inside biomolecules constitute integral parts of their structure and participate in the functions of the proteins. Some of the X-ray crystallographic data are insufficient for analyzing a series of ligand-protein complexes in the same condition. We theoretically investigated antibody binding abilities of saccharide ligands and the effects of the inner water molecules of ligand-antibody complexes. Classical molecular dynamics and quantum chemical simulations using a model with possible water molecules inside the protein were performed with saccharide ligands and Human Immunodeficiency Virus 1 neutralizing antibody 2G12 complexes to estimate how inner water molecules of the protein affect the dynamics of the complexes as well as the ligand-antibody interaction. Our results indicate the fact that d-fructose's strong affinity to the antibody was partly due to the good retentiveness of solvent water molecules of the ligand and its stability of the ligand's conformation and relative position in the active site. © 2016 Wiley Periodicals, Inc.

  11. 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.

  12. Cold stress affects H(+)-ATPase and phospholipase D activity in Arabidopsis.

    PubMed

    Muzi, Carlo; Camoni, Lorenzo; Visconti, Sabina; Aducci, Patrizia

    2016-11-01

    Low temperature is an environmental stress that greatly influences plant performance and distribution. Plants exposed to cold stress exhibit modifications of plasma membrane physical properties that can affect their functionality. Here it is reported the effect of low temperature exposure of Arabidopsis plants on the activity of phospholipase D and H(+)-ATPase, the master enzyme located at the plasma membrane. The H(+)-ATPase activity was differently affected, depending on the length of cold stress imposed. In particular, an exposure to 4 °C for 6 h determined the strong inhibition of the H(+)-ATPase activity, that correlates with a reduced association with the regulatory 14-3-3 proteins. A longer exposure first caused the full recovery of the enzymatic activity followed by a significant activation, in accordance with both the increased association with 14-3-3 proteins and induction of H(+)-ATPase gene transcription. Different time lengths of cold stress treatment were also shown to strongly stimulate the phospholipase D activity and affect the phosphatidic acid levels of the plasma membranes. Our results suggest a functional correlation between the activity of phospholipase D and H(+)-ATPase mediated by phosphatidic acid release during the cold stress response.

  13. Asymmetric frontal cortical activity and negative affective responses to ostracism.

    PubMed

    Peterson, Carly K; Gravens, Laura C; Harmon-Jones, Eddie

    2011-06-01

    Ostracism arouses negative affect. However, little is known about variables that influence the intensity of these negative affective responses. Two studies fill this void by incorporating work on approach- and withdrawal-related emotional states and their associated cortical activations. Study 1 found that following ostracism anger related directly to relative left frontal cortical activation. Study 2 used unilateral hand contractions to manipulate frontal cortical activity prior to an ostracizing event. Right-hand contractions, compared to left-hand contractions, caused greater relative left frontal cortical activation during the hand contractions as well as ostracism. Also, right-hand contractions caused more self-reported anger in response to being ostracized. Within-condition correlations revealed patterns of associations between ostracism-induced frontal asymmetry and emotive responses to ostracism consistent with Study 1. Taken together, these results suggest that asymmetrical frontal cortical activity is related to angry responses to ostracism, with greater relative left frontal cortical activity being associated with increased anger.

  14. Protein kinase C activators inhibit capillary endothelial cell growth

    SciTech Connect

    Doctrow, S.R.

    1986-05-01

    Phorbol 12,13-dibutyrate (PDBu) binds specifically to bovine capillary endothelial (BCE) cells (K/sub d/ = 8nM) and inhibits the proliferation (K/sub 50/ = 6 +/- 4 nM). Under similar conditions, PDBu does not inhibit the growth of bovine aortic endothelial or smooth muscle cells. PDBu markedly attenuates the response of BCE cells to purified human hepatoma-derived growth factor which, in the absence of PDBu, stimulates BCE cell growth by about 3-fold. Several observations suggest that the inhibition of BCE cell growth by PDBu is mediated by protein kinase C: (1) different phorbol compounds inhibit BCE cell growth according to the relative potencies as protein kinase C activators (12-tetradecanoylphorbol 13-acetate > PDBu >> phorbol 12,13-diacetate >>>..beta..-phorbol; ..cap alpha..-phorbol 12,13-didecanoate). (2) Specific binding of PDBu to BCE cells is displaced by sn-1,2-dioctanoylglycerol (diC/sub 8/), a protein kinase C activator and an analog of the putative second messenger activating this kinase in vivo. The weak protein kinase C activator, sn-1,2-dibutyrylglycerol, does not affect PDBu binding. (3) A cytosolic extract from BCE cells contains a Ca/sup 2 +//phosphatidylserine-dependent kinase that is activated by diC/sub 8/ and PDBu, but not by ..beta..-phorbol. These results support a role for protein kinase C in suppressing capillary endothelial cell growth and may therefore have implications in the intracellular regulation of angiogenesis.

  15. How does the anthropogenic activity affect the spring discharge?

    NASA Astrophysics Data System (ADS)

    Hao, Yonghong; Zhang, Juan; Wang, Jiaojiao; Li, Ruifang; Hao, Pengmei; Zhan, Hongbin

    2016-09-01

    Karst hydrological process has largely been altered by climate change and human activity. In many places throughout the world, human activity (e.g. groundwater pumping and dewatering from mining) has intensified and surpassed climate change, where human activity becomes the primary factor that affects groundwater system. But it is still largely unclear how the human activity affects spring discharge in magnitude and periodicity. This study investigates the effects of anthropogenic activity on spring discharge, using the Xin'an Springs of China as an example. The Xin'an Spring discharge were divided into two time periods: the pre-development period from 1956 to 1971 and the post-development period from 1972 to 2013. We confirm the dividing time (i.e. 1971) of these two periods using the Wilcoxon rank-sum test. Then the wavelet transform and wavelet coherence were used to analyze the karst hydrological processes for the two periods respectively. We analyze the correlations of precipitation and the Xin'an spring discharge with the monsoons including the Indian Summer Monsoon (ISM) and the West North Pacific Monsoon (WNPM) and the climate teleconnections including El Niño Southern Oscillation (ENSO) and Pacific Decadal Oscillation (PDO), respectively. The results indicated that the spring discharge was attenuated about 19.63% under the influence of human activity in the Xin'an Springs basin. However, human activity did not alter the size of the resonance frequencies between the spring discharge and the monsoons. In contrast, it reinforced the periodicities of the monsoons-driven spring discharge. It suggested that human has adapted to the major climate periodicities, and human activity had the same rhyme with the primary climate periodicity. In return, human activity enhances the correlation between the monsoons and the spring discharge.

  16. Factors affecting farm noise during common agricultural activities.

    PubMed

    Franklin, R C; Depczynski, J; Challinor, K; Williams, W; Fragar, L J

    2006-05-01

    Hearing injury due to exposure to excessive noise during common farming activities is a significant problem for farmers. The aim of this study was to investigate factors that affect the level of risk to hearing caused by common farming activities. Noise levels on farms were measured across a range of activities and producer groups, and situational factors that effect noise levels were also investigated. Older tractors were found to be 6 dB louder than newer tractors. Cabs reduced noise to the operator by 16 dB, which was halved to 8 dB if a door was open. Radios added between 3 and 5 dB to the noise in the cab. These variables significantly affect the noise level at the ear of operators and others in the workplace, and affect the subsequent exposure limits that are considered safe. Situational factors need to be considered in assessing the level of risk to farmers' hearing and in choosing noise management strategies on the farm. This information has been incorporated into material about hearing and discussions with farmers who participated in field day hearing screening programs in Australia.

  17. Prenatal caffeine intake differently affects synaptic proteins during fetal brain development.

    PubMed

    Mioranzza, Sabrina; Nunes, Fernanda; Marques, Daniela M; Fioreze, Gabriela T; Rocha, Andréia S; Botton, Paulo Henrique S; Costa, Marcelo S; Porciúncula, Lisiane O

    2014-08-01

    Caffeine is the psychostimulant most consumed worldwide. However, little is known about its effects during fetal brain development. In this study, adult female Wistar rats received caffeine in drinking water (0.1, 0.3 and 1.0 g/L) during the active cycle in weekdays, two weeks before mating and throughout pregnancy. Cerebral cortex and hippocampus from embryonic stages 18 or 20 (E18 or E20, respectively) were collected for immunodetection of the following synaptic proteins: brain-derived neurotrophic factor (BDNF), TrkB receptor, Sonic Hedgehog (Shh), Growth Associated Protein 43 (GAP-43) and Synaptosomal-associated Protein 25 (SNAP-25). Besides, the estimation of NeuN-stained nuclei (mature neurons) and non-neuronal nuclei was verified in both brain regions and embryonic periods. Caffeine (1.0 g/L) decreased the body weight of embryos at E20. Cortical BDNF at E18 was decreased by caffeine (1.0 g/L), while it increased at E20, with no major effects on TrkB receptors. In the hippocampus, caffeine decreased TrkB receptor only at E18, with no effects on BDNF. Moderate and high doses of caffeine promoted an increase in Shh in both brain regions at E18, and in the hippocampus at E20. Caffeine (0.3g/L) decreased GAP-43 only in the hippocampus at E18. The NeuN-stained nuclei increased in the cortex at E20 by lower dose and in the hippocampus at E18 by moderate dose. Our data revealed that caffeine transitorily affect synaptic proteins during fetal brain development. The increased number of NeuN-stained nuclei by prenatal caffeine suggests a possible acceleration of the telencephalon maturation. Although some modifications in the synaptic proteins were transient, our data suggest that caffeine even in lower doses may alter the fetal brain development.

  18. Sucrose Sensitivity of Honey Bees Is Differently Affected by Dietary Protein and a Neonicotinoid Pesticide

    PubMed Central

    Démares, Fabien J.; Crous, Kendall L.; Pirk, Christian W. W.; Nicolson, Susan W.; Human, Hannelie

    2016-01-01

    Over a decade, declines in honey bee colonies have raised worldwide concerns. Several potentially contributing factors have been investigated, e.g. parasites, diseases, and pesticides. Neonicotinoid pesticides have received much attention due to their intensive use in crop protection, and their adverse effects on many levels of honey bee physiology led the European Union to ban these compounds. Due to their neuronal target, a receptor expressed throughout the insect nervous system, studies have focused mainly on neuroscience and behaviour. Through the Geometric Framework of nutrition, we investigated effects of the neonicotinoid thiamethoxam on survival, food consumption and sucrose sensitivity of honey bees (Apis mellifera). Thiamethoxam did not affect protein and carbohydrate intake, but decreased responses to high concentrations of sucrose. Interestingly, when bees ate fixed unbalanced diets, dietary protein facilitated better sucrose detection. Both thiamethoxam and dietary protein influenced survival. These findings suggest that, in the presence of a pesticide and unbalanced food, honey bee health may be severely challenged. Consequences for foraging efficiency and colony activity, cornerstones of honey bee health, are also discussed. PMID:27272274

  19. Sucrose Sensitivity of Honey Bees Is Differently Affected by Dietary Protein and a Neonicotinoid Pesticide.

    PubMed

    Démares, Fabien J; Crous, Kendall L; Pirk, Christian W W; Nicolson, Susan W; Human, Hannelie

    2016-01-01

    Over a decade, declines in honey bee colonies have raised worldwide concerns. Several potentially contributing factors have been investigated, e.g. parasites, diseases, and pesticides. Neonicotinoid pesticides have received much attention due to their intensive use in crop protection, and their adverse effects on many levels of honey bee physiology led the European Union to ban these compounds. Due to their neuronal target, a receptor expressed throughout the insect nervous system, studies have focused mainly on neuroscience and behaviour. Through the Geometric Framework of nutrition, we investigated effects of the neonicotinoid thiamethoxam on survival, food consumption and sucrose sensitivity of honey bees (Apis mellifera). Thiamethoxam did not affect protein and carbohydrate intake, but decreased responses to high concentrations of sucrose. Interestingly, when bees ate fixed unbalanced diets, dietary protein facilitated better sucrose detection. Both thiamethoxam and dietary protein influenced survival. These findings suggest that, in the presence of a pesticide and unbalanced food, honey bee health may be severely challenged. Consequences for foraging efficiency and colony activity, cornerstones of honey bee health, are also discussed.

  20. Communication: Microsecond dynamics of the protein and water affect electron transfer in a bacterial bc{sub 1} complex

    SciTech Connect

    Martin, Daniel R.; Matyushov, Dmitry V.

    2015-04-28

    Cross-membrane electron transport between cofactors localized in proteins of mitochondrial respiration and bacterial photosynthesis is the source of all biological energy. The statistics and dynamics of nuclear fluctuations in these protein/membrane/water heterogeneous systems are critical for their energetic efficiency. The results of 13 μs of atomistic molecular dynamics simulations of the membrane-bound bc{sub 1} bacterial complex are analyzed here. The reaction is affected by a broad spectrum of nuclear modes, with the slowest dynamics in the range of time-scales ∼0.1-1.6 μs contributing half of the reaction reorganization energy. Two reorganization energies are required to describe protein electron transfer due to dynamical arrest of protein conformations on the observation window. This mechanistic distinction allows significant lowering of activation barriers for reactions in proteins.

  1. The CREB-binding protein affects the circadian regulation of behaviour.

    PubMed

    Maurer, Christian; Winter, Tobias; Chen, Siwei; Hung, Hsiu-Cheng; Weber, Frank

    2016-09-01

    Rhythmic changes in light and temperature conditions form the primary environmental cues that synchronize the molecular circadian clock of most species with the external cycles of day and night. Previous studies established a role for the CREB-binding protein (CBP) in molecular clock function by coactivation of circadian transcription. Here, we report that moderately increased levels of CBP strongly dampen circadian behavioural rhythms without affecting molecular oscillations of circadian transcription. Interestingly, light-dark cycles as well as high temperature facilitated a circadian control of behavioural activity. Based on these observations we propose that in addition to its coactivator function for circadian transcription, CBP is involved in the regulation of circadian behaviour down-stream of the circadian clock.

  2. Serotonin and Dopamine: Unifying Affective, Activational, and Decision Functions

    PubMed Central

    Cools, Roshan; Nakamura, Kae; Daw, Nathaniel D

    2011-01-01

    Serotonin, like dopamine (DA), has long been implicated in adaptive behavior, including decision making and reinforcement learning. However, although the two neuromodulators are tightly related and have a similar degree of functional importance, compared with DA, we have a much less specific understanding about the mechanisms by which serotonin affects behavior. Here, we draw on recent work on computational models of dopaminergic function to suggest a framework by which many of the seemingly diverse functions associated with both DA and serotonin—comprising both affective and activational ones, as well as a number of other functions not overtly related to either—can be seen as consequences of a single root mechanism. PMID:20736991

  3. Regulatory network controlling extracellular proteins in Erwinia carotovora subsp. carotovora: FlhDC, the master regulator of flagellar genes, activates rsmB regulatory RNA production by affecting gacA and hexA (lrhA) expression.

    PubMed

    Cui, Yaya; Chatterjee, Asita; Yang, Hailian; Chatterjee, Arun K

    2008-07-01

    Erwinia carotovora subsp. carotovora produces an array of extracellular proteins (i.e., exoproteins), including plant cell wall-degrading enzymes and Harpin, an effector responsible for eliciting hypersensitive reaction. Exoprotein genes are coregulated by the quorum-sensing signal, N-acyl homoserine lactone, plant signals, an assortment of transcriptional factors/regulators (GacS/A, ExpR1, ExpR2, KdgR, RpoS, HexA, and RsmC) and posttranscriptional regulators (RsmA, rsmB RNA). rsmB RNA production is positively regulated by GacS/A, a two-component system, and negatively regulated by HexA (PecT in Erwinia chrysanthemi; LrhA [LysR homolog A] in Escherichia coli) and RsmC, a putative transcriptional adaptor. While free RsmA, an RNA-binding protein, promotes decay of mRNAs of exoprotein genes, binding of RsmA with rsmB RNA neutralizes the RsmA effect. In the course of studies of GacA regulation, we discovered that a locus bearing strong homology to the flhDC operon of E. coli also controls extracellular enzyme production. A transposon insertion FlhDC(-) mutant produces very low levels of pectate lyase, polygalacturonase, cellulase, protease, and E. carotovora subsp. carotovora Harpin (Harpin(Ecc)) and is severely attenuated in its plant virulence. The production of these exoproteins is restored in the mutant carrying an FlhDC(+) plasmid. Sequence analysis and transcript assays disclosed that the flhD operon of E. carotovora subsp. carotovora, like those of other enterobacteria, consists of flhD and flhC. Complementation analysis revealed that the regulatory effect requires functions of both flhD and flhC products. The data presented here show that FlhDC positively regulates gacA, rsmC, and fliA and negatively regulates hexA (lrhA). Evidence shows that FlhDC controls extracellular protein production through cumulative effects on hexA and gacA. Reduced levels of GacA and elevated levels of HexA in the FlhDC(-) mutant are responsible for the inhibition of rsmB RNA

  4. Activities affecting surface water resources: A general overview

    SciTech Connect

    Not Available

    1990-01-01

    In November 1987, P.E.I. signed a federal/provincial work-sharing arrangement on water resource management focusing on groundwater pollution, surface water degradation and estuarine eutrophication. The surface water program was designed to identify current surface water uses and users within 12 major watersheds across the Island containing 26 individual rivers, as well as problems arising due to practices that degrade the quality of surface water and restricts its value to other user groups. This report presents a general overview of the program, covering the general characteristics of the Island; operations in agriculture, fish and wildlife, forestry, recreation, fisheries, and industry; alterations of natural features of waterways; wetlands; additional watershed activities such as hydrometric stations and subdivision development; and activities affecting surface water resources such as sedimentation sources, pollution point sources and instream obstructions.

  5. Intolerance of uncertainty correlates with insula activation during affective ambiguity

    PubMed Central

    Simmons, Alan; Matthews, Scott C.; Paulus, Martin P.; Stein, Murray B.

    2009-01-01

    Intolerance of uncertainty (IU), or the increased affective response to situations with uncertain outcomes, is an important component process of anxiety disorders. Increased IU is observed in panic disorder (PD), obsessive compulsive disorder (OCD) and generalized anxiety disorder (GAD), and is thought to relate to dysfunctional behaviors and thought patterns in these disorders. Identifying what brain systems are associated with IU would contribute to a comprehensive model of anxiety processing, and increase our understanding of the neurobiology of anxiety disorders. Here, we used a behavioral task, Wall of Faces (WOF), during functional magnetic resonance imaging (fMRI), which probes both affect and ambiguity, to examine the neural circuitry of IU in fourteen (10 females) college age (18.8 yrs) subjects. All subjects completed the Intolerance of Uncertainty Scale (IUS), Anxiety Sensitivity Index (ASI), and a measure of neuroticism (i.e. the NEO-N). IUS scores but neither ASI nor NEO-N scores, correlated positively with activation in bilateral insula during affective ambiguity. Thus, the experience of IU during certain types of emotion processing may relate to the degree to which bilateral insula processes uncertainty. Previously observed insula hyperactivity in anxiety disorder individuals may therefore be directly linked to altered processes of uncertainty. PMID:18079060

  6. Intolerance of uncertainty correlates with insula activation during affective ambiguity.

    PubMed

    Simmons, Alan; Matthews, Scott C; Paulus, Martin P; Stein, Murray B

    2008-01-10

    Intolerance of uncertainty (IU), or the increased affective response to situations with uncertain outcomes, is an important component process of anxiety disorders. Increased IU is observed in panic disorder (PD), obsessive compulsive disorder (OCD) and generalized anxiety disorder (GAD), and is thought to relate to dysfunctional behaviors and thought patterns in these disorders. Identifying what brain systems are associated with IU would contribute to a comprehensive model of anxiety processing, and increase our understanding of the neurobiology of anxiety disorders. Here, we used a behavioral task, Wall of Faces (WOFs), during functional magnetic resonance imaging (fMRI), which probes both affect and ambiguity, to examine the neural circuitry of IU in 14 (10 females) college age (18.8 years) subjects. All subjects completed the Intolerance of Uncertainty Scale (IUS), Anxiety Sensitivity Index (ASI), and a measure of neuroticism (i.e. the NEO-N). IUS scores but neither ASI nor NEO-N scores, correlated positively with activation in bilateral insula during affective ambiguity. Thus, the experience of IU during certain types of emotion processing may relate to the degree to which bilateral insula processes uncertainty. Previously observed insula hyperactivity in anxiety disorder individuals may therefore be directly linked to altered processes of uncertainty.

  7. Reduction of Cellular Expression Levels Is a Common Feature of Functionally Affected Pendrin (SLC26A4) Protein Variants

    PubMed Central

    de Moraes, Vanessa C S; Bernardinelli, Emanuele; Zocal, Nathalia; Fernandez, Jhonathan A; Nofziger, Charity; Castilho, Arthur M; Sartorato, Edi L; Paulmichl, Markus; Dossena, Silvia

    2016-01-01

    Sequence alterations in the pendrin gene (SLC26A4) leading to functionally affected protein variants are frequently involved in the pathogenesis of syndromic and nonsyndromic deafness. Considering the high number of SLC26A4 sequence alterations reported to date, discriminating between functionally affected and unaffected pendrin protein variants is essential in contributing to determine the genetic cause of deafness in a given patient. In addition, identifying molecular features common to the functionally affected protein variants can be extremely useful to design future molecule-directed therapeutic approaches. Here we show the functional and molecular characterization of six previously uncharacterized pendrin protein variants found in a cohort of 58 Brazilian deaf patients. Two variants (p.T193I and p.L445W) were undetectable in the plasma membrane, completely retained in the endoplasmic reticulum and showed no transport function; four (p.P142L, p.G149R, p.C282Y and p.Q413R) showed reduced function and significant, although heterogeneous, expression levels in the plasma membrane. Importantly, total expression levels of all of the functionally affected protein variants were significantly reduced with respect to the wild-type and a fully functional variant (p.R776C), regardless of their subcellular localization. Interestingly, reduction of expression may also reduce the transport activity of variants with an intrinsic gain of function (p.Q413R). As reduction of overall cellular abundance was identified as a common molecular feature of pendrin variants with affected function, the identification of strategies to prevent reduction in expression levels may represent a crucial step of potential future therapeutic interventions aimed at restoring the transport activity of dysfunctional pendrin variants. PMID:26752218

  8. Protein source and choice of anticoagulant decisively affect nanoparticle protein corona and cellular uptake

    NASA Astrophysics Data System (ADS)

    Schöttler, S.; Klein, Katja; Landfester, K.; Mailänder, V.

    2016-03-01

    Protein adsorption on nanoparticles has been a focus of the field of nanocarrier research in the past few years and more and more papers are dealing with increasingly detailed lists of proteins adsorbed to a plethora of nanocarriers. While there is an urgent need to understand the influence of this protein corona on nanocarriers' interactions with cells the strong impact of the protein source on corona formation and the consequence for interaction with different cell types are factors that are regularly neglected, but should be taken into account for a meaningful analysis. In this study, the importance of the choice of protein source used for in vitro protein corona analysis is concisely investigated. Major and decisive differences in cellular uptake of a polystyrene nanoparticle incubated in fetal bovine serum, human serum, human citrate and heparin plasma are reported. Furthermore, the protein compositions are determined for coronas formed in the respective incubation media. A strong influence of heparin, which is used as an anticoagulant for plasma generation, on cell interaction is demonstrated. While heparin enhances the uptake into macrophages, it prevents internalization into HeLa cells. Taken together we can give the recommendation that human plasma anticoagulated with citrate seems to give the most relevant results for in vitro studies of nanoparticle uptake.Protein adsorption on nanoparticles has been a focus of the field of nanocarrier research in the past few years and more and more papers are dealing with increasingly detailed lists of proteins adsorbed to a plethora of nanocarriers. While there is an urgent need to understand the influence of this protein corona on nanocarriers' interactions with cells the strong impact of the protein source on corona formation and the consequence for interaction with different cell types are factors that are regularly neglected, but should be taken into account for a meaningful analysis. In this study, the importance

  9. Activated protein C: biased for translation

    PubMed Central

    Zlokovic, Berislav V.; Mosnier, Laurent O.

    2015-01-01

    The homeostatic blood protease, activated protein C (APC), can function as (1) an antithrombotic on the basis of inactivation of clotting factors Va and VIIIa; (2) a cytoprotective on the basis of endothelial barrier stabilization and anti-inflammatory and antiapoptotic actions; and (3) a regenerative on the basis of stimulation of neurogenesis, angiogenesis, and wound healing. Pharmacologic therapies using recombinant human and murine APCs indicate that APC provides effective acute or chronic therapies for a strikingly diverse range of preclinical injury models. APC reduces the damage caused by the following: ischemia/reperfusion in brain, heart, and kidney; pulmonary, kidney, and gastrointestinal inflammation; sepsis; Ebola virus; diabetes; and total lethal body radiation. For these beneficial effects, APC alters cell signaling networks and gene expression profiles by activating protease-activated receptors 1 and 3. APC’s activation of these G protein–coupled receptors differs completely from thrombin’s activation mechanism due to biased signaling via either G proteins or β-arrestin-2. To reduce APC-associated bleeding risk, APC variants were engineered to lack >90% anticoagulant activity but retain normal cell signaling. Such a neuroprotective variant, 3K3A-APC (Lys191-193Ala), has advanced to clinical trials for ischemic stroke. A rich data set of preclinical knowledge provides a solid foundation for potential translation of APC variants to future novel therapies. PMID:25824691

  10. Diethyl pyrocarbonate reaction with the lactose repressor protein affects both inducer and DNA binding

    SciTech Connect

    Sams, C.F.; Matthews, K.S.

    1988-04-05

    Modification of the lactose repressor protein of Escherichia coli with diethyl pyrocarbonate (DPC) results in decreased inducer binding as well as operator and nonspecific DNA binding. Spectrophotometric measurements indicated a maximum of three histidines per subunit was modified, and quantitation of lysine residues with trinitrobenzenesulfonate revealed the modification of one lysine residue. The loss of DNA binding, both operator and nonspecific, was correlated with histidine modification; removal of the carbethoxy groups from the histidines by hydroxylamine was accompanied by significant recovery of DNA binding function. The presence of inducing sugars during the DPC reaction had no effect on histidine modification or the loss of DNA binding activity. In contrast, inducer binding was not recovered upon reversal of the histidine modification. However, the presence of inducer during reaction protected lysine from reaction and also prevented the decrease in inducer binding; these results indicate that reaction of the lysine residue(s) may correlate to the loss of sugar binding activity. Since no difference in incorporation of radiolabeled carbethoxy was observed following reaction with diethyl pyrocarbonate in the presence or absence of inducer, the reagent appears to function as a catalyst in the modification of the lysine. The formation of an amide bond between the affected lysine and a nearby carboxylic acid moiety provides a possible mechanism for the activity loss. Reaction of the isolated NH2-terminal domain resulted in loss of DNA binding with modification of the single histidine at position 29. Results from the modification of core domain paralleled observations with intact repressor.

  11. 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

  12. Lipid Dependent Mechanisms of Protein Pump Activity

    DTIC Science & Technology

    1989-05-23

    properties which result form the colligative interactions of many lipid molecules. Important materials properties include . . . i I I II II I i I 1 the...d identify by olock number) *This project is aime at investigating if a lipid elastic property , known as the spontaneous radius of curvature Ro’, is...a regulated membrane property and if its value modulates membrane protein activity. Specific aims reported on here include: 1) Correlation of ion pump

  13. Hyperosmotic and isosmotic shrinkage differentially affect protein phosphorylation and ion transport.

    PubMed

    Koltsova, Svetlana V; Akimova, Olga A; Kotelevtsev, Sergei V; Grygorczyk, Ryszard; Orlov, Sergei N

    2012-02-01

    In the present work, we compared the outcome of hyperosmotic and isosmotic shrinkage on ion transport and protein phosphorylation in C11-MDCK cells resembling intercalated cells from collecting ducts and in vascular smooth muscle cells (VSMC) from the rat aorta. Hyperosmotic shrinkage was triggered by cell exposure to hypertonic medium, whereas isosmotic shrinkage was evoked by cell transfer from an hypoosmotic to an isosmotic environment. Despite a similar cell volume decrease of 40%-50%, the consequences of hyperosmotic and isosmotic shrinkage on cellular functions were sharply different. In C11-MDCK and VSMC, hyperosmotic shrinkage completely inhibited Na(+),K(+)-ATPase and Na(+),P(i) cotransport. In contrast, in both types of cells isosmotic shrinkage slightly increased rather than suppressed Na(+),K(+)-ATPase and did not change Na(+),P(i) cotransport. In C11-MDCK cells, phosphorylation of JNK1/2 and Erk1/2 mitogen-activated protein kinases was augmented in hyperosmotically shrunken cells by ∼7- and 2-fold, respectively, but was not affected in cells subjected to isosmotic shrinkage. These results demonstrate that the data obtained in cells subjected to hyperosmotic shrinkage cannot be considered as sufficient proof implicating cell volume perturbations in the regulation of cellular functions under isosmotic conditions.

  14. Nanog RNA-binding proteins YBX1 and ILF3 affect pluripotency of embryonic stem cells.

    PubMed

    Guo, Chuanliang; Xue, Yan; Yang, Guanheng; Yin, Shang; Shi, Wansheng; Cheng, Yan; Yan, Xiaoshuang; Fan, Shuyue; Zhang, Huijun; Zeng, Fanyi

    2016-08-01

    Nanog is a well-known transcription factor that plays a fundamental role in stem cell self-renewal and the maintenance of their pluripotent cell identity. There remains a large data gap with respect to the spectrum of the key pluripotency transcription factors' interaction partners. Limited information is available concerning Nanog-associated RNA-binding proteins (RBPs), and the intrinsic protein-RNA interactions characteristic of the regulatory activities of Nanog. Herein, we used an improved affinity protocol to purify Nanog-interacting RBPs from mouse embryonic stem cells (ESCs), and 49 RBPs of Nanog were identified. Among them, the interaction of YBX1 and ILF3 with Nanog mRNA was further confirmed by in vitro assays, such as Western blot, RNA immunoprecipitation (RIP), and ex vivo methods, such as immunofluorescence staining and fluorescent in situ hybridization (FISH), MS2 in vivo biotin-tagged RNA affinity purification (MS2-BioTRAP). Interestingly, RNAi studies revealed that YBX1 and ILF3 positively affected the expression of Nanog and other pluripotency-related genes. Particularly, downregulation of YBX1 or ILF3 resulted in high expression of mesoderm markers. Thus, a reduction in the expression of YBX1 and ILF3 controls the expression of pluripotency-related genes in ESCs, suggesting their roles in further regulation of the pluripotent state of ESCs.

  15. ceRNA crosstalk stabilizes protein expression and affects the correlation pattern of interacting proteins.

    PubMed

    Martirosyan, Araks; De Martino, Andrea; Pagnani, Andrea; Marinari, Enzo

    2017-03-07

    Gene expression is a noisy process and several mechanisms, both transcriptional and post-transcriptional, can stabilize protein levels in cells. Much work has focused on the role of miRNAs, showing in particular that miRNA-mediated regulation can buffer expression noise for lowly expressed genes. Here, using in silico simulations and mathematical modeling, we demonstrate that miRNAs can exert a much broader influence on protein levels by orchestrating competition-induced crosstalk between mRNAs. Most notably, we find that miRNA-mediated cross-talk (i) can stabilize protein levels across the full range of gene expression rates, and (ii) modifies the correlation pattern of co-regulated interacting proteins, changing the sign of correlations from negative to positive. The latter feature may constitute a potentially robust signature of the existence of RNA crosstalk induced by endogenous competition for miRNAs in standard cellular conditions.

  16. ceRNA crosstalk stabilizes protein expression and affects the correlation pattern of interacting proteins

    PubMed Central

    Martirosyan, Araks; De Martino, Andrea; Pagnani, Andrea; Marinari, Enzo

    2017-01-01

    Gene expression is a noisy process and several mechanisms, both transcriptional and post-transcriptional, can stabilize protein levels in cells. Much work has focused on the role of miRNAs, showing in particular that miRNA-mediated regulation can buffer expression noise for lowly expressed genes. Here, using in silico simulations and mathematical modeling, we demonstrate that miRNAs can exert a much broader influence on protein levels by orchestrating competition-induced crosstalk between mRNAs. Most notably, we find that miRNA-mediated cross-talk (i) can stabilize protein levels across the full range of gene expression rates, and (ii) modifies the correlation pattern of co-regulated interacting proteins, changing the sign of correlations from negative to positive. The latter feature may constitute a potentially robust signature of the existence of RNA crosstalk induced by endogenous competition for miRNAs in standard cellular conditions. PMID:28266541

  17. Feeding modality affects muscle protein deposition by influencing protein synthesis, but not degradation in muscle of neonatal pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Neonatal pigs can serve as dual-use models for nutrition research in animal agriculture and biomedical fields. To determine how feeding modality by either intermittent bolus or continuous schedule affects protein anabolism and catabolism, neonatal pigs (n = 6/group, 9-d-old) were overnight fasted (F...

  18. Zinc supplementation affects the activity patterns of rural Guatemalan infants.

    PubMed

    Bentley, M E; Caulfield, L E; Ram, M; Santizo, M C; Hurtado, E; Rivera, J A; Ruel, M T; Brown, K H

    1997-07-01

    Zinc deficiency has been associated with growth deficits, reduced dietary intake and appetite, and has been hypothesized to result in reduced activity. This randomized, double-blind, placebo-controlled study examined whether 10 mg of oral zinc as zinc sulfate, given daily for up to 7 mo, affected activity patterns of 85 Guatemalan infants recruited at 6-9 mo of age. Infant activity was assessed by time sampling-observation method at 10-min intervals during a 12-h data collection period, at base line, 3 and 7 mo follow-up. Motor development and the percentage of time infants were observed in various positions (being carried, lying down, sitting, crawling, standing or walking) and engaged in various activities (eating, sleeping, resting, crying/whining or playing) were compared by treatment group. No differences in motor development were observed by treatment group. However, at follow-up 2 (after 7 mo of supplementation), zinc-supplemented infants were significantly more frequently observed sitting up compared with lying down, and were playing during 4.18 +/- 1.95% (P < 0.05) more observations than unsupplemented infants. They were also somewhat less likely to be observed crying or whining (P < 0.10) compared with those receiving the placebo. These effects are independent of other factors including infant age, motor development, sex, maternal education, family socioeconomic status and nutritional status at base line. Further research must be conducted to determine the long-term developmental importance of these differences in activity patterns associated with zinc supplementation in this setting.

  19. How Lipid Membranes Affect Pore Forming Toxin Activity.

    PubMed

    Rojko, Nejc; Anderluh, Gregor

    2015-12-15

    , events associated with pore formation can modulate properties of the lipid membrane and affect its organization. Model membranes do not necessarily reproduce the physicochemical properties of the native cellular membrane, and caution is needed when transferring results from model to native lipid membranes. In this context, the utilization of novel approaches that enable studying PFTs on living cells at a single molecule level should reveal complex protein-lipid membrane interactions in greater detail.

  20. 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

  1. Steric effects in peptide and protein exchange with activated disulfides.

    PubMed

    Kerr, Jason; Schlosser, Jessica L; Griffin, Donald R; Wong, Darice Y; Kasko, Andrea M

    2013-08-12

    Disulfide exchange is an important bioconjugation tool, enabling chemical modification of peptides and proteins containing free cysteines. We previously reported the synthesis of a macromer bearing an activated disulfide and its incorporation into hydrogels. Despite their ability to diffuse freely into hydrogels, larger proteins were unable to undergo in-gel disulfide exchange. In order to understand this phenomenon, we synthesized four different activated disulfide-bearing model compounds (Mn = 300 Da to 10 kDa) and quantified their rate of disulfide exchange with a small peptide (glutathione), a moderate-sized protein (β-lactoglobulin), and a large protein (bovine serum albumin) in four different pH solutions (6.0, 7.0, 7.4, and 8.0) to mimic biological systems. Rate constants of exchange depend significantly on the size and accessibility of the thiolate. pH also significantly affects the rate of reaction, with the faster reactions occurring at higher pH. Surprisingly, little difference in exchange rates is seen between macromolecular disulfides of varying size (Mn = 2 kDa - 10 kDa), although all undergo exchange more slowly than their small molecule analogue (MW = 300 g/mol). The maximum exchange efficiencies (% disulfides exchanged after 24 h) are not siginificantly affected by thiol size or pH, but somewhat affected by disulfide size. Therefore, while all three factors investigated (pH, disulfide size, and thiolate size) can influence the exchange kinetics and extent of reaction, the size of the thiolate and its accessibility plays the most significant role.

  2. Induced lung inflammation and dietary protein supply affect nitrogen retention and amino acid metabolism in growing pigs.

    PubMed

    Kampman-van de Hoek, Esther; Sakkas, Panagiotis; Gerrits, Walter J J; van den Borne, Joost J G C; van der Peet-Schwering, Carola M C; Jansman, Alfons J M

    2015-02-14

    It is hypothesised that during immune system activation, there is a competition for amino acids (AA) between body protein deposition and immune system functioning. The aim of the present study was to quantify the effect of immune system activation on N retention and AA metabolism in growing pigs, depending on dietary protein supply. A total of sixteen barrows received an adequate (Ad) or restricted (Res) amount of dietary protein, and were challenged at day 0 with intravenous complete Freund's adjuvant (CFA). At days - 5, 3 and 8, an irreversible loss rate (ILR) of eight AA was determined. CFA successfully activated the immune system, as indicated by a 2- to 4-fold increase in serum concentrations of acute-phase proteins (APP). Pre-challenge C-reactive protein concentrations were lower (P< 0·05) and pre- and post-challenge albumin tended to be lower in Res-pigs. These findings indicate that a restricted protein supply can limit the acute-phase response. CFA increased urinary N losses (P= 0·04) and tended to reduce N retention in Ad-pigs, but not in Res-pigs (P= 0·07). The ILR for Val was lower (P= 0·05) at day 8 than at day 3 in the post-challenge period. The ILR of most AA, except for Trp, were strongly affected by dietary protein supply and positively correlated with N retention. The correlations between the ILR and APP indices were absent or negative, indicating that changes in AA utilisation for APP synthesis were either not substantial or more likely outweighed by a decrease in muscle protein synthesis during immune system activation in growing pigs.

  3. Arginine depletion by arginine deiminase does not affect whole protein metabolism or muscle fractional protein synthesis rate in mice.

    PubMed

    Marini, Juan C; Didelija, Inka Cajo

    2015-01-01

    Due to the absolute need for arginine that certain cancer cells have, arginine depletion is a therapy in clinical trials to treat several types of cancers. Arginine is an amino acids utilized not only as a precursor for other important molecules, but also for protein synthesis. Because arginine depletion can potentially exacerbate the progressive loss of body weight, and especially lean body mass, in cancer patients we determined the effect of arginine depletion by pegylated arginine deiminase (ADI-PEG 20) on whole body protein synthesis and fractional protein synthesis rate in multiple tissues of mice. ADI-PEG 20 successfully depleted circulating arginine (<1 μmol/L), and increased citrulline concentration more than tenfold. Body weight and body composition, however, were not affected by ADI-PEG 20. Despite the depletion of arginine, whole body protein synthesis and breakdown were maintained in the ADI-PEG 20 treated mice. The fractional protein synthesis rate of muscle was also not affected by arginine depletion. Most tissues (liver, kidney, spleen, heart, lungs, stomach, small and large intestine, pancreas) were able to maintain their fractional protein synthesis rate; however, the fractional protein synthesis rate of brain, thymus and testicles was reduced due to the ADI-PEG 20 treatment. Furthermore, these results were confirmed by the incorporation of ureido [14C]citrulline, which indicate the local conversion into arginine, into protein. In conclusion, the intracellular recycling pathway of citrulline is able to provide enough arginine to maintain protein synthesis rate and prevent the loss of lean body mass and body weight.

  4. MAPKAP kinase-2; a novel protein kinase activated by mitogen-activated protein kinase.

    PubMed Central

    Stokoe, D; Campbell, D G; Nakielny, S; Hidaka, H; Leevers, S J; Marshall, C; Cohen, P

    1992-01-01

    A novel protein kinase, which was only active when phosphorylated by the mitogen-activated protein kinase (MAP kinase), has been purified 85,000-fold to homogeneity from rabbit skeletal muscle. This MAP kinase activated protein kinase, termed MAPKAP kinase-2, was distinguished from S6 kinase-II (MAPKAP kinase-1) by its response to inhibitors, lack of phosphorylation of S6 peptides and amino acid sequence. MAPKAP kinase-2 phosphorylated glycogen synthase at Ser7 and the equivalent serine (*) in the peptide KKPLNRTLS*VASLPGLamide whose sequence is similar to the N terminus of glycogen synthase. MAPKAP kinase-2 was resolved into two monomeric species of apparent molecular mass 60 and 53 kDa that had similar specific activities and substrate specificities. Peptide sequences of the 60 and 53 kDa species were identical, indicating that they are either closely related isoforms or derived from the same gene. MAP kinase activated the 60 and 53 kDa forms of MAPKAP kinase-2 by phosphorylating the first threonine residue in the sequence VPQTPLHTSR. Furthermore, Mono Q chromatography of extracts from rat phaeochromocytoma and skeletal muscle demonstrated that two MAP kinase isoforms (p42mapk and p44mapk) were the only enzymes in these cells that were capable of reactivating MAPKAP kinase-2. These results indicate that MAP kinase activates at least two distinct protein kinases, suggesting that it represents a point at which the growth factor-stimulated protein kinase cascade bifurcates. Images PMID:1327754

  5. Crumbs affects protein dynamics in anterior regions of the developing Drosophila embryo.

    PubMed

    Firmino, João; Tinevez, Jean-Yves; Knust, Elisabeth

    2013-01-01

    Maintenance of apico-basal polarity is essential for epithelial integrity and requires particular reinforcement during tissue morphogenesis, when cells are reorganised, undergo shape changes and remodel their junctions. It is well established that epithelial integrity during morphogenetic processes depends on the dynamic exchange of adherens junction components, but our knowledge on the dynamics of other proteins and their dynamics during these processes is still limited. The early Drosophila embryo is an ideal system to study membrane dynamics during morphogenesis. Here, morphogenetic activities differ along the anterior-posterior axis, with the extending germband showing a high degree of epithelial remodelling. We developed a Fluorescence Recovery After Photobleaching (FRAP) assay with a higher temporal resolution, which allowed the distinction between a fast and a slow component of recovery of membrane proteins during the germband extension stage. We show for the first time that the recovery kinetics of a general membrane marker, SpiderGFP, differs in the anterior and posterior parts of the embryo, which correlates well with the different morphogenetic activities of the respective embryonic regions. Interestingly, absence of crumbs, a polarity regulator essential for epithelial integrity in the Drosophila embryo, decreases the fast component of SpiderGFP and of the apical marker Stranded at Second-Venus specifically in the anterior region. We suggest that the defects in kinetics observed in crumbs mutant embryos are the first signs of tissue instability in this region, explaining the earlier breakdown of the head epidermis in comparison to that of the trunk, and that diffusion in the plasma membrane is affected by the absence of Crumbs.

  6. Morbillivirus and henipavirus attachment protein cytoplasmic domains differently affect protein expression, fusion support and particle assembly.

    PubMed

    Sawatsky, Bevan; Bente, Dennis A; Czub, Markus; von Messling, Veronika

    2016-05-01

    The amino-terminal cytoplasmic domains of paramyxovirus attachment glycoproteins include trafficking signals that influence protein processing and cell surface expression. To characterize the role of the cytoplasmic domain in protein expression, fusion support and particle assembly in more detail, we constructed chimeric Nipah virus (NiV) glycoprotein (G) and canine distemper virus (CDV) haemagglutinin (H) proteins carrying the respective heterologous cytoplasmic domain, as well as a series of mutants with progressive deletions in this domain. CDV H retained fusion function and was normally expressed on the cell surface with a heterologous cytoplasmic domain, while the expression and fusion support of NiV G was dramatically decreased when its cytoplasmic domain was replaced with that of CDV H. The cell surface expression and fusion support functions of CDV H were relatively insensitive to cytoplasmic domain deletions, while short deletions in the corresponding region of NiV G dramatically decreased both. In addition, the first 10 residues of the CDV H cytoplasmic domain strongly influence its incorporation into virus-like particles formed by the CDV matrix (M) protein, while the co-expression of NiV M with NiV G had no significant effect on incorporation of G into particles. The cytoplasmic domains of both the CDV H and NiV G proteins thus contribute differently to the virus life cycle.

  7. Gel-free proteomic analysis of soybean root proteins affected by calcium under flooding stress

    PubMed Central

    Oh, MyeongWon; Nanjo, Yohei; Komatsu, Setsuko

    2014-01-01

    Soybean is sensitive to flooding stress and exhibits reduced growth under flooding conditions. To better understand the flooding-responsive mechanisms of soybean, the effect of exogenous calcium on flooding-stressed soybeans was analyzed using proteomic technique. An increase in exogenous calcium levels enhanced soybean root elongation and suppressed the cell death of root tip under flooding stress. Proteins were extracted from the roots of 4-day-old soybean seedlings exposed to flooding stress without or with calcium for 2 days and analyzed using gel-free proteomic technique. Proteins involved in protein degradation/synthesis/posttranslational modification, hormone/cell wall metabolisms, and DNA synthesis were decreased by flooding stress; however, their reductions were recovered by calcium treatment. Development, lipid metabolism, and signaling-related proteins were increased in soybean roots when calcium was supplied under flooding stress. Fermentation and glycolysis-related proteins were increased in response to flooding; however, these proteins were not affected by calcium supplementation. Furthermore, urease and copper chaperone proteins exhibited similar profiles in 4-day-old untreated soybeans and 4-day-old soybeans exposed to flooding for 2 days in the presence of calcium. These results suggest that calcium might affect the cell wall/hormone metabolisms, protein degradation/synthesis, and DNA synthesis in soybean roots under flooding stress. PMID:25368623

  8. Activation and activities of the p53 tumour suppressor protein

    PubMed Central

    Bálint, É; Vousden, K H

    2001-01-01

    The p53 tumour suppressor protein inhibits malignant progression by mediating cell cycle arrest, apoptosis or repair following cellular stress. One of the major regulators of p53 function is the MDM2 protein, and multiple forms of cellular stress activate p53 by inhibiting the MDM2-mediated degradation of p53. Mutations in p53, or disruption of the pathways that allow activation of p53, seem to be a general feature of all cancers. Here we review recent advances in our understanding of the pathways that regulate p53 and the pathways that are induced by p53, as well as their implications for cancer therapy. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11747320

  9. Cholesterol-Lowering Activity of Tartary Buckwheat Protein.

    PubMed

    Zhang, Chengnan; Zhang, Rui; Li, Yuk Man; Liang, Ning; Zhao, Yimin; Zhu, Hanyue; He, Zouyan; Liu, Jianhui; Hao, Wangjun; Jiao, Rui; Ma, Ka Ying; Chen, Zhen-Yu

    2017-03-08

    Previous research has shown that Tartary buckwheat flour is capable of reducing plasma cholesterol. The present study was to examine the effect of rutin and Tartary buckwheat protein on plasma total cholesterol (TC) in hypercholesterolemia hamsters. In the first animal experiment, 40 male hamsters were divided into four groups fed either the control diet or one of the three experimental diets containing 8.2 mmol rutin, 8.2 mmol quercetin, or 2.5 g kg(-1) cholestyramine, respectively. Results showed that only cholestyramine but not rutin and its aglycone quercetin decreased plasma TC, which suggested that rutin was not the active ingredient responsible for plasma TC-lowering activity of Tartary buckwheat flour. In the second animal experiment, 45 male hamsters were divided into five groups fed either the control diet or one of the four experimental diets containing 24% Tartary buckwheat protein, 24% rice protein, 24% wheat protein, or 5 g kg(-1) cholestyramine, respectively. Tartary buckwheat protein reduced plasma TC more effectively than cholestyramine (45% versus 37%), while rice and wheat proteins only reduced plasma TC by 10-13%. Tartary buckwheat protein caused 108% increase in the fecal excretion of total neutral sterols and 263% increase in the fecal excretion of total acidic sterols. real-time polymerase chain reaction and Western blotting analyses showed that Tartary buckwheat protein affected the gene expression of intestinal Niemann-Pick C1-like protein 1 (NPC1L1), acyl CoA:cholesterol acyltransferase 2 (ACAT2), and ATP binding cassette transporters 5 and 8 (ABCG5/8) in a down trend, whereas it increased the gene expression of hepatic cholesterol-7α -hydroxylase (CYP7A1). It was concluded that Tartary buckwheat protein was at least one of the active ingredients in Tartary buckwheat flour to lower plasma TC, mainly mediated by enhancing the excretion of bile acids via up-regulation of hepatic CYP7A1 and also by inhibiting the absorption of dietary

  10. Inbreeding affects locomotor activity in Drosophila melanogaster at different ages.

    PubMed

    Manenti, Tommaso; Pertoldi, Cino; Moghadam, Neda Nasiri; Nasiri, Neda; Schou, Mads Fristrup; Kjærsgaard, Anders; Cavicchi, Sandro; Loeschcke, Volker

    2015-01-01

    The ability to move is essential for many behavioural traits closely related to fitness. Here we studied the effect of inbreeding on locomotor activity (LA) of Drosophila melanogaster at different ages under both dark and light regimes. We expected to find a decreased LA in inbred lines compared to control lines. We also predicted an increased differentiation between lines due to inbreeding. LA was higher in the dark compared to the light regime for both inbred and outbred control lines. As expected, inbreeding increased phenotypic variance in LA, with some inbred lines showing higher and some lower LA than control lines. Moreover, age per se did not affect LA neither in control nor in inbred lines, while we found a strong line by age interaction between inbred lines. Interestingly, inbreeding changed the daily activity pattern of the flies: these patterns were consistent across all control lines but were lost in some inbred lines. The departure in the daily pattern of LA in inbred lines may contribute to the inbreeding depression observed in inbred natural populations.

  11. White wine proteins: how does the pH affect their conformation at room temperature?

    PubMed

    Dufrechou, Marie; Vernhet, Aude; Roblin, Pierre; Sauvage, François-Xavier; Poncet-Legrand, Céline

    2013-08-20

    Our studies focused on the determination of aggregation mechanisms of proteins occurring in wine at room temperature. Even if the wine pH range is narrow (2.8 to 3.7), some proteins are affected by this parameter. At low pH, the formation of aggregates and the development of a haze due to proteins sometimes occur. The objective of this work was to determine if the pH impacted the conformational stability of wine proteins. Different techniques were used: circular dichroism and fluorescence spectroscopy to investigate the modification of their secondary and tertiary structure and also SAXS to determine their global shape. Four pure proteins were used, two considered to be stable (invertase and thaumatin-like proteins) and two considered to be unstable (two chitinase isoforms). Two pH values were tested to emphasize their behavior (pH 2.5 and 4.0). The present work highlighted the fact that the conformational stability of some wine proteins (chitinases) was impacted by partial modifications, related to the exposure of some hydrophobic sites. These modifications were enough to destabilize the native state of the protein. These modifications were not observed on wine proteins determined to be stable (invertase and thaumatin-like proteins).

  12. Interaction of Protein Inhibitor of Activated STAT (PIAS) Proteins with the TATA-binding Protein, TBP*

    PubMed Central

    Prigge, Justin R.; Schmidt, Edward E.

    2007-01-01

    Transcription activators often recruit promoter-targeted assembly of a pre-initiation complex; many repressors antagonize recruitment. These activities can involve direct interactions with proteins in the pre-initiation complex. We used an optimized yeast two-hybrid system to screen mouse pregnancy-associated libraries for proteins that interact with TATA-binding protein (TBP). Screens revealed an interaction between TBP and a single member of the zinc finger family of transcription factors, ZFP523. Two members of the protein inhibitor of activated STAT (PIAS) family, PIAS1 and PIAS3, also interacted with TBP in screens. Endogenous PIAS1 and TBP co-immunoprecipitated from nuclear extracts, suggesting the interaction occurred in vivo. In vitro-translated PIAS1 and TBP coimmunopreciptated, which indicated that other nuclear proteins were not required for the interaction. Deletion analysis mapped the PIAS-interacting domain of TBP to the conserved TBPCORE and the TBP-interacting domain on PIAS1 to a 39-amino acid C-terminal region. Mammals issue seven known PIAS proteins from four pias genes, pias1, pias3, piasx, and piasy, each with different cell type-specific expression patterns; the TBP-interacting domain reported here is the only part of the PIAS C-terminal region shared by all seven PIAS proteins. Direct analyses indicated that PIASx and PIASy also interacted with TBP. Our results suggest that all PIAS proteins might mediate situation-specific regulatory signaling at the TBP interface and that previously unknown levels of complexity could exist in the gene regulatory interplay between TBP, PIAS proteins, ZFP523, and other transcription factors. PMID:16522640

  13. Streptomycin affects the growth and photochemical activity of the alga Chlorella vulgaris.

    PubMed

    Perales-Vela, Hugo Virgilio; García, Roberto Velasco; Gómez-Juárez, Evelyn Alicia; Salcedo-Álvarez, Martha Ofelia; Cañizares-Villanueva, Rosa Olivia

    2016-10-01

    Antibiotics are increasingly being used in human and veterinary medicine, as well as pest control in agriculture. Recently, their emergence in the aquatic environment has become a global concern. The aim of this study was to evaluate the effect of streptomycin on growth and photosynthetic activity of Chlorella vulgaris after 72h exposure. We found that growth, photosynthetic activity and the content of the D1 protein of photosystem II decreased. Analysis of chlorophyll a fluorescence emission shows a reduction in the energy transfer between the antenna complex and reaction center. Also the activity of the oxygen evolution complex and electron flow between QA and QB were significantly reduced; in contrast, we found an increase in the reduction rate of the acceptor side of photosystem I. The foregoing can be attributed to the inhibition of the synthesis of the D1 protein and perhaps other coded chloroplast proteins that are part of the electron transport chain which are essential for the transformation of solar energy in the photosystems. We conclude that micromolar concentrations of streptomycin can affect growth and photosynthetic activity of Chlorella vulgaris. The accumulation of antibiotics in the environment can become an ecological problem for primary producers in the aquatic environment.

  14. Prolonged leucine infusion differentially affects tissue protein synthesis in neonatal pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Leucine (Leu) acutely stimulates protein synthesis by activating the mammalian target of rapamycin complex 1 (mTORC1) pathway. To determine whether Leu can stimulate protein synthesis in muscles of different fiber types and visceral tissues of the neonate for a prolonged period and to determine the ...

  15. Differential regulation of Gli proteins by Sufu in the lung affects PDGF signaling and myofibroblast development

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mammalian Hedgehog (Hh) signaling relies on three Gli transcription factors to mediate Hh responses. This process is controlled in part by a major negative regulator, Sufu, through its effects on Gli protein level, distribution and activity. In this report, we showed that Sufu regulates Gli1 protein...

  16. The Role of Mitogen-Activated Protein Kinase-Activated Protein Kinases (MAPKAPKs) in Inflammation

    PubMed Central

    Moens, Ugo; Kostenko, Sergiy; Sveinbjørnsson, Baldur

    2013-01-01

    Mitogen-activated protein kinase (MAPK) pathways are implicated in several cellular processes including proliferation, differentiation, apoptosis, cell survival, cell motility, metabolism, stress response and inflammation. MAPK pathways transmit and convert a plethora of extracellular signals by three consecutive phosphorylation events involving a MAPK kinase kinase, a MAPK kinase, and a MAPK. In turn MAPKs phosphorylate substrates, including other protein kinases referred to as MAPK-activated protein kinases (MAPKAPKs). Eleven mammalian MAPKAPKs have been identified: ribosomal-S6-kinases (RSK1-4), mitogen- and stress-activated kinases (MSK1-2), MAPK-interacting kinases (MNK1-2), MAPKAPK-2 (MK2), MAPKAPK-3 (MK3), and MAPKAPK-5 (MK5). The role of these MAPKAPKs in inflammation will be reviewed. PMID:24705157

  17. Activity-dependent Protein Dynamics Define Interconnected Cores of Co-regulated Postsynaptic Proteins*

    PubMed Central

    Trinidad, Jonathan C.; Thalhammer, Agnes; Burlingame, Alma L.; Schoepfer, Ralf

    2013-01-01

    Synapses are highly dynamic structures that mediate cell–cell communication in the central nervous system. Their molecular composition is altered in an activity-dependent fashion, which modulates the efficacy of subsequent synaptic transmission events. Whereas activity-dependent trafficking of individual key synaptic proteins into and out of the synapse has been characterized previously, global activity-dependent changes in the synaptic proteome have not been studied. To test the feasibility of carrying out an unbiased large-scale approach, we investigated alterations in the molecular composition of synaptic spines following mass stimulation of the central nervous system induced by pilocarpine. We observed widespread changes in relative synaptic abundances encompassing essentially all proteins, supporting the view that the molecular composition of the postsynaptic density is tightly regulated. In most cases, we observed that members of gene families displayed coordinate regulation even when they were not known to physically interact. Analysis of correlated synaptic localization revealed a tightly co-regulated cluster of proteins, consisting of mainly glutamate receptors and their adaptors. This cluster constitutes a functional core of the postsynaptic machinery, and changes in its size affect synaptic strength and synaptic size. Our data show that the unbiased investigation of activity-dependent signaling of the postsynaptic density proteome can offer valuable new information on synaptic plasticity. PMID:23035237

  18. The RecX protein interacts with the RecA protein and modulates its activity in Herbaspirillum seropedicae

    PubMed Central

    Galvão, C.W.; Souza, E.M.; Etto, R.M.; Pedrosa, F.O.; Chubatsu, L.S.; Yates, M.G.; Schumacher, J.; Buck, M.; Steffens, M.B.R.

    2012-01-01

    DNA repair is crucial to the survival of all organisms. The bacterial RecA protein is a central component in the SOS response and in recombinational and SOS DNA repairs. The RecX protein has been characterized as a negative modulator of RecA activity in many bacteria. The recA and recX genes of Herbaspirillum seropedicae constitute a single operon, and evidence suggests that RecX participates in SOS repair. In the present study, we show that the H. seropedicae RecX protein (RecXHs) can interact with the H. seropedicae RecA protein (RecAHs) and that RecAHs possesses ATP binding, ATP hydrolyzing and DNA strand exchange activities. RecXHs inhibited 90% of the RecAHs DNA strand exchange activity even when present in a 50-fold lower molar concentration than RecAHs. RecAHs ATP binding was not affected by the addition of RecX, but the ATPase activity was reduced. When RecXHs was present before the formation of RecA filaments (RecA-ssDNA), inhibition of ATPase activity was substantially reduced and excess ssDNA also partially suppressed this inhibition. The results suggest that the RecXHs protein negatively modulates the RecAHs activities by protein-protein interactions and also by DNA-protein interactions. PMID:23044625

  19. Eccentric exercise activates novel transcriptional regulation of hypertrophic signaling pathways not affected by hormone changes.

    PubMed

    MacNeil, Lauren G; Melov, Simon; Hubbard, Alan E; Baker, Steven K; Tarnopolsky, Mark A

    2010-05-18

    Unaccustomed eccentric exercise damages skeletal muscle tissue, activating mechanisms of recovery and remodeling that may be influenced by the female sex hormone 17beta-estradiol (E2). Using high density oligonucleotide based microarrays, we screened for differences in mRNA expression caused by E2 and eccentric exercise. After random assignment to 8 days of either placebo (CON) or E2 (EXP), eighteen men performed 150 single-leg eccentric contractions. Muscle biopsies were collected at baseline (BL), following supplementation (PS), +3 hours (3H) and +48 hours (48H) after exercise. Serum E2 concentrations increased significantly with supplementation (P<0.001) but did not affect microarray results. Exercise led to early transcriptional changes in striated muscle activator of Rho signaling (STARS), Rho family GTPase 3 (RND3), mitogen activated protein kinase (MAPK) regulation and the downstream transcription factor FOS. Targeted RT-PCR analysis identified concurrent induction of negative regulators of calcineurin signaling RCAN (P<0.001) and HMOX1 (P = 0.009). Protein contents were elevated for RND3 at 3H (P = 0.02) and FOS at 48H (P<0.05). These findings indicate that early RhoA and NFAT signaling and regulation are altered following exercise for muscle remodeling and repair, but are not affected by E2.

  20. Actin depolymerization affects stress-induced translational activity of potato tuber tissue

    PubMed

    Morelli; Zhou; Yu; Lu; Vayda

    1998-04-01

    Changes in polymerized actin during stress conditions were correlated with potato (Solanum tuberosum L.) tuber protein synthesis. Fluorescence microscopy and immunoblot analyses indicated that filamentous actin was nearly undetectable in mature, quiescent aerobic tubers. Mechanical wounding of postharvest tubers resulted in a localized increase of polymerized actin, and microfilament bundles were visible in cells of the wounded periderm within 12 h after wounding. During this same period translational activity increased 8-fold. By contrast, low-oxygen stress caused rapid reduction of polymerized actin coincident with acute inhibition of protein synthesis. Treatment of aerobic tubers with cytochalasin D, an agent that disrupts actin filaments, reduced wound-induced protein synthesis in vivo. This effect was not observed when colchicine, an agent that depolymerizes microtubules, was used. Neither of these drugs had a significant effect in vitro on run-off translation of isolated polysomes. However, cytochalasin D did reduce translational competence in vitro of a crude cellular fraction containing both polysomes and cytoskeletal elements. These results demonstrate the dependence of wound-induced protein synthesis on the integrity of microfilaments and suggest that the dynamics of the actin cytoskeleton may affect translational activity during stress conditions.

  1. 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

  2. Relaxation training affects success and activation on a teaching test.

    PubMed

    Helin, P; Hänninen, O

    1987-12-01

    We studied the effects of an audiocassette-relaxation training period (ART) and its timing on success at a teaching test (lecture type), on observed tension and on a number of physiological responses. The electrical activity of the upper trapezius muscle (EMG), heart rate (HR) and blood pressure (BP), of female and male instructor candidates, were examined before, during and after the teaching test as well as during its critique. The relaxation period (18 min) was presented either on the preceding night (ARTnt) or immediately before the teaching test (ARTimm). The influence of personality (types A-B and extrovert-introvert) was also studied. ART improved success at the teaching test in both sexes. In males (but not in females), ARTimm decreased EMG level during the test, but ARTnt increased EMG at the test period as compared to the control group. In females, both ARTnt and ARTimm lowered HR more than in the control group. ARTimm lowered systolic BP in both sexes. Personality types affected the ART responses; ART was more beneficial for type A than B subjects.

  3. The relationship between activating affects, inhibitory affects, and self-compassion in patients with Cluster C personality disorders.

    PubMed

    Schanche, Elisabeth; Stiles, Tore C; McCullough, Leigh; Svartberg, Martin; Nielsen, Geir Høstmark

    2011-09-01

    In the short-term dynamic psychotherapy model termed "Affect Phobia Treatment," it is assumed that increase in patients' defense recognition, decrease in inhibitory affects (e.g., anxiety, shame, guilt), and increase in the experience of activating affects (e.g., sadness, anger, closeness) are related to enhanced self-compassion across therapeutic approaches. The present study aimed to test this assumption on the basis of data from a randomized controlled trial, which compared a 40-session short-term dynamic psychotherapy (N = 25) with 40-session cognitive treatment (N = 25) for outpatients with Cluster C personality disorders. Patients' defense recognition, inhibitory affects, activating affects, and self-compassion were rated with the Achievement of Therapeutic Objectives Scale (McCullough et al., 2003b) in Sessions 6 and 36. Results showed that increase in self-compassion from early to late in therapy significantly predicted pre- to post-decrease in psychiatric symptoms, interpersonal problems, and personality pathology. Decrease in levels of inhibitory affects and increase in levels of activating affects during therapy were significantly associated with higher self-compassion toward the end of treatment. Increased levels of defense recognition did not predict higher self-compassion when changes in inhibitory and activating affects were statistically controlled for. There were no significant interaction effects with type of treatment. These findings support self-compassion as an important goal of psychotherapy and indicate that increase in the experience of activating affects and decrease in inhibitory affects seem to be worthwhile therapeutic targets when working to enhance self-compassion in patients with Cluster C personality disorders.

  4. Activated Protein C action in inflammation

    PubMed Central

    Sarangi, Pranita P.; Lee, Hyun-wook; Kim, Minsoo

    2010-01-01

    Summary Activated protein C (APC) is a natural anticoagulant that plays an important role in coagulation homeostasis by inactivating the procoagulation factor Va and VIIIa. In addition to its anticoagulation functions, APC also has cytoprotective effects such as anti-inflammatory, anti-apoptotic, and endothelial barrier protection. Recently, a recombinant form of human APC (rhAPC or drotrecogin alfa activated; known commercially as “Xigris”) was approved by the US Federal Drug Administration for treatment of severe sepsis associated with a high risk of mortality. Sepsis, also known as systemic inflammatory response syndrome (SIRS) resulting from infection, is a serious medical condition in critical care patients. In sepsis, hyperactive and dysregulated inflammatory responses lead to secretion of pro- and anti-inflammatory cytokines, activation and migration of leucocytes, activation of coagulation, inhibition of fibrinolysis, and increased apoptosis. Although initial hypotheses focused on antithrombotic and profibrinolytic functions of APC in sepsis, other agents with more potent anticoagulation functions were not effective in treating severe sepsis. Furthermore, APC therapy is also associated with the risk of severe bleeding in treated patients. Therefore, the cytoprotective effects, rather than the anticoagulant effect of APC are postulated to be responsible for the therapeutic benefit of APC in the treatment of severe sepsis. PMID:19995397

  5. Retroviral nucleocapsid proteins possess potent nucleic acid strand renaturation activity.

    PubMed Central

    Dib-Hajj, F.; Khan, R.; Giedroc, D. P.

    1993-01-01

    The nucleocapsid protein (NC) is the major genomic RNA binding protein that plays integral roles in the structure and replication of all animal retroviruses. In this report, select biochemical properties of recombinant Mason-Pfizer monkey virus (MPMV) and HIV-1 NCs are compared. Evidence is presented that two types of saturated Zn2 NC-polynucleotide complexes can be formed under conditions of low [NaCl] that differ in apparent site-size (n = 8 vs. n = 14). The formation of one or the other complex appears dependent on the molar ratio of NC to RNA nucleotide with the putative low site-size mode apparently predominating under conditions of protein excess. Both MPMV and HIV-1 NCs kinetically facilitate the renaturation of two complementary DNA strands, suggesting that this is a general property of retroviral NCs. NC proteins increase the second-order rate constant for renaturation of a 149-bp DNA fragment by more than four orders of magnitude over that obtained in the absence of protein at 37 degrees C. The protein-assisted rate is 100-200-fold faster than that obtained at 68 degrees C, 1 M NaCl, solution conditions considered to be optimal for strand renaturation. Provided that sufficient NC is present to coat all strands, the presence of 400-1,000-fold excess nonhomologous DNA does not greatly affect the reaction rate. The HIV-1 NC-mediated renaturation reaction functions stoichiometrically, requiring a saturated strand of DNA nucleotide:NC ratio of about 7-8, rather than 14. Under conditions of less protein, the rate acceleration is not realized. The finding of significant nucleic acid strand renaturation activity may have important implications for various events of reverse transcription particularly in initiation and cDNA strand transfer. PMID:8443601

  6. Mutations affecting export and activity of cytolysin A from Escherichia coli.

    PubMed

    Ludwig, Albrecht; Völkerink, Guido; von Rhein, Christine; Bauer, Susanne; Maier, Elke; Bergmann, Birgit; Goebel, Werner; Benz, Roland

    2010-08-01

    Cytolysin A (known as ClyA, HlyE, and SheA) is a cytolytic pore-forming protein toxin found in several Escherichia coli and Salmonella enterica strains. The structure of its water-soluble monomeric form and that of dodecameric ClyA pores is known, but the mechanisms of ClyA export from bacterial cells and of pore assembly are only partially understood. Here we used site-directed mutagenesis to study the importance of different regions of the E. coli ClyA protein for export and activity. The data indicate that ClyA translocation to the periplasm requires several protein segments located closely adjacent to each other in the "tail" domain of the ClyA monomer, namely, the N- and C-terminal regions and the hydrophobic sequence ranging from residues 89 to 101. Deletion of most of the "head" domain of the monomer (residues 181 to 203), on the other hand, did not strongly affect ClyA secretion, suggesting that the tail domain plays a particular role in export. Furthermore, we found that the N-terminal amphipathic helix alphaA1 of ClyA is crucial for the formation and the properties of the transmembrane channel, and hence for hemolytic activity. Several mutations affecting the C-terminal helix alphaG, the "beta-tongue" region in the head domain, or the hydrophobic region in the tail domain of the ClyA monomer strongly impaired the hemolytic activity and reduced the activity toward planar lipid bilayer membranes but did not totally prevent formation of wild-type-like channels in these artificial membranes. The latter regions thus apparently promote membrane interaction without being directly required for pore formation in a lipid bilayer.

  7. The outer membrane protein TolC from Sinorhizobium meliloti affects protein secretion, polysaccharide biosynthesis, antimicrobial resistance, and symbiosis.

    PubMed

    Cosme, Ana M; Becker, Anke; Santos, Mário R; Sharypova, Larissa A; Santos, Pedro M; Moreira, Leonilde M

    2008-07-01

    Sinorhizobium meliloti is capable of establishing a symbiotic nitrogen fixation relationship with Medicago sativa. During this process, it must cope with diverse environments and has evolved different types of transport systems that help its propagation in the plant roots. TolC protein family members are the outer-membrane components of several transport systems involved in the export of diverse molecules, playing an important role in bacterial survival. In this work, we have characterized the protein TolC from S. meliloti 2011. An insertional mutation in the tolC gene strongly affected the resistance phenotype to antimicrobial agents and induced higher susceptibility to osmotic and oxidative stresses. Immunodetection experiments and comparison of the extracellular proteins present in the supernatant of the wild-type versus tolC mutant strains showed that the calcium-binding protein ExpE1, the endoglycanase ExsH, and the product of open reading frame SMc04171, a putative hemolysin-type calcium-binding protein, are secreted by a TolC-dependent secretion system. In the absence of TolC, neither succinoglycan nor galactoglucan were detected in the culture supernatant. Moreover, S. meliloti tolC mutant induced a reduced number of nonfixing nitrogen nodules in M. sativa roots. Taken together, our results confirm the importance of TolC in protein secretion, exopolysaccharide biosynthesis, antimicrobials resistance, and symbiosis.

  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. Tetracycline treatment targeting Wolbachia affects expression of an array of proteins in Brugia malayi parasite.

    PubMed

    Dangi, Anil; Vedi, Satish; Nag, Jeetendra Kumar; Paithankar, Sameer; Singh, Mahendra Pratap; Kar, Santosh Kumar; Dube, Anuradha; Misra-Bhattacharya, Shailja

    2009-09-01

    Wolbachia is an intracellular endosymbiont of Brugia malayi parasite whose presence is essential for the survival of the parasite. Treatment of B. malayi-infected jirds with tetracycline eliminates Wolbachia, which affects parasite survival and fitness. In the present study we have tried to identify parasite proteins that are affected when Wolbachia is targeted by tetracycline. For this Wolbachia depleted parasites (B. malayi) were obtained by tetracycline treatment of infected Mongolian jirds (Meriones unguiculatus) and their protein profile after 2-DE separation was compared with that of untreated parasites harboring Wolbachia. Approximately 100 protein spots could be visualized followed by CBB staining of 2-D gel and included for comparative analysis. Of these, 54 showed differential expressions, while two new protein spots emerged (of 90.3 and 64.4 kDa). These proteins were subjected to further analysis by MALDI-TOF for their identification using Brugia coding sequence database composed of both genomic and EST sequences. Our study unravels two crucial findings: (i) the parasite or Wolbachia proteins, which disappeared/down-regulated appear be essential for parasite survival and may be used as drug targets and (ii) tetracycline treatment interferes with the regulatory machinery vital for parasites cellular integrity and defense and thus could possibly be a molecular mechanism for the killing of filarial parasite. This is the first proteomic study substantiating the wolbachial genome integrity with its nematode host and providing functional genomic data of human lymphatic filarial parasite B. malayi.

  10. Myelin basic protein is affected by reduced synthesis of myelin proteolipid protein in the jimpy mouse.

    PubMed Central

    Fannon, A M; Moscarello, M A

    1990-01-01

    Myelin basic proteins (MBPs) from 6-day-old, 10-day-old, 20-day-old and adult normal mouse brain were compared with those from 20-day-old jimpy (dysmyelinating mutant) mouse brain to determine the effect of reduced levels of proteolipid protein (PLP) on MBPs. Alkaline-urea-gel electrophoresis showed that 6-day-old and 10-day-old normal and jimpy MBPs lacked charge microheterogeneity, since C8 (the least cationic of the components; not be confused with complement component C8) was the only charge isomer present. In contrast, MBPs from 20-day-old and adult normal mouse brain displayed extensive charge microheterogeneity, having at least eight components. A 32 kDa MBP was the major isoform observed on immunoblots of acid-soluble protein from 6-day-old and 10-day-old normal and 20-day-old jimpy mouse brain. There were eight bands present in 20-day-old and adult normal mouse brain. Purified human MBP charge heteromers C1, C2, C3 and C4 reacted strongly with rat 14 kDa MBP antiserum, whereas the reaction with human C8 was weak. This suggested that MBPs from early-myelinating and jimpy mice did not react to MBP antisera because C8 was the major charge isomer in these animals. Purification of MBPs from normal and jimpy brain by alkaline-gel electrophoresis showed that both normal and jimpy MBPs have size heterogeneity when subjected to SDS/PAGE. However, the size isoforms in normal mouse brain (32, 21, 18.5, 17 and 14 kDa) differed from those in jimpy brain (32, 21, 20, 17, 15 and 14 kDa) in both size and relative amounts. Amino acid analyses of MBPs from jimpy brain showed an increase in glutamic acid, alanine and ornithine, and a decrease in histidine, arginine and proline. The changes in glutamic acid, ornithine and arginine are characteristic of the differences observed in human C8 when compared with C1. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:1693071

  11. Mutations that affect phosphorylation of the adenovirus DNA-binding protein alter its ability to enhance its own synthesis.

    PubMed Central

    Morin, N; Delsert, C; Klessig, D F

    1989-01-01

    The multifunctional adenovirus single-strand DNA-binding protein (DBP) is highly phosphorylated. Its phosphorylation sites are located in the amino-terminal domain of the protein, and its DNA- and RNA-binding activity resides in the carboxy-terminal half of the polypeptide. We have substituted cysteine or alanine for up to 10 of these potential phosphorylation sites by using oligonucleotide-directed mutagenesis. Alteration of one or a few of these sites had little effect on the viability of virus containing the mutated DBP. However, when eight or more sites were altered, viral growth decreased significantly. This suggests that the overall phosphorylation state of the protein was more important than whether any particular site was modified. The reduction in growth correlated with both depressed DNA replication and expression of late genes. This reduction was probably the result of lower DBP accumulation in mutant-infected cells. Interestingly, although the stability of the mutated DBP was not affected, DBP synthesis and the level of its mRNA were depressed 5- to 10-fold for the underphosphorylated protein. These results suggest that DBP enhances its own expression and imply that phosphorylation of the DBP may be important for this function. Similarities to several eucaryotic transcriptional activators, which are composed of negatively charged activating domains and separate binding domains, are discussed. Images PMID:2585602

  12. Platelet factor 4 stimulates thrombomodulin protein C-activating cofactor activity. A structure-function analysis.

    PubMed

    Slungaard, A; Key, N S

    1994-10-14

    Thrombomodulin (TM) is an anionic (pI approximately 4) protein cofactor that promotes thrombin (THR) cleavage of protein C to generate activated protein C (APC), a potent anticoagulant. We find that the cationic platelet alpha-granule protein platelet factor 4 (PF4) stimulates 4-25-fold the cofactor activity of rabbit TM and two differentially glycanated versions of an extracellular domain human TM polypeptide in which the glycosaminoglycan (GAG) is either present (GAG+ TM) or absent (GAG- TM) with an ED50 of 3.3-10 micrograms/ml. No such stimulation occurs in response to beta-thromboglobulin or thrombospondin, or when protein C lacking its gamma-carboxyglutamic acid (Gla) domain is the substrate. Heparin and chondroitin sulfates A and E reverse PF4 stimulation. PF4 minimally affects the Kd for THR but decreases 30-fold (from 8.3 to 0.3 microM) the Km for protein C of APC generation by GAG+ TM. PF4 also strikingly transforms the [Ca2+] dependence profile of rabbit and GAG+ TM to resemble that of GAG- TM. A potential explanation for this is that PF4, like Ca2+, induces heparin-reversible alterations in native (but not Gla-domainless) protein C conformation as assessed by autofluorescence emission analysis. We conclude that PF4 stimulates TM APC generation by interacting electrostatically with both the TM GAG and the protein C Gla domain to enhance markedly the affinity of the THR.TM complex for protein C. By this mechanism, PF4 may play a previously unsuspected role in the physiologic regulation of clotting.

  13. Protein kinase C activity in boar sperm.

    PubMed

    Teijeiro, J M; Marini, P E; Bragado, M J; Garcia-Marin, L J

    2017-03-01

    Male germ cells undergo different processes within the female reproductive tract to successfully fertilize the oocyte. These processes are triggered by different extracellular stimuli leading to activation of protein phosphorylation. Protein kinase C (PKC) is a key regulatory enzyme in signal transduction mechanisms involved in many cellular processes. Studies in boar sperm demonstrated a role for PKC in the intracellular signaling involved in motility and cellular volume regulation. Experiments using phorbol 12-myristate 13-acetate (PMA) showed increases in the Serine/Threonine phosphorylation of substrates downstream of PKC in boar sperm. In order to gain knowledge about those cellular processes regulated by PKC, we evaluate the effects of PMA on boar sperm motility, lipid organization of plasma membrane, integrity of acrosome membrane and sperm agglutination. Also, we investigate the crosstalk between PKA and PKC intracellular pathways in spermatozoa from this species. The results presented here reveal a participation of PKC in sperm motility regulation and membrane fluidity changes, which is probably associated to acrosome reaction and to agglutination. Also, we show the existence of a hierarchy in the kinases pathway. Previous works on boar sperm suggest a pathway in which PKA is positioned upstream to PKC and this new results support such model.

  14. Membrane transporters and protein traffic networks differentially affecting metal tolerance: a genomic phenotyping study in yeast

    PubMed Central

    Ruotolo, Roberta; Marchini, Gessica; Ottonello, Simone

    2008-01-01

    Background The cellular mechanisms that underlie metal toxicity and detoxification are rather variegated and incompletely understood. Genomic phenotyping was used to assess the roles played by all nonessential Saccharomyces cerevisiae proteins in modulating cell viability after exposure to cadmium, nickel, and other metals. Results A number of novel genes and pathways that affect multimetal as well as metal-specific tolerance were discovered. Although the vacuole emerged as a major hot spot for metal detoxification, we also identified a number of pathways that play a more general, less direct role in promoting cell survival under stress conditions (for example, mRNA decay, nucleocytoplasmic transport, and iron acquisition) as well as proteins that are more proximally related to metal damage prevention or repair. Most prominent among the latter are various nutrient transporters previously not associated with metal toxicity. A strikingly differential effect was observed for a large set of deletions, the majority of which centered on the ESCRT (endosomal sorting complexes required for transport) and retromer complexes, which - by affecting transporter downregulation and intracellular protein traffic - cause cadmium sensitivity but nickel resistance. Conclusion The data show that a previously underestimated variety of pathways are involved in cadmium and nickel tolerance in eukaryotic cells. As revealed by comparison with five additional metals, there is a good correlation between the chemical properties and the cellular toxicity signatures of various metals. However, many conserved pathways centered on membrane transporters and protein traffic affect cell viability with a surprisingly high degree of metal specificity. PMID:18394190

  15. Construction and genetic selection of small transmembrane proteins that activate the human erythropoietin receptor.

    PubMed

    Cammett, Tobin J; Jun, Susan J; Cohen, Emily B; Barrera, Francisco N; Engelman, Donald M; Dimaio, Daniel

    2010-02-23

    This work describes a genetic approach to isolate small, artificial transmembrane (TM) proteins with biological activity. The bovine papillomavirus E5 protein is a dimeric, 44-amino acid TM protein that transforms cells by specifically binding and activating the platelet-derived growth factor beta receptor (PDGFbetaR). We used the E5 protein as a scaffold to construct a retrovirus library expressing approximately 500,000 unique 44-amino acid proteins with randomized TM domains. We screened this library to select small, dimeric TM proteins that were structurally unrelated to erythropoietin (EPO), but specifically activated the human EPO receptor (hEPOR). These proteins did not activate the murine EPOR or the PDGFbetaR. Genetic studies with one of these activators suggested that it interacted with the TM domain of the hEPOR. Furthermore, this TM activator supported erythroid differentiation of primary human hematopoietic progenitor cells in vitro in the absence of EPO. Thus, we have changed the specificity of a protein so that it no longer recognizes its natural target but, instead, modulates an entirely different protein. This represents a novel strategy to isolate small artificial proteins that affect diverse membrane proteins. We suggest the word "traptamer" for these transmembrane aptamers.

  16. The Regulatory Protein RosR Affects Rhizobium leguminosarum bv. trifolii Protein Profiles, Cell Surface Properties, and Symbiosis with Clover

    PubMed Central

    Rachwał, Kamila; Boguszewska, Aleksandra; Kopcińska, Joanna; Karaś, Magdalena; Tchórzewski, Marek; Janczarek, Monika

    2016-01-01

    Rhizobium leguminosarum bv. trifolii is capable of establishing a symbiotic relationship with plants from the genus Trifolium. Previously, a regulatory protein encoded by rosR was identified and characterized in this bacterium. RosR possesses a Cys2-His2-type zinc finger motif and belongs to Ros/MucR family of rhizobial transcriptional regulators. Transcriptome profiling of the rosR mutant revealed a role of this protein in several cellular processes, including the synthesis of cell-surface components and polysaccharides, motility, and bacterial metabolism. Here, we show that a mutation in rosR resulted in considerable changes in R. leguminosarum bv. trifolii protein profiles. Extracellular, membrane, and periplasmic protein profiles of R. leguminosarum bv. trifolii wild type and the rosR mutant were examined, and proteins with substantially different abundances between these strains were identified. Compared with the wild type, extracellular fraction of the rosR mutant contained greater amounts of several proteins, including Ca2+-binding cadherin-like proteins, a RTX-like protein, autoaggregation protein RapA1, and flagellins FlaA and FlaB. In contrast, several proteins involved in the uptake of various substrates were less abundant in the mutant strain (DppA, BraC, and SfuA). In addition, differences were observed in membrane proteins of the mutant and wild-type strains, which mainly concerned various transport system components. Using atomic force microscopy (AFM) imaging, we characterized the topography and surface properties of the rosR mutant and wild-type cells. We found that the mutation in rosR gene also affected surface properties of R. leguminosarum bv. trifolii. The mutant cells were significantly more hydrophobic than the wild-type cells, and their outer membrane was three times more permeable to the hydrophobic dye N-phenyl-1-naphthylamine. The mutation of rosR also caused defects in bacterial symbiotic interaction with clover plants. Compared with

  17. Hsp90 protein interacts with phosphorothioate oligonucleotides containing hydrophobic 2'-modifications and enhances antisense activity.

    PubMed

    Liang, Xue-Hai; Shen, Wen; Sun, Hong; Kinberger, Garth A; Prakash, Thazha P; Nichols, Joshua G; Crooke, Stanley T

    2016-05-05

    RNase H1-dependent antisense oligonucleotides (ASOs) are chemically modified to enhance pharmacological properties. Major modifications include phosphorothioate (PS) backbone and different 2'-modifications in 2-5 nucleotides at each end (wing) of an ASO. Chemical modifications can affect protein binding and understanding ASO-protein interactions is important for better drug design. Recently we identified many intracellular ASO-binding proteins and found that protein binding could affect ASO potency. Here, we analyzed the structure-activity-relationships of ASO-protein interactions and found 2'-modifications significantly affected protein binding, including La, P54nrb and NPM. PS-ASOs containing more hydrophobic 2'-modifications exhibit higher affinity for proteins in general, although certain proteins, e.g. Ku70/Ku80 and TCP1, are less affected by 2'-modifications. We found that Hsp90 protein binds PS-ASOs containing locked-nucleic-acid (LNA) or constrained-ethyl-bicyclic-nucleic-acid ((S)-cEt) modifications much more avidly than 2'-O-methoxyethyl (MOE). ASOs bind the mid-domain of Hsp90 protein. Hsp90 interacts with more hydrophobic 2' modifications, e.g. (S)-cEt or LNA, in the 5'-wing of the ASO. Reduction of Hsp90 protein decreased activity of PS-ASOs with 5'-LNA or 5'-cEt wings, but not with 5'-MOE wing. Together, our results indicate Hsp90 protein enhances the activity of PS/LNA or PS/(S)-cEt ASOs, and imply that altering protein binding of ASOs using different chemical modifications can improve therapeutic performance of PS-ASOs.

  18. Nonconsensus Protein Binding to Repetitive DNA Sequence Elements Significantly Affects Eukaryotic Genomes

    PubMed Central

    Barber-Zucker, Shiran; Gordân, Raluca; Lukatsky, David B.

    2015-01-01

    Recent genome-wide experiments in different eukaryotic genomes provide an unprecedented view of transcription factor (TF) binding locations and of nucleosome occupancy. These experiments revealed that a large fraction of TF binding events occur in regions where only a small number of specific TF binding sites (TFBSs) have been detected. Furthermore, in vitro protein-DNA binding measurements performed for hundreds of TFs indicate that TFs are bound with wide range of affinities to different DNA sequences that lack known consensus motifs. These observations have thus challenged the classical picture of specific protein-DNA binding and strongly suggest the existence of additional recognition mechanisms that affect protein-DNA binding preferences. We have previously demonstrated that repetitive DNA sequence elements characterized by certain symmetries statistically affect protein-DNA binding preferences. We call this binding mechanism nonconsensus protein-DNA binding in order to emphasize the point that specific consensus TFBSs do not contribute to this effect. In this paper, using the simple statistical mechanics model developed previously, we calculate the nonconsensus protein-DNA binding free energy for the entire C. elegans and D. melanogaster genomes. Using the available chromatin immunoprecipitation followed by sequencing (ChIP-seq) results on TF-DNA binding preferences for ~100 TFs, we show that DNA sequences characterized by low predicted free energy of nonconsensus binding have statistically higher experimental TF occupancy and lower nucleosome occupancy than sequences characterized by high free energy of nonconsensus binding. This is in agreement with our previous analysis performed for the yeast genome. We suggest therefore that nonconsensus protein-DNA binding assists the formation of nucleosome-free regions, as TFs outcompete nucleosomes at genomic locations with enhanced nonconsensus binding. In addition, here we perform a new, large-scale analysis using

  19. Contaminant loading in remote Arctic lakes affects cellular stress-related proteins expression in feral charr.

    USGS Publications Warehouse

    Wiseman, Steve; Jorgensen, Even H.; Maule, Alec G.; Vijayan, Mathilakath M.

    2011-01-01

    The remote Arctic lakes on Bjornoya Island, Norway, offer a unique opportunity to study possible affect of lifelong contaminant exposure in wild populations of landlocked Arctic charr (Salvelinus alpinus). This is because Lake Ellasjoen has persistent organic pollutant (POP) levels that are significantly greater than in the nearby Lake Oyangen. We examined whether this differential contaminant loading was reflected in the expression of protein markers of exposure and effect in the native fish. We assessed the expressions of cellular stress markers, including cytochrome P4501A (Cyp1A), heat shock protein 70 (hsp70), and glucocorticoid receptor (GR) in feral charr from the two lakes. The average polychlorinated biphenyl (PCB) load in the charr liver from Ellasjoen was approximately 25-fold higher than in individuals from Oyangen. Liver Cyp1A protein expression was significantly higher in individuals from Ellasjoen compared with Oyangen, confirming differential PCB exposure. There was no significant difference in hsp70 protein expression in charr liver between the two lakes. However, brain hsp70 protein expression was significantly elevated in charr from Ellasjoen compared with Oyangen. Also, liver GR protein expression was significantly higher in the Ellasjoen charr compared with Oyangen charr. Taken together, our results suggest changes to cellular stress-related protein expression as a possible adaptation to chronic-contaminant exposure in feral charr in the Norwegian high-Arctic.

  20. Amount and distribution of dietary protein affects clinical response to levodopa in Parkinson's disease.

    PubMed

    Carter, J H; Nutt, J G; Woodward, W R; Hatcher, L F; Trotman, T L

    1989-04-01

    Reducing dietary protein improves the effectiveness of levodopa (LD) but the most effective distribution of a low-protein diet (0.8 g/kg) is unclear. We compared a 1.6 g/kg protein diet, a 0.8 g/kg diet with protein evenly distributed between meals, and a 0.8 g/kg diet with protein restricted to the evening meal in 5 parkinsonian patients with motor fluctuations. We monitored clinical response, plasma LD, and plasma large amino acids (LNAAs) hourly throughout the day. Mean "on" times were 51% (1.6 g/kg diet), 67% (0.8 g/kg evenly distributed), and 77% (0.8 g/kg restricted). Hourly averages of plasma LD did not differ between the diets. The mean plasma LNAAs were 732 nmol/ml (1.6 g/kg diet), 640 (0.8 g/kg distributed), and 542 (0.8 g/kg restricted), and the diurnal pattern reflected the distribution of protein intake. In conclusion, the amount and distribution of dietary protein affect clinical response to LD. These effects are not related to LD absorption but are explained by the variation in plasma LNAAs.

  1. Protein and lipid sources affect cholesterol concentrations of juvenile Pacific white shrimp, Litopenaeus vannamei (Boone).

    PubMed

    Cheng, Z J; Hardy, R W

    2004-04-01

    Two experiments were conducted to evaluate the effects of protein and lipid sources on cholesterol, AA, and fatty acid content, and on biological performance of juvenile Pacific white shrimp, Litopenaeus vannamei (Boone). In Exp. 1, seven isonitrogenous and isocaloric diets were prepared using fish meal; soybean meal; casein; fish meal + soybean meal; fish meal + casein; soybean meal + casein; and fish meal + soybean meal + casein. In Exp. 2, seven isonitrogenous and isocaloric diets were prepared using fish oil; soy oil; poultry fat; fish oil + soy oil; fish oil + poultry fat; soy oil + poultry fat; and fish oil + soy oil + poultry fat. Nine shrimp (average BW 570 mg) were stocked per 60-L tank, with three tanks per diet in each experiment. Shrimp were fed to apparent satiation twice daily for 28 d. Protein sources affected shrimp cholesterol, feed consumption, feed efficiency, protein consumption, protein efficiency ratio, and crude body fat (P < or = 0.05), but not weight gain, survival, hepatosomatic index, body protein, ash, and AA composition. Body (without hepatopancreas) cholesterol concentrations were the highest in shrimp fed the diet containing fish meal (0.81%), lowest for those fed the casein diet (0.64%), and intermediate in the other dietary treatment groups (range 0.71 to 0.74%). Lipid source also affected shrimp body cholesterol, body fatty acid profiles, and fatty acid profiles in the hepatopancreas (P < or = 0.05), but not growth performance, body protein, fat, ash, and cholesterol concentrations in the hepatopancreas. Shrimp fed the fish oil diet had the highest body cholesterol (0.75%), whereas those fed the soy oil or poultry fat diets were lowest (0.66 and 0.65%, respectively). Results indicate that by replacing fish meal and fish oil with soybean meal and soy oil, shrimp growth performance is not affected, but body cholesterol concentration is reduced.

  2. Protein corona composition of gold nanoparticles/nanorods affects amyloid beta fibrillation process

    NASA Astrophysics Data System (ADS)

    Mirsadeghi, Somayeh; Dinarvand, Rassoul; Ghahremani, Mohammad Hossein; Hormozi-Nezhad, Mohammad Reza; Mahmoudi, Zohreh; Hajipour, Mohammad Javad; Atyabi, Fatemeh; Ghavami, Mahdi; Mahmoudi, Morteza

    2015-03-01

    Protein fibrillation process (e.g., from amyloid beta (Aβ) and α-synuclein) is the main cause of several catastrophic neurodegenerative diseases such as Alzheimer's and Parkinson diseases. During the past few decades, nanoparticles (NPs) were recognized as one of the most promising tools for inhibiting the progress of the disease by controlling the fibrillation kinetic process; for instance, gold NPs have a strong capability to inhibit Aβ fibrillations. It is now well understood that a layer of biomolecules would cover the surface of NPs (so called ``protein corona'') upon the interaction of NPs with protein sources. Due to the fact that the biological species (e.g., cells and amyloidal proteins) ``see'' the protein corona coated NPs rather than the pristine coated particles, one should monitor the fibrillation process of amyloidal proteins in the presence of corona coated NPs (and not pristine coated ones). Therefore, the previously obtained data on NPs effects on the fibrillation process should be modified to achieve a more reliable and predictable in vivo results. Herein, we probed the effects of various gold NPs (with different sizes and shapes) on the fibrillation process of Aβ in the presence and absence of protein sources (i.e., serum and plasma). We found that the protein corona formed a shell at the surface of gold NPs, regardless of their size and shape, reducing the access of Aβ to the gold inhibitory surface and, therefore, affecting the rate of Aβ fibril formation. More specifically, the anti-fibrillation potencies of various corona coated gold NPs were strongly dependent on the protein source and their concentrations (10% serum/plasma (simulation of an in vitro milieu) and 100% serum/plasma (simulation of an in vivo milieu)).Protein fibrillation process (e.g., from amyloid beta (Aβ) and α-synuclein) is the main cause of several catastrophic neurodegenerative diseases such as Alzheimer's and Parkinson diseases. During the past few decades

  3. Newly identified protein Imi1 affects mitochondrial integrity and glutathione homeostasis in Saccharomyces cerevisiae.

    PubMed

    Kowalec, Piotr; Grynberg, Marcin; Pająk, Beata; Socha, Anna; Winiarska, Katarzyna; Fronk, Jan; Kurlandzka, Anna

    2015-09-01

    Glutathione homeostasis is crucial for cell functioning. We describe a novel Imi1 protein of Saccharomyces cerevisiae affecting mitochondrial integrity and involved in controlling glutathione level. Imi1 is cytoplasmic and, except for its N-terminal Flo11 domain, has a distinct solenoid structure. A lack of Imi1 leads to mitochondrial lesions comprising aberrant morphology of cristae and multifarious mtDNA rearrangements and impaired respiration. The mitochondrial malfunctioning is coupled to significantly decrease the level of intracellular reduced glutathione without affecting oxidized glutathione, which decreases the reduced/oxidized glutathione ratio. These defects are accompanied by decreased cadmium sensitivity and increased phytochelatin-2 level.

  4. Comparative Proteomics Identifies Host Immune System Proteins Affected by Infection with Mycobacterium bovis.

    PubMed

    López, Vladimir; Villar, Margarita; Queirós, João; Vicente, Joaquín; Mateos-Hernández, Lourdes; Díez-Delgado, Iratxe; Contreras, Marinela; Alves, Paulo C; Alberdi, Pilar; Gortázar, Christian; de la Fuente, José

    2016-03-01

    Mycobacteria of the Mycobacterium tuberculosis complex (MTBC) greatly impact human and animal health worldwide. The mycobacterial life cycle is complex, and the mechanisms resulting in pathogen infection and survival in host cells are not fully understood. Eurasian wild boar (Sus scrofa) are natural reservoir hosts for MTBC and a model for mycobacterial infection and tuberculosis (TB). In the wild boar TB model, mycobacterial infection affects the expression of innate and adaptive immune response genes in mandibular lymph nodes and oropharyngeal tonsils, and biomarkers have been proposed as correlates with resistance to natural infection. However, the mechanisms used by mycobacteria to manipulate host immune response are not fully characterized. Our hypothesis is that the immune system proteins under-represented in infected animals, when compared to uninfected controls, are used by mycobacteria to guarantee pathogen infection and transmission. To address this hypothesis, a comparative proteomics approach was used to compare host response between uninfected (TB-) and M. bovis-infected young (TB+) and adult animals with different infection status [TB lesions localized in the head (TB+) or affecting multiple organs (TB++)]. The results identified host immune system proteins that play an important role in host response to mycobacteria. Calcium binding protein A9, Heme peroxidase, Lactotransferrin, Cathelicidin and Peptidoglycan-recognition protein were under-represented in TB+ animals when compared to uninfected TB- controls, but protein levels were higher as infection progressed in TB++ animals when compared to TB- and/or TB+ adult wild boar. MHCI was the only protein over-represented in TB+ adult wild boar when compared to uninfected TB- controls. The results reported here suggest that M. bovis manipulates host immune response by reducing the production of immune system proteins. However, as infection progresses, wild boar immune response recovers to limit pathogen

  5. Comparative Proteomics Identifies Host Immune System Proteins Affected by Infection with Mycobacterium bovis

    PubMed Central

    López, Vladimir; Villar, Margarita; Queirós, João; Vicente, Joaquín; Mateos-Hernández, Lourdes; Díez-Delgado, Iratxe; Contreras, Marinela; Alves, Paulo C.; Alberdi, Pilar; Gortázar, Christian; de la Fuente, José

    2016-01-01

    Mycobacteria of the Mycobacterium tuberculosis complex (MTBC) greatly impact human and animal health worldwide. The mycobacterial life cycle is complex, and the mechanisms resulting in pathogen infection and survival in host cells are not fully understood. Eurasian wild boar (Sus scrofa) are natural reservoir hosts for MTBC and a model for mycobacterial infection and tuberculosis (TB). In the wild boar TB model, mycobacterial infection affects the expression of innate and adaptive immune response genes in mandibular lymph nodes and oropharyngeal tonsils, and biomarkers have been proposed as correlates with resistance to natural infection. However, the mechanisms used by mycobacteria to manipulate host immune response are not fully characterized. Our hypothesis is that the immune system proteins under-represented in infected animals, when compared to uninfected controls, are used by mycobacteria to guarantee pathogen infection and transmission. To address this hypothesis, a comparative proteomics approach was used to compare host response between uninfected (TB-) and M. bovis-infected young (TB+) and adult animals with different infection status [TB lesions localized in the head (TB+) or affecting multiple organs (TB++)]. The results identified host immune system proteins that play an important role in host response to mycobacteria. Calcium binding protein A9, Heme peroxidase, Lactotransferrin, Cathelicidin and Peptidoglycan-recognition protein were under-represented in TB+ animals when compared to uninfected TB- controls, but protein levels were higher as infection progressed in TB++ animals when compared to TB- and/or TB+ adult wild boar. MHCI was the only protein over-represented in TB+ adult wild boar when compared to uninfected TB- controls. The results reported here suggest that M. bovis manipulates host immune response by reducing the production of immune system proteins. However, as infection progresses, wild boar immune response recovers to limit pathogen

  6. Barley chloroplast glutamine synthetase activity is not affected by CO sub 2 -concentration

    SciTech Connect

    Avila, C.; Forde, B.; Wallsgrove, R. )

    1990-05-01

    It has been reported that when photorespiration is suppressed by raising the concentration of CO{sub 2}, the expression of the chloroplast glutamine synthetase (GS2) gene in pea leaves is reduced (Plant Cell, 1, 241). We have examined this effect in barley (Hordeum vulgare), and confirm that plants grown continuously in 0.8% CO{sub 2}, or transferred to such conditions after growth in air, appear to have a reduced GS2 mRNA abundance. However, we were unable to detect any significant difference in the extractable GS2 activity, or any change in amount of GS2 protein (judged by Western blots). Whatever controls are operating on gS2 mRNA expression in response to changes is external CO{sub 2}, they do not affect the activity or amount of the enzyme in barley.

  7. Levodopa influences striatal activity but does not affect cortical hyper-activity in Parkinson's disease.

    PubMed

    Martinu, K; Degroot, C; Madjar, C; Strafella, A P; Monchi, O

    2012-02-01

    Motor studies of Parkinson's disease (PD) have shown cortical hypo-activity in relation to nigrostriatal dopamine depletion. Cognitive studies also identified increased cortical activity in PD. We have previously suggested that the hypo-activity/hyper-activity patterns observed in PD are related to the striatal contribution. Tasks that recruit the striatum in control participants are associated with cortical hypo-activity in patients with PD, whereas tasks that do not result in cortical hyper-activity. The putamen, a structure affected by the neurodegeneration observed in PD, shows increased activation for externally-triggered (ET) and self-initiated (SI) movements. The first goal of this study was to evaluate the effect of levodopa on the putamen's response to ET and SI movements. Our second goal was to assess the effect of levodopa on the hypo-activity/hyper-activity patterns in cortical areas. Patients with PD on and off levodopa and healthy volunteers performed SI, ET and control finger movements during functional magnetic resonance imaging. Healthy participants displayed significant differences in putamen activity in ET and SI movements. These differences were reduced in patients off medication, with non-task-specific increases in activity after levodopa administration. Furthermore, the ventrolateral prefrontal cortex showed significant increases in activity during SI movements in healthy controls, whereas it was hypo-active in PD. This region showed significantly increased activity during ET movements in patients off medication. Levodopa had no effect on this discrepancy. Our results suggest that dopamine replacement therapy has a non-task-specific effect on motor corticostriatal regions, and support the hypothesis that increases and decreases in cortical activity in PD are related to the mesocortical dopamine pathway imbalance.

  8. Real-World Affected Upper Limb Activity in Chronic Stroke: An Examination of Potential Modifying Factors

    PubMed Central

    Bailey, Ryan R.; Birkenmeier, Rebecca L.; Lang, Catherine E.

    2015-01-01

    BACKGROUND Despite improvement in motor function after intervention, adults with chronic stroke experience disability in everyday activity. Factors other than motor function may influence affected upper limb (UL) activity. OBJECTIVE To characterize affected UL activity and examine potential modifying factors of affected UL activity in community-dwelling adults with chronic stroke. METHODS Forty-six adults with chronic stroke wore accelerometers on both ULs for 25 hours and provided information about potential modifying factors (time spent in sedentary activity, cognitive impairment, depressive symptomatology, number of comorbidities, motor dysfunction of the affected UL, age, activities of daily living (ADL) status, and living arrangement). Accelerometry was used to quantify duration of affected and unaffected UL activity. The ratio of affected-to-unaffected UL activity was also calculated. Associations within and between accelerometry-derived variables and potential modifying factors were examined. RESULTS Mean hours of affected and unaffected UL activity were 5.0 ± 2.2 and 7.6 ± 2.1 hours, respectively. The ratio of affected-to-unaffected UL activity was 0.64 ± 0.19, and hours of affected and unaffected UL activity were strongly correlated (r=0.78). Increased severity of motor dysfunction and dependence in ADLs were associated with decreased affected UL activity. No other factors were associated with affected UL activity. CONCLUSIONS Severity of motor dysfunction and ADL status should be taken into consideration when setting goals for UL activity in people with chronic stroke. Given the strong, positive correlation between affected and unaffected UL activity, encouragement to increase activity of the unaffected UL may increase affected UL activity. PMID:25776118

  9. Protein-Protein Interactions Suggest Novel Activities of Human Cytomegalovirus Tegument Protein pUL103

    PubMed Central

    Ortiz, Daniel A.; Glassbrook, James E.

    2016-01-01

    ABSTRACT Human cytomegalovirus (HCMV) is an enveloped double-stranded DNA virus that causes severe disease in newborns and immunocompromised patients. During infection, the host cell endosecretory system is remodeled to form the cytoplasmic virion assembly complex (cVAC). We and others previously identified the conserved, multifunctional HCMV virion tegument protein pUL103 as important for cVAC biogenesis and efficient secondary envelopment. To help define its mechanisms of action and predict additional functions, we used two complementary methods, coimmunoprecipitation (co-IP) and proximity biotinylation (BioID), to identify viral and cellular proteins that interact with pUL103. By using the two methods in parallel and applying stringent selection criteria, we identified potentially high-value interactions of pUL103 with 13 HCMV and 18 cellular proteins. Detection of the previously identified pUL103-pUL71 interaction, as well as verification of several interactions by reverse co-IP, supports the specificity of our screening process. As might be expected for a tegument protein, interactions were identified that suggest distinct roles for pUL103 across the arc of lytic infection, including interactions with proteins involved in cellular antiviral responses, nuclear activities, and biogenesis and transport of cytoplasmic vesicles. Further analysis of some of these interactions expands our understanding of the multifunctional repertoire of pUL103: we detected HCMV pUL103 in nuclei of infected cells and identified an ALIX-binding domain within the pUL103 sequence. IMPORTANCE Human cytomegalovirus (HCMV) is able to reconfigure the host cell machinery to establish a virion production factory, the cytoplasmic virion assembly complex (cVAC). cVAC biogenesis and operation represent targets for development of novel HCMV antivirals. We previously showed that the HCMV tegument protein pUL103 is required for cVAC biogenesis. Using pUL103 as bait, we investigated viral and

  10. Special AT-rich Binding Protein-2 (SATB2) Differentially Affects Disease-causing p63 Mutant Proteins*

    PubMed Central

    Chung, Jacky; Grant, R. Ian; Kaplan, David R.; Irwin, Meredith S.

    2011-01-01

    p63, a p53 family member, is critical for proper skin and limb development and directly regulates gene expression in the ectoderm. Mice lacking p63 exhibit skin and craniofacial defects including cleft palate. In humans p63 mutations are associated with several distinct developmental syndromes. p63 sterile-α-motif domain, AEC (ankyloblepharon-ectodermal dysplasia-clefting)-associated mutations are associated with a high prevalence of orofacial clefting disorders, which are less common in EEC (ectrodactyly-ectodermal dysplasia-clefting) patients with DNA binding domain p63 mutations. However, the mechanisms by which these mutations differentially influence p63 function remain unclear, and interactions with other proteins implicated in craniofacial development have not been identified. Here, we show that AEC p63 mutations affect the ability of the p63 protein to interact with special AT-rich binding protein-2 (SATB2), which has recently also been implicated in the development of cleft palate. p63 and SATB2 are co-expressed early in development in the ectoderm of the first and second branchial arches, two essential sites where signaling is required for craniofacial patterning. SATB2 attenuates p63-mediated gene expression of perp (p53 apoptosis effector related to PMP-22), a critical downstream target gene during development, and specifically decreases p63 perp promoter binding. Interestingly, AEC but not EEC p63 mutations affect the ability of p63 to interact with SATB2 and the inhibitory effects of SATB2 on p63 transactivation of perp are most pronounced for AEC-associated p63 mutations. Our findings reveal a novel gain-of-function property of AEC-causing p63 mutations and identify SATB2 as the first p63 binding partner that differentially influences AEC and EEC p63 mutant proteins. PMID:21965674

  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

    2011-02-08

    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.

  12. Stable complex formation between HIV Rev and the nucleosome assembly protein, NAP1, affects Rev function

    SciTech Connect

    Cochrane, Alan; Murley, Laura Lea; Gao Mian; Wong, Raymond; Clayton, Kiera; Brufatto, Nicole; Canadien, Veronica; Mamelak, Daniel; Chen, Tricia; Richards, Dawn; Zeghouf, Mahel; Greenblatt, Jack; Burks, Christian; Frappier, Lori

    2009-05-25

    The Rev protein of HIV-1 is essential for HIV-1 proliferation due to its role in exporting viral RNA from the nucleus. We used a modified version of tandem affinity purification (TAP) tagging to identify proteins interacting with HIV-1 Rev in human cells and discovered a prominent interaction between Rev and nucleosome assembly protein 1 (Nap1). This interaction was also observed by specific retention of Nap1 from human cell lysates on a Rev affinity column. Nap1 was found to bind Rev through the Rev arginine-rich domain and altered the oligomerization state of Rev in vitro. Overexpression of Nap1 stimulated the ability of Rev to export RNA, reduced the nucleolar localization of Rev, and affected Rev nuclear import rates. The results suggest that Nap-1 may influence Rev function by increasing the availability of Rev.

  13. Defects in Protein Folding Machinery Affect Cell Wall Integrity and Reduce Ethanol Tolerance in S. cerevisiae.

    PubMed

    Narayanan, Aswathy; Pullepu, Dileep; Reddy, Praveen Kumar; Uddin, Wasim; Kabir, M Anaul

    2016-07-01

    The chaperonin complex CCT/TRiC (chaperonin containing TCP-1/TCP-1 ring complex) participates in the folding of many crucial proteins including actin and tubulin in eukaryotes. Mutations in genes encoding its subunits can affect protein folding and in turn, the physiology of the organism. Stress response in Saccharomyces cerevisiae is important in fermentation reactions and operates through overexpression and underexpression of genes, thus altering the protein profile. Defective protein folding machinery can disturb this process. In this study, the response of cct mutants to stress conditions in general and ethanol in specific was investigated. CCT1 mutants showed decreased resistance to different conditions tested including osmotic stress, metal ions, surfactants, reducing and oxidising agents. Cct1-3 mutant with the mutation in the conserved ATP-binding region showed irreversible defects than other mutants. These mutants were found to have inherent cell wall defects and showed decreased ethanol tolerance. This study reveals that cell wall defects and ethanol sensitivity are linked. Genetic and proteomic analyses showed that the yeast genes RPS6A (ribosomal protein), SCL1 (proteasomal subunit) and TDH3 (glyceraldehyde-3-phosphate dehydrogenase) on overexpression, improved the growth of cct1-3 mutant on ethanol. We propose the breakdown of common stress response pathways caused by mutations in CCT complex and the resulting scarcity of functional stress-responsive proteins, affecting the cell's defence against different stress agents in cct mutants. Defective cytoskeleton and perturbed cell wall integrity reduce the ethanol tolerance in the mutants which are rescued by the extragenic suppressors.

  14. Rapid and Quantitative Assay of Amyloid-Seeding Activity in Human Brains Affected with Prion Diseases

    PubMed Central

    Takatsuki, Hanae; Satoh, Katsuya; Sano, Kazunori; Fuse, Takayuki; Nakagaki, Takehiro; Mori, Tsuyoshi; Ishibashi, Daisuke; Mihara, Ban; Takao, Masaki; Iwasaki, Yasushi; Yoshida, Mari; Atarashi, Ryuichiro; Nishida, Noriyuki

    2015-01-01

    The infectious agents of the transmissible spongiform encephalopathies are composed of amyloidogenic prion protein, PrPSc. Real-time quaking-induced conversion can amplify very small amounts of PrPSc seeds in tissues/body fluids of patients or animals. Using this in vitro PrP-amyloid amplification assay, we quantitated the seeding activity of affected human brains. End-point assay using serially diluted brain homogenates of sporadic Creutzfeldt–Jakob disease patients demonstrated that 50% seeding dose (SD50) is reached approximately 1010/g brain (values varies 108.79–10.63/g). A genetic case (GSS-P102L) yielded a similar level of seeding activity in an autopsy brain sample. The range of PrPSc concentrations in the samples, determined by dot-blot assay, was 0.6–5.4 μg/g brain; therefore, we estimated that 1 SD50 unit was equivalent to 0.06–0.27 fg of PrPSc. The SD50 values of the affected brains dropped more than three orders of magnitude after autoclaving at 121°C. This new method for quantitation of human prion activity provides a new way to reduce the risk of iatrogenic prion transmission. PMID:26070208

  15. Multifunctional Thioredoxin-Like Protein from the Gastrointestinal Parasitic Nematodes Strongyloides ratti and Trichuris suis Affects Mucosal Homeostasis

    PubMed Central

    Hansmann, Jan; Winter, Dominic; Schramm, Guido; Erttmann, Klaus D.; Liebau, Eva

    2016-01-01

    The cellular redox state is important for the regulation of multiple functions and is essential for the maintenance of cellular homeostasis and antioxidant defense. In the excretory/secretory (E/S) products of Strongyloides ratti and Trichuris suis sequences for thioredoxin (Trx) and Trx-like protein (Trx-lp) were identified. To characterize the antioxidant Trx-lp and its interaction with the parasite's mucosal habitat, S. ratti and T. suis Trx-lps were cloned and recombinantly expressed. The primary antioxidative activity was assured by reduction of insulin and IgM. Further analysis applying an in vitro mucosal 3D-cell culture model revealed that the secreted Trx-lps were able to bind to monocytic and intestinal epithelial cells and induce the time-dependent release of cytokines such as TNF-α, IL-22, and TSLP. In addition, the redox proteins also possessed chemotactic activity for monocytic THP-1 cells and fostered epithelial wound healing activity. These results confirm that the parasite-secreted Trx-lps are multifunctional proteins that can affect the host intestinal mucosa. PMID:27872753

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

    SciTech Connect

    Scalise, F.W.

    1988-01-01

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

  17. Nitrogen Assimilation and Protein Synthesis in Wheat Seedlings As Affected by Mineral Nutrition. I. Macronutrients 1

    PubMed Central

    Harper, James E.; Paulsen, Gary M.

    1969-01-01

    Deficiencies of each macronutrient (N, P, K, Ca. Mg, S, and Fe) decreased the specific activity of nitrate reductase from Triticum aestivum L. seedlings. Nitrate content was decreased by N, P, K, Ca, and Mg deficiencies and unaffected by S and Fe deficiencies. Glutamic acid dehydrogenase activity was decreased by N, P, and S deficiencies, unchanged by K deficiency, and increased by Ca, Mg, and Fe deficiencies. Glutamine synthetase activity closely paralleled nitrate reductase activity and was decreased by deficiencies of N, P, K, Ca, Mg, and S. Glutamic-oxaloacetic transaminase was not sensitive to macronutrient deficiencies. High 14C-leucine incorporation into tissue sections of N-, P-, K-, Ca-, and S-deficient seedlings did not appear indicative of protein synthesis rates in intact seedlings. Nutritional deficiencies apparently depleted endogenous amino acid pools and caused less inhibition of exogenous 14C-leucine incorporation into protein. PMID:16657034

  18. Metallothionein 2A affects the cell respiration by suppressing the expression of mitochondrial protein cytochrome c oxidase subunit II.

    PubMed

    Bragina, Olga; Gurjanova, Karina; Krishtal, Jekaterina; Kulp, Maria; Karro, Niina; Tõugu, Vello; Palumaa, Peep

    2015-06-01

    Metallothioneins (MT) are involved in a broad range of cellular processes and play a major role in protection of cells towards various stressors. Two functions of MTs, namely the maintaining of the homeostasis of transition metal ions and the redox balance, are directly linked to the functioning of mitochondria. Dyshomeostasis of MTs is often related with malfunctioning of mitochondria; however, the mechanism by which MTs affect the mitochondrial respiratory chain is still unknown. We demonstrated that overexpression of MT-2A in HEK cell line decreased the oxidative phosphorylation capacity of the cells. HEK cells overexpressing MT-2A demonstrated reduced oxygen consumption and lower cellular ATP levels. MT-2A did not affect the number of mitochondria, but reduced specifically the level of cytochrome c oxidase subunit II protein, which resulted in lower activity of the complex IV.

  19. Transcription activation by the adenovirus E1a protein

    NASA Astrophysics Data System (ADS)

    Lillie, James W.; Green, Michael R.

    1989-03-01

    The adenovirus Ela protein stimulates transcription of a wide variety of viral and cellular genes. It is shown here that Ela has the two functions characteristic of a typical cellular activator: one direct Ela to the promoter, perhaps by interacting with a DMA-bound protein, and the other, an activating region, enables the bound activator to stimulate transcription.

  20. DNA helicase activity in purified human RECQL4 protein.

    PubMed

    Suzuki, Takahiro; Kohno, Toshiyuki; Ishimi, Yukio

    2009-09-01

    Human RECQL4 protein was expressed in insect cells using a baculovirus protein expression system and it was purified to near homogeneity. The protein sedimented at a position between catalase (230 kDa) and ferritin (440 kDa) in glycerol gradient centrifugation, suggesting that it forms homo-multimers. Activity to displace annealed 17-mer oligonucleotide in the presence of ATP was co-sedimented with hRECQL4 protein. In ion-exchange chromatography, both DNA helicase activity and single-stranded DNA-dependent ATPase activity were co-eluted with hRECQL4 protein. The requirements of ATP and Mg for the helicase activity were different from those for the ATPase activity. The data suggest that the helicase migrates on single-stranded DNA in a 3'-5' direction. These results suggest that the hRECQL4 protein exhibits DNA helicase activity.

  1. [The effect of goal framing on the activation of affective representations].

    PubMed

    Takehashi, Hiroki; Karasawa, Kaori

    2007-10-01

    Guided by regulatory focus theory, this study examined the effects of goal framing on the subjective experience of affect and the accessibility of affective representations. Study I examined lay persons' beliefs concerning the relationship between goal framing and certain kinds of affective experiences. The results indicated that a promotion focus was associated with happiness and disappointment, whereas a prevention focus was associated with relaxation and tension. Study 2 examined the effect of goal framing on the activation of affective representations, and found that a promotion focus activated both gain-related representations (happy and disappointment) and loss-related representations (relaxation and tension), whereas a prevention focus activated only loss-related representations. These results suggest that goal framing activates particular affective representations, and the activated affective representations may influence the interpretation of positive or negative experiences. The discussion considered the function of the activation of affective representations as a mediator between goal framing and its cognitive and behavioral consequences.

  2. T Cell Activation Thresholds are Affected by Gravitational

    NASA Technical Reports Server (NTRS)

    Adams, Charley; Gonzalez, M.; Nelman-Gonzalez, M.

    1999-01-01

    T cells stimulated in space flight by various mitogenic signals show a dramatic reduction in proliferation and expression of early activation markers. Similar results are also obtained in a ground based model of microgravity, clinorotation, which provides a vector-averaged reduction of the apparent gravity on cells without significant shear force. Here we demonstrate that T cell inhibition is due to an increase in the required threshold for activation. Dose response curves indicate that cells activated during clinorotation require higher stimulation to achieve the same level of activation, as measured by CD69 expression. Interleukin 2 receptor expression, and DNA synthesis. The amount of stimulation necessary for 50% activation is 5 fold in the clinostat relative to static. Correlation of TCR internalization with activation also exhibit a dramatic right shift in clinorotation, demonstrating unequivocally that signal transduction mechanism independent of TCR triggering account for the increased activation threshold. Previous results from space flight experiments are consistent with the dose response curves obtained for clinorotation. Activation thresholds are important aspects of T cell memory, autoimmunity and tolerance Clinorotation is a useful, noninvasive tool for the study of cellular and biochemical event regulating T cell activation threshold and the effects of gravitation forces on these systems.

  3. An amino acid substitution in the pyruvate dehydrogenase E1{alpha} gene, affecting mitochondrial import of the precursor protein

    SciTech Connect

    Takakubo, F.; Thorburn, D.R.; Dahl, H.H.M.

    1995-10-01

    A mutation in the mitochondrial targeting sequence was characterized in a male patient with X chromosome-linked pyruvate dehydrogenase E1{alpha} deficiency. The mutation was a base substitution of G by C at nucleotide 134 in the mitochondrial targeting sequence of the PDHA1 gene, resulting in an arginine-to-proline substitution at codon 10 (R10P). Pyruvate dehydrogenase activity in cultured skin fibroblasts was 28% of the control value, and immunoblot analysis revealed a decreased level of pyruvate dehydrogenase E1{alpha}immunoreactivity. Chimeric constructs in which the normal and mutant pyruvate dehydrogenase E1{alpha} targeting sequences were attached to the mitochondrial matrix protein ornithine transcarbamylase were synthesized in a cell free translation system, and mitochondrial import of normal and mutant proteins was compared in vitro. The results show that ornithine transcarbamylase targeted by the mutant pyruvate dehydrogenase E1{alpha} sequence was translocated into the mitochondrial matrix at a reduced rate, suggesting that defective import is responsible for the reduced pyruvate dehydrogenase level in mitochondria. The mutation was also present in an affected brother and the mildly affected mother. The clinical presentations of this X chromosome-linked disorder in affected family members are discussed. To our knowledge, this is the first report of an amino acid substitution in a mitochondrial targeting sequence resulting in a human genetic disease. 58 refs., 5 figs., 1 tab.

  4. Determinants affecting physical activity levels in animal models

    NASA Technical Reports Server (NTRS)

    Tou, Janet C L.; Wade, Charles E.

    2002-01-01

    Weight control is dependent on energy balance. Reduced energy expenditure (EE) associated with decreased physical activity is suggested to be a major underlying cause in the increasing prevalence of weight gain and obesity. Therefore, a better understanding of the biological determinants involved in the regulation of physical activity is essential. To facilitate interpretation in humans, it is helpful to consider the evidence from animal studies. This review focuses on animal studies examining the biological determinants influencing activity and potential implications to human. It appears that physical activity is influenced by a number of parameters. However, regardless of the parameter involved, body weight appears to play an underlying role in the regulation of activity. Furthermore, the regulation of activity associated with body weight appears to occur only after the animal achieves a critical weight. This suggests that activity levels are a consequence rather than a contributor to weight control. However, the existence of an inverse weight-activity relationship remains inconclusive. Confounding the results are the multifactorial nature of physical activity and the lack of appropriate measuring devices. Furthermore, many determinants of body weight are closely interlocked, making it difficult to determine whether a single, combination, or interaction of factors is important for the regulation of activity. For example, diet-induced obesity, aging, lesions to the ventral medial hypothalamus, and genetics all produce hypoactivity. Providing a better understanding of the biological determinants involved in the regulation of activity has important implications for the development of strategies for the prevention of weight gain leading to obesity and subsequent morbidity and mortality in the human population.

  5. Determinants Affecting Physical Activity Levels In Animal Models

    NASA Technical Reports Server (NTRS)

    Tou, Janet C. L.; Wade, Charles E.; Dalton, Bonnie P. (Technical Monitor)

    2001-01-01

    Weight control is dependent on energy balance. Reduced energy expenditure (EE) associated with decreased physical activity is suggested to be a major underlying cause in the increasing prevalence of weight gain and obesity. Therefore, a better understanding of the biological determinants involved in the regulation of physical activity is essential. To facilitate interpretation in humans, it is helpful to consider the evidence from animal studies. This review focuses on animal studies examining the biological determinants influencing activity and potential implications to human. It appears that physical activity is influenced by a number of parameters. However, regardless of the parameter involved, body weight appears to play all underlying role in the regulation of activity. Furthermore, the regulation of activity associated with body weight appears to occur only after the animal achieves a critical weight. This suggests that activity levels are a consequence rather than a contributor to weight control. However, the existence of an inverse weight-activity relationship remains inconclusive. Confounding the results are the multi-factorial nature of physical activity and the lack of appropriate measuring devices. Furthermore, many determinants of body weight are closely interlocked making it difficult to determine whether a single, combination or interaction of factors is important for the regulation of activity. For example, diet-induced obesity, aging, lesions to tile ventral medial hypothalamus and genetics all produce hypoactivity. Providing a better understanding of the biological determinants involved in the regulation of activity has important implications for the development of strategies for the prevention of weight gain leading to obesity and subsequent morbidity and mortality in the human population.

  6. Trithorax group proteins: switching genes on and keeping them active.

    PubMed

    Schuettengruber, Bernd; Martinez, Anne-Marie; Iovino, Nicola; Cavalli, Giacomo

    2011-11-23

    Cellular memory is provided by two counteracting groups of chromatin proteins termed Trithorax group (TrxG) and Polycomb group (PcG) proteins. TrxG proteins activate transcription and are perhaps best known because of the involvement of the TrxG protein MLL in leukaemia. However, in terms of molecular analysis, they have lived in the shadow of their more famous counterparts, the PcG proteins. Recent advances have improved our understanding of TrxG protein function and demonstrated that the heterogeneous group of TrxG proteins is of critical importance in the epigenetic regulation of the cell cycle, senescence, DNA damage and stem cell biology.

  7. Facets of dynamic positive affect: differentiating joy, interest, and activation in the positive and negative affect schedule (PANAS).

    PubMed

    Egloff, Boris; Schmukle, Stefan C; Burns, Lawrence R; Kohlmann, Carl-Walter; Hock, Michael

    2003-09-01

    This article proposes the differentiation of Joy, Interest, and Activation in the Positive Affect (PA) scale of the Positive and Negative Affect Schedule (PANAS; D. Watson, L. A. Clark, & A. Tellegen, 1988). Study 1 analyzed the dynamic course of PA before, during, and after an exam and established the differentiation of the three facets. Study 2 used a multistate-multitrait analysis to confirm this structure. Studies 3-5 used success-failure experiences, speaking tasks, and feedback of exam results to further examine PA facets in affect-arousing settings. All studies provide convincing evidence for the benefit of differentiating three facets of PA in the PANAS: Joy, Interest, and Activation do have distinct and sometimes even opposite courses that make their separation meaningful and rewarding.

  8. Factors affecting yield and safety of protein production from cassava by Cephalosporium eichhorniae

    SciTech Connect

    Mikami, Y.; Gregory, K.F.; Levadoux, W.L.; Balagopalan, C.; Whitwill, S.T.

    1982-01-01

    The properties of C. eichhorniae 152 (ATCC 38255) affecting protein production from cassava carbohydrate, for use as an animal feed, were studied. This strain is a true thermophile, showing optimum growth at 45-47 degrees, maximum protein yield at 45 degrees, and no growth at 25 degrees. It has an optimum pH of approximately 3.8 and is obligately acidophilic, being unable to sustain growth at pH of more than or equal to 6.0 in a liquid medium, or pH of more than or equal to 7.0 on solid media. The optimum growth conditions of pH 3.8 and 45 degrees were strongly inhibitive to potential contaminants. It rapidly hydrolyzed cassava starch. It did not utilize sucrose, but approximately 16% of the small sucrose component of cassava was chemically hydrolyzed during the process. Growth with cassava meal (50 g/l) was complete in approximately 20 h, yielding 22.5 g/l (dry biomass), containing 41% crude protein (48-50% crude protein in the mycelium) and 31% true protein (7.0 g/l). Resting and germinating spores (10 to the power of 6 - 10 to the power of 8 per animal) injected by various routes into normal and gamma-irradiated 6-week-old mice and 7-day-old chickens failed to initiate infections.

  9. Hemoglobin S and C affect protein export in Plasmodium falciparum-infected erythrocytes

    PubMed Central

    Kilian, Nicole; Srismith, Sirikamol; Dittmer, Martin; Ouermi, Djeneba; Bisseye, Cyrille; Simpore, Jacques; Cyrklaff, Marek; Sanchez, Cecilia P.; Lanzer, Michael

    2015-01-01

    ABSTRACT Malaria is a potentially deadly disease. However, not every infected person develops severe symptoms. Some people are protected by naturally occurring mechanisms that frequently involve inheritable modifications in their hemoglobin. The best studied protective hemoglobins are the sickle cell hemoglobin (HbS) and hemoglobin C (HbC) which both result from a single amino acid substitution in β-globin: glutamic acid at position 6 is replaced by valine or lysine, respectively. How these hemoglobinopathies protect from severe malaria is only partly understood. Models currently proposed in the literature include reduced disease-mediating cytoadherence of parasitized hemoglobinopathic erythrocytes, impaired intraerythrocytic development of the parasite, dampened inflammatory responses, or a combination thereof. Using a conditional protein export system and tightly synchronized Plasmodium falciparum cultures, we now show that export of parasite-encoded proteins across the parasitophorous vacuolar membrane is delayed, slower, and reduced in amount in hemoglobinopathic erythrocytes as compared to parasitized wild type red blood cells. Impaired protein export affects proteins targeted to the host cell cytoplasm, Maurer's clefts, and the host cell plasma membrane. Impaired protein export into the host cell compartment provides a mechanistic explanation for the reduced cytoadherence phenotype associated with parasitized hemoglobinopathic erythrocytes. PMID:25701664

  10. Physical Activity Affects Brain Integrity in HIV + Individuals

    PubMed Central

    Ortega, Mario; Baker, Laurie M.; Vaida, Florin; Paul, Robert; Basco, Brian; Ances, Beau M.

    2015-01-01

    Prior research has suggested benefits of aerobic physical activity (PA) on cognition and brain volumes in HIV uninfected (HIV−) individuals, however, few studies have explored the relationships between PA and brain integrity (cognition and structural brain volumes) in HIV-infected (HIV +) individuals. Seventy HIV + individuals underwent neuropsychological testing, structural neuroimaging, laboratory tests, and completed a PA questionnaire, recalling participation in walking, running, and jogging activities over the last year. A PA engagement score of weekly metabolic equivalent (MET) hr of activity was calculated using a compendium of PAs. HIV + individuals were classified as physically active (any energy expended above resting expenditure, n = 22) or sedentary (n = 48). Comparisons of neuropsychological performance, grouped by executive and motor domains, and brain volumes were completed between groups. Physically active and sedentary HIV + individuals had similar demographic and laboratory values, but the active group had higher education (14.0 vs. 12.6 years, p = .034). Physically active HIV + individuals performed better on executive (p = .040, unadjusted; p = .043, adjusted) but not motor function (p = .17). In addition, among the physically active group the amount of physical activity (METs) positively correlated with executive (Pearson’s r = 0.45, p = 0.035) but not motor (r = 0.21; p = .35) performance. In adjusted analyses the physically active HIV + individuals had larger putamen volumes (p = .019). A positive relationship exists between PA and brain integrity in HIV + individuals. Results from the present study emphasize the importance to conduct longitudinal interventional investigation to determine if PA improves brain integrity in HIV + individuals. PMID:26581799

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

    PubMed

    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

    2013-04-01

    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.

  12. 75 FR 62634 - Proposed Information Collection (Obligation to Report Factors Affecting Entitlement) Activity...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-10-12

    ... AFFAIRS Proposed Information Collection (Obligation to Report Factors Affecting Entitlement) Activity... techniques or the use of other forms of information technology. Title: Obligation to Report Factors Affecting... entitlement factors. Individual factors such as income, marital status, and the beneficiary's number...

  13. 78 FR 46418 - Proposed Information Collection (Obligation To Report Factors Affecting Entitlement) Activity...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-07-31

    ... AFFAIRS Proposed Information Collection (Obligation To Report Factors Affecting Entitlement) Activity... techniques or the use of other forms of information technology. Title: Obligation to Report Factors Affecting... entitlement factors. Individual factors such as income, marital status, and the beneficiary's number...

  14. Oxidative Activity of Heated Coal Affected by Antypirogens

    NASA Astrophysics Data System (ADS)

    Torosyan, V. F.; Torosyan, E. S.; Borovikov, I. F.; Yakutova, V. A.

    2016-04-01

    The effect of antypirogens on chemical activity of heated coal is studied. It is proved that ammonium sulfate, calcium phosphate, calcium chloride, calcium nitrate and acid fluoride are the most effective antypirogens.

  15. ANALYSIS OF DISCRIMINATING FACTORS IN HUMAN ACTIVITIES THAT AFFECT EXPOSURE

    EPA Science Inventory

    Accurately modeling exposure to particulate matter (PM) and other pollutants ultimately involves the utilization of human location-activity databases to assist in understanding the potential variability of microenvironmental exposures. This paper critically considers and stati...

  16. Factors affecting antibacterial activity of hop compounds and their derivatives.

    PubMed

    Simpson, W J; Smith, A R

    1992-04-01

    The antibacterial effect of weak acids derived from the hop plant (Humulus lupulus L.) increased with decreasing pH. Analysis of the minimum inhibitory concentration of such compounds against Lactobacillus brevis IFO 3960 over pH 4-7 suggests that undissociated molecules were mainly responsible for inhibition of bacterial growth. The antibacterial activity of trans-isohumulone was ca 20 times greater than that of humulone, 11 times greater than that of colupulone and nine times greater than that of trans-humulinic acid when the degree of ionization was taken into account. Monovalent cations (K+, Na+, NH4+, Rb+, Li+) stimulated antibacterial activity of trans-isohumulone but the effect was smaller than that observed with H+. The response to divalent cations varied: Ca2+ had little effect on antibacterial activity, whereas Mg2+ reduced activity. Lipid materials and beta-cyclodextrin also antagonized the antibacterial action of trans-isohumulone.

  17. Technology trends, energy prices affect worldwide rig activity

    SciTech Connect

    Rappold, K.

    1995-09-25

    The major worldwide offshore rig markets have improved slightly this year, while the onshore markets generally lagged slightly. Offshore rig utilization rates have remained strong worldwide, with some areas reaching nearly 100%. Total worldwide offshore rig (jack ups, semisubmersible, drillships, submersibles, and barges) utilization was about 86%. Offshore drilling activity is driven primarily by oil and natural gas price expectations. Natural gas prices tend to drive North American offshore drilling activity, including the shallow waters in the Gulf of Mexico. International offshore drilling activity and deepwater projects in the Gulf of Mexico are more closely tied to oil prices. The paper discusses US rig count, directional drilling activity, jack up rig demand, semisubmersibles demand, rig replacement costs, and new construction.

  18. Protein acetylation affects acetate metabolism, motility and acid stress response in Escherichia coli

    PubMed Central

    Castaño-Cerezo, Sara; Bernal, Vicente; Post, Harm; Fuhrer, Tobias; Cappadona, Salvatore; Sánchez-Díaz, Nerea C; Sauer, Uwe; Heck, Albert JR; Altelaar, AF Maarten; Cánovas, Manuel

    2014-01-01

    Although protein acetylation is widely observed, it has been associated with few specific regulatory functions making it poorly understood. To interrogate its functionality, we analyzed the acetylome in Escherichia coli knockout mutants of cobB, the only known sirtuin-like deacetylase, and patZ, the best-known protein acetyltransferase. For four growth conditions, more than 2,000 unique acetylated peptides, belonging to 809 proteins, were identified and differentially quantified. Nearly 65% of these proteins are related to metabolism. The global activity of CobB contributes to the deacetylation of a large number of substrates and has a major impact on physiology. Apart from the regulation of acetyl-CoA synthetase, we found that CobB-controlled acetylation of isocitrate lyase contributes to the fine-tuning of the glyoxylate shunt. Acetylation of the transcription factor RcsB prevents DNA binding, activating flagella biosynthesis and motility, and increases acid stress susceptibility. Surprisingly, deletion of patZ increased acetylation in acetate cultures, which suggests that it regulates the levels of acetylating agents. The results presented offer new insights into functional roles of protein acetylation in metabolic fitness and global cell regulation. PMID:25518064

  19. Habitat degradation affects the summer activity of polar bears.

    PubMed

    Ware, Jasmine V; Rode, Karyn D; Bromaghin, Jeffrey F; Douglas, David C; Wilson, Ryan R; Regehr, Eric V; Amstrup, Steven C; Durner, George M; Pagano, Anthony M; Olson, Jay; Robbins, Charles T; Jansen, Heiko T

    2017-02-28

    Understanding behavioral responses of species to environmental change is critical to forecasting population-level effects. Although climate change is significantly impacting species' distributions, few studies have examined associated changes in behavior. Polar bear (Ursus maritimus) subpopulations have varied in their near-term responses to sea ice decline. We examined behavioral responses of two adjacent subpopulations to changes in habitat availability during the annual sea ice minimum using activity data. Location and activity sensor data collected from 1989 to 2014 for 202 adult female polar bears in the Southern Beaufort Sea (SB) and Chukchi Sea (CS) subpopulations were used to compare activity in three habitat types varying in prey availability: (1) land; (2) ice over shallow, biologically productive waters; and (3) ice over deeper, less productive waters. Bears varied activity across and within habitats with the highest activity at 50-75% sea ice concentration over shallow waters. On land, SB bears exhibited variable but relatively high activity associated with the use of subsistence-harvested bowhead whale carcasses, whereas CS bears exhibited low activity consistent with minimal feeding. Both subpopulations had fewer observations in their preferred shallow-water sea ice habitats in recent years, corresponding with declines in availability of this substrate. The substantially higher use of marginal habitats by SB bears is an additional mechanism potentially explaining why this subpopulation has experienced negative effects of sea ice loss compared to the still-productive CS subpopulation. Variability in activity among, and within, habitats suggests that bears alter their behavior in response to habitat conditions, presumably in an attempt to balance prey availability with energy costs.

  20. [Increased fibrinolytic activity during cardiopulmonary bypass is caused by activated protein C system].

    PubMed

    Gando, S; Tedo, I; Masio, H; Goda, Y; Kawahigashi, H

    1994-06-01

    To examine the hypothesis that activated protein C system during cardiopulmonary bypass surgery may increase fibrinolytic activity during cardiopulmonary bypass, protein C activity, protein C antigen and thrombomodulin of sixteen patients undergoing elective cardiopulmonary bypass surgery were investigated after induction of anesthesia, before and after cardiopulmonary bypass, and at the end of operation. Protein C activity decreased and thrombomodulin increased significantly after the cardiopulmonary bypass. There were no significant correlations of thrombomodulin with protein C activity and protein C antigen. In conclusion, we have demonstrated that protein C system is activated and circulating thrombomodulin appears in the systemic circulation during cardiopulmonary bypass surgery and this enhanced activation of protein C system is possibly related to the reported increase of fibrinolytic activity during cardiopulmonary bypass.

  1. Acute-phase protein concentration and metabolic status affect the outcome of treatment in cows with clinical and subclinical endometritis.

    PubMed

    Heidarpour, M; Mohri, M; Fallah-Rad, A H; Dehghan Shahreza, F; Mohammadi, M

    2012-09-01

    The aim of this study was to investigate the role of acute-phase protein concentration and metabolic status in the establishment and resistance of clinical endometritis (CE) and subclinical endometritis (SE) in dairy cows. We also characterised the treatment-related changes in the concentration of acute-phase proteins and metabolic variables in dairy cows affected by CE and SE. Cows of the SE and CE groups presented a significantly higher β-hydroxybutyrate (BHB), haptoglobin and total sialic acid (TSA) concentrations compared with a healthy group of animals. A significantly lower serum calcium concentration, and a significantly higher serum aspartate aminotransferase activity in the CE group, were observed when compared with SE and healthy groups. The comparison of parameters before treatment indicated that cows suffering from CE or SE with lower concentrations of hepatic and inflammatory markers showed a better response to further treatment, and endometritis was not detected in the second examination. Moreover, decreased concentrations of BHB, acute-phase proteins and hepatic markers were observed after successful treatment for endometritis in CE and SE cows. The results obtained in this study suggest that improved liver function and a decrease in the acute-phase protein concentration might favour the resolution of endometritis after treatment.

  2. The nucleoporin Nup153 affects spindle checkpoint activity due to an association with Mad1

    PubMed Central

    Shimi, Takeshi

    2010-01-01

    The nucleoporin Nup153 is known to play pivotal roles in nuclear import and export in interphase cells and as the cell transitions into mitosis, Nup153 is involved in nuclear envelope breakdown. In this study, we demonstrate that the interaction of Nup153 with the spindle assembly checkpoint protein Mad1 is important in the regulation of the spindle checkpoint. Overexpression of human Nup153 in HeLa cells leads to the appearance of multinucleated cells and induces the formation of multipolar spindles. Importantly, it causes inactivation of the spindle checkpoint due to hypophosphorylation of Mad1. Depletion of Nup153 using RNA interference results in the decline of Mad1 at nuclear pores during interphase and more significantly causes a delayed dissociation of Mad1 from kinetochores in metaphase and an increase in the number of unresolved midbodies. In the absence of Nup153 the spindle checkpoint remains active. In vitro studies indicate direct binding of Mad1 to the N-terminal domain of Nup153. Importantly, Nup153 binding to Mad1 affects Mad1's phosphorylation status, but not its ability to interact with Mad2. Our data suggest that Nup153 levels regulate the localization of Mad1 during the metaphase/anaphase transition thereby affecting its phoshorylation status and in turn spindle checkpoint activity and mitotic exit. PMID:21327106

  3. Structural stability and surface activity of sunflower 2S albumins and nonspecific lipid transfer protein.

    PubMed

    Berecz, Bernadett; Mills, E N Clare; Tamás, László; Láng, Ferenc; Shewry, Peter R; Mackie, Alan R

    2010-05-26

    The structural and interfacial properties of five different fractions of sunflower ( Helianthus annuus L.) seed storage proteins were studied. The fractions comprised lipid transfer protein (LTP), the methionine-rich 2S albumin SFA8 (sunflower albumin 8), and three mixtures of non-methionine-rich 2S albumins called Alb1 and Alb2 proteins (sunflower albumins 1 and 2). Heating affected all of the proteins studied, with SFA8 and LTP becoming more surface active than the native proteins after heating and cooling. LTP appeared to be less thermostable than homologous LTPs from other plant species. SFA8 generated the greatest elastic modulus and formed the most stable emulsions, whereas LTP showed poorer emulsification properties. The mixed 2S albumin fractions showed moderate levels of surface activity but had the poorest emulsification properties among the proteins studied.

  4. Ionizing Radiation Impairs T Cell Activation by Affecting Metabolic Reprogramming.

    PubMed

    Li, Heng-Hong; Wang, Yi-Wen; Chen, Renxiang; Zhou, Bin; Ashwell, Jonathan D; Fornace, Albert J

    2015-01-01

    Ionizing radiation has a variety of acute and long-lasting adverse effects on the immune system. Whereas measureable effects of radiation on immune cell cytotoxicity and population change have been well studied in human and animal models, little is known about the functional alterations of the surviving immune cells after ionizing radiation. The objective of this study was to delineate the effects of radiation on T cell function by studying the alterations of T cell receptor activation and metabolic changes in activated T cells isolated from previously irradiated animals. Using a global metabolomics profiling approach, for the first time we demonstrate that ionizing radiation impairs metabolic reprogramming of T cell activation, which leads to substantial decreases in the efficiency of key metabolic processes required for activation, such as glucose uptake, glycolysis, and energy metabolism. In-depth understanding of how radiation impacts T cell function highlighting modulation of metabolism during activation is not only a novel approach to investigate the pivotal processes in the shift of T cell homeostasis after radiation, it also may lead to new targets for therapeutic manipulation in the combination of radiotherapy and immune therapy. Given that appreciable effects were observed with as low as 10 cGy, our results also have implications for low dose environmental exposures.

  5. Does lunisolar gravitational tide affect the activity of animals?

    NASA Astrophysics Data System (ADS)

    Deshcherevskii, A. V.; Sidorin, A. Ya.

    2010-12-01

    Multiyear time series obtained by the continuous instrumental monitoring of the electrical activity (EA) of weakly electric fish Gnathonemus leopoldianus and the motor activity (MA) of the freshwater catfish Hoplosternum thoracatum and the cockroach Blaberus craniifer are compared to the parameters of the lunisolar gravitational tide. These curves are observed to be very similar for a large number of time intervals. However, a more detailed analysis shows this to be only a superficial resemblance caused by the closeness of the periods of diurnal and semidiurnal rhythms of bioindicator activity (the dominant rhythms in EA and MA patterns) and the periods of main gravitational tidal waves. It is concluded that the lunisolar gravitational tide has no significant effect on animal behavior in our experiment.

  6. Identification of highly active flocculant proteins in bovine blood.

    PubMed

    Piazza, George J; Nuñez, Alberto; Garcia, Rafael A

    2012-03-01

    Synthetic polymeric flocculants are used extensively for wastewater remediation, soil stabilization, and reduction in water leakage from unlined canals. Sources of highly active, inexpensive, renewable flocculants are needed to replace synthetic flocculants. High kaolin flocculant activity was documented for bovine blood (BB) and blood plasma with several anticoagulant treatments. BB serum also had high flocculant activity. To address the hypothesis that some blood proteins have strong flocculating activity, the BB proteins were separated by SEC. Then, the major proteins of the flocculant-active fractions were separated by SDS-PAGE. Identity of the major protein components was determined by tryptic digestion and peptide analysis by MALDI TOF MS. The sequence of selected peptides was confirmed using TOF/TOF-MS/MS fragmentation. Hemoglobin dimer (subunits α and β) was identified as the major protein component of the active fraction in BB; its high flocculation activity was confirmed by testing a commercial sample of hemoglobin. In the same manner, three proteins from blood plasma (fibrinogen, γ-globulin, α-2-macroglobulin) were found to be highly active flocculants, but bovine serum albumin, α-globulin, and β-globulin were not flocculants. On a mass basis, hemoglobin, γ-globulin, α-2-macroglobulin were as effective as anionic polyacrylamide (PAM), a widely used synthetic flocculant. The blood proteins acted faster than PAM, and unlike PAM, the blood proteins flocculants did not require calcium salts for their activity.

  7. SR-like RNA-binding protein Slr1 affects Candida albicans filamentation and virulence.

    PubMed

    Ariyachet, Chaiyaboot; Solis, Norma V; Liu, Yaoping; Prasadarao, Nemani V; Filler, Scott G; McBride, Anne E

    2013-04-01

    Candida albicans causes both mucosal and disseminated infections, and its capacity to grow as both yeast and hyphae is a key virulence factor. Hyphal formation is a type of polarized growth, and members of the SR (serine-arginine) family of RNA-binding proteins influence polarized growth of both Saccharomyces cerevisiae and Aspergillus nidulans. Therefore, we investigated whether SR-like proteins affect filamentous growth and virulence of C. albicans. BLAST searches with S. cerevisiae SR-like protein Npl3 (ScNpl3) identified two C. albicans proteins: CaNpl3, an apparent ScNpl3 ortholog, and Slr1, another SR-like RNA-binding protein with no close S. cerevisiae ortholog. Whereas ScNpl3 was critical for growth, deletion of NPL3 in C. albicans resulted in few phenotypic changes. In contrast, the slr1Δ/Δ mutant had a reduced growth rate in vitro, decreased filamentation, and impaired capacity to damage epithelial and endothelial cells in vitro. Mice infected intravenously with the slr1Δ/Δ mutant strain had significantly prolonged survival compared to that of mice infected with the wild-type or slr1Δ/Δ mutant complemented with SLR1 (slr1Δ/Δ+SLR1) strain, without a concomitant decrease in kidney fungal burden. Histopathology, however, revealed differential localization of slr1Δ/Δ hyphal and yeast morphologies within the kidney. Mice infected with slr1Δ/Δ cells also had an increased brain fungal burden, which correlated with increased invasion of brain, but not umbilical vein, endothelial cells in vitro. The enhanced brain endothelial cell invasion was likely due to the increased surface exposure of the Als3 adhesin on slr1Δ/Δ cells. Our results indicate that Slr1 is an SR-like protein that influences C. albicans growth, filamentation, host cell interactions, and virulence.

  8. Dietary lipid and gross energy affect protein utilization in the rare minnow Gobiocypris rarus

    NASA Astrophysics Data System (ADS)

    Wu, Benli; Xiong, Xiaoqin; Xie, Shouqi; Wang, Jianwei

    2016-07-01

    An 8-week feeding trial was conducted to detect the optimal dietary protein and energy, as well as the effects of protein to energy ratio on growth, for the rare minnow ( Gobiocypris rarus), which are critical to nutrition standardization for model fish. Twenty-four diets were formulated to contain three gross energy (10, 12.5, 15 kJ/g), four protein (20%, 25%, 30%, 35%), and two lipid levels (3%, 6%). The results showed that optimal dietary E/P was 41.7-50 kJ/g for maximum growth in juvenile rare minnows at 6% dietary crude lipid. At 3% dietary lipid, specific growth rate (SGR) increased markedly when E/P decreased from 62.5 kJ/g to 35.7 kJ/g and gross energy was 12.5 kJ/g, and from 75 kJ/g to 42.9 kJ/g when gross energy was 15.0 kJ/g. The optimal gross energy was estimated at 12.5 kJ/g and excess energy decreased food intake and growth. Dietary lipid exhibited an apparent protein-sparing effect. Optimal protein decreased from 35% to 25%-30% with an increase in dietary lipid from 3% to 6% without adversely effecting growth. Dietary lipid level affects the optimal dietary E/P ratio. In conclusion, recommended dietary protein and energy for rare minnow are 20%-35% and 10-12.5 kJ/g, respectively.

  9. Nicotine reward and affective nicotine withdrawal signs are attenuated in calcium/calmodulin-dependent protein kinase IV knockout mice.

    PubMed

    Jackson, Kia J; Sanjakdar, Sarah S; Chen, Xiangning; Damaj, M Imad

    2012-01-01

    The influx of Ca(2+) through calcium-permeable nicotinic acetylcholine receptors (nAChRs) leads to activation of various downstream processes that may be relevant to nicotine-mediated behaviors. The calcium activated protein, calcium/calmodulin-dependent protein kinase IV (CaMKIV) phosphorylates the downstream transcription factor cyclic AMP response element binding protein (CREB), which mediates nicotine responses; however the role of CaMKIV in nicotine dependence is unknown. Given the proposed role of CaMKIV in CREB activation, we hypothesized that CaMKIV might be a crucial molecular component in the development of nicotine dependence. Using male CaMKIV genetically modified mice, we found that nicotine reward is attenuated in CaMKIV knockout (-/-) mice, but cocaine reward is enhanced in these mice. CaMKIV protein levels were also increased in the nucleus accumbens of C57Bl/6 mice after nicotine reward. In a nicotine withdrawal assessment, anxiety-related behavior, but not somatic signs or the hyperalgesia response are attenuated in CaMKIV -/- mice. To complement our animal studies, we also conducted a human genetic association analysis and found that variants in the CaMKIV gene are associated with a protective effect against nicotine dependence. Taken together, our results support an important role for CaMKIV in nicotine reward, and suggest that CaMKIV has opposing roles in nicotine and cocaine reward. Further, CaMKIV mediates affective, but not physical nicotine withdrawal signs, and has a protective effect against nicotine dependence in human genetic association studies. These findings further indicate the importance of calcium-dependent mechanisms in mediating behaviors associated with drugs of abuse.

  10. Monocyte chemoattractant protein-1 in dogs affected with neoplasia or inflammation.

    PubMed

    Ishioka, Katsumi; Suzuki, Yumi; Tajima, Kana; Ohtaki, Sumire; Miyabe, Masahiro; Takasaki, Mariko; Mori, Akihiro; Momota, Yutaka; Azakami, Daigo; Sako, Toshinori

    2013-02-01

    Monocyte chemoattractant protein-1 (MCP-1) is a member of the C-C family chemokines, which mobilizes monocytes from bone marrow to the site of inflammation. To evaluate the clinical utility of canine MCP-1 as a blood test item, we measured serum MCP-1 concentrations in normal and ill dogs. Reference interval of canine MCP-1 was established as 115.6-176.9 pg/ml. Serum MCP-1 concentrations increased in the dogs affected with neoplastic (518.0 ± 84.8 pg/ml), inflammatory (257.0 ± 42.5 pg/ml) or other diseases (360.3 ± 45.2 pg/ml). The results showed high sensitivity of MCP-1 to detect neoplasia and inflammation. Moreover, MCP-1 increased in some cases in which C-reactive protein didn't increase. MCP-1 might be helpful as a screening blood test marker for detection of neoplasia and inflammation in dogs.

  11. Dimerization between aequorea fluorescent proteins does not affect interaction between tagged estrogen receptors in living cells.

    PubMed

    Kofoed, Eric M; Guerbadot, Martin; Schaufele, Fred

    2008-01-01

    Forster resonance energy transfer (FRET) detection of protein interaction in living cells is commonly measured following the expression of interacting proteins genetically fused to the cyan (CFP) and yellow (YFP) derivatives of the Aequorea victoria fluorescent protein (FP). These FPs can dimerize at mM concentrations, which may introduce artifacts into the measurement of interaction between proteins that are fused with the FPs. Here, FRET analysis of the interaction between estrogen receptors (alpha isoform, ERalpha) labeled with "wild-type" CFP and YFP is compared with that of ERalpha labeled with "monomeric" A206K mutants of CFP and YFP. The intracellular equilibrium dissociation constant for the hormone-induced ERalpha-ERalpha interaction is similar for ERalpha labeled with wild-type or monomeric FPs. However, the measurement of energy transfer measured for ERalpha-ERalpha interaction in each cell is less consistent with the monomeric FPs. Thus, dimerization of the FPs does not affect the kinetics of ERalpha-ERalpha interaction but, when brought close together via ERalpha-ERalpha interaction, FP dimerization modestly improves FRET measurement.

  12. Lethal Factor Active-Site Mutations Affect Catalytic Activity In Vitro

    PubMed Central

    Hammond, S. E.; Hanna, P. C.

    1998-01-01

    The lethal factor (LF) protein of Bacillus anthracis lethal toxin contains the thermolysin-like active-site and zinc-binding consensus motif HEXXH (K. R. Klimpel, N. Arora, and S. H. Leppla, Mol. Microbiol. 13:1093–1100, 1994). LF is hypothesized to act as a Zn2+ metalloprotease in the cytoplasm of macrophages, but no proteolytic activities have been previously shown on any target substrate. Here, synthetic peptides are hydrolyzed by LF in vitro. Mass spectroscopy and peptide sequencing of isolated cleavage products separated by reverse-phase high-pressure liquid chromatography indicate that LF seems to prefer proline-containing substrates. Substitution mutations within the consensus active-site residues completely abolish all in vitro catalytic functions, as does addition of 1,10-phenanthroline, EDTA, and certain amino acid hydroxamates, including the novel zinc metalloprotease inhibitor ZINCOV. In contrast, the protease inhibitors bestatin and lysine CMK, previously shown to block LF activity on macrophages, did not block LF activity in vitro. These data provide the first direct evidence that LF may act as an endopeptidase. PMID:9573135

  13. Inhibition of hsp70 by methylene blue affects signaling protein function and ubiquitination and modulates polyglutamine protein degradation.

    PubMed

    Wang, Adrienne M; Morishima, Yoshihiro; Clapp, Kelly M; Peng, Hwei-Ming; Pratt, William B; Gestwicki, Jason E; Osawa, Yoichi; Lieberman, Andrew P

    2010-05-21

    The Hsp90/Hsp70-based chaperone machinery regulates the activity and degradation of many signaling proteins. Cycling with Hsp90 stabilizes client proteins, whereas Hsp70 interacts with chaperone-dependent E3 ubiquitin ligases to promote protein degradation. To probe these actions, small molecule inhibitors of Hsp70 would be extremely useful; however, few have been identified. Here we test the effects of methylene blue, a recently described inhibitor of Hsp70 ATPase activity, in three well established systems of increasing complexity. First, we demonstrate that methylene blue inhibits the ability of the purified Hsp90/Hsp70-based chaperone machinery to enable ligand binding by the glucocorticoid receptor and show that this effect is due to specific inhibition of Hsp70. Next, we establish that ubiquitination of neuronal nitric-oxide synthase by the native ubiquitinating system of reticulocyte lysate is dependent upon both Hsp70 and the E3 ubiquitin ligase CHIP and is blocked by methylene blue. Finally, we demonstrate that methylene blue impairs degradation of the polyglutamine expanded androgen receptor, an Hsp90 client mutated in spinal and bulbar muscular atrophy. In contrast, degradation of an amino-terminal fragment of the receptor, which lacks the ligand binding domain and, therefore, is not a client of the Hsp90/Hsp70-based chaperone machinery, is enhanced through homeostatic induction of autophagy that occurs when Hsp70-dependent proteasomal degradation is inhibited by methylene blue. Our data demonstrate the utility of methylene blue in defining Hsp70-dependent functions and reveal divergent effects on polyglutamine protein degradation depending on whether the substrate is an Hsp90 client.

  14. Early life stress affects limited regional brain activity in depression.

    PubMed

    Du, Lian; Wang, Jingjie; Meng, Ben; Yong, Na; Yang, Xiangying; Huang, Qingling; Zhang, Yan; Yang, Lingling; Qu, Yuan; Chen, Zhu; Li, Yongmei; Lv, Fajin; Hu, Hua

    2016-05-03

    Early life stress (ELS) can alter brain function and increases the risk of major depressive disorder (MDD) in later life. This study investigated whether ELS contributes to differences in regional brain activity between MDD patients and healthy controls (HC), as measured by amplitude of low-frequency fluctuation (ALFF)/fractional (f)ALFF. Eighteen first-episode, treatment-naïve MDD patients and HC were assessed with the Childhood Trauma Questionnaire and resting-state functional magnetic resonance imaging. We compared ALFF/fALFF between MDD patients and HC, with or without controlling for ELS, and determined whether ELS level was correlated with regional brain activity in each group. After regressing out ELS, we found that ALFF increased in bilateral amygdala and left orbital/cerebellum, while fALFF decreased in left inferior temporal and right middle frontal gyri in MDD patients relative to controls. ELS positively correlated with regional activity in the left cerebellum in MDD and in the right post-central/inferior temporal/superior frontal cingulate, inferior frontal gyrus and bilateral cerebellum in HC. Our findings indicate that there is only very limited region showing correlation between ELS and brain activity in MDD, while diverse areas in HC, suggesting ELS has few impacts on MDD patients.

  15. Early life stress affects limited regional brain activity in depression

    PubMed Central

    Du, Lian; Wang, Jingjie; Meng, Ben; Yong, Na; Yang, Xiangying; Huang, Qingling; Zhang, Yan; Yang, Lingling; Qu, Yuan; Chen, Zhu; Li, Yongmei; Lv, Fajin; Hu, Hua

    2016-01-01

    Early life stress (ELS) can alter brain function and increases the risk of major depressive disorder (MDD) in later life. This study investigated whether ELS contributes to differences in regional brain activity between MDD patients and healthy controls (HC), as measured by amplitude of low-frequency fluctuation (ALFF)/fractional (f)ALFF. Eighteen first-episode, treatment-naïve MDD patients and HC were assessed with the Childhood Trauma Questionnaire and resting-state functional magnetic resonance imaging. We compared ALFF/fALFF between MDD patients and HC, with or without controlling for ELS, and determined whether ELS level was correlated with regional brain activity in each group. After regressing out ELS, we found that ALFF increased in bilateral amygdala and left orbital/cerebellum, while fALFF decreased in left inferior temporal and right middle frontal gyri in MDD patients relative to controls. ELS positively correlated with regional activity in the left cerebellum in MDD and in the right post-central/inferior temporal/superior frontal cingulate, inferior frontal gyrus and bilateral cerebellum in HC. Our findings indicate that there is only very limited region showing correlation between ELS and brain activity in MDD, while diverse areas in HC, suggesting ELS has few impacts on MDD patients. PMID:27138376

  16. 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.

  17. The affected gene underlying the class K glycosylphosphatidylinositol (GPI) surface protein defect codes for the GPI transamidase

    PubMed Central

    Yu, Jianliang; Nagarajan, Shanmugam; Knez, Jansen J.; Udenfriend, Sidney; Chen, Rui; Medof, M. Edward

    1997-01-01

    The final step in glycosylphosphatidylinositol (GPI) anchoring of cell surface proteins consists of a transamidation reaction in which preassembled GPI donors are substituted for C-terminal signal sequences in nascent polypeptides. In previous studies we described a human K562 cell mutant, termed class K, that accumulates fully assembled GPI units but is unable to transfer them to N-terminally processed proproteins. In further work we showed that, unlike wild-type microsomes, microsomes from these cells are unable to support C-terminal interaction of proproteins with the small nucleophiles hydrazine or hydroxylamine, and that the cells thus are defective in transamidation. In this study, using a modified recombinant vaccinia transient transfection system in conjunction with a composite cDNA prepared by 5′ extension of an existing GenBank sequence, we found that the genetic element affected in these cells corresponds to the human homolog of yGPI8, a gene affected in a yeast mutant strain exhibiting similar accumulation of GPI donors without transfer. hGPI8 gives rise to mRNAs of 1.6 and 1.9 kb, both encoding a protein of 395 amino acids that varies in cells with their ability to couple GPIs to proteins. The gene spans ≈25 kb of DNA on chromosome 1. Reconstitution of class K cells with hGPI8 abolishes their accumulation of GPI precursors and restores C-terminal processing of GPI-anchored proteins. Also, hGPI8 restores the ability of microsomes from the mutant cells to yield an active carbonyl in the presence of a proprotein which is considered to be an intermediate in catalysis by a transamidase. PMID:9356492

  18. Partial calcium depletion during membrane filtration affects gelation of reconstituted milk protein concentrates.

    PubMed

    Eshpari, H; Jimenez-Flores, R; Tong, P S; Corredig, M

    2015-12-01

    Milk protein concentrate powders (MPC) with improved rehydration properties are often manufactured using processing steps, such as acidification and high-pressure processing, and with addition of other ingredients, such as sodium chloride, during their production. These steps are known to increase the amount of serum caseins or modify the mineral equilibrium, hence improving solubility of the retentates. The processing functionality of the micelles may be affected. The aim of this study was to investigate the effects of partial acidification by adding glucono-δ-lactone (GDL) to skim milk during membrane filtration on the structural changes of the casein micelles by observing their chymosin-induced coagulation behavior, as such coagulation is affected by both the supramolecular structure of the caseins and calcium equilibrium. Milk protein concentrates were prepared by preacidification with GDL to pH 6 using ultrafiltration (UF) and diafiltration (DF) followed by spray-drying. Reconstituted UF and DF samples (3.2% protein) treated with GDL showed significantly increased amounts of soluble calcium and nonsedimentable caseins compared with their respective controls, as measured by ion chromatography and sodium dodecyl sulfate-PAGE electrophoresis, respectively. The primary phase of chymosin-induced gelation was not significantly different between treatments as measured by the amount of caseino-macropeptide released. The rheological properties of the reconstituted MPC powders were determined immediately after addition of chymosin, both before and after dialysis against skim milk, to ensure similar serum composition for all samples. Reconstituted samples before dialysis showed no gelation (defined as tan δ=1), and after re-equilibration only control UF and DF samples showed gelation. The gelation properties of reconstituted MPC powders were negatively affected by the presence of soluble casein, and positively affected by the amount of both soluble and insoluble

  19. An allosteric role for receptor activity-modifying proteins in defining GPCR pharmacology

    PubMed Central

    J Gingell, Joseph; Simms, John; Barwell, James; Poyner, David R; Watkins, Harriet A; Pioszak, Augen A; Sexton, Patrick M; Hay, Debbie L

    2016-01-01

    G protein-coupled receptors are allosteric proteins that control transmission of external signals to regulate cellular response. Although agonist binding promotes canonical G protein signalling transmitted through conformational changes, G protein-coupled receptors also interact with other proteins. These include other G protein-coupled receptors, other receptors and channels, regulatory proteins and receptor-modifying proteins, notably receptor activity-modifying proteins (RAMPs). RAMPs have at least 11 G protein-coupled receptor partners, including many class B G protein-coupled receptors. Prototypic is the calcitonin receptor, with altered ligand specificity when co-expressed with RAMPs. To gain molecular insight into the consequences of this protein–protein interaction, we combined molecular modelling with mutagenesis of the calcitonin receptor extracellular domain, assessed in ligand binding and functional assays. Although some calcitonin receptor residues are universally important for peptide interactions (calcitonin, amylin and calcitonin gene-related peptide) in calcitonin receptor alone or with receptor activity-modifying protein, others have RAMP-dependent effects, whereby mutations decreased amylin/calcitonin gene-related peptide potency substantially only when RAMP was present. Remarkably, the key residues were completely conserved between calcitonin receptor and AMY receptors, and between subtypes of AMY receptor that have different ligand preferences. Mutations at the interface between calcitonin receptor and RAMP affected ligand pharmacology in a RAMP-dependent manner, suggesting that RAMP may allosterically influence the calcitonin receptor conformation. Supporting this, molecular dynamics simulations suggested that the calcitonin receptor extracellular N-terminal domain is more flexible in the presence of receptor activity-modifying protein 1. Thus, RAMPs may act in an allosteric manner to generate a spectrum of unique calcitonin receptor

  20. Jun Dimerization Protein 2 Activates Mc2r Transcriptional Activity: Role of Phosphorylation and SUMOylation

    PubMed Central

    Wang, Chiung-Min; Wang, Raymond X.; Liu, Runhua; Yang, Wei-Hsiung

    2017-01-01

    Jun dimerization protein 2 (JDP2), a basic leucine zipper transcription factor, is involved in numerous biological and cellular processes such as cancer development and regulation, cell-cycle regulation, skeletal muscle and osteoclast differentiation, progesterone receptor signaling, and antibacterial immunity. Though JDP2 is widely expressed in mammalian tissues, its function in gonads and adrenals (such as regulation of steroidogenesis and adrenal development) is largely unknown. Herein, we find that JDP2 mRNA and proteins are expressed in mouse adrenal gland tissues. Moreover, overexpression of JDP2 in Y1 mouse adrenocortical cancer cells increases the level of melanocortin 2 receptor (MC2R) protein. Notably, Mc2r promoter activity is activated by JDP2 in a dose-dependent manner. Next, by mapping the Mc2r promoter, we show that cAMP response elements (between −1320 and −720-bp) are mainly required for Mc2r activation by JDP2 and demonstrate that −830-bp is the major JDP2 binding site by real-time chromatin immunoprecipitation (ChIP) analysis. Mutations of cAMP response elements on Mc2r promoter disrupts JDP2 effect. Furthermore, we demonstrate that removal of phosphorylation of JDP2 results in attenuated transcriptional activity of Mc2r. Finally, we show that JDP2 is a candidate for SUMOylation and SUMOylation affects JDP2-mediated Mc2r transcriptional activity. Taken together, JDP2 acts as a novel transcriptional activator of the mouse Mc2r gene, suggesting that JDP2 may have physiological functions as a novel player in MC2R-mediated steroidogenesis as well as cell signaling in adrenal glands. PMID:28146118

  1. Factors affecting the adsorption of chromium (VI) on activated carbon

    SciTech Connect

    Yavuz, R.; Orbak, I.; Karatepe, N.

    2006-09-15

    The aim of this investigation was to determine the adsorption behavior of chromium (VI) on two different activated carbon samples produced from Tuncbilek lignite. The effects of the initial chromium (VI) concentration (250-1000 mg/L), temperature (297-323 K) and pH (2.0-9.5) on adsorption were investigated systematically. The effectiveness of the parameters on chromium adsorption was found to be in the order of pH, the initial Cr(VI) concentration and the temperature. Increasing the pH from 2.0 to 9.5 caused a decrease in adsorption. However, the adsorption was increased by increasing the initial Cr(VI) concentration and temperature. The multilinear mathematical model was also developed to predict the Cr(VI) adsorption on activated carbon samples within the experimental conditions.

  2. How absorbed hydrogen affects the catalytic activity of transition metals.

    PubMed

    Aleksandrov, Hristiyan A; Kozlov, Sergey M; Schauermann, Swetlana; Vayssilov, Georgi N; Neyman, Konstantin M

    2014-12-01

    Heterogeneous catalysis is commonly governed by surface active sites. Yet, areas just below the surface can also influence catalytic activity, for instance, when fragmentation products of catalytic feeds penetrate into catalysts. In particular, H absorbed below the surface is required for certain hydrogenation reactions on metals. Herein, we show that a sufficient concentration of subsurface hydrogen, H(sub) , may either significantly increase or decrease the bond energy and the reactivity of the adsorbed hydrogen, H(ad) , depending on the metal. We predict a representative reaction, ethyl hydrogenation, to speed up on Pd and Pt, but to slow down on Ni and Rh in the presence of H(sub) , especially on metal nanoparticles. The identified effects of subsurface H on surface reactivity are indispensable for an atomistic understanding of hydrogenation processes on transition metals and interactions of hydrogen with metals in general.

  3. Interaction of Berberine derivative with protein POT1 affect telomere function in cancer cells

    SciTech Connect

    Xiao, Nannan; Chen, Siqi; Ma, Yan; Qiu, Jun; Tan, Jia-Heng; Ou, Tian-Miao; Gu, Lian-Quan; Huang, Zhi-Shu; Li, Ding

    2012-03-16

    Highlights: Black-Right-Pointing-Pointer The protein POT1 plays an important role in telomere protection. Black-Right-Pointing-Pointer Functional POT1 was overexpressed in Escherichia coli for the first time, and purified. Black-Right-Pointing-Pointer Compound Sysu-00692 was found to be the first POT1-binding ligand. Black-Right-Pointing-Pointer Sysu-00692 could interfere with the binding activity of POT1 in vivo. Black-Right-Pointing-Pointer Sysu-00692 had inhibition on telomerase and cell proliferation. -- Abstract: The protein POT1 plays an important role in telomere protection, which is related with telomere elongation and cell immortality. The protein has been recognized as a promising drug target for cancer treatment. In the present study, we cloned, overexpressed in Escherichia coli for the first time, and purified recombinant human POT1. The protein was proved to be active through filter binding assay, FRET and CD experiments. In the initial screening for protein binding ligands using SPR, compound Sysu-00692 was found to bind well with the POT1, which was confirmed with EMSA. Its in vivo activity study showed that compound Sysu-00692 could interfere with the binding between human POT1 and the telomeric DNA through chromatin immunoprecipitation. Besides, the compound showed mild inhibition on telomerase and cell proliferation. As we know, compound Sysu-00692 is the first reported POT1-binding ligand, which could serve as a lead compound for further improvement. This work offered a potentially new approach for drug design for the treatment of cancers.

  4. Cigarette smoke extract affects functional activity of MRP1 in bronchial epithelial cells.

    PubMed

    van der Deen, Margaretha; de Vries, Elisabeth G E; Visserman, Hylke; Zandbergen, Wouter; Postma, Dirkje S; Timens, Wim; Timmer-Bosscha, Hetty

    2007-01-01

    Cigarette smoke is the principal risk factor for development of chronic obstructive pulmonary disease (COPD). Multidrug resistance-associated protein 1 (MRP1) is a member of the ATP-binding cassette (ABC) superfamily of transporters, which transport physiologic and toxic substrates across cell membranes. MRP1 is highly expressed in lung epithelium. This study aims to analyze the effect of cigarette smoke extract (CSE) on MRP1 activity. In the human bronchial epithelial cell line 16HBE14o-, MRP1 function was studied flow cytometrically by cellular retention of carboxyfluorescein (CF) after CSE incubation and MRP1 downregulation by RNA interference (siRNA). Cell survival was measured by the MTT assay. Immunocytochemically, it was shown that 16HBE14o(-) expressed MRP1 and breast cancer resistance protein. Coincubation of CSE IC50 (1.53% +/- 0.22%) with MK571 further decreased cell survival 31% (p, = 0.018). CSE increased cellular CF retention dose dependently from 1.7-fold at 5% CSE to 10.3-fold at 40% CSE (both p < 0.05). siRNA reduced MRP1 RNA expression with 49% and increased CF accumulation 67% versus control transfected cells. CSE exposure further increased CF retention 24% (p = 0.031). A linear positive relation between MRP1 function and CSE-modulating effects (r = 0.99, p =0.089) was shown in untransfected, control transfected, and MRP1 downregulated 16HBE14o- cells analogous to blocking effects with MRP1 inhibitor MK571 (r = 0.99, p = 0.034). In conclusion, cigarette smoke extract affects MRP1 activity probably competitively in bronchial epithelial cells. Inhibition of MRP1 in turn results in higher CSE toxicity. We propose that MRP1 may be a protective protein for COPD development.

  5. Impaired suppressor activity in children affected by coeliac disease.

    PubMed Central

    Pignata, C; Troncone, R; Monaco, G; Ciriaco, M; Farris, E; Carminati, G; Auricchio, S

    1985-01-01

    Immunoregulatory cells were enumerated in 19 coeliac disease children on a gluten free diet by means of monoclonal antibodies that define total T lymphocytes (T3), helper/inducer T cells (T4), suppressor/cytotoxic T cells (T8) and monocytes (M1), as well as by means of surface receptors for Fc fragments of IgM and IgG (T mu and T gamma, respectively). In addition, suppressor cell function was assessed in 17 coeliac disease patients by examining the ability of concanavalin-A (Con-A)-activated suppressor cells to inhibit autologous cell response to mitogenic stimulus as compared with age-matched controls. No statistically significant differences were found in the percentages of subsets defined by monoclonal antibodies between coeliac disease patients and age-matched controls, whereas coeliac disease patients had a significant decrease of the subpopulation bearing membrane receptor for Fc fragment of IgG. Mean value was 8.5% in coeliac patients versus 13.4% in age-matched controls. In the functional assay, mononuclear cells from 10 out of 17 coeliac disease patients either totally or partially failed to suppress responder cells after Con-A-activation. This defect is not related to HLA-DR status, because no difference was found between patients-HLA-matched and unmatched normal individuals. In this assay, mononuclear cells of three coeliac disease patients with low suppressor activity were able to inhibit responder cells to the same extent as controls, when indomethacin was used to block prostaglandin production in the induction phase of Con-A-activated suppressor cells. Our results suggest that an abnormality in immunoregulation may play a role in the pathogenesis of coeliac disease. PMID:3156076

  6. Seasonal Pacing - Match Importance Affects Activity in Professional Soccer.

    PubMed

    Link, Daniel; de Lorenzo, Michael F

    2016-01-01

    This research explores the influence of match importance on player activity in professional soccer. Therefore, we used an observational approach and analyzed 1,211 matches of German Bundesliga and 2nd Bundesliga. The importance measurement employed is based on post season consequences of teams involved in a match. This means, if a match result could potentially influence the final rank, and this rank would lead to different consequences for a team, such as qualification for Champions League opposed to qualification for Europe League, then this match is classified as important; otherwise not. Activity was quantified by TOTAL DISTANCE COVERED, SPRINTS, FAST RUNS, DUELS, FOULS and ATTEMPTS. Running parameters were recorded using a semi-automatic optical tracking system, while technical variables were collected by professional data loggers. Based on our importance classification, low important matches occurred at the beginning of round 29. A two-way ANOVA indicates significantly increased FAST RUNS (+4%, d = 0.3), DUELS (+16%, d = 1.0) and FOULS (+36%, d = 1.2) in important matches compared to low important ones. For FAST RUNS and FOULS, this effect only exists in Bundesliga. A comparison of the two leagues show that TOTAL DISTANCE COVERED (+3%, d = 0.9), SPRINTS (+25%, d = 1.4) and FAST RUNS (+15%, d = 1.4) are higher compared to 2nd Bundesliga, whilst FOULS is less in Bundesliga (-7%, d = 0.3). No difference in player activity was found between matches at the beginning of a season (round 1-6) and at the end of a season (round 29-34). We conclude that match importance influences player activity in German professional soccer. The most reasonable explanation is a conscious or unconscious pacing strategy, motivated by preserving abilities or preventing injury. Since this tendency mainly exists in Bundesliga, this may suggest that more skilled players show a higher awareness for the need of pacing.

  7. Factors affecting the adsorption of xenon on activated carbon

    SciTech Connect

    Underhill, D.W.; DiCello, D.C.; Scaglia, L.A.; Watson, J.A.

    1986-08-01

    The presence of water vapor was found to interfere strongly with the dynamic adsorption of /sup 133/Xe on coconut-base activated charcoal. The percent loss in the xenon adsorption coefficient was similar to values reported earlier for the adsorption of krypton on humidified charcoal. Attempts to increase the adsorption of xenon by (a) using a petroleum-based adsorbent with an extremely high surface area and (b) by impregnation of the adsorbent with iodine were not successful.

  8. Seasonal Pacing - Match Importance Affects Activity in Professional Soccer

    PubMed Central

    Link, Daniel; de Lorenzo, Michael F.

    2016-01-01

    This research explores the influence of match importance on player activity in professional soccer. Therefore, we used an observational approach and analyzed 1,211 matches of German Bundesliga and 2nd Bundesliga. The importance measurement employed is based on post season consequences of teams involved in a match. This means, if a match result could potentially influence the final rank, and this rank would lead to different consequences for a team, such as qualification for Champions League opposed to qualification for Europe League, then this match is classified as important; otherwise not. Activity was quantified by TOTAL DISTANCE COVERED, SPRINTS, FAST RUNS, DUELS, FOULS and ATTEMPTS. Running parameters were recorded using a semi-automatic optical tracking system, while technical variables were collected by professional data loggers. Based on our importance classification, low important matches occurred at the beginning of round 29. A two-way ANOVA indicates significantly increased FAST RUNS (+4%, d = 0.3), DUELS (+16%, d = 1.0) and FOULS (+36%, d = 1.2) in important matches compared to low important ones. For FAST RUNS and FOULS, this effect only exists in Bundesliga. A comparison of the two leagues show that TOTAL DISTANCE COVERED (+3%, d = 0.9), SPRINTS (+25%, d = 1.4) and FAST RUNS (+15%, d = 1.4) are higher compared to 2nd Bundesliga, whilst FOULS is less in Bundesliga (-7%, d = 0.3). No difference in player activity was found between matches at the beginning of a season (round 1–6) and at the end of a season (round 29–34). We conclude that match importance influences player activity in German professional soccer. The most reasonable explanation is a conscious or unconscious pacing strategy, motivated by preserving abilities or preventing injury. Since this tendency mainly exists in Bundesliga, this may suggest that more skilled players show a higher awareness for the need of pacing. PMID:27281051

  9. Disturbances of electrodynamic activity affect abortion in human

    NASA Astrophysics Data System (ADS)

    Jandová, A.; Nedbalová, M.; Kobilková, J.; Čoček, A.; Dohnalová, A.; Cifra, M.; Pokorný, J.

    2011-12-01

    Biochemical research of biological systems is highly developed, and it has disclosed a spectrum of chemical reactions, genetic processes, and the pathological development of various diseases. The fundamental hypothesis of physical processes in biological systems, in particular of coherent electrically polar vibrations and electromagnetic activity, was formulated by H. Fröhlich he assumed connection of cancer process with degradation of coherent electromagnetic activity. But the questions of cellular structures capable of the coherent electrical polar oscillation, mechanisms of energy supply, and the specific role of the endogenous electromagnetic fields in transport, organisation, interactions, and information transfer remained open. The nature of physical disturbances caused by some diseases (including the recurrent abortion in humans and the cancer) was unknown. We have studied the reasons of recurrent abortions in humans by means of the cell mediated immunity (using immunologic active RNA prepared from blood of inbred laboratory mice strain C3H/H2K, infected with the lactate dehydrogenase elevating virus-LD V) and the cytogenetic examination from karyotype pictures. The recurrent abortion group contained women with dg. spontaneous abortion (n = 24) and the control group was composed of 30 healthy pregnant women. Our hypothesis was related to quality of endometrium in relation to nidation of the blastocyst. The energetic insufficiency (ATP) inhibits normal development of fetus and placenta. We hope that these ideas might have impact on further research, which could provide background for effective interdisciplinary cooperation of malignant and non-malignant diseases.

  10. Implications of mitogen-activated protein kinase signaling in glioma.

    PubMed

    Pandey, Vimal; Bhaskara, Vasantha Kumar; Babu, Phanithi Prakash

    2016-02-01

    Gliomas are the most common primary central nervous system tumors. Gliomas originate from astrocytes, oligodendrocytes, and neural stem cells or their precursors. According to WHO classification, gliomas are classified into four different malignant grades ranging from grade I to grade IV based on histopathological features and related molecular aberrations. The induction and maintenance of these tumors can be attributed largely to aberrant signaling networks. In this regard, the mitogen-activated protein kinase (MAPK) network has been widely studied and is reported to be severely altered in glial tumors. Mutations in MAPK pathways most frequently affect RAS and B-RAF in the ERK, c-Jun N-terminal kinase (JNK), and p38 pathways leading to malignant transformation. Also, it is linked to both inherited and sequential accumulations of mutations that control receptor tyrosine kinase (RTK)-activated signal transduction pathways, cell cycle growth arrest pathways, and nonresponsive cell death pathways. Genetic alterations that modulate RTK signaling can also alter several downstream pathways, including RAS-mediated MAP kinases along with JNK pathways, which ultimately regulate cell proliferation and cell death. The present review focuses on recent literature regarding important deregulations in the RTK-activated MAPK pathway during gliomagenesis and progression.

  11. Glutamine enema regulates colonic ubiquitinated proteins but not proteasome activities during TNBS-induced colitis leading to increased mitochondrial activity.

    PubMed

    Bertrand, Julien; Marion-Letellier, Rachel; Azhar, Saïda; Chan, Philippe; Legrand, Romain; Goichon, Alexis; Ghouzali, Ibtissem; Aziz, Moutaz; Vaudry, David; Savoye, Guillaume; Déchelotte, Pierre; Coëffier, Moïse

    2015-07-01

    Ubiquitin proteasome system contributes to the regulation of intestinal inflammatory response as its inhibition is associated with tissue damage improvement. We aimed to evaluate whether glutamine is able to limit inflammation by targeting ubiquitin proteasome system in experimental colitis. Colitis was induced in male rats by intrarectal instillation of 2-4-6-trinitrobenzen sulfonic acid (TNBS) at day 1. From day 2 to day 6, rats daily received either an intrarectal instillation of PBS (TNBS/PBS group) or glutamine (TNBS/Gln). Rats were euthanized at day 7 and colonic samples were taken to evaluate ubiqutinated proteins by proteomic approach combining 2D electrophoresis and immunoblots directed against ubiquitin. Results were then confirmed by evaluating total expression of proteins and mRNA levels. Survival rate, TNFα, and IL-1β mRNA were improved in TNBS/Gln compared with TNBS/PBS (p < 0.05). Proteasome activities were affected by TNBS but not by glutamine. We identified eight proteins that were less ubiquitinated in TNBS/PBS compared with controls with no effect of glutamine. Four proteins were more ubiquitinated in TNBS/PBS group and restored in TNBS/Gln group. Finally, 12 ubiquitinated proteins were only affected by glutamine. Among proteins affected by glutamine, eight proteins (GFPT1, Gapdh, Pkm2, LDH, Bcat2, ATP5a1, Vdac1, and Vdac2) were involved in metabolic pathways. In conclusion, glutamine may regulate ubiquitination process during intestinal inflammation.

  12. Antitussive and immunomodulating activities of instant coffee arabinogalactan-protein.

    PubMed

    Nosáľová, G; Prisenžňáková, L; Paulovičová, E; Capek, P; Matulová, M; Navarini, L; Liverani, F Suggi

    2011-11-01

    A low molecular mass arabinogalactan-protein (AGP) composed of galactose and arabinose with a low protein content, isolated from the instant coffee powder of Coffea arabica beans, has been tested on antitussive (in vivo) and immunomodulating (ex vivo) activities. The results of antitussive tests revealed a significant dose dependant cough-suppressive effect of coffee AGP. It was observed 30 or 60 min after AGP administration and its efficacy lasted during the entire experiment course. Immunological tests showed that AGP affected some mediators of immunocompetent cells of immune system as TNF-α, IFN-γ and IL-2 cytokines. It seems that coffee AGP is a good inductor of both pro-inflammatory cytokines TNF-α and IFN-γ, however, less potent in TNF-α induction in comparison with that of β-D-glucan. Evident induction of TNF-α, IL-2 and IFN-γ cytokines, pro-TH1 polarization supports our conclusion about bio-immunological efficacy of AGP with an emphasis on the cellular immunity.

  13. Polycomb Protein OsFIE2 Affects Plant Height and Grain Yield in Rice

    PubMed Central

    Sheng, Zhonghua; Jiao, Guiai; Tang, Shaoqing; Luo, Ju; Hu, Peisong

    2016-01-01

    Polycomb group (PcG) proteins have been shown to affect growth and development in plants. To further elucidate their role in these processes in rice, we isolated and characterized a rice mutant which exhibits dwarfism, reduced seed setting rate, defective floral organ, and small grains. Map-based cloning revealed that abnormal phenotypes were attributed to a mutation of the Fertilization Independent Endosperm 2 (OsFIE2) protein, which belongs to the PcG protein family. So we named the mutant as osfie2-1. Histological analysis revealed that the number of longitudinal cells in the internodes decreased in osfie2-1, and that lateral cell layer of the internodes was markedly thinner than wild-type. In addition, compared to wild-type, the number of large and small vascular bundles decreased in osfie2-1, as well as cell number and cell size in spikelet hulls. OsFIE2 is expressed in most tissues and the coded protein localizes in both nucleus and cytoplasm. Yeast two-hybrid and bimolecular fluorescence complementation assays demonstrated that OsFIE2 interacts with OsiEZ1 which encodes an enhancer of zeste protein previously identified as a histone methylation enzyme. RNA sequencing-based transcriptome profiling and qRT-PCR analysis revealed that some homeotic genes and genes involved in endosperm starch synthesis, cell division/expansion and hormone synthesis and signaling are differentially expressed between osfie2-1 and wild-type. In addition, the contents of IAA, GA3, ABA, JA and SA in osfie2-1 are significantly different from those in wild-type. Taken together, these results indicate that OsFIE2 plays an important role in the regulation of plant height and grain yield in rice. PMID:27764161

  14. Ice recrystallization inhibition in ice cream as affected by ice structuring proteins from winter wheat grass.

    PubMed

    Regand, A; Goff, H D

    2006-01-01

    Ice recrystallization in quiescently frozen sucrose solutions that contained some of the ingredients commonly found in ice cream and in ice cream manufactured under commercial conditions, with or without ice structuring proteins (ISP) from cold-acclimated winter wheat grass extract (AWWE), was assessed by bright field microscopy. In sucrose solutions, critical differences in moisture content, viscosity, ionic strength, and other properties derived from the presence of other ingredients (skim milk powder, corn syrup solids, locust bean gum) caused a reduction in ice crystal growth. Significant ISP activity in retarding ice crystal growth was observed in all solutions (44% for the most complex mix) containing 0.13% total protein from AWWE. In heat-shocked ice cream, ice recrystallization rates were significantly reduced 40 and 46% with the addition of 0.0025 and 0.0037% total protein from AWWE. The ISP activity in ice cream was not hindered by its inclusion in mix prior to pasteurization. A synergistic effect between ISP and stabilizer was observed, as ISP activity was reduced in the absence of stabilizer in ice cream formulations. A remarkably smoother texture for ice creams containing ISP after heat-shock storage was evident by sensory evaluation. The efficiency of ISP from AWWE in controlling ice crystal growth in ice cream has been demonstrated.

  15. Cooperative hydration effect causes thermal unfolding of proteins and water activity plays a key role in protein stability in solutions.

    PubMed

    Miyawaki, Osato; Dozen, Michiko; Hirota, Kaede

    2016-08-01

    The protein unfolding process observed in a narrow temperature range was clearly explained by evaluating the small difference in the enthalpy of hydrogen-bonding between amino acid residues and the hydration of amino acid residue separately. In aqueous solutions, the effect of cosolute on the protein stability is primarily dependent on water activity, aw, the role of which has been long neglected in the literature. The effect of aw on protein stability works as a power law so that a small change in aw is amplified substantially through the cooperative hydration effect. In the present approach, the role of hydrophobic interaction stands behind. This affects protein stability indirectly through the change in solution structure caused by the existence of cosolute.

  16. Myotonic dystrophy CTG expansion affects synaptic vesicle proteins, neurotransmission and mouse behaviour

    PubMed Central

    Hernández-Hernández, Oscar; Guiraud-Dogan, Céline; Sicot, Géraldine; Huguet, Aline; Luilier, Sabrina; Steidl, Esther; Saenger, Stefanie; Marciniak, Elodie; Obriot, Hélène; Chevarin, Caroline; Nicole, Annie; Revillod, Lucile; Charizanis, Konstantinos; Lee, Kuang-Yung; Suzuki, Yasuhiro; Kimura, Takashi; Matsuura, Tohru; Cisneros, Bulmaro; Swanson, Maurice S.; Trovero, Fabrice; Buisson, Bruno; Bizot, Jean-Charles; Hamon, Michel; Humez, Sandrine; Bassez, Guillaume; Metzger, Friedrich; Buée, Luc; Munnich, Arnold; Sergeant, Nicolas; Gourdon, Geneviève

    2013-01-01

    Myotonic dystrophy type 1 is a complex multisystemic inherited disorder, which displays multiple debilitating neurological manifestations. Despite recent progress in the understanding of the molecular pathogenesis of myotonic dystrophy type 1 in skeletal muscle and heart, the pathways affected in the central nervous system are largely unknown. To address this question, we studied the only transgenic mouse line expressing CTG trinucleotide repeats in the central nervous system. These mice recreate molecular features of RNA toxicity, such as RNA foci accumulation and missplicing. They exhibit relevant behavioural and cognitive phenotypes, deficits in short-term synaptic plasticity, as well as changes in neurochemical levels. In the search for disease intermediates affected by disease mutation, a global proteomics approach revealed RAB3A upregulation and synapsin I hyperphosphorylation in the central nervous system of transgenic mice, transfected cells and post-mortem brains of patients with myotonic dystrophy type 1. These protein defects were associated with electrophysiological and behavioural deficits in mice and altered spontaneous neurosecretion in cell culture. Taking advantage of a relevant transgenic mouse of a complex human disease, we found a novel connection between physiological phenotypes and synaptic protein dysregulation, indicative of synaptic dysfunction in myotonic dystrophy type 1 brain pathology. PMID:23404338

  17. Myotonic dystrophy CTG expansion affects synaptic vesicle proteins, neurotransmission and mouse behaviour.

    PubMed

    Hernández-Hernández, Oscar; Guiraud-Dogan, Céline; Sicot, Géraldine; Huguet, Aline; Luilier, Sabrina; Steidl, Esther; Saenger, Stefanie; Marciniak, Elodie; Obriot, Hélène; Chevarin, Caroline; Nicole, Annie; Revillod, Lucile; Charizanis, Konstantinos; Lee, Kuang-Yung; Suzuki, Yasuhiro; Kimura, Takashi; Matsuura, Tohru; Cisneros, Bulmaro; Swanson, Maurice S; Trovero, Fabrice; Buisson, Bruno; Bizot, Jean-Charles; Hamon, Michel; Humez, Sandrine; Bassez, Guillaume; Metzger, Friedrich; Buée, Luc; Munnich, Arnold; Sergeant, Nicolas; Gourdon, Geneviève; Gomes-Pereira, Mário

    2013-03-01

    Myotonic dystrophy type 1 is a complex multisystemic inherited disorder, which displays multiple debilitating neurological manifestations. Despite recent progress in the understanding of the molecular pathogenesis of myotonic dystrophy type 1 in skeletal muscle and heart, the pathways affected in the central nervous system are largely unknown. To address this question, we studied the only transgenic mouse line expressing CTG trinucleotide repeats in the central nervous system. These mice recreate molecular features of RNA toxicity, such as RNA foci accumulation and missplicing. They exhibit relevant behavioural and cognitive phenotypes, deficits in short-term synaptic plasticity, as well as changes in neurochemical levels. In the search for disease intermediates affected by disease mutation, a global proteomics approach revealed RAB3A upregulation and synapsin I hyperphosphorylation in the central nervous system of transgenic mice, transfected cells and post-mortem brains of patients with myotonic dystrophy type 1. These protein defects were associated with electrophysiological and behavioural deficits in mice and altered spontaneous neurosecretion in cell culture. Taking advantage of a relevant transgenic mouse of a complex human disease, we found a novel connection between physiological phenotypes and synaptic protein dysregulation, indicative of synaptic dysfunction in myotonic dystrophy type 1 brain pathology.

  18. Characterization of Factors Affecting Nanoparticle Tracking Analysis Results With Synthetic and Protein Nanoparticles.

    PubMed

    Krueger, Aaron B; Carnell, Pauline; Carpenter, John F

    2016-04-01

    In many manufacturing and research areas, the ability to accurately monitor and characterize nanoparticles is becoming increasingly important. Nanoparticle tracking analysis is rapidly becoming a standard method for this characterization, yet several key factors in data acquisition and analysis may affect results. Nanoparticle tracking analysis is prone to user input and bias on account of a high number of parameters available, contains a limited analysis volume, and individual sample characteristics such as polydispersity or complex protein solutions may affect analysis results. This study systematically addressed these key issues. The integrated syringe pump was used to increase the sample volume analyzed. It was observed that measurements recorded under flow caused a reduction in total particle counts for both polystyrene and protein particles compared to those collected under static conditions. In addition, data for polydisperse samples tended to lose peak resolution at higher flow rates, masking distinct particle populations. Furthermore, in a bimodal particle population, a bias was seen toward the larger species within the sample. The impacts of filtration on an agitated intravenous immunoglobulin sample and operating parameters including "MINexps" and "blur" were investigated to optimize the method. Taken together, this study provides recommendations on instrument settings and sample preparations to properly characterize complex samples.

  19. Soil biological activity as affected by tillage intensity

    NASA Astrophysics Data System (ADS)

    Gajda, A.; Przewłoka, B.

    2012-02-01

    The effect of tillage intensity on changes of microbiological activity and content of particulate organic matter in soil under winter wheat duirng 3 years was studied. Microbial response related to the tillage-induced changes in soil determined on the content of biomass C and N, the rate of CO2 evolution, B/F ratio, the activity of dehydrogenases, acid and alkaline phosphatases, soil C/N ratio and microbial biomass C/N ratio confirmed the high sensitivity of soil microbial populations to the tillage system applied. After three year studies, the direct sowing system enhanced the increase of labile fraction of organic matter content in soil. There were no significant changes in the labile fraction quantity observed in soil under conventional tillage. Similar response related to the tillage intensity was observed in particulate organic matter quantities expressed as a percentage of total organic matter in soil. A high correlation coefficients calculated between contents of soil microbial biomass C and N, particulate organic matter and potentially mineralizable N, and the obtained yields of winter wheat grown on experimental fields indicated on a high importance of biological quality of status of soil for agricultural crop production.

  20. Disturbances of electrodynamic activity affect abortion in animals

    NASA Astrophysics Data System (ADS)

    Nedbalova, M.; Jandova, A.; Dohnalova, A.

    2011-12-01

    A specific kind of intracellular organelles, the mitochondria, is the place of metabolic energy production by oxidative mechanism. We used cell mediated immunity method for verification of the energy metabolism (ATP production). The antigen (immunological functional RNA) was obtained from blood of inbred laboratory mice strain C3H/H2K, infected with the lactate dehydrogenase elevating virus (LDV) and prepared by the high pressure gel chromatography (HPGC). We have studied the immunological adaptability of LDH viral antigen in 62 pigs (12 parents and 50 piglings). Exitus of piglings was in case of positive imunological response on LDV. The statement results from a comparison of the relative frequency of an incidence of identical findings in male piglets and sows and from identical findings in female piglets and pigs. The efficient elaboration and utilization of energy in cell may be damaged by the changes of energy production systems and also by long-term parasitary depletion of ATP energy. Biological activity is based not only on biochemical but also on biophysical mechanisms. Biophysical processes are also involved in the transfer of information and its processing for making decisions and providing control, which are important parts of biological activity. These experimental results were used for the same study in human.

  1. Sucrose increases calcium-dependent protein kinase and phosphatase activities in potato plants.

    PubMed

    Raíces, M; MacIntosh, G C; Ulloa, R M; Gargantini, P R; Vozza, N F; Téllez-Inón, M T

    2003-09-01

    The effect of sucrose on tuber formation, calcium-dependent protein kinase (CDPK) and phosphatase activities was analysed using in vitro cultured potato plants. In short treatments, sucrose induced CDPK and phosphatase activities. In long treatments, sucrose induced tuber formation in the absence of other tuber inducing stimuli. Sorbitol caused a minor increase in CDPK activity and affected plant morphology but did not induce tuber development. The addition of the protein kinase inhibitor Staurosporine precluded sucrose-induced tuberization. Altogether, our results suggest that phosphorylation/dephosphorylation events are involved in sucrose-induced tuber development.

  2. Control of mechanically activated polymersome fusion: Factors affecting fusion

    DOE PAGES

    Henderson, Ian M.; Paxton, Walter F.

    2014-12-15

    Previously we have studied the mechanically-activated fusion of extruded (200 nm) polymer vesicles into giant polymersomes using agitation in the presence of salt. In this study we have investigated several factors contributing to this phenomenon, including the effects of (i) polymer vesicle concentration, (ii) agitation speed and duration, and iii) variation of the salt and its concentration. It was found that increasing the concentration of the polymer dramatically increases the production of giant vesicles through the increased collisions of polymersomes. Our investigations also found that increasing the frequency of agitation increased the efficiency of fusion, though ultimately limited the sizemore » of vesicle which could be produced due to the high shear involved. Finally it was determined that salt-mediation of the fusion process was not limited to NaCl, but is instead a general effect facilitated by the presence of solvated ionic compounds, albeit with different salts initiating fusion at different concentration.« less

  3. Control of mechanically activated polymersome fusion: Factors affecting fusion

    SciTech Connect

    Henderson, Ian M.; Paxton, Walter F.

    2014-12-15

    Previously we have studied the mechanically-activated fusion of extruded (200 nm) polymer vesicles into giant polymersomes using agitation in the presence of salt. In this study we have investigated several factors contributing to this phenomenon, including the effects of (i) polymer vesicle concentration, (ii) agitation speed and duration, and iii) variation of the salt and its concentration. It was found that increasing the concentration of the polymer dramatically increases the production of giant vesicles through the increased collisions of polymersomes. Our investigations also found that increasing the frequency of agitation increased the efficiency of fusion, though ultimately limited the size of vesicle which could be produced due to the high shear involved. Finally it was determined that salt-mediation of the fusion process was not limited to NaCl, but is instead a general effect facilitated by the presence of solvated ionic compounds, albeit with different salts initiating fusion at different concentration.

  4. 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.

  5. Error-prone and error-restrictive mutations affecting ribosomal protein S12.

    PubMed

    Agarwal, Deepali; Gregory, Steven T; O'Connor, Michael

    2011-07-01

    Ribosomal protein S12 plays a pivotal role in decoding functions on the ribosome. X-ray crystallographic analyses of ribosomal complexes have revealed that S12 is involved in the inspection of codon-anticodon pairings in the ribosomal A site, as well as in the succeeding domain rearrangements of the 30S subunit that are essential for accommodation of aminoacyl-tRNA. A role for S12 in tRNA selection is also well supported by classical genetic analyses; mutations affecting S12 are readily isolated in bacteria and organelles, since specific alterations in S12 confer resistance to the error-inducing antibiotic streptomycin, and the ribosomes from many such streptomycin-resistant S12 mutants display decreased levels of miscoding. However, substitutions that confer resistance to streptomycin likely represent a very distinct class of all possible S12 mutants. Until recently, the technical difficulties in generating random, unselectable mutations in essential genes in complex operons have generally precluded the analysis of other classes of S12 alterations. Using a recombineering approach, we have targeted the Escherichia coli rpsL gene, encoding S12, for random mutagenesis and screened the resulting mutants for effects on decoding fidelity. We have recovered over 40 different substitutions located throughout the S12 protein that alter the accuracy of translation without substantially affecting the sensitivity to streptomycin. Moreover, this collection includes mutants that promote miscoding, as well as those that restrict decoding errors. These results affirm the importance of S12 in decoding processes and indicate that alterations in this essential protein can have diverse effects on the accuracy of decoding.

  6. Protein v. carbohydrate intake differentially affects liking- and wanting-related brain signalling.

    PubMed

    Born, Jurriaan M; Martens, Mieke J I; Lemmens, Sofie G T; Goebel, Rainer; Westerterp-Plantenga, Margriet S

    2013-01-28

    Extreme macronutrient intakes possibly lead to different brain signalling. The aim of the present study was to determine the effects of ingesting high-protein v. high-carbohydrate food on liking and wanting task-related brain signalling (TRS) and subsequent macronutrient intake. A total of thirty female subjects (21.6 (SD 2.2) years, BMI 25.0 (SD 3.7) kg/m²) completed four functional MRI scans: two fasted and two satiated on two different days. During the scans, subjects rated all food items for liking and wanting, thereby choosing the subsequent meal. The results show that high-protein (PROT) v. high-carbohydrate (CARB) conditions were generated using protein or carbohydrate drinks at the first meal. Energy intake and hunger were recorded. PROT (protein: 53.7 (SD 2.1) percentage of energy (En%); carbohydrate: 6.4 (SD 1.3) En%) and CARB conditions (protein: 11.8 (SD 0.6) En%; carbohydrate: 70.0 (SD 2.4) En%) were achieved during the first meal, while the second meals were not different between the conditions. Hunger, energy intake, and behavioural liking and wanting ratings were decreased after the first meal (P< 0.001). Comparing the first with the second meal, the macronutrient content changed: carbohydrate -26.9 En% in the CARB condition, protein -37.8 En% in the PROT condition. After the first meal in the CARB condition, wanting TRS was increased in the hypothalamus. After the first meal in the PROT condition, liking TRS was decreased in the putamen (P< 0.05). The change in energy intake from the first to the second meal was inversely related to the change in liking TRS in the striatum and hypothalamus in the CARB condition and positively related in the PROT condition (P< 0.05). In conclusion, wanting and liking TRS were affected differentially with a change in carbohydrate or protein intake, underscoring subsequent energy intake and shift in macronutrient composition.

  7. AKAP-Lbc nucleates a protein kinase D activation scaffold.

    PubMed

    Carnegie, Graeme K; Smith, F Donelson; McConnachie, George; Langeberg, Lorene K; Scott, John D

    2004-09-24

    The transmission of cellular signals often proceeds through multiprotein complexes where enzymes are positioned in proximity to their upstream activators and downstream substrates. In this report we demonstrate that the A-kinase anchoring protein AKAP-Lbc assembles an activation complex for the lipid-dependent enzyme protein kinase D (PKD). Using a combination of biochemical, enzymatic, and immunofluorescence techniques, we show that the anchoring protein contributes to PKD activation in two ways: it recruits an upstream kinase PKCeta and coordinates PKA phosphorylation events that release activated protein kinase D. Thus, AKAP-Lbc synchronizes PKA and PKC activities in a manner that leads to the activation of a third kinase. This configuration illustrates the utility of kinase anchoring as a mechanism to constrain the action of broad-spectrum enzymes.

  8. Immunological responses as affected by dietary protein and arginine concentrations in starting broiler chicks.

    PubMed

    Jahanian, R

    2009-09-01

    The study presented here aimed to investigate the effect of dietary protein content on Arg needs and immunological responses of broiler chicks during the starter period. A total of 715 one-day-old male Ross broiler chicks were randomly assigned to 5 replicate pens for each of 11 experimental diets during a 21-d feeding trial. The dietary treatments included a corn-soybean meal control diet or experimental diets (corn-soybean meal-corn gluten meal) containing 5 dietary Arg levels of 80, 90, 100, 110, or 120% of NRC recommendations and 2 dietary protein levels of 19 and 22.35% of diet. Increasing dietary CP content significantly (P<0.001) increased daily feed consumption and weight gain. Also, feeding diets deficient in Arg to the chicks led to a noticeable decline in feed intake, and dietary Arg supplementation overcame decreased feed consumption and weight gain observed in Arg-deficient chicks. Feed efficiency was affected only by dietary Arg concentration so that chicks on Arg-deficient diets markedly (P<0.001) increased feed conversion ratio. Contrast comparisons showed that the highly variable responses of chicks to dietary Arg level were mainly attributed to dietary protein concentration: more dietary protein content and higher Arg demands. Among lymphoid organs, thymus (P<0.001) and spleen (P<0.05) were affected by dietary Arg deficiency, whereas diets low in CP content decreased (P<0.001) relative weights of thymus and bursa of Fabricius. Increase in dietary CP level from 19 to 22.35% caused an increase (P<0.001) in the proportion of lymphocytes and consequently lower (P<0.05) heterophil-to-lymphocyte ratio. Broiler chicks on Arg-deficient diets decreased the proportion of heterophils in peripheral blood. Furthermore, skin reaction to phytohemagglutinin P was impaired when the diets were low in CP and Arg contents. Similarly, a decrease in dietary CP and Arg levels diminished the antibody production response to Newcastle disease virus. The broken

  9. Microvillus inclusion disease: a genetic defect affecting apical membrane protein traffic in intestinal epithelium.

    PubMed

    Ameen, N A; Salas, P J

    2000-01-01

    The striking similarities between microvillus inclusions (MIs) in enterocytes in microvillus inclusion disease (MID) and vacuolar apical compartment in tissue culture epithelial cells, led us to analyze endoscopic biopsies of duodenal mucosa of a patient after the samples were used for diagnostic procedures. Samples from another patient with an unrelated disease were used as controls. The MID enterocytes showed a decrease in the thickness of the apical F-actin layer, and normal microtubules. The immunofluorescence analysis of the distribution of five apical membrane markers (sucrase isomaltase, alkaline phosphatase, NHE-3 Na+/H+ exchanger, cGMP-dependent protein kinase, and cystic fibrosis trans-membrane conductance regulator), showed low levels of these proteins in their standard localization at the apical membrane as compared with normal duodenal epithelium processed in parallel. Instead, four of these markers were found in a diffuse distribution in the apical cytoplasm, below the terminal web (as indicated by co-localization with F-actin and cytokeratin 19), and in MIs as well. The basolateral protein Na(+)-K+ATPase, in contrast, was normally localized. These results support the hypothesis that MID may represent the first genetic defect affecting apical membrane traffic, possibly in a late step of apical exocytosis.

  10. PMT family of Candida albicans: five protein mannosyltransferase isoforms affect growth, morphogenesis and antifungal resistance.

    PubMed

    Prill, Stephan K-H; Klinkert, Birgit; Timpel, Claudia; Gale, Cheryl A; Schröppel, Klaus; Ernst, Joachim F

    2005-01-01

    Protein O-mannosyltransferases (Pmt proteins) initiate O-mannosylation of secretory proteins. The PMT gene family of the human fungal pathogen Candida albicans consists of PMT1 and PMT6, as well as three additional PMT genes encoding Pmt2, Pmt4 and Pmt5 isoforms described here. Both PMT2 alleles could not be deleted and growth of conditional strains, containing PMT2 controlled by the MET3- or tetOScHOP1-promoters, was blocked in non-permissive conditions, indicating that PMT2 is essential for growth. A homozygous pmt4 mutant was viable, but synthetic lethality of pmt4 was observed in combination with pmt1 mutations. Hyphal morphogenesis of a pmt4 mutant was defective under aerobic induction conditions, yet increased in embedded or hypoxic conditions, suggesting a role of Pmt4p-mediated O-glycosylation for environment-specific morphogenetic signalling. Although a PMT5 transcript was detected, a homozygous pmt5 mutant was phenotypically silent. All other pmt mutants showed variable degrees of supersensitivity to antifungals and to cell wall-destabilizing agents. Cell wall composition was markedly affected in pmt1 and pmt4 mutants, showing a significant decrease in wall mannoproteins. In a mouse model of haematogenously disseminated infection, PMT4 was required for full virulence of C. albicans. Functional analysis of the first complete PMT gene family in a fungal pathogen indicates that Pmt isoforms have variable and specific roles for in vitro and in vivo growth, morphogenesis and antifungal resistance.

  11. Mutations in the West Nile prM protein affect VLP and virion secretion in vitro.

    PubMed

    Calvert, Amanda E; Huang, Claire Y-H; Blair, Carol D; Roehrig, John T

    2012-11-10

    Mutation of the West Nile virus-like particle (WN VLP) prM protein (T20D, K31A, K31V, or K31T) results in undetectable VLP secretion from transformed COS-1 cells. K31 mutants formed intracellular prM-E heterodimers; however these proteins remained in the ER and ER-Golgi intermediary compartments of transfected cells. The T20D mutation affected glycosylation, heterodimer formation, and WN VLP secretion. When infectious viruses bearing the same mutations were used to infect COS-1 cells, K31 mutant viruses exhibited delayed growth and reduced infectivity compared to WT virus. Epitope maps of WN VLP and WNV prM were also different. These results suggest that while mutations in the prM protein can reduce or eliminate secretion of WN VLPs, they have less effect on virus. This difference may be due to the quantity of prM in WN VLPs compared to WNV or to differences in maturation, structure, and symmetry of these particles.

  12. Regulation of the activity of protein kinases by endogenous heat stable protein inhibitors.

    PubMed

    Szmigielski, A

    1985-01-01

    Protein kinase activities are regulated by endogenous thermostable protein inhibitors. Type I inhibitor is a protein of MW 22,000-24,000 which inhibits specifically cyclic AMP-(cAMP) dependent protein kinase (APK) as a competitive inhibitor of catalytic subunits of the enzyme. Type I inhibitor activity changes inversely according to the activation of adenylate cyclase and the changes in cAMP content in tissues. It seems that type I inhibitor serves as a factor preventing spontaneous cAMP-dependent phosphorylation in unstimulated cell. The other thermostable protein which inhibits APK activity has been found in Sertoli cell-enriched testis (testis inhibitor). Physiological role of the testis inhibitor is unknown. Type II inhibitor is a protein of MW 15,000 which blocks phosphorylation mediated by cAMP and cyclic GMP (cGMP) dependent (APK and GPK) and cyclic nucleotide independent protein kinases as a competitive inhibitor of substrate proteins. Activity of this inhibitor specifically changes in reciprocal manner to the changes in cGMP content. It seems that type II inhibitor serves as a factor preventing the phosphorylation catalyzed by GPK when cGMP content is low. Stimulation of guanylate cyclase and activation of GPK is followed by a decrease of type II inhibitor activity. This change in relationship between activities of GPK and type II inhibitor allows for effective phosphorylation catalyzed by this enzyme when cGMP content is increased.

  13. Variability in affective activation predicts non-suicidal self-injury in eating disorders.

    PubMed

    Vansteelandt, Kristof; Claes, Laurence; Muehlenkamp, Jennifer; De Cuyper, Kathleen; Lemmens, Jos; Probst, Michel; Vanderlinden, Johan; Pieters, Guido

    2013-03-01

    We examined whether affective variability can predict non-suicidal self-injury (NSSI) in eating disorders. Affect was represented by valence (positive versus negative) and activation (high versus low). Twenty-one patients with anorexia nervosa-restricting type, 18 patients with anorexia nervosa-binge-purging type and 20 patients with bulimia nervosa reported their momentary affect at nine random times a day during a one week period using a hand-held computer. Affective variability was calculated as the within-person standard deviation of valence and activation over time. Results indicate that patients displaying greater variability in activation and using selective serotonin reuptake inhibitors have a higher probability to engage in lifetime NSSI after adjustment for depression and borderline personality disorder. Neither variability of valence nor mean level of valence and activation had any predictive association with engaging in NSSI. It is suggested that the treatment of NSSI should focus on affect stabilization rather than reducing negative affect.

  14. The Evolutionarily Conserved Protein PHOTOSYNTHESIS AFFECTED MUTANT71 Is Required for Efficient Manganese Uptake at the Thylakoid Membrane in Arabidopsis

    PubMed Central

    Steinberger, Iris; Herdean, Andrei; Gandini, Chiara; Labs, Mathias; Flügge, Ulf-Ingo; Geimer, Stefan; Schmidt, Sidsel Birkelund; Husted, Søren; Spetea, Cornelia; Leister, Dario

    2016-01-01

    In plants, algae, and cyanobacteria, photosystem II (PSII) catalyzes the light-driven oxidation of water. The oxygen-evolving complex of PSII is a Mn4CaO5 cluster embedded in a well-defined protein environment in the thylakoid membrane. However, transport of manganese and calcium into the thylakoid lumen remains poorly understood. Here, we show that Arabidopsis thaliana PHOTOSYNTHESIS AFFECTED MUTANT71 (PAM71) is an integral thylakoid membrane protein involved in Mn2+ and Ca2+ homeostasis in chloroplasts. This protein is required for normal operation of the oxygen-evolving complex (as evidenced by oxygen evolution rates) and for manganese incorporation. Manganese binding to PSII was severely reduced in pam71 thylakoids, particularly in PSII supercomplexes. In cation partitioning assays with intact chloroplasts, Mn2+ and Ca2+ ions were differently sequestered in pam71, with Ca2+ enriched in pam71 thylakoids relative to the wild type. The changes in Ca2+ homeostasis were accompanied by an increased contribution of the transmembrane electrical potential to the proton motive force across the thylakoid membrane. PSII activity in pam71 plants and the corresponding Chlamydomonas reinhardtii mutant cgld1 was restored by supplementation with Mn2+, but not Ca2+. Furthermore, PAM71 suppressed the Mn2+-sensitive phenotype of the yeast mutant Δpmr1. Therefore, PAM71 presumably functions in Mn2+ uptake into thylakoids to ensure optimal PSII performance. PMID:27020959

  15. The specific activation of TRPC4 by Gi protein subtype.

    PubMed

    Jeon, Jae-Pyo; Lee, Kyu Pil; Park, Eun Jung; Sung, Tae Sik; Kim, Byung Joo; Jeon, Ju-Hong; So, Insuk

    2008-12-12

    The classical type of transient receptor potential channel (TRPC) is a molecular candidate for Ca(2+)-permeable cation channels in mammalian cells. Especially, TRPC4 has the similar properties to Ca(2+)-permeable nonselective cation channels (NSCCs) activated by muscarinic stimulation in visceral smooth muscles. In visceral smooth muscles, NSCCs activated by muscarinic stimulation were blocked by anti-Galphai/o antibodies. However, there is still no report which Galpha proteins are involved in the activation process of TRPC4. Among Galpha proteins, only Galphai protein can activate TRPC4 channel. The activation effect of Galphai was specific for TRPC4 because Galphai has no activation effect on TRPC5, TRPC6 and TRPV6. Coexpression with muscarinic receptor M2 induced TRPC4 current activation by muscarinic stimulation with carbachol, which was inhibited by pertussis toxin. These results suggest that Galphai is involved specifically in the activation of TRPC4.

  16. Tasting calories differentially affects brain activation during hunger and satiety.

    PubMed

    van Rijn, Inge; de Graaf, Cees; Smeets, Paul A M

    2015-02-15

    An important function of eating is ingesting energy. Our objectives were to assess whether oral exposure to caloric and non-caloric stimuli elicits discriminable responses in the brain and to determine in how far these responses are modulated by hunger state and sweetness. Thirty women tasted three stimuli in two motivational states (hunger and satiety) while their brain responses were measured using functional magnetic resonance imaging in a randomized crossover design. Stimuli were solutions of sucralose (sweet, no energy), maltodextrin (non-sweet, energy) and sucralose+maltodextrin (sweet, energy). We found no main effect of energy content and no interaction between energy content and sweetness. However, there was an interaction between hunger state and energy content in the median cingulate (bilaterally), ventrolateral prefrontal cortex, anterior insula and thalamus. This indicates that the anterior insula and thalamus, areas in which hunger state and taste of a stimulus are integrated, also integrate hunger state with caloric content of a taste stimulus. Furthermore, in the median cingulate and ventrolateral prefrontal cortex, tasting energy resulted in more activation during satiety compared to hunger. This finding indicates that these areas, which are known to be involved in processes that require approach and avoidance, are also involved in guiding ingestive behavior. In conclusion, our results suggest that energy sensing is a hunger state dependent process, in which the median cingulate, ventrolateral prefrontal cortex, anterior insula and thalamus play a central role by integrating hunger state with stimulus relevance.

  17. Might as Well Jump: Sound Affects Muscle Activation in Skateboarding

    PubMed Central

    Cesari, Paola; Camponogara, Ivan; Papetti, Stefano; Rocchesso, Davide; Fontana, Federico

    2014-01-01

    The aim of the study is to reveal the role of sound in action anticipation and performance, and to test whether the level of precision in action planning and execution is related to the level of sensorimotor skills and experience that listeners possess about a specific action. Individuals ranging from 18 to 75 years of age - some of them without any skills in skateboarding and others experts in this sport - were compared in their ability to anticipate and simulate a skateboarding jump by listening to the sound it produces. Only skaters were able to modulate the forces underfoot and to apply muscle synergies that closely resembled the ones that a skater would use if actually jumping on a skateboard. More importantly we showed that only skaters were able to plan the action by activating anticipatory postural adjustments about 200 ms after the jump event. We conclude that expert patterns are guided by auditory events that trigger proper anticipations of the corresponding patterns of movements. PMID:24619134

  18. Social decisions affect neural activity to perceived dynamic gaze

    PubMed Central

    Latinus, Marianne; Love, Scott A.; Rossi, Alejandra; Parada, Francisco J.; Huang, Lisa; Conty, Laurence; George, Nathalie; James, Karin

    2015-01-01

    Gaze direction, a cue of both social and spatial attention, is known to modulate early neural responses to faces e.g. N170. However, findings in the literature have been inconsistent, likely reflecting differences in stimulus characteristics and task requirements. Here, we investigated the effect of task on neural responses to dynamic gaze changes: away and toward transitions (resulting or not in eye contact). Subjects performed, in random order, social (away/toward them) and non-social (left/right) judgment tasks on these stimuli. Overall, in the non-social task, results showed a larger N170 to gaze aversion than gaze motion toward the observer. In the social task, however, this difference was no longer present in the right hemisphere, likely reflecting an enhanced N170 to gaze motion toward the observer. Our behavioral and event-related potential data indicate that performing social judgments enhances saliency of gaze motion toward the observer, even those that did not result in gaze contact. These data and that of previous studies suggest two modes of processing visual information: a ‘default mode’ that may focus on spatial information; a ‘socially aware mode’ that might be activated when subjects are required to make social judgments. The exact mechanism that allows switching from one mode to the other remains to be clarified. PMID:25925272

  19. Social decisions affect neural activity to perceived dynamic gaze.

    PubMed

    Latinus, Marianne; Love, Scott A; Rossi, Alejandra; Parada, Francisco J; Huang, Lisa; Conty, Laurence; George, Nathalie; James, Karin; Puce, Aina

    2015-11-01

    Gaze direction, a cue of both social and spatial attention, is known to modulate early neural responses to faces e.g. N170. However, findings in the literature have been inconsistent, likely reflecting differences in stimulus characteristics and task requirements. Here, we investigated the effect of task on neural responses to dynamic gaze changes: away and toward transitions (resulting or not in eye contact). Subjects performed, in random order, social (away/toward them) and non-social (left/right) judgment tasks on these stimuli. Overall, in the non-social task, results showed a larger N170 to gaze aversion than gaze motion toward the observer. In the social task, however, this difference was no longer present in the right hemisphere, likely reflecting an enhanced N170 to gaze motion toward the observer. Our behavioral and event-related potential data indicate that performing social judgments enhances saliency of gaze motion toward the observer, even those that did not result in gaze contact. These data and that of previous studies suggest two modes of processing visual information: a 'default mode' that may focus on spatial information; a 'socially aware mode' that might be activated when subjects are required to make social judgments. The exact mechanism that allows switching from one mode to the other remains to be clarified.

  20. Modulation of endogenous Cysteine Protease Inhibitor (ICP) 1 expression in Entamoeba histolytica affects amoebic adhesion to Extracellular Matrix proteins.

    PubMed

    Lee, Young Ah; Saito-Nakano, Yumiko; Kim, Kyeong Ah; Min, Arim; Nozaki, Tomoyoshi; Shin, Myeong Heon

    2015-02-01

    Entamoeba histolytica is an enteric tissue-invading protozoan parasite that causes amoebic colitis and occasionally liver abscess in humans. During tissue invasion, amoebic adhesion to host components is an important event for host cell death leading to successful invasion and infection. Among amoebic virulence factors, Gal/GalNAc lectin is known to be major adhesion factor to host cells. In this study, we investigated the role of amoebic secreted CP (Cysteine Proteases) in amoebic adhesion to extracellular matrix (ECM) protein using CP inhibitor and E. histolytica strains in which the endogenous inhibitor of cysteine protease (ICP) 1 gene was overexpressed (ICP1(+)) or repressed by antisense small RNA-mediated gene silencing (ICP1(-)). We found that pretreatment of wild-type amoebae with CP inhibitor E64, or thiol-group modifiers such as diamide and N-Ethylmaleimide resulted in a significant decrease in adhesion to laminin and collagen ECM proteins. Furthermore, ICP1(+) strain, with a reduction of secreted CP activity, exhibited reduced ability by 40% to adhere to laminin. In contrast, ICP1(-) strain, with a 1.9-fold increase of secreted CP activity, showed a two-fold increase in amoebic adherence to laminin compared to the control strain. In addition, total amount of secreted CP5 was decreased in ICP1(+) amoeba. Conversely, total amount of secreted CP1 and mature-form CP5 were increased in ICP1(-) amoeba. We also found that ICP1 was secreted into extracellular milieu. These results suggest that secreted CP activity by E. histolytica may be an important factor affecting adhesion to host proteins, and regulation of CP secretion by ICP plays a major role in pathogenesis. This study provides insight into the CP-mediated tissue pathogenesis in amoeba-invaded lesions during human amoebiasis.

  1. Modulation of Leishmania major aquaglyceroporin activity by a mitogen-activated protein kinase

    PubMed Central

    Mandal, Goutam; Sharma, Mansi; Kruse, Martin; Sander-Juelch, Claudia; Munro, Laura Anne; Wang, Yong; Vilg, Jenny Veide; Tamás, Markus J; Bhattacharjee, Hiranmoy; Wiese, Martin; Mukhopadhyay, Rita

    2012-01-01

    Summary Leishmania major aquaglyceroporin (LmjAQP1) adventitiously facilitates the uptake of antimonite [Sb(III)], an active form of Pentostam® or Glucantime®, which are the first line of defense against all forms of leishmaniasis. The present paper shows that LmjAQP1 activity is modulated by the mitogen-activated protein kinase, LmjMPK2. Leishmania parasites co-expressing LmjAQP1 and LmjMPK2 show increased Sb(III) uptake and increased Sb(III) sensitivity. When subjected to a hypo-osmotic stress, these cells show faster volume recovery than cells expressing LmjAQP1 alone. LmjAQP1 is phosphorylated in vivo at Thr197 and this phosphorylation requires LmjMPK2 activity. Lys42 of LmjMPK2 is critical for its kinase activity. Cells expressing altered T197A LmjAQP1 or K42A LmjMPK2 showed decreased Sb(III) influx and a slower volume recovery than cells expressing wild type proteins. Phosphorylation of LmjAQP1 led to a decrease in its turnover rate affecting LmjAQP1 activity. Although LmjAQP1 is localized to the flagellum of promastigotes, upon phosphorylation, it is relocalized to the entire surface of the parasite. L. mexicana promastigotes with an MPK2 deletion showed reduced Sb(III) uptake and slower volume recovery than wild type cells. This is the first report where a parasite aquaglyceroporin activity is post-translationally modulated by a MAP kinase. PMID:22779703

  2. HMGA proteins as modulators of chromatin structure during transcriptional activation

    PubMed Central

    Ozturk, Nihan; Singh, Indrabahadur; Mehta, Aditi; Braun, Thomas; Barreto, Guillermo

    2013-01-01

    High mobility group (HMG) proteins are the most abundant non-histone chromatin associated proteins. HMG proteins bind to DNA and nucleosome and alter the structure of chromatin locally and globally. Accessibility to DNA within chromatin is a central factor that affects DNA-dependent nuclear processes, such as transcription, replication, recombination, and repair. HMG proteins associate with different multi-protein complexes to regulate these processes by mediating accessibility to DNA. HMG proteins can be subdivided into three families: HMGA, HMGB, and HMGN. In this review, we will focus on recent advances in understanding the function of HMGA family members, specifically their role in gene transcription regulation during development and cancer. PMID:25364713

  3. Peroxisome proliferator-activated receptor gamma activators affect the maturation of human monocyte-derived dendritic cells.

    PubMed

    Gosset, P; Charbonnier, A S; Delerive, P; Fontaine, J; Staels, B; Pestel, J; Tonnel, A B; Trottein, F

    2001-10-01

    Peroxisome proliferator-activated receptor gamma (PPARgamma ), a member of the nuclear receptor superfamily, has recently been described as a modulator of macrophage functions and as an inhibitor of T cell proliferation. Here, we investigated the role of PPARgamma in dendritic cells (DC), the most potent antigen-presenting cells. We showed that PPARgamma is highly expressed in immature human monocyte-derived DC (MDDC) and that it may affect the immunostimulatory function of MDDC stimulated with lipopolysaccharide (LPS) or via CD40 ligand (CD40L). We found that the synthetic PPARgamma agonist rosiglitazone (as well as pioglitazone and troglitazone) significantly increases on LPS- and CD40L-activated MDDC, the surface expression of CD36 (by 184% and 104%, respectively) and CD86 (by 54% and 48%), whereas it reduces the synthesis of CD80 (by 42% and 42%). Moreover, activation of PPARgamma resulted in a dramatic decreased secretion of the Th1-promoting factor IL-12 in LPS- and CD40L-stimulated cells (by 47% and 62%), while the production of IL-1beta, TNF-alpha, IL-6 and IL-10 was unaffected. Finally, PPARgamma ligands down-modulate the synthesis of IFN-gamma -inducible protein-10 (recently termed as CXCL10) and RANTES (CCL5), both chemokines involved in the recruitment of Th1 lymphocytes (by 49% and 30%), but not the levels of the Th2 cell-attracting chemokines,macrophage-derived chemokine (CCL22) and thymus and activation regulated chemokine (CCL17), in mature MDDC. Taken together, our data suggest that activation of PPARgamma in human DC may have an impact in the orientation of primary and secondary immune responses by favoring type 2 responses.

  4. Genetic variants in ABCA1 promoter affect transcription activity and plasma HDL level in pigs.

    PubMed

    Dang, Xiao-yong; Chu, Wei-wei; Shi, Heng-chuan; Yu, Shi-gang; Han, Hai-yin; Gu, Shu-Hua; Chen, Jie

    2015-01-25

    Excess accumulation of cholesterol in plasma may result in coronary artery disease. Numerous studies have demonstrated that ATP-binding cassette protein A1 (ABCA1) mediates the efflux of cholesterol and phospholipids to apolipoproteins, a process necessary for plasma high density lipoprotein (HDL) formation. Higher plasma levels of HDL are associated with lower risk for cardiovascular disease. Studies of human disease and animal models had shown that an increased hepatic ABCA1 activity relates to an enhanced plasma HDL level. In this study, we hypothesized that functional mutations in the ABCA1 promoter in pigs may affect gene transcription activity, and consequently the HDL level in plasma. The promoter region of ABCA1 was comparatively scanned by direct sequencing with pool DNA of high- and low-HDL groups (n=30 for each group). Two polymorphisms, c. - 608A>G and c. - 418T>A, were revealed with reverse allele distribution in the two groups. The two polymorphisms were completely linked and formed only G-A or A-T haplotypes when genotyped in a larger population (n=526). Furthermore, we found that the G-A/G-A genotype was associated with higher HDL and ABCA1 mRNA level than A-T/A-T genotype. Luciferase assay also revealed that G-A haplotype promoter had higher activity than A-T haplotype. Single-nucleotide mutant assay showed that c.-418T>A was the causal mutation for ABCA1 transcription activity alteration. Conclusively, we identified two completely linked SNPs in porcine ABCA1 promoter region which have influence on the plasma HDL level by altering ABCA1 gene transcriptional activity.

  5. Protein Crystal Growth Activities on STS-42

    NASA Technical Reports Server (NTRS)

    1992-01-01

    The Protein Crystal Growth (PCG) middeck payload is currently manifested to fly on STS-42 in January 1992. This payload is a joint effort between NASA s Office of Commercial Programs (OCP) and Office of Space Science and Applications (OSSA). The PCG experiments are managed by the Center for Macromolecular Crystallography (CMC), a NASA Center for the Commercial Development of Space (CCDS) based at the University of Alabama at Birmingham (UAB). This is the eighth flight of a payload in the PCG program that is jointly sponsored by the OCP and the OSSA. The flight hardware for STS-42 includes six Vapor Diffusion Apparatus (VDA) trays stored in two Refrigerator/Incubator Modules (R/TM s). The VDA trays will simultaneously conduct 120 experiments involving 15 different protein compounds, four of which are sponsored by the OCP, the UAB CCDS, and four co-investigators.

  6. 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.

  7. Leukocyte adhesion deficiency-III is caused by mutations in KINDLIN3 affecting integrin activation

    PubMed Central

    Svensson, Lena; Howarth, Kimberley; McDowall, Alison; Patzak, Irene; Evans, Rachel; Ussar, Siegfried; Moser, Markus; Metin, Ayse; Fried, Mike; Tomlinson, Ian; Hogg, Nancy

    2009-01-01

    Integrins are the major adhesion receptors of leukocytes and platelets. β1 and β2 integrin function on leukocytes is crucial for a successful immune response and the platelet integrin αIIbβ3 initiates the process of blood clotting through binding fibrinogen1-3. Integrins on circulating cells bind poorly to their ligands but become active after ‘inside-out’ signaling through other membrane receptors4,5. Subjects with leukocyte adhesion deficiency-1 (LAD-I) do not express β2 integrins because of mutations in the gene specifying the β2 subunit, and they suffer recurrent bacterial infections6,7. Mutations affecting αIIbβ3 integrin cause the bleeding disorder termed Glanzmann’s thrombasthenia3. Subjects with LAD-III show symptoms of both LAD-I and Glanzmann’s thrombasthenia. Their hematopoietically-derived cells express β1, β2 and β3 integrins, but defective inside-out signaling causes immune deficiency and bleeding problems8. The LAD-III lesion has been attributed to a C→A mutation in the gene encoding calcium and diacylglycerol guanine nucleotide exchange factor (CALDAGGEF1; official symbol RASGRP2) specifying the CALDAG-GEF1 protein9, but we show that this change is not responsible for the LAD-III disorder. Instead, we identify mutations in the KINDLIN3 (official symbol FERMT3) gene specifying the KINDLIN-3 protein as the cause of LAD-III in Maltese and Turkish subjects. Two independent mutations result in decreased KINDLIN3 messenger RNA levels and loss of protein expression. Notably, transfection of the subjects’ lymphocytes with KINDLIN3 complementary DNA but not CALDAGGEF1 cDNA reverses the LAD-III defect, restoring integrin-mediated adhesion and migration. PMID:19234463

  8. Nuclear localization of γ-tubulin affects E2F transcriptional activity and S-phase progression

    PubMed Central

    Höög, Greta; Zarrizi, Reihaneh; von Stedingk, Kristoffer; Jonsson, Kristina; Alvarado-Kristensson, Maria

    2011-01-01

    We show that the centrosome- and microtubule-regulating protein γ-tubulin interacts with E2 promoter binding factors (E2Fs) to modulate E2F transcriptional activity and thereby control cell cycle progression. γ-Tubulin contains a C-terminal signal that results in its translocation to the nucleus during late G1 to early S phase. γ-Tubulin mutants showed that the C terminus interacts with the transcription factor E2F1 and that the E2F1–γ-tubulin complex is formed during the G1/S transition, when E2F1 is transcriptionally active. Furthermore, E2F transcriptional activity is altered by reduced expression of γ-tubulin or by complex formation between γ-tubulin and E2F1, E2F2, or E2F3, but not E2F6. In addition, the γ-tubulin C terminus encodes a DNA-binding domain that interacts with E2F-regulated promoters, resulting in γ-tubulin-mediated transient activation of E2Fs. Thus, we report a novel mechanism regulating the activity of E2Fs, which can help explain how these proteins affect cell cycle progression in mammalian cells.—Höög, G., Zarrizi, R., von Stedingk, K., Jonsson, K., Alvarado-Kristensson, M. Nuclear localization of γ-tubulin affects E2F transcriptional activity and S-phase progression. PMID:21788450

  9. 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

  10. New constitutive latex osmotin-like proteins lacking antifungal activity.

    PubMed

    Freitas, Cleverson D T; Silva, Maria Z R; Bruno-Moreno, Frederico; Monteiro-Moreira, Ana C O; Moreira, Renato A; Ramos, Márcio V

    2015-11-01

    Proteins that share similar primary sequences to the protein originally described in salt-stressed tobacco cells have been named osmotins. So far, only two osmotin-like proteins were purified and characterized of latex fluids. Osmotin from Carica papaya latex is an inducible protein lacking antifungal activity, whereas the Calotropis procera latex osmotin is a constitutive antifungal protein. To get additional insights into this subject, we investigated osmotins in latex fluids of five species. Two potential osmotin-like proteins in Cryptostegia grandiflora and Plumeria rubra latex were detected by immunological cross-reactivity with polyclonal antibodies produced against the C. procera latex osmotin (CpOsm) by ELISA, Dot Blot and Western Blot assays. Osmotin-like proteins were not detected in the latex of Thevetia peruviana, Himatanthus drasticus and healthy Carica papaya fruits. Later, the two new osmotin-like proteins were purified through immunoaffinity chromatography with anti-CpOsm immobilized antibodies. Worth noting the chromatographic efficiency allowed for the purification of the osmotin-like protein belonging to H. drasticus latex, which was not detectable by immunoassays. The identification of the purified proteins was confirmed after MS/MS analyses of their tryptic digests. It is concluded that the constitutive osmotin-like proteins reported here share structural similarities to CpOsm. However, unlike CpOsm, they did not exhibit antifungal activity against Fusarium solani and Colletotrichum gloeosporioides. These results suggest that osmotins of different latex sources may be involved in distinct physiological or defensive events.

  11. Phosphorylation of Staphylococcus aureus Protein-Tyrosine Kinase Affects the Function of Glucokinase and Biofilm Formation

    PubMed Central

    Vasu, Dudipeta; Kumar, Pasupuleti Santhosh; Prasad, Uppu Venkateswara; Swarupa, Vimjam; Yeswanth, Sthanikam; Srikanth, Lokanathan; Sunitha, Manne Mudhu; Choudhary, Abhijith; Krishna Sarma, Potukuchi Venkata Gurunadha

    2017-01-01

    Background: When Staphylococcus aureus is grown in the presence of high concentration of external glucose, this sugar is phosphorylated by glucokinase (glkA) to form glucose-6-phosphate. This product subsequently enters into anabolic phase, which favors biofilm formation. The presence of ROK (repressor protein, open reading frame, sugar kinase) motif, phosphate-1 and -2 sites, and tyrosine kinase sites in glkA of S. aureus indicates that phosphorylation must regulate the glkA activity. The aim of the present study was to identify the effect of phosphorylation on the function of S. aureus glkA and biofilm formation. Methods: Pure glkA and protein-tyrosine kinase (BYK) of S. aureus ATCC 12600 were obtained by fractionating the cytosolic fractions of glkA1 and BYK-1 expressing recombinant clones through nickel metal chelate column. The pure glkA was used as a substrate for BYK, and the phosphorylation of glkA was confirmed by treating with reagent A and resolving in SDS-PAGE, as well as staining with reagent A. The kinetic parameters of glkA and phosphorylated glkA were determined spectrophotometrically, and in silico tools were used for validation. S. aureus was grown in brain heart infusion broth, which was supplemented with glucose, and then biofilm units were calculated. Results: Fourfold elevated glkA activity was observed upon the phosphorylation by BYK. Protein-protein docking analysis revealed that glkA structure docked close to the adenosine triphosphate-binding site of BYK structure corroborating the kinetic results. Further, S. aureus grown in the presence of elevated glucose concentration exhibited an increase in the rate of biofilm formation. Conclusion: The elevated function of glkA is an essential requirement for increased biofilm units in S. aureus, a key pathogenic factor that helps its survival and the progress of infection. PMID:27695030

  12. On the role of phosphatidylethanolamine in the inhibition of activated protein C activity by antiphospholipid antibodies.

    PubMed Central

    Smirnov, M D; Triplett, D T; Comp, P C; Esmon, N L; Esmon, C T

    1995-01-01

    Phosphatidylethanolamine (PE) is an important membrane component for supporting activated protein C anticoagulant activity but has little influence on prothrombin activation. This difference constitutes a potential mechanism for selective inhibition of the protein C anticoagulant pathway by lupus anticoagulants and/or antiphospholipid antibodies. In this study, we demonstrate that the presence of PE augments lupus anticoagulant activity. In the plasma of some patients with lupus anticoagulants, activated protein C anticoagulant activity is more potently inhibited than prothrombin activation. As a result, in the presence of activated protein C and PE, these patient plasmas clot faster than normal plasma. Patients with minimal lupus anticoagulant activity are identified whose plasma potently inhibits activated protein C anticoagulant activity. This process is also PE dependent. In three patient plasmas, these phenomena are shown to be due to immunoglobulins. The PE requirement in the expression of activated protein C anticoagulant activity and the PE dependence of some antiphospholipid antibodies provide a mechanistic basis for the selective inhibition of the protein C pathway. Inhibition of activated protein C function may be a common mechanism contributing to increased thrombotic risk in certain patients with antiphospholipid antibodies. PMID:7814631

  13. Adaptor protein Nck1 interacts with p120 Ras GTPase-activating protein and regulates its activity.

    PubMed

    Ger, Marija; Zitkus, Zigmantas; Valius, Mindaugas

    2011-10-01

    Adaptor protein Nck1 binds a number of intracellular proteins and influences various signaling pathways. Here we show that Nck1 directly binds and activates the GTPase-activating protein of Ras (RasGAP), which is responsible for the down-regulation of Ras. The first and the third SH3 domains of Nck1 and the NH(2)-terminal proline-rich sequence of RasGAP contribute most to the complex formation causing direct molecular interaction between the two proteins. Cell adhesion to the substrate is obligatory for the Nck1 and RasGAP association, as cell detachment makes RasGAP incapable of associating with Nck1. This leads to the complex dissipation, decrease of RasGAP activity and the increase of H-Ras-GTP level in the detached cells. Our findings reveal unexpected feature of adaptor protein Nck1 as the regulator of RasGAP activity.

  14. The importance of physical activity and sleep for affect on stressful days: Two intensive longitudinal studies.

    PubMed

    Flueckiger, Lavinia; Lieb, Roselind; Meyer, Andrea H; Witthauer, Cornelia; Mata, Jutta

    2016-06-01

    We investigated the potential stress-buffering effect of 3 health behaviors-physical activity, sleep quality, and snacking-on affect in the context of everyday life in young adults. In 2 intensive longitudinal studies with up to 65 assessment days over an entire academic year, students (Study 1, N = 292; Study 2, N = 304) reported stress intensity, sleep quality, physical activity, snacking, and positive and negative affect. Data were analyzed using multilevel regression analyses. Stress and positive affect were negatively associated; stress and negative affect were positively associated. The more physically active than usual a person was on a given day, the weaker the association between stress and positive affect (Study 1) and negative affect (Studies 1 and 2). The better than usual a person's sleep quality had been during the previous night, the weaker the association between stress and positive affect (Studies 1 and 2) and negative affect (Study 2). The association between daily stress and positive or negative affect did not differ as a function of daily snacking (Studies 1 and 2). On stressful days, increasing physical activity or ensuring high sleep quality may buffer adverse effects of stress on affect in young adults. These findings suggest potential targets for health-promotion and stress-prevention programs, which could help reduce the negative impact of stress in young adults. (PsycINFO Database Record

  15. Advanced Glycation End Products Affect Osteoblast Proliferation and Function by Modulating Autophagy Via the Receptor of Advanced Glycation End Products/Raf Protein/Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase Kinase/Extracellular Signal-regulated Kinase (RAGE/Raf/MEK/ERK) Pathway.

    PubMed

    Meng, Hong-Zheng; Zhang, Wei-Lin; Liu, Fei; Yang, Mao-Wei

    2015-11-20

    The interaction between advanced glycation end products (AGEs) and receptor of AGEs (RAGE) is associated with the development and progression of diabetes-associated osteoporosis, but the mechanisms involved are still poorly understood. In this study, we found that AGE-modified bovine serum albumin (AGE-BSA) induced a biphasic effect on the viability of hFOB1.19 cells; cell proliferation was stimulated after exposure to low dose AGE-BSA, but cell apoptosis was stimulated after exposure to high dose AGE-BSA. The low dose AGE-BSA facilitates proliferation of hFOB1.19 cells by concomitantly promoting autophagy, RAGE production, and the Raf/MEK/ERK signaling pathway activation. Furthermore, we investigated the effects of AGE-BSA on the function of hFOB1.19 cells. Interestingly, the results suggest that the short term effects of low dose AGE-BSA increase osteogenic function and decrease osteoclastogenic function, which are likely mediated by autophagy and the RAGE/Raf/MEK/ERK signal pathway. In contrast, with increased treatment time, the opposite effects were observed. Collectively, AGE-BSA had a biphasic effect on the viability of hFOB1.19 cells in vitro, which was determined by the concentration of AGE-BSA and treatment time. A low concentration of AGE-BSA activated the Raf/MEK/ERK signal pathway through the interaction with RAGE, induced autophagy, and regulated the proliferation and function of hFOB1.19 cells.

  16. Proteinase 3 Is a Phosphatidylserine-binding Protein That Affects the Production and Function of Microvesicles.

    PubMed

    Martin, Katherine R; Kantari-Mimoun, Chahrazade; Yin, Min; Pederzoli-Ribeil, Magali; Angelot-Delettre, Fanny; Ceroi, Adam; Grauffel, Cédric; Benhamou, Marc; Reuter, Nathalie; Saas, Philippe; Frachet, Philippe; Boulanger, Chantal M; Witko-Sarsat, Véronique

    2016-05-13

    Proteinase 3 (PR3), the autoantigen in granulomatosis with polyangiitis, is expressed at the plasma membrane of resting neutrophils, and this membrane expression increases during both activation and apoptosis. Using surface plasmon resonance and protein-lipid overlay assays, this study demonstrates that PR3 is a phosphatidylserine-binding protein and this interaction is dependent on the hydrophobic patch responsible for membrane anchorage. Molecular simulations suggest that PR3 interacts with phosphatidylserine via a small number of amino acids, which engage in long lasting interactions with the lipid heads. As phosphatidylserine is a major component of microvesicles (MVs), this study also examined the consequences of this interaction on MV production and function. PR3-expressing cells produced significantly fewer MVs during both activation and apoptosis, and this reduction was dependent on the ability of PR3 to associate with the membrane as mutating the hydrophobic patch restored MV production. Functionally, activation-evoked MVs from PR3-expressing cells induced a significantly larger respiratory burst in human neutrophils compared with control MVs. Conversely, MVs generated during apoptosis inhibited the basal respiratory burst in human neutrophils, and those generated from PR3-expressing cells hampered this inhibition. Given that membrane expression of PR3 is increased in patients with granulomatosis with polyangiitis, MVs generated from neutrophils expressing membrane PR3 may potentiate oxidative damage of endothelial cells and promote the systemic inflammation observed in this disease.

  17. Polymorphism of the prion protein gene (PRNP) in Polish cattle affected by classical bovine spongiform encephalopathy.

    PubMed

    Gurgul, Artur; Czarnik, Urszula; Urszula, Czarnik; Larska, Magdalena; Polak, Mirosław P; Strychalski, Janusz; Słota, Ewa

    2012-05-01

    Recent attempts to discover genetic factors affecting cattle resistance/susceptibility to bovine spongiform encephalopathy (BSE) have led to the identification of two insertion/deletion (indel) polymorphisms, located within the promoter and intron 1 of the prion protein gene PRNP, showing a significant association with the occurrence of classical form of the disease. Because the effect of the polymorphisms was studied only in few populations, in this study we investigated whether previously described association of PRNP indel polymorphisms with BSE susceptibility in cattle is also present in Polish cattle population. We found a significant relation between the investigated PRNP indel polymorphisms (23 and 12 bp indels), and susceptibility of Polish Holstein-Friesian cattle to classical BSE (P < 0.05). The deletion variants of both polymorphisms were related to increased susceptibility, whereas insertion variants were protective against BSE.

  18. Does hyperketonemia affect protein or glucose kinetics in postabsorptive or traumatized man

    SciTech Connect

    Crowe, P.J.; Royle, G.T.; Wagner, D.; Burke, J.F. )

    1989-10-01

    Leucine and glucose turnover were measured using simultaneous infusions of (13C)leucine and (2H)glucose before and during an infusion of Na DL-hydroxybutyrate (Na DL-HB) in overnight-fasted patients the day before and 3 days after total hip replacement. The ketone body infusion before surgery resulted in a significant increase in plasma leucine concentration and leucine turnover, while glucose concentration and turnover decreased. Surgery increased leucine turnover. Ketone body infusion after surgery caused a further increased leucine turnover while turnover fell as before surgery. We suggest that exogenous ketone bodies decrease hepatic glucose production and probably stimulate a rise in protein synthesis above breakdown leading to a decreased nitrogen excretion as observed by other investigators. Despite the metabolic adaptation to trauma, this response was not affected by surgery.

  19. In situ protein folding and activation in bacterial inclusion bodies.

    PubMed

    Gonzalez-Montalban, Nuria; Natalello, Antonino; García-Fruitós, Elena; Villaverde, Antonio; Doglia, Silvia Maria

    2008-07-01

    Recent observations indicate that bacterial inclusion bodies formed in absence of the main chaperone DnaK result largely enriched in functional, properly folded recombinant proteins. Unfortunately, the molecular basis of this intriguing fact, with obvious biotechnological interest, remains unsolved. We have explored here two non-excluding physiological mechanisms that could account for this observation, namely selective removal of inactive polypeptides from inclusion bodies or in situ functional activation of the embedded proteins. By combining structural and functional analysis, we have not observed any preferential selection of inactive and misfolded protein species by the dissagregating machinery during inclusion body disintegration. Instead, our data strongly support that folding intermediates aggregated as inclusion bodies could complete their natural folding process once deposited in protein clusters, which conduces to significant functional activation. In addition, in situ folding and protein activation in inclusion bodies is negatively regulated by the chaperone DnaK.

  20. Protein stability and enzyme activity at extreme biological temperatures.

    PubMed

    Feller, Georges

    2010-08-18

    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.

  1. CIPK23 is involved in iron acquisition of Arabidopsis by affecting ferric chelate reductase activity.

    PubMed

    Tian, Qiuying; Zhang, Xinxin; Yang, An; Wang, Tianzuo; Zhang, Wen-Hao

    2016-05-01

    Iron deficiency is one of the major limiting factors affecting quality and production of crops in calcareous soils. Numerous signaling molecules and transcription factors have been demonstrated to play a regulatory role in adaptation of plants to iron deficiency. However, the mechanisms underlying the iron deficiency-induced physiological processes remain to be fully dissected. Here, we demonstrated that the protein kinase CIPK23 was involved in iron acquisition. Lesion of CIPK23 rendered Arabidopsis mutants hypersensitive to iron deficiency, as evidenced by stronger chlorosis in young leaves and lower iron concentration than wild-type plants under iron-deficient conditions by down-regulating ferric chelate reductase activity. We found that iron deficiency evoked an increase in cytosolic Ca(2+) concentration and the elevated Ca(2+) would bind to CBL1/CBL9, leading to activation of CIPK23. These novel findings highlight the involvement of calcium-dependent CBL-CIPK23 complexes in the regulation of iron acquisition. Moreover, mutation of CIPK23 led to changes in contents of mineral elements, suggesting that CBL-CIPK23 complexes could be as "nutritional sensors" to sense and regulate the mineral homeostasis in Arabisopsis.

  2. Dietary electrolyte balance affects growth performance, amylase activity and metabolic response in the meagre (Argyrosomus regius).

    PubMed

    Magnoni, Leonardo J; Salas-Leiton, Emilio; Peixoto, Maria-João; Pereira, Luis; Silva-Brito, Francisca; Fontinha, Filipa; Gonçalves, José F M; Wilson, Jonathan M; Schrama, Johan W; Ozório, Rodrigo O A

    2017-03-16

    Dietary ion content is known to alter the acid-base balance in freshwater fish. The current study investigated the metabolic impact of acid-base disturbances produced by differences in dietary electrolyte balance (DEB) in the meagre (Argyrosomus regius), an euryhaline species. Changes in fish performance, gastric chyme characteristics, pH and ion concentrations in the bloodstream, digestive enzyme activities and metabolic rates were analyzed in meagre fed ad libitum two experimental diets (DEB 200 or DEB 700mEq/kg) differing in the Na2CO3 content for 69days. Fish fed the DEB 200 diet had 60-66% better growth performance than the DEB 700 group. Meagre consuming the DEB 200 diet were 90-96% more efficient than fish fed the DEB 700 diet at allocating energy from feed into somatic growth. The pH values in blood were significantly lower in the DEB 700 group 2h after feeding when compared to DEB 200, indicating that acid-base balance in meagre was affected by electrolyte balance in diet. Osmolality, and Na(+) and K(+) concentrations in plasma did not vary with the dietary treatment. Gastric chyme in the DEB 700 group had higher pH values, dry matter, protein and energy contents, but lower lipid content than in the DEB 200 group. Twenty-four hours after feeding, amylase activity was higher in the gastrointestinal tract of DEB 700 group when compared to the DEB 200 group. DEB 700 group had lower routine metabolic (RMR) and standard metabolic (SMR) rates, indicating a decrease in maintenance energy expenditure 48h after feeding the alkaline diet. The current study demonstrates that feeding meagre with an alkaline diet not only causes acid-base imbalance, but also negatively affects digestion and possibly nutrient assimilation, resulting in decreased growth performance.

  3. Affective dysfunction in a mouse model of Rett syndrome: Therapeutic effects of environmental stimulation and physical activity.

    PubMed

    Kondo, Mari A; Gray, Laura J; Pelka, Gregory J; Leang, Sook-Kwan; Christodoulou, John; Tam, Patrick P L; Hannan, Anthony J

    2016-02-01

    Rett syndrome (RTT) is a neurodevelopmental disorder associated with mutations in the X-linked gene encoding methyl-CpG-binding protein 2 (MeCP2) and consequent dysregulation of brain maturation. Patients suffer from a range of debilitating physical symptoms, however, behavioral and emotional symptoms also severely affect their quality of life. Here, we present previously unreported and clinically relevant affective dysfunction in the female heterozygous Mecp2(tm1Tam) mouse model of RTT (129sv and C57BL6 mixed background). The affective dysfunction and aberrant anxiety-related behavior of the Mecp2(+/-) mice were found to be reversible with environmental enrichment (EE) from 4 weeks of age. The effect of exercise alone (via wheel running) was also explored, providing the first evidence that increased voluntary physical activity in an animal model of RTT is beneficial for some phenotypes. Mecp2(+/-) mutants displayed elevated corticosterone despite decreased Crh expression, demonstrating hypothalamic-pituitary-adrenal axis dysregulation. EE of Mecp2(+/-) mice normalized basal serum corticosterone and hippocampal BDNF protein levels. The enrichment-induced rescue appears independent of the transcriptional regulation of the MeCP2 targets Bdnf exon 4 and Crh. These findings provide new insight into the neurodevelopmental role of MeCP2 and pathogenesis of RTT, in particular the affective dysfunction. The positive outcomes of environmental stimulation and physical exercise have implications for the development of therapies targeting the affective symptoms, as well as behavioral and cognitive dimensions, of this devastating neurodevelopmental disorder.

  4. 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.

  5. The signaling protein MucG negatively affects the production and the molecular mass of alginate in Azotobacter vinelandii.

    PubMed

    Ahumada-Manuel, Carlos Leonel; Guzmán, Josefina; Peña, Carlos; Quiroz-Rocha, Elva; Espín, Guadalupe; Núñez, Cinthia

    2017-02-01

    Azotobacter vinelandii is a soil bacterium that produces the polysaccharide alginate. In this work, we identified a miniTn5 mutant, named GG9, which showed increased alginate production of higher molecular mass, and increased expression of the alginate biosynthetic genes algD and alg8 when compared to its parental strain. The miniTn5 was inserted within ORF Avin07920 encoding a hypothetical protein. Avin07910, located immediately downstream and predicted to form an operon with Avin07920, encodes an inner membrane multi-domain signaling protein here named mucG. Insertional inactivation of mucG resulted in a phenotype of increased alginate production of higher molecular mass similar to that of mutant GG9. The MucG protein contains a periplasmic and putative HAMP and PAS domains, which are linked to GGDEF and EAL domains. The last two domains are potentially involved in the synthesis and degradation, respectively, of bis-(3'-5')-cyclic dimeric GMP (c-di-GMP), a secondary messenger that has been reported to be essential for alginate production. Therefore, we hypothesized that the negative effect of MucG on the production of this polymer could be explained by the putative phosphodiesterase activity of the EAL domain. Indeed, we found that alanine replacement mutagenesis of the MucG EAL motif or deletion of the entire EAL domain resulted in increased alginate production of higher molecular mass similar to the GG9 and mucG mutants. To our knowledge, this is the first reported protein that simultaneous affects the production of alginate and its molecular mass.

  6. Mutations in domain a′ of protein disulfide isomerase affect the folding pathway of bovine pancreatic ribonuclease A

    PubMed Central

    Ruoppolo, Margherita; Orrù, Stefania; Talamo, Fabio; Ljung, Johanna; Pirneskoski, Annamari; Kivirikko, Kari I.; Marino, Gennaro; Koivunen, Peppi

    2003-01-01

    Protein disulfide isomerase (PDI, EC 5.3.4.1), an enzyme and chaperone, catalyses disulfide bond formation and rearrangements in protein folding. It is also a subunit in two proteins, the enzyme collagen prolyl 4-hydroxylase and the microsomal triglyceride transfer protein. It consists of two catalytically active domains, a and a′, and two inactive ones, b and b′, all four domains having the thioredoxin fold. Domain b′ contains the primary peptide binding site, but a′ is also critical for several of the major PDI functions. Mass spectrometry was used here to follow the folding pathway of bovine pancreatic ribonuclease A (RNase A) in the presence of three PDI mutants, F449R, Δ455–457, and abb′, and the individual domains a and a′. The first two mutants contained alterations in the last α helix of domain a′, while the third lacked the entire domain a′. All mutants produced genuine, correctly folded RNase A, but the appearance rate of 50% of the product, as compared to wild-type PDI, was reduced 2.5-fold in the case of PDI Δ455–457, 7.5-fold to eightfold in the cases of PDI F449R and PDI abb′, and over 15-fold in the cases of the individual domains a and a′. In addition, PDI F449R and PDI abb′ affected the distribution of folding intermediates. Domains a and a′ catalyzed the early steps in the folding but no disulfide rearrangements, and therefore the rate observed in the presence of these individual domains was similar to that of the spontaneous process. PMID:12717017

  7. Hypoxia Affects Neprilysin Expression Through Caspase Activation and an APP Intracellular Domain-dependent Mechanism

    PubMed Central

    Kerridge, Caroline; Kozlova, Daria I.; Nalivaeva, Natalia N.; Turner, Anthony J.

    2015-01-01

    While gene mutations in the amyloid precursor protein (APP) and the presenilins lead to an accumulation of the amyloid β-peptide (Aβ) in the brain causing neurodegeneration and familial Alzheimer's disease (AD), over 95% of all AD cases are sporadic. Despite the pathologies being indistinguishable, relatively little is known about the mechanisms affecting generation of Aβ in the sporadic cases. Vascular disorders such as ischaemia and stroke are well established risk factors for the development of neurodegenerative diseases and systemic hypoxic episodes have been shown to increase Aβ production and accumulation. We have previously shown that hypoxia causes a significant decrease in the expression of the major Aβ-degrading enzyme neprilysin (NEP) which might deregulate Aβ clearance. Aβ itself is derived from the transmembrane APP along with several other biologically active metabolites including the C-terminal fragment (CTF) termed the APP intracellular domain (AICD), which regulates the expression of NEP and some other genes in neuronal cells. Here we show that in hypoxia there is a significantly increased expression of caspase-3, 8, and 9 in human neuroblastoma NB7 cells, which can degrade AICD. Using chromatin immunoprecipitation we have revealed that there was also a reduction of AICD bound to the NEP promoter region which underlies the decreased expression and activity of the enzyme under hypoxic conditions. Incubation of the cells with a caspase-3 inhibitor Z-DEVD-FMK could rescue the effect of hypoxia on NEP activity protecting the levels of AICD capable of binding the NEP promoter. These data suggest that activation of caspases might play an important role in regulation of NEP levels in the brain under pathological conditions such as hypoxia and ischaemia leading to a deficit of Aβ clearance and increasing the risk of development of AD. PMID:26617481

  8. Dobesilate diminishes activation of the mitogen - activated protein kinase ERK1/2 in glioma cells

    PubMed Central

    Cuevas, P; Diaz-González, Diana; Garcia-Martin-Córdova, C; Sánchez, I; Lozano, Rosa Maria; Giménez-Gallego, G; Dujovny, M

    2006-01-01

    Fibroblast growth factors (FGFs) and their receptors, regularly expressed at high levels in gliomas, are further upregulated during the transition of the tumor from low- to high-grade malignancy, and are essential for glioma progression. FGFs induce upregulation of the mitogen-activated protein kinase (MAPK) signaling cascade in cultured glioma cells, which suggests that MAPK pathway participates in the FGF-dependent glioma development. Recently, it has been shown that dobesilate, an inhibitor of FGF mitogenic activity, shows antiproliferative and proapoptotic activities in glioma cell cultures. Accordingly, it should be expected this new synthetic FGF inhibitor to affect the activation levels of MAPK. Here we report that immunocytochemical and Western blot data unequivocally show that treatment of cell cultures with dobesilate causes a significant decrease of the intracellular levels of ERK1/2 activation, one of the components of the MAPK signalling cascade. This finding supports an important role for dobesilate in glioma growth, suggesting that dobesilate should be a treatment to be born in mind for glioma management. PMID:16563234

  9. The Slx5-Slx8 complex affects sumoylation of DNA repair proteins and negatively regulates recombination.

    PubMed

    Burgess, Rebecca C; Rahman, Sadia; Lisby, Michael; Rothstein, Rodney; Zhao, Xiaolan

    2007-09-01

    Recombination is important for repairing DNA lesions, yet it can also lead to genomic rearrangements. This process must be regulated, and recently, sumoylation-mediated mechanisms were found to inhibit Rad51-dependent recombination. Here, we report that the absence of the Slx5-Slx8 complex, a newly identified player in the SUMO (small ubiquitin-like modifier) pathway, led to increased Rad51-dependent and Rad51-independent recombination. The increases were most striking during S phase, suggesting an accumulation of DNA lesions during replication. Consistent with this view, Slx8 protein localized to replication centers. In addition, like SUMO E2 mutants, slx8Delta mutants exhibited clonal lethality, which was due to the overamplification of 2 microm, an extrachromosomal plasmid. Interestingly, in both SUMO E2 and slx8Delta mutants, clonal lethality was rescued by deleting genes required for Rad51-independent recombination but not those involved in Rad51-dependent events. These results suggest that sumoylation negatively regulates Rad51-independent recombination, and indeed, the Slx5-Slx8 complex affected the sumoylation of several enzymes involved in early steps of Rad51-independent recombination. We propose that, during replication, the Slx5-Slx8 complex helps prevent DNA lesions that are acted upon by recombination. In addition, the complex inhibits Rad51-independent recombination via modulating the sumoylation of DNA repair proteins.

  10. Membrane protein assembly: two cytoplasmic phosphorylated serine sites of Vpu from HIV-1 affect oligomerization

    PubMed Central

    Chen, Chin-Pei; Lin, Meng-Han; Chan, Ya-Ting; Chen, Li-Chyong; Ma, Che; Fischer, Wolfgang B.

    2016-01-01

    Viral protein U (Vpu) encoded by human immunodeficiency virus type 1 (HIV-1) is a short integral membrane protein which is known to self-assemble within the lipid membrane and associate with host factors during the HIV-1 infectivity cycle. In this study, full-length Vpu (M group) from clone NL4-3 was over-expressed in human cells and purified in an oligomeric state. Various single and double mutations were constructed on its phosphorylation sites to mimic different degrees of phosphorylation. Size exclusion chromatography of wild-type Vpu and mutants indicated that the smallest assembly unit of Vpu was a dimer and over time Vpu formed higher oligomers. The rate of oligomerization increased when (i) the degree of phosphorylation at serines 52 and 56 was decreased and (ii) when the ionic strength was increased indicating that the cytoplasmic domain of Vpu affects oligomerization. Coarse-grained molecular dynamic simulations with models of wild-type and mutant Vpu in a hydrated lipid bilayer supported the experimental data in demonstrating that, in addition to a previously known role in downregulation of host factors, the phosphorylation sites of Vpu also modulate oligomerization. PMID:27353136

  11. Pathophysiological changes that affect drug disposition in protein-energy malnourished children

    PubMed Central

    2009-01-01

    Protein-energy malnutrition (PEM) is a major public health problem affecting a high proportion of infants and older children world-wide and accounts for a high childhood morbidity and mortality in the developing countries. The epidemiology of PEM has been extensively studied globally and management guidelines formulated by the World Health Organization (WHO). A wide spectrum of infections such as measles, malaria, acute respiratory tract infection, intestinal parasitosis, tuberculosis and HIV/AIDS may complicate PEM with two or more infections co-existing. Thus, numerous drugs may be required to treat the patients. In-spite of abundant literature on the epidemiology and management of PEM, focus on metabolism and therapeutic drug monitoring is lacking. A sound knowledge of pathophysiology of PEM and pharmacology of the drugs frequently used for their treatment is required for safe and rational treatment. In this review, we discuss the pathophysiological changes in children with PEM that may affect the disposition of drugs frequently used for their treatment. This review has established abnormal disposition of drugs in children with PEM that may require dosage modification. However, the relevance of these abnormalities to the clinical management of PEM remains inconclusive. At present, there are no good indications for drug dosage modification in PEM; but for drug safety purposes, further studies are required to accurately determine dosages of drugs frequently used for children with PEM. PMID:19951418

  12. Induced autoimmunity against gonadal proteins affects gonadal development in juvenile zebrafish.

    PubMed

    Presslauer, Christopher; Nagasawa, Kazue; Dahle, Dalia; Babiak, Joanna; Fernandes, Jorge M O; Babiak, Igor

    2014-01-01

    A method to mitigate or possibly eliminate reproduction in farmed fish is highly demanded. The existing approaches have certain applicative limitations. So far, no immunization strategies affecting gonadal development in juvenile animals have been developed. We hypothesized that autoimmune mechanisms, occurring spontaneously in a number of diseases, could be induced by targeted immunization. We have asked whether the immunization against specific targets in a juvenile zebrafish gonad will produce an autoimmune response, and, consequently, disturbance in gonadal development. Gonadal soma-derived factor (Gsdf), growth differentiation factor (Gdf9), and lymphocyte antigen 75 (Cd205/Ly75), all essential for early gonad development, were targeted with 5 immunization tests. Zebrafish (n = 329) were injected at 6 weeks post fertilization, a booster injection was applied 15 days later, and fish were sampled at 30 days. We localized transcripts encoding targeted proteins by in situ hybridization, quantified expression of immune-, apoptosis-, and gonad-related genes with quantitative real-time PCR, and performed gonadal histology and whole-mount immunohistochemistry for Bcl2-interacting-killer (Bik) pro-apoptotic protein. The treatments resulted in an autoimmune reaction, gonad developmental retardation, intensive apoptosis, cell atresia, and disturbed transcript production. Testes were remarkably underdeveloped after anti-Gsdf treatments. Anti-Gdf9 treatments promoted apoptosis in testes and abnormal development of ovaries. Anti-Cd205 treatment stimulated a strong immune response in both sexes, resulting in oocyte atresia and strong apoptosis in supporting somatic cells. The effect of immunization was FSH-independent. Furthermore, immunization against germ cell proteins disturbed somatic supporting cell development. This is the first report to demonstrate that targeted autoimmunity can disturb gonadal development in a juvenile fish. It shows a straightforward potential

  13. The two CcdA proteins of Bacillus anthracis differentially affect virulence gene expression and sporulation.

    PubMed

    Han, Hesong; Wilson, Adam C

    2013-12-01

    The cytochrome c maturation system influences the expression of virulence factors in Bacillus anthracis. B. anthracis carries two copies of the ccdA gene, encoding predicted thiol-disulfide oxidoreductases that contribute to cytochrome c maturation, while the closely related organism Bacillus subtilis carries only one copy of ccdA. To investigate the roles of the two ccdA gene copies in B. anthracis, strains were constructed without each ccdA gene, and one strain was constructed without both copies simultaneously. Loss of both ccdA genes results in a reduction of cytochrome c production, an increase in virulence factor expression, and a reduction in sporulation efficiency. Complementation and expression analyses indicate that ccdA2 encodes the primary CcdA in B. anthracis, active in all three pathways. While CcdA1 retains activity in cytochrome c maturation and virulence control, it has completely lost its activity in the sporulation pathway. In support of this finding, expression of ccdA1 is strongly reduced when cells are grown under sporulation-inducing conditions. When the activities of CcdA1 and CcdA2 were analyzed in B. subtilis, neither protein retained activity in cytochrome c maturation, but CcdA2 could still function in sporulation. These observations reveal the complexities of thiol-disulfide oxidoreductase function in pathways relevant to virulence and physiology.

  14. Effect of membrane-associated f1 bacteriophage coat protein upon the activity of Escherichia coli phosphatidylserine synthetase.

    PubMed Central

    Chamberlain, B K; Webster, R E

    1978-01-01

    The effects of insertion of the major coat protein of f1 bacteriophage into Escherichia coli membranes were investigated under conditions allowing in vivo analysis of phosphatidylserine synthesis. An E. coli strain possessing a temperature-sensitive phosphatidylserine decarboxylase was utilized under conditions in which the decarboxylase activity was reduced but nonlethal. The presence of the coat protein in the host membranes inhibits the activity of the phosphatidylserine synthetase and perhaps affects the activity of the phosphatidylserine decarboxylase. PMID:211116

  15. A Gammaherpesvirus Complement Regulatory Protein Promotes Initiation of Infection by Activation of Protein Kinase Akt/PKB

    PubMed Central

    Steer, Beatrix; Adler, Barbara; Jonjic, Stipan; Stewart, James P.; Adler, Heiko

    2010-01-01

    Background Viruses have evolved to evade the host's complement system. The open reading frames 4 (ORF4) of gammaherpesviruses encode homologs of regulators of complement activation (RCA) proteins, which inhibit complement activation at the level of C3 and C4 deposition. Besides complement regulation, these proteins are involved in heparan sulfate and glycosaminoglycan binding, and in case of MHV-68, also in viral DNA synthesis in macrophages. Methodology/Principal Findings Here, we made use of MHV-68 to study the role of ORF4 during infection of fibroblasts. While attachment and penetration of virions lacking the RCA protein were not affected, we observed a delayed delivery of the viral genome to the nucleus of infected cells. Analysis of the phosphorylation status of a variety of kinases revealed a significant reduction in phosphorylation of the protein kinase Akt in cells infected with ORF4 mutant virus, when compared to cells infected with wt virus. Consistent with a role of Akt activation in initial stages of infection, inhibition of Akt signaling in wt virus infected cells resulted in a phenotype resembling the phenotype of the ORF4 mutant virus, and activation of Akt by addition of insulin partially reversed the phenotype of the ORF4 mutant virus. Importantly, the homologous ORF4 of KSHV was able to rescue the phenotype of the MHV-68 ORF4 mutant, indicating that ORF4 is functionally conserved and that ORF4 of KSHV might have a similar function in infection initiation. Conclusions/Significance In summary, our studies demonstrate that ORF4 contributes to efficient infection by activation of the protein kinase Akt and thus reveal a novel function of a gammaherpesvirus RCA protein. PMID:20657771

  16. Tomato Plant Proteins Actively Responding to Fungal Applications and Their Role in Cell Physiology

    PubMed Central

    Bashir, Zoobia; Shafique, Sobiya; Ahmad, Aqeel; Shafique, Shazia; Yasin, Nasim A.; Ashraf, Yaseen; Ibrahim, Asma; Akram, Waheed; Noreen, Sibgha

    2016-01-01

    The pattern of protein induction in tomato plants has been investigated after the applications of pathogenic and non-pathogenic fungal species. Moreover, particular roles of the most active protein against biological applications were also determined using chromatographic techniques. Alternaria alternata and Penicillium oxalicum were applied as a pathogenic and non-pathogenic fungal species, respectively. Protein profile analysis revealed that a five protein species (i.e., protein 1, 6, 10, 12, and 13) possessed completely coupled interaction with non-pathogenic inducer application (P. oxalicum). However, three protein species (i.e., 10, 12, and 14) recorded a strong positive interaction with both fungal species. Protein 14 exhibited the maximum interaction with fungal applications, and its role in plant metabolism was studied after its identification as protein Q9M1W6. It was determined that protein Q1M1W6 was involved in guaiacyl lignin biosynthesis, and its inhibition increased the coumarin contents in tomato plants. Moreover, it was also observed that the protein Q9M1W6 takes significant part in the biosynthesis of jasmonic acid and Indole acetic acid contents, which are defense and growth factors of tomato plants. The study will help investigators to design fundamental rules of plant proteins affecting cell physiology under the influence of external fungal applications. PMID:27445848

  17. Growth hormone, but not insulin, activates STAT5 proteins in adipocytes in vitro and in vivo.

    PubMed

    Zvonic, Sanjin; Story, David J; Stephens, Jacqueline M; Mynatt, Randall L

    2003-03-07

    STAT 5 proteins are latent transcription factors which have been shown to be activated by growth hormone (GH) in many cell types. However, some recent studies also suggest that STAT 5B is a physiological substrate of the insulin receptor. In our studies, we have shown that physiological levels of insulin do not induce STAT 5 tyrosine phosphorylation or affect the nuclear distribution of STATs 5A or 5B in 3T3-L1 adipocytes. Moreover, we did not observe the activation of STAT 5 in the adipose tissue or skeletal muscle of mice following an acute intraperitoneal injection of insulin. However, acute GH administration, both in vitro and in vivo, resulted in the activation of STAT 5 proteins. In summary, our results indicate that STAT 5 proteins are not activated by physiological levels of insulin in adipose tissue.

  18. Suppression of DNA-dependent protein kinase sensitize cells to radiation without affecting DSB repair.

    PubMed

    Gustafsson, Ann-Sofie; Abramenkovs, Andris; Stenerlöw, Bo

    2014-11-01

    Efficient and correct repair of DNA double-strand break (DSB) is critical for cell survival. Defects in the DNA repair may lead to cell death, genomic instability and development of cancer. The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an essential component of the non-homologous end joining (NHEJ) which is the major DSB repair pathway in mammalian cells. In the present study, by using siRNA against DNA-PKcs in four human cell lines, we examined how low levels of DNA-PKcs affected cellular response to ionizing radiation. Decrease of DNA-PKcs levels by 80-95%, induced by siRNA treatment, lead to extreme radiosensitivity, similar to that seen in cells completely lacking DNA-PKcs and low levels of DNA-PKcs promoted cell accumulation in G2/M phase after irradiation and blocked progression of mitosis. Surprisingly, low levels of DNA-PKcs did not affect the repair capacity and the removal of 53BP1 or γ-H2AX foci and rejoining of DSB appeared normal. This was in strong contrast to cells completely lacking DNA-PKcs and cells treated with the DNA-PKcs inhibitor NU7441, in which DSB repair were severely compromised. This suggests that there are different mechanisms by which loss of DNA-PKcs functions can sensitize cells to ionizing radiation. Further, foci of phosphorylated DNA-PKcs (T2609 and S2056) co-localized with DSB and this was independent of the amount of DNA-PKcs but foci of DNA-PKcs was only seen in siRNA-treated cells. Our study emphasizes on the critical role of DNA-PKcs for maintaining survival after radiation exposure which is uncoupled from its essential function in DSB repair. This could have implications for the development of therapeutic strategies aiming to radiosensitize tumors by affecting the DNA-PKcs function.

  19. Antibacterial peptides and mitochondrial presequences affect mitochondrial coupling, respiration and protein import.

    PubMed

    Hugosson, M; Andreu, D; Boman, H G; Glaser, E

    1994-08-01

    Cecropins A and P1, antibacterial peptides from insects and from pig and some related peptides released respiratory control, inhibited protein import and at higher concentrations also inhibited respiration. However, PR-39, an antibacterial peptide from pig intestine, was found to be almost inert towards mitochondria. The concentrations at which the three mitochondrial functions were effected varied for different peptides. Melittin, magainin and Cecropin-A-(1,13)-Melittin(1,13)-NH2, a hybrid between cecropin A and melittin, were most potent, while the two cecropins acted at higher concentrations. The biosynthesis of cecropin A is known and the intermediates are synthesized. We have used four peptides from this pathway to investigate their effects on coupling, respiration and protein import into mitochondria. Mature cecropin A followed by the preproprotein were most aggressive whereas the intermediates were less active or inert. The efficiency of different derivatives of cecropin A as uncouplers correlates well with their capacity to release membrane potential measured as fluorescence quenching of Rhodamine 123. Inhibition of respiration was found to be dependent on membrane potential and was most pronounced with mature cecropin A, less so with its three precursors. We also found that three peptides derived from mitochondrial presequences showed antibacterial activity. It is concluded that, there are similarities in the functions of antibacterial peptides and mitochondrial presequences, uncoupling activity in mitochondria cannot be correlated with the antibacterial activity (contrary to a previous suggestion), the processing of preprocecropin A may have evolved in such a way that there is a minimum of membrane damage from the intermediates in the pathway, and peptides produced for delivery outside of an animal have evolved to be more aggressive against mitochondria than peptides for delivery inside of the animal.

  20. Differentially expressed proteins in gill and skin mucus of Atlantic salmon (Salmo salar) affected by amoebic gill disease.

    PubMed

    Valdenegro-Vega, Victoria A; Crosbie, Phil; Bridle, Andrew; Leef, Melanie; Wilson, Richard; Nowak, Barbara F

    2014-09-01

    The external surfaces of fish, such as gill and skin, are covered by mucus, which forms a thin interface between the organism and water. Amoebic gill disease (AGD) is a parasitic condition caused by Neoparamoeba perurans that affects salmonids worldwide. This disease induces excessive mucus production in the gills. The host immune response to AGD is not fully understood, and research tools such as genomics and proteomics could be useful in providing further insight. Gill and skin mucus samples were obtained from Atlantic salmon (Salmo salar) which were infected with N. perurans on four successive occasions. NanoLC tandem mass spectrometry (MS/MS) was used to identify proteins in gill and skin mucus of Atlantic salmon affected by AGD. A total of 186 and 322 non-redundant proteins were identified in gill and skin mucus respectively, based on stringent filtration criteria, and statistics demonstrated that 52 gill and 42 skin mucus proteins were differentially expressed in mucus samples from AGD-affected fish. By generating protein-protein interaction networks, some of these proteins formed part of cell to cell signalling and inflammation pathways, such as C-reactive protein, apolipoprotein 1, granulin, cathepsin, angiogenin-1. In addition to proteins that were entirely novel in the context in the host response to N. perurans, our results have confirmed the presence of protein markers in mucus that have been previously predicted on the basis of modified mRNA expression, such as anterior gradient-2 protein, annexin A-1 and complement C3 factor. This first proteomic analysis of AGD-affected salmon provides new information on the effect of AGD on protein composition of gill and skin mucus. Future research should focus on better understanding of the role these components play in the response against infection with N. perurans.

  1. 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.

  2. Resistance exercise volume affects myofibrillar protein synthesis and anabolic signalling molecule phosphorylation in young men

    PubMed Central

    Burd, Nicholas A; Holwerda, Andrew M; Selby, Keegan C; West, Daniel W D; Staples, Aaron W; Cain, Nathan E; Cashaback, Joshua G A; Potvin, James R; Baker, Steven K; Phillips, Stuart M

    2010-01-01

    We aimed to determine if any mechanistic differences exist between a single set (1SET) and multiple sets (i.e. 3 sets; 3SET) of resistance exercise by utilizing a primed constant infusion of [ring-13C6]phenylalanine to determine myofibrillar protein synthesis (MPS) and Western blot analysis to examine anabolic signalling molecule phosphorylation following an acute bout of resistance exercise. Eight resistance-trained men (24 ± 5 years, BMI = 25 ± 4 kg m−2) were randomly assigned to perform unilateral leg extension exercise at 70% concentric one repetition maximum (1RM) until volitional fatigue for 1SET or 3SET. Biopsies from the vastus lateralis were taken in the fasted state (Fast) and fed state (Fed; 20 g of whey protein isolate) at rest, 5 h Fed, 24 h Fast and 29 h Fed post-exercise. Fed-state MPS was transiently elevated above rest at 5 h for 1SET (2.3-fold) and returned to resting levels by 29 h post-exercise. However, the exercise induced increase in MPS following 3SET was superior in amplitude and duration as compared to 1SET at both 5 h (3.1-fold above rest) and 29 h post-exercise (2.3-fold above rest). Phosphorylation of 70 kDa S6 protein kinase (p70S6K) demonstrated a coordinated increase with MPS at 5 h and 29 h post-exercise such that the extent of p70S6K phosphorylation was related to the MPS response (r = 0.338, P = 0.033). Phosphorylation of 90 kDa ribosomal S6 protein kinase (p90RSK) and ribosomal protein S6 (rps6) was similar for 1SET and 3SET at 24 h Fast and 29 h Fed, respectively. However, 3SET induced a greater activation of eukaryotic translation initiation factor 2Bɛ (eIF2Bɛ) and rpS6 at 5 h Fed. These data suggest that 3SET of resistance exercise is more anabolic than 1SET and may lead to greater increases in myofibrillar protein accretion over time. PMID:20581041

  3. [Soil enzyme activities under two forest types as affected by different levels of nitrogen deposition].

    PubMed

    Zhao, Yu-tao; Li, Xue-feng; Han, Shi-jie; Hu, Yan-ling

    2008-12-01

    A simulation test was conducted to study the change trends of soil cellulase, polyphenol oxidase, and sucrase activities under natural broadleaf-Korean pine (Pinus koraiensis) and secondary poplar (Populus davidiana) -birch (Betula platyphylla) mixed forests as affected by 0, 25, and 50 kg x hm(-2) x a(-1) of N deposition. The results showed that the effects of elevated N deposition on test enzyme activities varied with forest type, and short-term nitrogen addition could significantly affect the test enzyme activities. High N deposition decreased soil polyphyneol oxidase activity, and correspondingly, soil cellulase and sucrase activities also had a trend of decrease.

  4. Do government brochures affect physical activity cognition? A pilot study of Canada's physical activity guide to healthy active living.

    PubMed

    Kliman, Aviva M; Rhodes, Ryan

    2008-08-01

    Health Canada has published national physical activity (PA) guidelines, which are included in their 26-page Physical Activity Guide to Healthy Active Living (CPAG). To date, the use of CPAG as a motivational instrument for PA promotion has not been evaluated. The purpose of this study was to determine whether reading CPAG 1) increased motivational antecedents to engage in regular PA, and 2) increased regular PA intention and behaviour over 1 month. Participants included 130 randomly sampled Canadian adults (18 years or older) who were randomly mailed pack ages consisting of either 1) a questionnaire and a copy of CPAG, or 2) a questionnaire. Questionnaire items pertained to participants' sociodemographics, previous PA behaviours (Godin Leisure-Time Questionnaire) and PA motivation (theory of planned behaviour). Participants were then sent a follow-up questionnaire pertaining to their PA behaviours throughout the previous month. Results revealed significant interactions between the guide condition and previous activity status on instrumental behavioural beliefs about strength activities and subjective norms about endurance activities (p < 0.05), but all other factors were not significantly different. It was concluded that among previously inactive people, receiving this guide may change some informational/motivational constructs, but key motivational antecedents (affective attitude, perceived behavioural control) and outcomes (intention, behaviour) seem unaffected.

  5. Duality of G protein-coupled mechanisms for beta-adrenergic activation of NKCC activity in skeletal muscle.

    PubMed

    Gosmanov, Aidar R; Wong, Jennifer A; Thomason, Donald B

    2002-10-01

    Skeletal muscle Na(+)-K(+)-2Cl(-) cotransporter (NKCC) activity provides a potential mechanism for regulated K(+) uptake. beta-Adrenergic receptor (beta-AR) activation stimulates skeletal muscle NKCC activity in a MAPK pathway-dependent manner. We examined potential G protein-coupled pathways for beta-AR-stimulated NKCC activity. Inhibition of G(s)-coupled PKA blocked isoproterenol-stimulated NKCC activity in both the slow-twitch soleus muscle and the fast-twitch plantaris muscle. However, the PKA-activating agents cholera toxin, forskolin, and 8-bromo-cAMP (8-BrcAMP) were not sufficient to activate NKCC in the plantaris and partially stimulated NKCC activity in the soleus. Isoproterenol-stimulated NKCC activity in the soleus was abolished by pretreatment with pertussis toxin (PTX), indicating a G(i)-coupled mechanism. PTX did not affect the 8-BrcAMP-stimulated NKCC activity. PTX treatment also precluded the isoproterenol-mediated ERK1/2 MAPK phosphorylation in the soleus, consistent with NKCC's MAPK dependency. Inhibition of isoproterenol-stimulated ERK activity by PTX treatment was associated with an increase in Akt activation and phosphorylation of Raf-1 on the inhibitory residue Ser(259). These results demonstrate a novel, muscle phenotype-dependent mechanism for beta-AR-mediated NKCC activation that involves both G(s) and G(i) protein-coupled mechanisms.

  6. The cyanobacterial Fluorescence Recovery Protein has two distinct activities: Orange Carotenoid Protein amino acids involved in FRP interaction.

    PubMed

    Thurotte, Adrien; Bourcier de Carbon, Céline; Wilson, Adjélé; Talbot, Léa; Cot, Sandrine; López-Igual, Rocio; Kirilovsky, Diana

    2017-04-01

    To deal with fluctuating light condition, cyanobacteria have developed a photoprotective mechanism which, under high light conditions, decreases the energy arriving at the photochemical centers. It relies on a photoswitch, the Orange Carotenoid Protein (OCP). Once photoactivated, OCP binds to the light harvesting antenna, the phycobilisome (PBS), and triggers the thermal dissipation of the excess energy absorbed. Deactivation of the photoprotective mechanism requires the intervention of a third partner, the Fluorescence Recovery Protein (FRP). FRP by interacting with the photoactivated OCP accelerates its conversion to the non-active form and its detachment from the phycobilisome. We have studied the interaction of FRP with free and phycobilisome-bound OCP. Several OCP variants were constructed and characterized. In this article we show that OCP amino acid F299 is essential and D220 important for OCP deactivation mediated by FRP. Mutations of these amino acids did not affect FRP activity as helper to detach OCP from phycobilisomes. In addition, while mutated R60L FRP is inactive on OCP deactivation, its activity on the detachment of the OCP from the phycobilisomes is not affected. Thus, our results demonstrate that FRP has two distinct activities: it accelerates OCP detachment from phycobilisomes and then it helps deactivation of the OCP. They also suggest that different OCP and FRP amino acids could be involved in these two activities.

  7. Cajal body proteins differentially affect the processing of box C/D scaRNPs.

    PubMed

    Enwerem, Isioma I; Wu, Guowei; Yu, Yi Tao; Hebert, Michael D

    2015-01-01

    Small nuclear ribonucleoproteins (snRNPs), which are required for pre-mRNA splicing, contain extensively modified snRNA. Small Cajal body-specific ribonucleoproteins (scaRNPs) mediate these modifications. It is unknown how the box C/D class of scaRNPs localizes to Cajal Bodies (CBs). The processing of box C/D scaRNA is also unclear. Here, we explore the processing of box C/D scaRNA 2 and 9 by coilin. We also broaden our investigation to include WRAP53 and SMN, which accumulate in CBs, play a role in RNP biogenesis and associate with coilin. These studies demonstrate that the processing of an ectopically expressed scaRNA2 is altered upon the reduction of coilin, WRAP53 or SMN, but the extent and direction of this change varies depending on the protein reduced. We also show that box C/D scaRNP activity is reduced in a cell line derived from coilin knockout mice. Collectively, the findings presented here further implicate coilin as being a direct participant in the formation of box C/D scaRNPs, and demonstrate that WRAP53 and SMN may also play a role, but the activity of these proteins is divergent to coilin.

  8. Proteasome affects the expression of aryl hydrocarbon receptor-regulated proteins.

    PubMed

    Ishida, Takumi; Kawakami, Masayo; Baba, Hiroko; Yahata, Masahiro; Mutoh, Junpei; Takeda, Shuso; Fujita, Hideaki; Tanaka, Yoshitaka; Ishii, Yuji; Yamada, Hideyuki

    2008-11-01

    The effect of proteasome inhibition with N-acetyl-leucyl-leucyl-norleucinal (ALLN) on the protein expression regulated by aryl hydrocarbon receptor (AhR) was studied in T47D breast tumor cells. The luciferase reporter gene assay using a construct which has the xenobiotic responsive element showed that the inducible expression of the reporter with AhR ligands was significantly reduced by co-treatment with ALLN. The same suppressive effect by ALLN was observed for ethoxyresorufin O-deethylase (EROD) activity induced by an AhR ligand, 3-methylcholanthrene (3MC). Despite the above effects, the induced expression of CYP1A1 and CYP1B1 mRNAs was unaffected by ALLN. While lactacystin, another proteasome inhibitor, exhibited the same effect as ALLN on EROD activity induced by 3MC, leupeptin, which is one of the cysteine protease inhibitors, had no such effect. Based on the evidence obtained, it appears that proteasome inhibition results in a reduction in the expression of AhR-regulated proteins.

  9. Regional brain activation as a biological marker of affective responsivity to acute exercise: influence of fitness.

    PubMed

    Petruzzello, S J; Hall, E E; Ekkekakis, P

    2001-01-01

    Previous research has shown that regional brain activation, assessed via frontal electroencephalographic (EEG) asymmetry, predicts affective responsivity to aerobic exercise. To replicate and extend this work, in the present study we examined whether resting brain activation was associated with affective responses to an acute bout of aerobic exercise and the extent to which aerobic fitness mediated this relationship. Participants (high-fit, n = 22; low/moderate-fit, n = 45) ran on a treadmill for 30 min at 75% VO2max. EEG and affect were assessed pre- and 0-, 10-, 20-, and 30-min postexercise. Resting EEG asymmetry predicted positive affect (as measured by the energetic arousal subscale of the Activation Deactivation Adjective Check List) postexercise. Furthermore, resting frontal EEG asymmetry predicted affect only in the high-fit group, suggesting the effect might be mediated by some factor related to fitness. It was also shown that subjects with relatively greater left frontal activation had significantly more energy (i.e., activated pleasant affect) following exercise than subjects with relatively greater right frontal activation. In conclusion, aerobic fitness influenced the relationship between resting frontal asymmetry and exercise-related affective responsivity.

  10. Regional brain activity and strenuous exercise: predicting affective responses using EEG asymmetry.

    PubMed

    Hall, Eric E; Ekkekakis, Panteleimon; Petruzzello, Steven J

    2007-05-01

    Previous research using the model proposed by Davidson has shown that resting frontal electroencephalographic (EEG) asymmetry can predict affective responses to aerobic exercise at moderate intensities. Specifically, greater relative left frontal activity has been shown to predict positive affect (i.e., energy) following exercise. The purpose of this study was to determine if resting frontal EEG asymmetry would predict affective responses following strenuous exercise. Thirty participants (13 women, 17 men) completed a maximal graded exercise test on a treadmill. EEG was recorded prior to exercise. Affect was measured by the Activation Deactivation Adjective Check List prior to the graded exercise test, immediately following, 10 and 20-min following exercise. Greater relative left frontal activity predicted tiredness and calmness during recovery from exercise, but not tension or energy. Tiredness and calmness following exercise covaried, suggesting that tiredness following exercise might not have been linked with displeasure. These findings offer further support for the link between EEG asymmetry and affective responses to exercise.

  11. Ozone-derived Oxysterols Affect Liver X Receptor (LXR) Signaling: A POTENTIAL ROLE FOR LIPID-PROTEIN ADDUCTS.

    PubMed

    Speen, Adam M; Kim, Hye-Young H; Bauer, Rebecca N; Meyer, Megan; Gowdy, Kymberly M; Fessler, Michael B; Duncan, Kelly E; Liu, Wei; Porter, Ned A; Jaspers, Ilona

    2016-11-25

    When inhaled, ozone (O3) interacts with cholesterols of airway epithelial cell membranes or the lung-lining fluid, generating chemically reactive oxysterols. The mechanism by which O3-derived oxysterols affect molecular function is unknown. Our data show that in vitro exposure of human bronchial epithelial cells to O3 results in the formation of oxysterols, epoxycholesterol-α and -β and secosterol A and B (Seco A and Seco B), in cell lysates and apical washes. Similarly, bronchoalveolar lavage fluid obtained from human volunteers exposed to O3 contained elevated levels of these oxysterol species. As expected, O3-derived oxysterols have a pro-inflammatory effect and increase NF-κB activity. Interestingly, expression of the cholesterol efflux pump ATP-binding cassette transporter 1 (ABCA1), which is regulated by activation of the liver X receptor (LXR), was suppressed in epithelial cells exposed to O3 Additionally, exposure of LXR knock-out mice to O3 enhanced pro-inflammatory cytokine production in the lung, suggesting LXR inhibits O3-induced inflammation. Using alkynyl surrogates of O3-derived oxysterols, our data demonstrate adduction of LXR with Seco A. Similarly, supplementation of epithelial cells with alkynyl-tagged cholesterol followed by O3 exposure causes observable lipid-LXR adduct formation. Experiments using Seco A and the LXR agonist T0901317 (T09) showed reduced expression of ABCA1 as compared with stimulation with T0901317 alone, indicating that Seco A-LXR protein adduct formation inhibits LXR activation by traditional agonists. Overall, these data demonstrate that O3-derived oxysterols have pro-inflammatory functions and form lipid-protein adducts with LXR, thus leading to suppressed cholesterol regulatory gene expression and providing a biochemical mechanism mediating O3-derived formation of oxidized lipids in the airways and subsequent adverse health effects.

  12. Metaproteomics: Evaluation of protein extraction from activated sludge.

    PubMed

    Hansen, Susan Hove; Stensballe, Allan; Nielsen, Per Halkjaer; Herbst, Florian-Alexander

    2014-11-01

    Metaproteomic studies of full-scale activated sludge systems require reproducible protein extraction methods. A systematic evaluation of three different extractions protocols, each in combination with three different methods of cell lysis, and a commercial kit were evaluated. Criteria used for comparison of each method included the extracted protein concentration and the number of identified proteins and peptides as well as their phylogenetic, cell localization and functional distribution and quantitative reproducibility. Furthermore, the advantage of using specific metagenomes and a 2-step database approach was illustrated. The results recommend a protocol for protein extraction from activated sludge based on the protein extraction reagent B-Per and bead beating. The data have been deposited to the ProteomeXchange with identifier PXD000862 (http://proteomecentral.proteomexchange.org/dataset/PXD000862).

  13. 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

  14. Stabilizing effects of G protein on the active conformation of adenosine A1 receptor differ depending on G protein type.

    PubMed

    Tateyama, Michihiro; Kubo, Yoshihiro

    2016-10-05

    G protein coupled receptors (GPCRs) trigger various cellular and physiological responses upon the ligand binding. The ligand binding induces conformational change in GPCRs which allows G protein to interact with the receptor. The interaction of G protein also affects the active conformation of GPCRs. In this study, we have investigated the effects of Gαi1, Gαo and chimeric Gαqi5 on the active conformation of the adenosine A1 receptor, as each Gα showed difference in the interaction with adenosine A1 receptor. The conformational changes in the adenosine A1 receptor were detected as the agonist-induced decreases in efficiency of Förster resonance energy transfer (FRET) between fluorescent proteins (FPs) fused at the two intracellular domains of the adenosine A1 receptor. Amplitudes of the agonist-induced FRET decreases were subtle when the FP-tagged adenosine A1 receptor was expressed alone, whereas they were significantly enhanced when co-expressed with Gαi1Gβ1Gγ22 (Gi1) or Gαqi5Gβ1Gγ22 (Gqi5) but not with GαοGβ1Gγ22 (Go). The enhancement of the agonist-induced FRET decrease in the presence of Gqi5 was significantly larger than that of Gi1. Furthermore, the FRET recovery upon the agonist removal in the presence of Gqi5 was significantly slower than that of Gi1. From these results it was revealed that the agonist-bound active conformation of adenosine A1 receptor is unstable without the binding of G protein and that the stabilizing effects of G protein differ depending on the types of G protein.

  15. Depletion of WRN protein causes RACK1 to activate several protein kinase C isoforms.

    PubMed

    Massip, L; Garand, C; Labbé, A; Perreault, E; Turaga, R V N; Bohr, V A; Lebel, M

    2010-03-11

    Werner's syndrome (WS) is a rare autosomal disease characterized by the premature onset of several age-associated pathologies. The protein defective in patients with WS (WRN) is a helicase/exonuclease involved in DNA repair, replication, transcription and telomere maintenance. In this study, we show that a knock down of the WRN protein in normal human fibroblasts induces phosphorylation and activation of several protein kinase C (PKC) enzymes. Using a tandem affinity purification strategy, we found that WRN physically and functionally interacts with receptor for activated C-kinase 1 (RACK1), a highly conserved anchoring protein involved in various biological processes, such as cell growth and proliferation. RACK1 binds strongly to the RQC domain of WRN and weakly to its acidic repeat region. Purified RACK1 has no impact on the helicase activity of WRN, but selectively inhibits WRN exonuclease activity in vitro. Interestingly, knocking down RACK1 increased the cellular frequency of DNA breaks. Depletion of the WRN protein in return caused a fraction of nuclear RACK1 to translocate out of the nucleus to bind and activate PKCdelta and PKCbetaII in the membrane fraction of cells. In contrast, different DNA-damaging treatments known to activate PKCs did not induce RACK1/PKCs association in cells. Overall, our results indicate that a depletion of the WRN protein in normal fibroblasts causes the activation of several PKCs through translocation and association of RACK1 with such kinases.

  16. Depletion of WRN protein causes RACK1 to activate several protein kinase C isoforms

    PubMed Central

    Massip, L; Garand, C; Labbé, A; Perreault, È; Turaga, RVN; Bohr, VA; Lebel, M

    2015-01-01

    Werner’s syndrome (WS) is a rare autosomal disease characterized by the premature onset of several age-associated pathologies. The protein defective in patients with WS (WRN) is a helicase/exonuclease involved in DNA repair, replication, transcription and telomere maintenance. In this study, we show that a knock down of the WRN protein in normal human fibroblasts induces phosphorylation and activation of several protein kinase C (PKC) enzymes. Using a tandem affinity purification strategy, we found that WRN physically and functionally interacts with receptor for activated C-kinase 1 (RACK1), a highly conserved anchoring protein involved in various biological processes, such as cell growth and proliferation. RACK1 binds strongly to the RQC domain of WRN and weakly to its acidic repeat region. Purified RACK1 has no impact on the helicase activity of WRN, but selectively inhibits WRN exonuclease activity in vitro. Interestingly, knocking down RACK1 increased the cellular frequency of DNA breaks. Depletion of the WRN protein in return caused a fraction of nuclear RACK1 to translocate out of the nucleus to bind and activate PKCδ and PKCβII in the membrane fraction of cells. In contrast, different DNA-damaging treatments known to activate PKCs did not induce RACK1/PKCs association in cells. Overall, our results indicate that a depletion of the WRN protein in normal fibroblasts causes the activation of several PKCs through translocation and association of RACK1 with such kinases. PMID:19966859

  17. SARS-CoV nucleocapsid protein interacts with cellular pyruvate kinase protein and inhibits its activity.

    PubMed

    Wei, Wei-Yen; Li, Hui-Chun; Chen, Chiung-Yao; Yang, Chee-Hing; Lee, Shen-Kao; Wang, Chia-Wen; Ma, Hsin-Chieh; Juang, Yue-Li; Lo, Shih-Yen

    2012-04-01

    The pathogenesis of SARS-CoV remains largely unknown. To study the function of the SARS-CoV nucleocapsid protein, we have conducted a yeast two-hybrid screening experiment to identify cellular proteins that may interact with the SARS-CoV nucleocapsid protein. Pyruvate kinase (liver) was found to interact with SARS-CoV nucleocapsid protein in this experiment. The binding domains of these two proteins were also determined using the yeast two-hybrid system. The physical interaction between the SARS-CoV nucleocapsid and cellular pyruvate kinase (liver) proteins was further confirmed by GST pull-down assay, co-immunoprecipitation assay and confocal microscopy. Cellular pyruvate kinase activity in hepatoma cells was repressed by SARS-CoV nucleocapsid protein in either transiently transfected or stably transfected cells. PK deficiency in red blood cells is known to result in human hereditary non-spherocytic hemolytic anemia. It is reasonable to assume that an inhibition of PKL activity due to interaction with SARS-CoV N protein is likely to cause the death of the hepatocytes, which results in the elevation of serum alanine aminotransferase and liver dysfunction noted in most SARS patients. Thus, our results suggest that SARS-CoV could reduce pyruvate kinase activity via its nucleocapsid protein, and this may in turn cause disease.

  18. Retention of OsNMD3 in the cytoplasm disturbs protein synthesis efficiency and affects plant development in rice.

    PubMed

    Shi, Yanyun; Liu, Xiangling; Li, Rui; Gao, Yaping; Xu, Zuopeng; Zhang, Baocai; Zhou, Yihua

    2014-07-01

    The ribosome is the basic machinery for translation, and biogenesis of ribosomes involves many coordinated events. However, knowledge about ribosomal dynamics in higher plants is very limited. This study chose a highly conserved trans-factor, the 60S ribosomal subunit nuclear export adaptor NMD3, to characterize the mechanism of ribosome biogenesis in the monocot plant Oryza sativa (rice). O. sativa NMD3 (OsNMD3) shares all the common motifs and shuttles between the nucleus and cytoplasm via CRM1/XPO1. A dominant negative form of OsNMD3 with a truncated nuclear localization sequence (OsNMD3(ΔNLS)) was retained in the cytoplasm, consequently interfering with the release of OsNMD3 from pre-60S particles and disturbing the assembly of ribosome subunits. Analyses of the transactivation activity and cellulose biosynthesis level revealed low protein synthesis efficiency in the transgenic plants compared with the wild-type plants. Pharmaceutical treatments demonstrated structural alterations in ribosomes in the transgenic plants. Moreover, global expression profiles of the wild-type and transgenic plants were investigated using the Illumina RNA sequencing approach. These expression profiles suggested that overexpression of OsNMD3(ΔNLS) affected ribosome biogenesis and certain basic pathways, leading to pleiotropic abnormalities in plant growth. Taken together, these results strongly suggest that OsNMD3 is important for ribosome assembly and the maintenance of normal protein synthesis efficiency.

  19. Design and Development of an Affective Interface for Supporting Energy-saving Activities and its Evaluation

    NASA Astrophysics Data System (ADS)

    Ito, Kyoko; Tomita, Daisuke; Imaki, Tomotaka; Hongo, Taishiro; Yoshikawa, Hidekazu

    Toward a sustainable society, energy and environmental issues are very important and controversial problems, and it is expected to support various human activities for the measures by using Information Technology. The purpose of this study is to develop an affective interface for supporting people's energy-saving activities. First, a model for supporting people's energy-saving activities involving affective elements has been constructed for supporting people's energy-saving activities, based on social psychological approaches. Based on the proposed model, the requirements on an affective interface for people's energy-saving activities have been considered. In this study, the affective interface presents suitable energy-saving activities and current electric energy consumption by a character agent with a graphical shape and synthesized voice. The character agent recommends people's energy-saving activities, tells the method of energy-saving activities and the effectiveness, and so on. The affective interface for supporting energy-saving activities has been designed in detail and developed. Then, the evaluation experiment of the developed interface has been conducted, and the results of the experiments were analyzed.

  20. Amino acid changes within the E protein hinge region that affect dengue virus type 2 infectivity and fusion

    SciTech Connect

    Butrapet, Siritorn; Childers, Thomas; Moss, Kelley J.; Erb, Steven M.; Luy, Betty E.; Calvert, Amanda E.; Blair, Carol D.; Roehrig, John T.; Huang, Claire Y.-H.

    2011-04-25

    Fifteen mutant dengue viruses were engineered and used to identify AAs in the molecular hinge of the envelope protein that are critical to viral infection. Substitutions at Q52, A54, or E133 reduced infectivity in mammalian cells and altered the pH threshold of fusion. Mutations at F193, G266, I270, or G281 affected viral replication in mammalian and mosquito cells, but only I270W had reduced fusion activity. T280Y affected the pH threshold for fusion and reduced replication in C6/36 cells. Three different mutations at L135 were lethal in mammalian cells. Among them, L135G abrogated fusion and reduced replication in C6/36 cells, but only slightly reduced the mosquito infection rate. Conversely, L135W replicated well in C6/36 cells, but had the lowest mosquito infection rate. Possible interactions between hinge residues 52 and 277, or among 53, 135, 170, 186, 265, and 276 required for hinge function were discovered by sequence analysis to identify compensatory mutations.

  1. Specific modulation of protein activity by using a bioorthogonal reaction.

    PubMed

    Warner, John B; Muthusamy, Anand K; Petersson, E James

    2014-11-24

    Unnatural amino acids with bioorthogonal reactive groups have the potential to provide a rapid and specific mechanism for covalently inhibiting a protein of interest. Here, we use mutagenesis to insert an unnatural amino acid containing an azide group (Z) into the target protein at positions such that a "click" reaction with an alkyne modulator (X) will alter the function of the protein. This bioorthogonally reactive pair can engender specificity of X for the Z-containing protein, even if the target is otherwise identical to another protein, allowing for rapid target validation in living cells. We demonstrate our method using inhibition of the Escherichia coli enzyme aminoacyl transferase by both active-site occlusion and allosteric mechanisms. We have termed this a "clickable magic bullet" strategy, and it should be generally applicable to studying the effects of protein inhibition, within the limits of unnatural amino acid mutagenesis.

  2. Protein-Binding RNA Aptamers Affect Molecular Interactions Distantly from Their Binding Sites

    PubMed Central

    Dupont, Daniel M.; Thuesen, Cathrine K.; Bøtkjær, Kenneth A.; Behrens, Manja A.; Dam, Karen; Sørensen, Hans P.; Pedersen, Jan S.; Ploug, Michael; Jensen, Jan K.; Andreasen, Peter A.

    2015-01-01

    Nucleic acid aptamer selection is a powerful strategy for the development of regulatory agents for molecular intervention. Accordingly, aptamers have proven their diligence in the intervention with serine protease activities, which play important roles in physiology and pathophysiology. Nonetheless, there are only a few studies on the molecular basis underlying aptamer-protease interactions and the associated mechanisms of inhibition. In the present study, we use site-directed mutagenesis to delineate the binding sites of two 2´-fluoropyrimidine RNA aptamers (upanap-12 and upanap-126) with therapeutic potential, both binding to the serine protease urokinase-type plasminogen activator (uPA). We determine the subsequent impact of aptamer binding on the well-established molecular interactions (plasmin, PAI-1, uPAR, and LRP-1A) controlling uPA activities. One of the aptamers (upanap-126) binds to the area around the C-terminal α-helix in pro-uPA, while the other aptamer (upanap-12) binds to both the β-hairpin of the growth factor domain and the kringle domain of uPA. Based on the mapping studies, combined with data from small-angle X-ray scattering analysis, we construct a model for the upanap-12:pro-uPA complex. The results suggest and highlight that the size and shape of an aptamer as well as the domain organization of a multi-domain protein such as uPA, may provide the basis for extensive sterical interference with protein ligand interactions considered distant from the aptamer binding site. PMID:25793507

  3. 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

  4. 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.

  5. Arabidopsis acyl-CoA-binding protein ACBP6 localizes in the phloem and affects jasmonate composition.

    PubMed

    Ye, Zi-Wei; Lung, Shiu-Cheung; Hu, Tai-Hua; Chen, Qin-Fang; Suen, Yung-Lee; Wang, Mingfu; Hoffmann-Benning, Susanne; Yeung, Edward; Chye, Mee-Len

    2016-12-01

    Arabidopsis thaliana ACYL-COA-BINDING PROTEIN6 (AtACBP6) encodes a cytosolic 10-kDa AtACBP. It confers freezing tolerance in transgenic Arabidopsis, possibly by its interaction with lipids as indicated by the binding of acyl-CoA esters and phosphatidylcholine to recombinant AtACBP6. Herein, transgenic Arabidopsis transformed with an AtACBP6 promoter-driven β-glucuronidase (GUS) construct exhibited strong GUS activity in the vascular tissues. Immunoelectron microscopy using anti-AtACBP6 antibodies showed AtACBP6 localization in the phloem especially in the companion cells and sieve elements. Also, the presence of gold grains in the plasmodesmata indicated its potential role in systemic trafficking. The AtACBP6 protein, but not its mRNA, was found in phloem exudate of wild-type Arabidopsis. Fatty acid profiling using gas chromatography-mass spectrometry revealed an increase in the jasmonic acid (JA) precursor, 12-oxo-cis,cis-10,15-phytodienoic acid (cis-OPDA), and a reduction in JA and/or its derivatives in acbp6 phloem exudates in comparison to the wild type. Quantitative real-time PCR showed down-regulation of COMATOSE (CTS) in acbp6 rosettes suggesting that AtACBP6 affects CTS function. AtACBP6 appeared to affect the content of JA and/or its derivatives in the sieve tubes, which is consistent with its role in pathogen-defense and in its wound-inducibility of AtACBP6pro::GUS. Taken together, our results suggest the involvement of AtACBP6 in JA-biosynthesis in Arabidopsis phloem tissues.

  6. Active and Passive Commuting to School: Influences on Affect in Primary School Children

    ERIC Educational Resources Information Center

    Hulley, Angela; Bentley, Nick; Clough, Catherine; Fishlock, Adelle; Morrell, Frances; O'Brien, James; Radmore, Joseph

    2008-01-01

    Active commuting among school children is being encouraged for physical and environmental reasons, but little is known about its influence on affect. The aim of this study was to test the hypothesis that children who walk further to school experience increased arousal and affective valence compared with children who walk a short distance. This was…

  7. 75 FR 80114 - Agency Information Collection (Obligation To Report Factors Affecting Entitlement) Activity Under...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-12-21

    ... AFFAIRS Agency Information Collection (Obligation To Report Factors Affecting Entitlement) Activity Under... INFORMATION: Title: Obligation to Report Factors Affecting Entitlement (38 CFR 3.204(a)(1), 38 CFR 3.256(a... compensation benefits must report changes in their entitlement factors. Individual factors such as...

  8. Familial Vulnerability to ADHD Affects Activity in the Cerebellum in Addition to the Prefrontal Systems

    ERIC Educational Resources Information Center

    Mulder, Martijn J.; Baeyens, Dieter; Davidson, Matthew C.; Casey, B. J.; Van Den Ban, Els; Van Engeland, Herman; Durston, Sarah

    2008-01-01

    The study examines whether cerebellar systems are sensitive to familial risk for ADHD in addition to frontostriatal circuitry. The results conclude that familial vulnerability to ADHD affects activity in both the prefrontal cortex and cerebellum.

  9. Relaxin Affects Smooth Muscle Biophysical Properties and Mechanical Activity of the Female Mouse Colon.

    PubMed

    Squecco, Roberta; Garella, Rachele; Idrizaj, Eglantina; Nistri, Silvia; Francini, Fabio; Baccari, Maria Caterina

    2015-12-01

    The hormone relaxin (RLX) has been reported to influence gastrointestinal motility in mice. However, at present, nothing is known about the effects of RLX on the biophysical properties of the gastrointestinal smooth muscle cells (SMCs). Other than extending previous knowledge of RLX on colonic motility, the purpose of this study was to investigate the ability of the hormone to induce changes in resting membrane potential (RMP) and on sarcolemmal ion channels of colonic SMCs of mice that are related to its mechanical activity. To this aim, we used a combined mechanical and electrophysiological approach. In the mechanical experiments, we observed that RLX caused a decay of the basal tone coupled to an increase of the spontaneous contractions, completely abolished by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]-quinoxalin-1-one (ODQ). The electrophysiological results indicate for the first time that RLX directly affects the SMC biophysical properties inducing hyperpolarization of RMP and cycles of slow hyperpolarization/depolarization oscillations. The effects of RLX on RMP were abolished by ODQ as well as by a specific inhibitor of the cGMP-dependent protein kinase, KT5823. RLX reduced Ca(2+) entry through the voltage-dependent L-type channels and modulated either voltage- or ATP-dependent K(+) channels. These effects were abolished by ODQ, suggesting the involvement of the nitric oxide/guanylate cyclase pathway in the effects of RLX on RMP and ion channel modulation. These actions of RLX on membrane properties may contribute to the regulation of the proximal colon motility by the nitric oxide/cGMP/cGMP-dependent protein kinase pathway.

  10. 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

  11. Situational Motivation and Perceived Intensity: Their Interaction in Predicting Changes in Positive Affect from Physical Activity

    PubMed Central

    Guérin, Eva; Fortier, Michelle S.

    2012-01-01

    There is evidence that affective experiences surrounding physical activity can contribute to the proper self-regulation of an active lifestyle. Motivation toward physical activity, as portrayed by self-determination theory, has been linked to positive affect, as has the intensity of physical activity, especially of a preferred nature. The purpose of this experimental study was to examine the interaction between situational motivation and intensity [i.e., ratings of perceived exertion (RPE)] in predicting changes in positive affect following an acute bout of preferred physical activity, namely, running. Fourty-one female runners engaged in a 30-minute self-paced treadmill run in a laboratory context. Situational motivation for running, pre- and post-running positive affect, and RPE were assessed via validated self-report questionnaires. Hierarchical regression analyses revealed a significant interaction effect between RPE and introjection (P < .05) but not between RPE and identified regulation or intrinsic motivation. At low levels of introjection, the influence of RPE on the change in positive affect was considerable, with higher RPE ratings being associated with greater increases in positive affect. The implications of the findings in light of SDT principles as well as the potential contingencies between the regulations and RPE in predicting positive affect among women are discussed. PMID:22778914

  12. Influenza B virus non-structural protein 1 counteracts ISG15 antiviral activity by sequestering ISGylated viral proteins

    PubMed Central

    Zhao, Chen; Sridharan, Haripriya; Chen, Ran; Baker, Darren P.; Wang, Shanshan; Krug, Robert M.

    2016-01-01

    The ubiquitin-like protein ISG15 and its conjugation to proteins (ISGylation) are strongly induced by type I interferon. Influenza B virus encodes non-structural protein 1 (NS1B) that binds human ISG15 and provides an appropriate model for determining how ISGylation affects virus replication in human cells. Here using a recombinant virus encoding a NS1B protein defective in ISG15 binding, we show that NS1B counteracts ISGylation-mediated antiviral activity by binding and sequestering ISGylated viral proteins, primarily ISGylated viral nucleoprotein (NP), in infected cells. ISGylated NP that is not sequestered by mutant NS1B acts as a dominant-negative inhibitor of oligomerization of the more abundant unconjugated NP. Consequently formation of viral ribonucleoproteins that catalyse viral RNA synthesis is inhibited, causing decreased viral protein synthesis and virus replication. We verify that ISGylated NP is largely responsible for inhibition of viral RNA synthesis by generating recombinant viruses that lack known ISGylation sites in NP. PMID:27587337

  13. Genetic Variants in the STMN1 Transcriptional Regulatory Region Affect Promoter Activity and Fear Behavior in English Springer Spaniels

    PubMed Central

    Zhang, Hanying; Xu, Yinxue

    2016-01-01

    Stathmin 1 (STMN1) is a neuronal growth-associated protein that is involved in microtubule dynamics and plays an important role in synaptic outgrowth and plasticity. Given that STMN1 affects fear behavior, we hypothesized that genetic variations in the STMN1 transcriptional regulatory region affect gene transcription activity and control fear behavior. In this study, two single nucleotide polymorphisms (SNPs), g. -327 A>G and g. -125 C>T, were identified in 317 English Springer Spaniels. A bioinformatics analysis revealed that both were loci located in the canine STMN1 putative promoter region and affected transcription factor binding. A statistical analysis revealed that the TT genotype at g.-125 C>T produced a significantly greater fear level than that of the CC genotype (P < 0.05). Furthermore, the H4H4 (GTGT) haplotype combination was significantly associated with canine fear behavior (P < 0.01). Using serially truncated constructs of the STMN1 promoters and the luciferase reporter, we found that a 395 bp (−312 nt to +83 nt) fragment constituted the core promoter region. The luciferase assay also revealed that the H4 (GT) haplotype promoter had higher activity than that of other haplotypes. Overall, our results suggest that the two SNPs in the canine STMN1 promoter region could affect canine fear behavior by altering STMN1 transcriptional activity. PMID:27390866

  14. PII Overexpression in Lotus japonicus Affects Nodule Activity in Permissive Low-Nitrogen Conditions and Increases Nodule Numbers in High Nitrogen Treated Plants.

    PubMed

    D'Apuzzo, Enrica; Valkov, Vladimir Totev; Parlati, Aurora; Omrane, Selim; Barbulova, Ani; Sainz, Maria Martha; Lentini, Marco; Esposito, Sergio; Rogato, Alessandra; Chiurazzi, Maurizio

    2015-04-01

    We report here the first characterization of a GLNB1 gene coding for the PII protein in leguminous plants. The main purpose of this work was the investigation of the possible roles played by this multifunctional protein in nodulation pathways. The Lotus japonicus LjGLB1 gene shows a significant transcriptional regulation during the light-dark cycle and different nitrogen availability, conditions that strongly affect nodule formation, development, and functioning. We also report analysis of the spatial profile of expression of LjGLB1 in root and nodule tissues and of the protein's subcellular localization. Transgenic L. japonicus lines overexpressing the PII protein were obtained and tested for the analysis of the symbiotic responses in different conditions. The uncoupling of PII from its native regulation affects nitrogenase activity and nodule polyamine content. Furthermore, our results suggest the involvement of PII in the signaling of the nitrogen nutritional status affecting the legumes' predisposition for nodule formation.

  15. Glycosaminoglycan Sulphation Affects the Seeded Misfolding of a Mutant Prion Protein

    PubMed Central

    Lawson, Victoria A.; Lumicisi, Brooke; Welton, Jeremy; Machalek, Dorothy; Gouramanis, Katrina; Klemm, Helen M.; Stewart, James D.; Masters, Colin L.; Hoke, David E.; Collins, Steven J.; Hill, Andrew F.

    2010-01-01

    Background The accumulation of protease resistant conformers of the prion protein (PrPres) is a key pathological feature of prion diseases. Polyanions, including RNA and glycosaminoglycans have been identified as factors that contribute to the propagation, transmission and pathogenesis of prion disease. Recent studies have suggested that the contribution of these cofactors to prion propagation may be species specific. Methodology/Principal Finding In this study a cell-free assay was used to investigate the molecular basis of polyanion stimulated PrPres formation using brain tissue or cell line derived murine PrP. Enzymatic depletion of endogenous nucleic acids or heparan sulphate (HS) from the PrPC substrate was found to specifically prevent PrPres formation seeded by mouse derived PrPSc. Modification of the negative charge afforded by the sulphation of glycosaminoglycans increased the ability of a familial PrP mutant to act as a substrate for PrPres formation, while having no effect on PrPres formed by wildtype PrP. This difference may be due to the observed differences in the binding of wild type and mutant PrP for glycosaminoglycans. Conclusions/Significance Cofactor requirements for PrPres formation are host species and prion strain specific and affected by disease associated mutations of the prion protein. This may explain both species and strain dependent propagation characteristics and provide insights into the underlying mechanisms of familial prion disease. It further highlights the challenge of designing effective therapeutics against a disease which effects a range of mammalian species, caused by range of aetiologies and prion strains. PMID:20808809

  16. Phenosafranin inhibits nuclear localization of transglutaminase 2 without affecting its transamidase activity.

    PubMed

    Furutani, Yutaka; Toguchi, Mariko; Shrestha, Rajan; Kojima, Soichi

    2017-03-01

    Transglutaminase 2 (TG2) localizes to the nucleus and induces apoptosis through a crosslinking inactivation of Sp1 in JHH-7 cells treated with acyclic retinoid. We screened an inhibitor suppressing transamidase activity in the nucleus without affecting transamidase activity itself. Phenosafranin was found to inhibit nuclear localization of EGFP-tagged TG2 and dose-dependently reduce nuclear transamidase activity without affecting the activity in a tube. We concluded that phenosafranin was a novel TG2 inhibitor capable of suppressing its nuclear localization.

  17. Auto-phosphorylation Represses Protein Kinase R Activity

    PubMed Central

    Wang, Die; de Weerd, Nicole A.; Willard, Belinda; Polekhina, Galina; Williams, Bryan R. G.; Sadler, Anthony J.

    2017-01-01

    The central role of protein kinases in controlling disease processes has spurred efforts to develop pharmaceutical regulators of their activity. A rational strategy to achieve this end is to determine intrinsic auto-regulatory processes, then selectively target these different states of kinases to repress their activation. Here we investigate auto-regulation of the innate immune effector protein kinase R, which phosphorylates the eukaryotic initiation factor 2α to inhibit global protein translation. We demonstrate that protein kinase R activity is controlled by auto-inhibition via an intra-molecular interaction. Part of this mechanism of control had previously been reported, but was then controverted. We account for the discrepancy and extend our understanding of the auto-inhibitory mechanism by identifying that auto-inhibition is paradoxically instigated by incipient auto-phosphorylation. Phosphor-residues at the amino-terminus instigate an intra-molecular interaction that enlists both of the N-terminal RNA-binding motifs of the protein with separate surfaces of the C-terminal kinase domain, to co-operatively inhibit kinase activation. These findings identify an innovative mechanism to control kinase activity, providing insight for strategies to better regulate kinase activity. PMID:28281686

  18. HMG Proteins and DNA Flexibility in Transcription Activation

    PubMed Central

    Ross, Eric D.; Hardwidge, Philip R.; Maher, L. James

    2001-01-01

    The relative stiffness of naked DNA is evident from measured values of longitudinal persistence length (∼150 bp) and torsional persistence length (∼180 bp). These parameters predict that certain arrangements of eukaryotic transcription activator proteins in gene promoters should be much more effective than others in fostering protein-protein interactions with the basal RNA polymerase II transcription apparatus. Thus, if such interactions require some kind of DNA looping, DNA loop energies should depend sensitively on helical phasing of protein binding sites, loop size, and intrinsic DNA curvature within the loop. Using families of artificial transcription templates where these parameters were varied, we were surprised to find that the degree of transcription activation by arrays of Gal4-VP1 transcription activators in HeLa cell nuclear extract was sensitive only to the linear distance separating a basal promoter from an array of bound activators on DNA templates. We now examine the hypothesis that this unexpected result is due to factors in the extract that act to enhance apparent DNA flexibility. We demonstrate that HeLa cell nuclear extract is rich in a heat-resistant activity that dramatically enhances apparent DNA longitudinal and torsional flexibility. Recombinant mammalian high-mobility group 2 (HMG-2) protein can substitute for this activity. We propose that the abundance of HMG proteins in eukaryotic nuclei provides an environment in which DNA is made sufficiently flexible to remove many constraints on protein binding site arrangements that would otherwise limit efficient transcription activation to certain promoter geometries. PMID:11533247

  19. 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.

  20. Steady-state compartmentalization of lipid membranes by active proteins.

    PubMed Central

    Sabra, M C; Mouritsen, O G

    1998-01-01

    Using a simple microscopic model of lipid-protein interactions, based on the hydrophobic matching principle, we study some generic aspects of lipid-membrane compartmentalization controlled by a dispersion of active integral membrane proteins. The activity of the proteins is simulated by conformational excitations governed by an external drive, and the deexcitation is controlled by interaction of the protein with its lipid surroundings. In response to the flux of energy into the proteins from the environment and the subsequent dissipation of energy into the lipid bilayer, the lipid-protein assembly reorganizes into a steady-state structure with a typical length scale determined by the strength of the external drive. In the specific case of a mixed dimyristoylphosphatidylcholine-distearoylphosphatidylcholine bilayer in the gel-fluid coexistence region, it is shown explicitly by computer simulation that the activity of an integral membrane protein can lead to a compartmentalization of the lipid-bilayer membrane. The compartmentalization is related to the dynamical process of phase separation and lipid domain formation. PMID:9533687

  1. Walk on the bright side: physical activity and affect in major depressive disorder.

    PubMed

    Mata, Jutta; Thompson, Renee J; Jaeggi, Susanne M; Buschkuehl, Martin; Jonides, John; Gotlib, Ian H

    2012-05-01

    Although prescribed exercise has been found to improve affect and reduce levels of depression, we do not know how self-initiated everyday physical activity influences levels of positive affect (PA) and negative affect (NA) in depressed persons. Fifty-three individuals diagnosed with Major Depressive Disorder (MDD) and 53 never-depressed controls participated in a seven-day experience sampling study. Participants were prompted randomly eight times per day and answered questions about their physical activity and affective state. Over the week, the two groups of participants did not differ in average level of physical activity. As expected, participants with MDD reported lower average PA and higher average NA than did never-depressed controls. Both participants with MDD and controls reported higher levels of PA at prompts after physical activity than at prompts after inactive periods; moreover, for both groups of participants, PA increased from a prompt after an inactive period to a subsequent prompt at which activity was reported. Depressed participants in particular showed a dose-response effect of physical activity on affect: longer duration and/or higher intensity of physical activity increased their PA significantly more than did short duration and/or lower intensity physical activity. Physical activity did not influence NA in either group. In contrast to previous treatment studies that examined the effects of prescribed structured exercise, this investigation showed that self-initiated physical activity influences PA. These findings also underscore the importance of distinguishing between PA and NA to gain a more comprehensive understanding of the effects of physical activity on affect in MDD.

  2. Increased activity of rat liver nucleolar protein kinase following triiodothyronine administration.

    PubMed

    Fugassa, E; Gallo, G; Pertica, M; Voci, A; Orunesu, M

    1977-12-08

    Triiodothyronine (T3) administration to thyroidectomized rats induces a significant increase in the nucleolus-associated protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) activity. The general properties of the protein kinase solubilized from liver nucleoli have been investigated. Mg2+ (20 mM) is essential for the reaction and an appropriate concentration of NaCl (100 mM) is required to achieve maximal phosphorylation rates. The optimal pH for casein phosphorylation is 7.6. The kinase phosphorylates casein more efficiently than phosvitin and displays an almost undetectable activity towards histones and protamine. No significant stimulation of the kinase activity by cyclic AMP has been detected. The apparent Km values for casein and ATP are 1.5 mg/ml and 1.5-10(-5) M, respectively, and are not affected by the hormone administration.

  3. 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%.

  4. Modeling the SHG activities of diverse protein crystals

    PubMed Central

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

    2012-01-01

    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. PMID:23090400

  5. Gene expression in primary cultured astrocytes affected by aluminum: alteration of chaperons involved in protein folding

    PubMed Central

    Aremu, David A.; Ezomo, Ojeiru F.

    2010-01-01

    Objectives Aluminum is notorious as a neurotoxic metal. The aim of our study was to determine whether endoplasmic reticulum (ER) stress is involved in aluminum-induced apoptosis in astrocytes. Methods Mitochondrial RNA (mRNA) was analyzed by reverse transcription (RT)-PCR following pulse exposure of aluminum glycinate to primary cultured astrocytes. Tunicamycin was used as a positive control. Results Gene expression analysis revealed that Ire1β was up-regulated in astrocytes exposed to aluminum while Ire1α was up-regulated by tunicamycin. Exposure to aluminum glycinate, in contrast to tunicamycin, seemed to down-regulate mRNA expression of many genes, including the ER resident molecular chaperone BiP/Grp78 and Ca2+-binding chaperones (calnexin and calreticulin), as well as stanniocalcin 2 and OASIS. The down-regulation or non-activation of the molecular chaperons, whose expressions are known to be protective by increasing protein folding, may spell doom for the adaptive response. Exposure to aluminum did not have any significant effects on the expression of Bax and Bcl2 in astrocytes. Conclusions The results of this study demonstrate that aluminum may induce apoptosis in astrocytes via ER stress by impairing the protein-folding machinery. PMID:21432213

  6. Inhibition of protein kinase C affects on mode of synaptic vesicle exocytosis due to cholesterol depletion

    SciTech Connect

    Petrov, Alexey M. Zakyrjanova, Guzalija F. Yakovleva, Anastasia A. Zefirov, Andrei L.

    2015-01-02

    Highlights: • We examine the involvement of PKC in MCD induced synaptic vesicle exocytosis. • PKC inhibitor does not decrease the effect MCD on MEPP frequency. • PKC inhibitor prevents MCD induced FM1-43 unloading. • PKC activation may switch MCD induced exocytosis from kiss-and-run to a full mode. • Inhibition of phospholipase C does not lead to similar change in exocytosis. - Abstract: Previous studies demonstrated that depletion of membrane cholesterol by 10 mM methyl-beta-cyclodextrin (MCD) results in increased spontaneous exocytosis at both peripheral and central synapses. Here, we investigated the role of protein kinase C in the enhancement of spontaneous exocytosis at frog motor nerve terminals after cholesterol depletion using electrophysiological and optical methods. Inhibition of the protein kinase C by myristoylated peptide and chelerythrine chloride prevented MCD-induced increases in FM1-43 unloading, whereas the frequency of spontaneous postsynaptic events remained enhanced. The increase in FM1-43 unloading still could be observed if sulforhodamine 101 (the water soluble FM1-43 quencher that can pass through the fusion pore) was added to the extracellular solution. This suggests a possibility that exocytosis of synaptic vesicles under these conditions could occur through the kiss-and-run mechanism with the formation of a transient fusion pore. Inhibition of phospholipase C did not lead to similar change in MCD-induced exocytosis.

  7. Functional properties of whey proteins affected by heat treatment and hydrodynamic high-pressure shearing.

    PubMed

    Dissanayake, M; Vasiljevic, T

    2009-04-01

    Two batches of native whey proteins (WP) were subjected to microfluidization or heat denaturation accompanied by microfluidization, followed by spray drying. Powders were assessed for their solubility, heat stability, coagulation time, and emulsifying and foaming properties. Effects of denaturation and shearing were examined by particle size analysis, differential scanning calorimetry, reducing and nonreducing sodium dodecyl sulfate-PAGE, and size exclusion-HPLC. Heat treatment significantly decreased solubility, whereas the number of microfluidization passes markedly improved solubility. The combined effect of heat and pressure significantly increased heat coagulation time. Emulsifying activity index substantially increased upon heat denaturation and was further enhanced by microfluidization. Emulsion stability appeared unaffected by the combined treatment, but the concentration of adsorbed protein on fat droplets was significantly increased. Foaming properties were diminished by heating. Particle size distribution patterns, sodium dodecyl sulfate-PAGE, and size exclusion-HPLC revealed disappearance of major WP and creation of relatively higher, as well as smaller, molecular weight aggregates as a result of the 2 treatments. The use of heat and microfluidization in combination could be used to stabilize WP against heat by producing microparticulated species that have different surface and colloidal properties compared with native WP. These results have implications for the use of WP as an additive in heat-processed foods.

  8. Changes in gravity affect gene expression, protein modulation and metabolite pools of arabidopsis

    NASA Astrophysics Data System (ADS)

    Hampp, R.; Martzivanou, M.; Maier, R. M.; Magel, E.

    Callus cultures of Arabidopsis thaliana (cv. Columbia) in Petri dishes / suspension cultures were exposed to altered g-forces by centrifugation (1 to 10 g), klinorotation, and μ g (sounding rocket flights). Using semi-quantitative RT-PCR, transcripts of genes coding for metabolic key enzymes (ADP-glucose pyrophosphorylase, ADPG-PP; ß-amylase, fructose-1,6-bisphosphatase, FBPase; glyceraldehyde-P dehydrogenase, GAPDH; hydroxymethylglutaryl-CoA reductase, HMG; phenylalanine-ammonium-lyase, PAL; PEP carboxylase, PEPC) were used to monitor threshold conditions for g-number (all) and time of exposure (ß-amylase) which led to altered amounts of the gene product. Exposure to approx. 5 g and higher for 1h resulted in altered transcript levels: transcripts of ß-amylase, PAL, and PEPC were increased, those of ADPG-PP decreased, while those of FBPase, GAPDH, and HMG were not affected. This probably indicates a shift from starch synthesis to starch degradation and increased rates of anaplerosis (PEPC: supply of ketoacids for amino acid synthesis). In order to get more information about g-related effects on gene expression, we used a 1h-exposure to 7 g for a microarray analysis. Transcripts of more than 200 genes were significantly increased in amount (ratio 7g / 1g control; 21.6 and larger). They fall into several categories. Transcripts coding for enzymes of major pathways form the largest group (25%), followed by gene products involved in cellular organisation and cell wall formation / rearrangement (17%), signalling, phosphorylation/dephosphorylation (12%), proteolysis and transport (10% each), hormone synthesis plus related events (8%), defense (4%), stress-response (2%), and gravisensing (2%). Many of the alterations are part of a general stress response, but some changes related to the synthesis / rearrangement of cell wall components could be more hyper-g-specific. Using macroarrays with selected genes according to our hypergravity study (metabolism / signalling

  9. 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.

  10. Utilizing avidity to improve antifreeze protein activity: a type III antifreeze protein trimer exhibits increased thermal hysteresis activity.

    PubMed

    Can, Özge; Holland, Nolan B

    2013-12-03

    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.

  11. Ethanolic Extract of Melia Fructus Has Anti-influenza A Virus Activity by Affecting Viral Entry and Viral RNA Polymerase

    PubMed Central

    Jin, Young-Hee; Choi, Jang-Gi; Cho, Won-Kyung; Ma, Jin Yeul

    2017-01-01

    Meliae Fructus (MF) is the dried ripe fruit of Melia toosendan Siebold et Zuccarini, Meliaceae family. MF is widely used in traditional medicine to treat inflammation and helminthic infection and has anti-bacterial, anti-oxidant, anti-cancer, anti-inflammatory, and analgesic activities. However, potential anti-influenza properties of MF have yet to be investigated. We determined whether an ethanolic extract of MF (EMF) has anti-viral activity via an EMF pre-, co-, and post-treatment assay, using the Influenza A/PR/8/34 and H3N2 virus on Madin-Darby canine kidney (MDCK) cells. The EMF had anti-influenza virus activity in pre- and co-treated cells in a dose-dependent manner, but not in post-treated cell. EMF inhibited the activity of hemagglutinin (HA) and neuraminidase (NA) of influenza virus. EMF inhibited viral HA, nucleoprotein (NP), matrix protein 2 (M2), non-structural protein 1 (NS1), polymerase acidic protein (PA), polymerase basic protein 1 (PB1), and polymerase basic protein 2 (PB2) mRNA synthesis at 5 h post infection (hpi), however, the levels of PA, PB1, and PB2 mRNA were increased in pre- and co-EMF treated cells compared with control virus-infected and EMF post-treated cells at 18 hpi. The level of M2 protein expression was also decreased upon pre- and co-treatment with EMF. The PA protein was accumulated and localized in not only the nucleus but also the cytoplasm of virus-infected MDCK cells at 18 hpi. Pre-EMF treatment inhibited the expression of pAKT, which is induced by influenza virus infection, at the stage of virus entry. We also found that treatment of EMF up-regulated the antiviral protein Mx1, which may play a partial role in inhibiting influenza virus infection in pre- and co-EMF treated MDCK cells. In summary, these results strongly suggested that an ethanolic extract of Meliae Fructus inhibited influenza A virus infection by affecting viral entry, PA proteins of the RNA polymerase complex, and Mx1 induction and may be a potential and

  12. Bone morphogenetic protein Smads signaling in mesenchymal stem cells affected by osteoinductive calcium phosphate ceramics.

    PubMed

    Tang, Zhurong; Wang, Zhe; Qing, Fangzhu; Ni, Yilu; Fan, Yujiang; Tan, Yanfei; Zhang, Xingdong

    2015-03-01

    Porous calcium phosphate ceramics (CaP ceramics) could induce ectopic bone formation which was regulated by various signal molecules. In this work, bone marrow mesenchymal stem cells (MSCs) were cultured on the surface of osteoinductive hydroxyapatite (HA) and biphasic calcium phosphate (BCP) ceramics in comparison with control (culture plate) for up to 14 days to detect the signal molecules which might be affected by the CaP ceramics. Without adding osteogenic factors, MSCs cultured on HA and BCP both expressed higher Runx2, Osterix, collagen type I, osteopontin, bone sialoprotein, and osteocalcin at various stages compared with control, thus confirmed the osteoblastic differentiation of MSCs. Later study demonstrated the messenger RNA level of bone morphogenetic protein 2 (BMP2) and BMP4 were also significantly enhanced by HA and BCP. Furthermore, Smad1, 4, 5, and Dlx5, the main molecules in the BMP/Smads signaling pathway, were upregulated by HA and BCP. Moreover, the higher expression of Smads and BMP2, 4 in BCP over HA, corresponded to the better performance of BCP in stimulating in vitro osteoblastic differentiation of MSCs. This was in accordance with the better osteoinductivity of BCP over HA in vivo. Altogether, these results implied that the CaP ceramics may initiate the osteoblastic differentiation of MSCs by influencing the expression of molecules in BMP/Smads pathway.

  13. Distribution of abnormal prion protein in a sheep affected with L-type bovine spongiform encephalopathy.

    PubMed

    Matsuura, Y; Iwamaru, Y; Masujin, K; Imamura, M; Mohri, S; Yokoyama, T; Okada, H

    2013-07-01

    To investigate the topographical distribution and patterns of deposition of immunolabelled abnormal prion protein (PrP(Sc)), interspecies transmission of atypical L-type bovine spongiform encephalopathy (BSE) to Cheviot ewes (ARQ/ARQ genotype) was performed. L-type BSE was successfully transmitted via the intracerebral route to a ewe, with an incubation period of 1,562 days. Minimal vacuolar change was detected in the basal ganglia, thalamus and brainstem, and PrP(Sc) accumulated throughout the brain. The L-type BSE-affected sheep was characterized by conspicuous fine particulate deposits in the neuropil, particulate and/or granular intraneuronal and intraglial deposits, and the absence of PrP(Sc) plaques or stellate deposits. In addition, immunohistochemical and western blot analyses revealed that PrP(Sc) accumulation was present in peripheral nervous tissues (including the trigeminal ganglia and dorsal root ganglion) and adrenal glands, but was absent in lymphoid tissues. These results suggest that L-type BSE has distinct and distinguishable characteristics as well as PrP(Sc) tissue tropism in sheep.

  14. RNA editing differently affects protein-coding genes in D. melanogaster and H. sapiens.

    PubMed

    Grassi, Luigi; Leoni, Guido; Tramontano, Anna

    2015-07-14

    When an RNA editing event occurs within a coding sequence it can lead to a different encoded amino acid. The biological significance of these events remains an open question: they can modulate protein functionality, increase the complexity of transcriptomes or arise from a loose specificity of the involved enzymes. We analysed the editing events in coding regions that produce or not a change in the encoded amino acid (nonsynonymous and synonymous events, respectively) in D. melanogaster and in H. sapiens and compared them with the appropriate random models. Interestingly, our results show that the phenomenon has rather different characteristics in the two organisms. For example, we confirm the observation that editing events occur more frequently in non-coding than in coding regions, and report that this effect is much more evident in H. sapiens. Additionally, in this latter organism, editing events tend to affect less conserved residues. The less frequently occurring editing events in Drosophila tend to avoid drastic amino acid changes. Interestingly, we find that, in Drosophila, changes from less frequently used codons to more frequently used ones are favoured, while this is not the case in H. sapiens.

  15. RNA editing differently affects protein-coding genes in D. melanogaster and H. sapiens

    PubMed Central

    Grassi, Luigi; Leoni, Guido; Tramontano, Anna

    2015-01-01

    When an RNA editing event occurs within a coding sequence it can lead to a different encoded amino acid. The biological significance of these events remains an open question: they can modulate protein functionality, increase the complexity of transcriptomes or arise from a loose specificity of the involved enzymes. We analysed the editing events in coding regions that produce or not a change in the encoded amino acid (nonsynonymous and synonymous events, respectively) in D. melanogaster and in H. sapiens and compared them with the appropriate random models. Interestingly, our results show that the phenomenon has rather different characteristics in the two organisms. For example, we confirm the observation that editing events occur more frequently in non-coding than in coding regions, and report that this effect is much more evident in H. sapiens. Additionally, in this latter organism, editing events tend to affect less conserved residues. The less frequently occurring editing events in Drosophila tend to avoid drastic amino acid changes. Interestingly, we find that, in Drosophila, changes from less frequently used codons to more frequently used ones are favoured, while this is not the case in H. sapiens. PMID:26169954

  16. Contribution of HN protein length diversity to Newcastle disease virus virulence, replication and biological activities

    PubMed Central

    Jin, Jihui; Zhao, Jing; Ren, Yingchao; Zhong, Qi; Zhang, Guozhong

    2016-01-01

    To evaluate the contribution of length diversity in the hemagglutinin-neuraminidase (HN) protein to the pathogenicity, replication and biological characteristics of Newcastle disease virus (NDV), we used reverse genetics to generate a series of recombinant NDVs containing truncated or extended HN proteins based on an infectious clone of genotype VII NDV (SG10 strain). The mean death times and intracerebral pathogenicity indices of these viruses showed that the different length mutations in the HN protein did not alter the virulence of NDV. In vitro studies of recombinant NDVs containing truncated or extended HN proteins revealed that the extension of HN protein increased its hemagglutination titer, receptor-binding ability and impaired its neuraminidase activity, fusogenic activity and replication ability. Furthermore, the hemadsorption, neuraminidase and fusogenic promotion activities at the protein level were consistent with those of viral level. Taken together, our results demonstrate that the HN biological activities affected by the C-terminal extension are associated with NDV replication but not the virulence. PMID:27833149

  17. 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.

  18. 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

  19. Enzymatic Activity of the Scaffold Protein Rapsyn for Synapse Formation.

    PubMed

    Li, Lei; Cao, Yu; Wu, Haitao; Ye, Xinchun; Zhu, Zhihui; Xing, Guanglin; Shen, Chengyong; Barik, Arnab; Zhang, Bin; Xie, Xiaoling; Zhi, Wenbo; Gan, Lin; Su, Huabo; Xiong, Wen-Cheng; Mei, Lin

    2016-12-07

    Neurotransmission is ensured by a high concentration of neurotransmitter receptors at the postsynaptic membrane. This is mediated by scaffold proteins that bridge the receptors with cytoskeleton. One such protein is rapsyn (receptor-associated protein at synapse), which is essential for acetylcholine receptor (AChR) clustering and NMJ (neuromuscular junction) formation. We show that the RING domain of rapsyn contains E3 ligase activity. Mutation of the RING domain that abolishes the enzyme activity inhibits rapsyn- as well as agrin-induced AChR clustering in heterologous and muscle cells. Further biological and genetic studies support a working model where rapsyn, a classic scaffold protein, serves as an E3 ligase to induce AChR clustering and NMJ formation, possibly by regulation of AChR neddylation. This study identifies a previously unappreciated enzymatic function of rapsyn and a role of neddylation in synapse formation, and reveals a potential target of therapeutic intervention for relevant neurological disorders.

  20. The role of adapter proteins in T cell activation.

    PubMed

    Koretzky, G A; Boerth, N J

    1999-12-01

    Engagement of antigen receptors on lymphocytes leads to a myriad of complex signal transduction cascades. Recently, work from several laboratories has led to the identification and characterization of novel adapter molecules, proteins with no intrinsic enzymatic activity but which integrate signal transduction pathways by mediating protein-protein interactions. Interestingly, it appears that many of these adapter proteins play as critical a role as the effector enzymes themselves in both lymphocyte development and activation. This review describes some of the biochemical and molecular features of several of these newly identified hematopoietic cell-specific adapter molecules highlighting their importance in regulating (both positively and negatively) signal transduction mediated by the T cell antigen receptor.

  1. Thrombopoietin potentiates agonist-stimulated activation of p38 mitogen-activated protein kinase in human platelets.

    PubMed

    Ezumi, Y; Nishida, E; Uchiyama, T; Takayama, H

    1999-07-22

    Thrombopoietin (TPO) plays a crucial role in megakaryocyte differentiation and platelet production. c-Mpl, a receptor for TPO, is also expressed in terminally differentiated platelets. We investigated the effects of TPO on activation of p38 mitogen-activated protein kinase in human platelets. Thrombin, a thrombin receptor agonist peptide, a thromboxane A(2) analogue, collagen, crosslinking the glycoprotein VI, ADP, and epinephrine, but not phorbol 12, 13-dibutyrate activated p38. TPO did not activate p38 by itself, whereas TPO pretreatment potentiated the agonist-induced activation of p38. TPO did not promote phosphorylation of Hsp27 and cytosolic phospholipase A(2) by itself, but enhanced thrombin-induced phosphorylation of them. The specific p38 inhibitor SB203580 strongly inhibited such phosphorylation. Thus, TPO possesses the priming effect on p38 activation in human platelets and could affect platelet functions through the p38 pathway.

  2. The regulation of AMP-activated protein kinase by phosphorylation.

    PubMed Central

    Stein, S C; Woods, A; Jones, N A; Davison, M D; Carling, D

    2000-01-01

    The AMP-activated protein kinase (AMPK) cascade is activated by an increase in the AMP/ATP ratio within the cell. AMPK is regulated allosterically by AMP and by reversible phosphorylation. Threonine-172 within the catalytic subunit (alpha) of AMPK (Thr(172)) was identified as the major site phosphorylated by the AMP-activated protein kinase kinase (AMPKK) in vitro. We have used site-directed mutagenesis to study the role of phosphorylation of Thr(172) on AMPK activity. Mutation of Thr(172) to an aspartic acid residue (T172D) in either alpha1 or alpha2 resulted in a kinase complex with approx. 50% the activity of the corresponding wild-type complex. The activity of wild-type AMPK decreased by greater than 90% following treatment with protein phosphatases, whereas the activity of the T172D mutant complex fell by only 10-15%. Mutation of Thr(172) to an alanine residue (T172A) almost completely abolished kinase activity. These results indicate that phosphorylation of Thr(172) accounts for most of the activation by AMPKK, but that other sites are involved. In support of this we have shown that AMPKK phosphorylates at least two other sites on the alpha subunit and one site on the beta subunit. Furthermore, we provide evidence that phosphorylation of Thr(172) may be involved in the sensitivity of the AMPK complex to AMP. PMID:10642499

  3. Factors affecting interactions between sulphonate-terminated dendrimers and proteins: A three case study.

    PubMed

    González-García, Estefanía; Maly, Marek; de la Mata, Francisco Javier; Gómez, Rafael; Marina, María Luisa; García, María Concepción

    2017-01-01

    This work proposes a deep study on the interactions between sulphonate-terminated carbosilane dendrimers and proteins. Three different proteins with different molecular weights and isoelectric points were employed and different pHs, dendrimer concentrations and generations were tested. Variations in fluorescence intensity and emission wavelength were used as protein-dendrimer interaction probes. Interaction between dendrimers and proteins greatly depended on the protein itself and pH. Other important issues were the dendrimer concentration and generation. Protein-dendrimer interactions were favored under acidic working conditions when proteins were positively charged. Moreover, in general, high dendrimer generations promoted these interactions. Modeling of protein-dendrimer interactions allowed to understand the different behaviors observed for every protein.

  4. Effect of gold nanoparticle conjugation on the activity and stability of functional proteins.

    PubMed

    Bailes, Julian; Gazi, Sara; Ivanova, Rositsa; Soloviev, Mikhail

    2012-01-01

    Immobilization of functional proteins such as enzymes on solid surfaces produces a variety of effects ranging from the reversal and strong inhibition to the enhancement of protein stability and function. Such effects are protein-dependent and are affected by the physical and chemical properties of the surfaces. Functional consequences of protein immobilization on the surface of gold nanoparticles (AuNPs) are protein-dependent and require thorough investigation using suitable functional tests. However, traditional approaches to making control samples, i.e., immobilized protein vs. protein in solution in absence of any nanoparticles do not provide sufficiently identical reaction conditions and complicate interpretation of the results. This report provides advice and methods for preparing AuNP-conjugated preparations generally suitable for studying the effects of immobilization on the activity and stability of different functional proteins. We use bovine catalase to illustrate our approach, but the methods are easily adaptable to any other enzyme or protein. The AuNP-immobilized enzyme showed increased stability at elevated temperatures compared to the same enzyme in solution.

  5. CARMA3 deficiency abrogates G protein-coupled receptor-induced NF-κB activation

    PubMed Central

    Grabiner, Brian C.; Blonska, Marzenna; Lin, Pei-Chun; You, Yun; Wang, Donghai; Sun, Jiyuan; Darnay, Bryant G.; Dong, Chen; Lin, Xin

    2007-01-01

    G protein-coupled receptors (GPCRs) play pivotal roles in regulating various cellular functions. Although many GPCRs induce NF-κB activation, the molecular mechanism of GPCR-induced NF-κB activation remains largely unknown. CARMA3 (CARD and MAGUK domain-containing protein 3) is a scaffold molecule with unknown biological functions. By generating CARMA3 knockout mice using the gene targeting approach, here we show CARMA3 is required for GPCR-induced NF-κB activation. Mechanistically, we found that CARMA3 deficiency impairs GPCR-induced IκB kinase (IKK) activation, although it does not affect GPCR-induced IKKα/β phosphorylation, indicating that inducible phosphorylation of IKKα/β alone is not sufficient to induce its kinase activity. We also found that CARMA3 is physically associated with NEMO/IKKγ, and induces polyubiquitination of an unknown protein(s) that associates with NEMO, likely by linking NEMO to TRAF6. Consistently, we found TRAF6 deficiency also abrogates GPCR-induced NF-κB activation. Together, our results provide the genetic evidence that CARMA3 is required for GPCR-induced NF-κB activation. PMID:17438001

  6. Activator of G-Protein Signaling 3-Induced Lysosomal Biogenesis Limits Macrophage Intracellular Bacterial Infection.

    PubMed

    Vural, Ali; Al-Khodor, Souhaila; Cheung, Gordon Y C; Shi, Chong-Shan; Srinivasan, Lalitha; McQuiston, Travis J; Hwang, Il-Young; Yeh, Anthony J; Blumer, Joe B; Briken, Volker; Williamson, Peter R; Otto, Michael; Fraser, Iain D C; Kehrl, John H

    2016-01-15

    Many intracellular pathogens cause disease by subverting macrophage innate immune defense mechanisms. Intracellular pathogens actively avoid delivery to or directly target lysosomes, the major intracellular degradative organelle. In this article, we demonstrate that activator of G-protein signaling 3 (AGS3), an LPS-inducible protein in macrophages, affects both lysosomal biogenesis and activity. AGS3 binds the Gi family of G proteins via its G-protein regulatory (GoLoco) motif, stabilizing the Gα subunit in its GDP-bound conformation. Elevated AGS3 levels in macrophages limited the activity of the mammalian target of rapamycin pathway, a sensor of cellular nutritional status. This triggered the nuclear translocation of transcription factor EB, a known activator of lysosomal gene transcription. In contrast, AGS3-deficient macrophages had increased mammalian target of rapamycin activity, reduced transcription factor EB activity, and a lower lysosomal mass. High levels of AGS3 in macrophages enhanced their resistance to infection by Burkholderia cenocepacia J2315, Mycobacterium tuberculosis, and methicillin-resistant Staphylococcus aureus, whereas AGS3-deficient macrophages were more susceptible. We conclude that LPS priming increases AGS3 levels, which enhances lysosomal function and increases the capacity of macrophages to eliminate intracellular pathogens.

  7. Properties of sweetened Indian yogurt (mishti dohi) as affected by added tryptic whey protein hydrolysate.

    PubMed

    Chatterjee, Alok; Kanawjia, S K; Khetra, Yogesh

    2016-01-01

    Utilization of Indian sweetened yogurt (colloquially termed as Mishti Dohi), as vehicle for ACE inhibition and antioxidant activity, by added tryptic whey protein hydrolysate (TWPH) (@ 1, 2, 3 % v/milk), was attempted. Yogurt with 3 % TWPH exhibited non-significant (p > 0.05) difference for sensory attributes; but for body & texture; and maximum biofunctional properties, electing it for storage study (5 ± 1 °C). Flavor and body & texture scores registered significant (p < 0.05) decline under 14 days storage. ACE inhibition and antioxidant activity of control increased by 47.95 and 13.18 % and of experimental 24.58 and 13.43 %, correspondingly. Acidity rose to 1.18 % LA. Control samples conveyed 18.07 % and experimental of 20.77 % escalation for wheying-off. Tyrosine value was 27.04 μg.mL(-1). Among rheological attributes, firmness, quantified by texture analyzer TA-XT2i, dropped (p < 0.05), due to decrease of gel rigidity whereas work of adhesion revealed non-significant difference (p > 0.05), throughout.

  8. Protein modifications affecting triplet energy transfer in bacterial photosynthetic reaction centers.

    PubMed Central

    Laible, P D; Chynwat, V; Thurnauer, M C; Schiffer, M; Hanson, D K; Frank, H A

    1998-01-01

    The efficiency of triplet energy transfer from the special pair (P) to the carotenoid (C) in photosynthetic reaction centers (RCs) from a large family of mutant strains has been investigated. The mutants carry substitutions at positions L181 and/or M208 near chlorophyll-based cofactors on the inactive and active sides of the complex, respectively. Light-modulated electron paramagnetic resonance at 10 K, where triplet energy transfer is thermally prohibited, reveals that the mutations do not perturb the electronic distribution of P. At temperatures > or = 70 K, we observe reduced signals from the carotenoid in most of the RCs with L181 substitutions. In particular, triplet transfer efficiency is reduced in all RCs in which a lysine at L181 donates a sixth ligand to the monomeric bacteriochlorophyll B(B). Replacement of the native Tyr at M208 on the active side of the complex with several polar residues increased transfer efficiency. The difference in the efficiencies of transfer in the RCs demonstrates the ability of the protein environment to influence the electronic overlap of the chromophores and thus the thermal barrier for triplet energy transfer. PMID:9591686

  9. Neural Activation Underlying Cognitive Control in the Context of Neutral and Affectively Charged Pictures in Children

    ERIC Educational Resources Information Center

    Lamm, Connie; White, Lauren K.; McDermott, Jennifer Martin; Fox, Nathan A.

    2012-01-01

    The neural correlates of cognitive control for typically developing 9-year-old children were examined using dense-array ERPs and estimates of cortical activation (LORETA) during a go/no-go task with two conditions: a neutral picture condition and an affectively charged picture condition. Activation was estimated for the entire cortex after which…

  10. Protein profiling of mouse livers with peroxisome proliferator-activated receptor alpha activation.

    PubMed

    Chu, Ruiyin; Lim, Hanjo; Brumfield, Laura; Liu, Hong; Herring, Chris; Ulintz, Peter; Reddy, Janardan K; Davison, Matthew

    2004-07-01

    Peroxisome proliferator-activated receptor alpha (PPARalpha) is important in the induction of cell-specific pleiotropic responses, including the development of liver tumors, when it is chronically activated by structurally diverse synthetic ligands such as Wy-14,643 or by unmetabolized endogenous ligands resulting from the disruption of the gene encoding acyl coenzyme A (CoA) oxidase (AOX). Alterations in gene expression patterns in livers with PPARalpha activation were delineated by using a proteomic approach to analyze liver proteins of Wy-14,643-treated and AOX(-/-) mice. We identified 46 differentially expressed proteins in mouse livers with PPARalpha activation. Up-regulated proteins, including acetyl-CoA acetyltransferase, farnesyl pyrophosphate synthase, and carnitine O-octanoyltransferase, are involved in fatty acid metabolism, whereas down-regulated proteins, including ketohexokinase, formiminotransferase-cyclodeaminase, fructose-bisphosphatase aldolase B, sarcosine dehydrogenase, and cysteine sulfinic acid decarboxylase, are involved in carbohydrate and amino acid metabolism. Among stress response and xenobiotic metabolism proteins, selenium-binding protein 2 and catalase showed a dramatic approximately 18-fold decrease in expression and a modest approximately 6-fold increase in expression, respectively. In addition, glycine N-methyltransferase, pyrophosphate phosphohydrolase, and protein phosphatase 1D were down-regulated with PPARalpha activation. These observations establish proteomic profiles reflecting a common and predictable pattern of differential protein expression in livers with PPARalpha activation. We conclude that livers with PPARalpha activation are transcriptionally geared towards fatty acid combustion.

  11. 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

  12. [Antimodification activity of the ArdA and Ocr proteins].

    PubMed

    Zavil'gel'skiĭ, G V; Kotova, V Iu; Rastorguev, S M

    2011-02-01

    The ArdA and Ocr antirestriction proteins, whose genes are in transmissible plasmids (ardA) and bacteriophage genomes (0.3 (ocr)), specifically inhibit type I restriction-modification enzymes. The Ocr protein (T7 bacteriophage) was shown to inhibit both restriction (endonuclease) and modification (methylase) activities of the EcoKI enzyme in a broad range of intracellular concentrations (starting from 10-20 molecules per cell). In contrast to Ocr, the ArdA protein (ColIb-P9 transmissible plasmid) inhibited both of the EcoKI activities only at high intracellular concentrations (30000-40000 molecules per cell). When the ArdA concentration was several fold lower, only endonuclease activity of EcoKI was inhibited. It was assumed that a poorer ArdA ability to inhibit EcoKI modification activity is related to the substantial difference in life cycle between transmissible plasmids (symbiosis with the bacterial cell) and bacteriophages (infection and lysis of bacteria). The Ocr and ArdA mutants that inhibited exclusively endonuclease activity of EcoKI were obtained. Antirestriction proteins incapable of homodimerization were assumed to inhibit only endonuclease activity of type I restriction-modification enzymes.

  13. 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.

  14. [Regulation of G protein-coupled receptor kinase activity].

    PubMed

    Haga, T; Haga, K; Kameyama, K; Nakata, H

    1994-09-01

    Recent progress on the activation of G protein-coupled receptor kinases is reviewed. beta-Adrenergic receptor kinase (beta ARK) is activated by G protein beta gamma -subunits, which interact with the carboxyl terminal portion of beta ARK. Muscarinic receptor m2-subtypes are phosphorylated by beta ARK1 in the central part of the third intracellular loop (I3). Phosphorylation of I3-GST fusion protein by beta ARK1 is synergistically stimulated by the beta gamma -subunits and mastoparan or a peptide corresponding to portions adjacent to the transmembrane segments of m2-receptors or by beta gamma -subunits and the agonist-bound I3-deleted m2 variant. These results indicate that agonist-bound receptors serve as both substrates and activators of beta ARK.

  15. Acute intrastriatal administration of quinolinic acid affects the expression of the coat protein AP-2 and its interaction with membranes.

    PubMed

    Borgonovo, Janina; Seltzer, Alicia; Sosa, Miguel Angel

    2009-10-01

    Clathrin-coated vesicle endocytosis is thought to be crucial for the maintenance of synaptic transmission and for the cell plasticity at the nervous system. In this study, we demonstrated that acute intrastriatal administration of quinolinic acid (QUIN), an agonist of the N-methyl-D: -aspartate receptor, induces a decrease of the coat protein AP-2 expression and affects their interaction with membranes. By western blot analysis we observed that at 24 h after QUIN intrastriatal injection, alpha1 subunit of AP-2 and alpha2, at lesser extent, were reduced in the striatal membranes. The decrease of both subunits expression was extended to 48 h after treatment, although the soluble proteins were mostly affected. Other areas of the brain were not affected by the treatment, except the cerebellum, where a significant increase of soluble AP-2 (both subunits) was observed at 48 h after injection. Another coat protein, as the phosphoprotein AP-180, was not affected by the injection of QUIN. We also confirmed that QUIN injection causes increasing loss of striatal neurons after the administration of the toxin. We concluded that QUIN may affect the endocytotic machinery of the striatum, by inducing changes in the AP-2 behaviour. Consequently, the internalization of NMDAR and/or AMPAR may be affected, by QUIN, contributing to the excitotoxic effect of the drug.

  16. Decavanadate inhibits the cell-free activation of neutrophil NADPH oxidase without affecting tyrosine phosphorylation.

    PubMed

    Okamura, N; Sakai, T; Nishimura, Y; Sakai, M; Araki, S; Yamaguchi, M; Ishibashi, S

    1999-08-01

    NADPH oxidase was activated by arachidonate in a cell-free system consisting of membrane and cytosol fractions prepared from guinea pig neutrophils. Vanadate apparently inhibited the NADPH oxidase activity in the cell-free system (IC50=2 microM) without phosphotyrosine accumulation. The pH dependency and stability of the inhibitory effect observed for vanadate solution indicated that decavanadate, an isopolyanion of vanadate, was responsible for the inhibition. Pervanadate (vanadyl hydroperoxide) also inhibited the oxidase activity but at a higher concentration (IC50=0.2 mM). Decavanadate lowered the Vmax but did not affect the Km value of NADPH oxidase for NADPH. Decavanadate inhibited the activation process of NADPH oxidase but not the oxidase activity itself. Decavanadate-pretreatment of membrane and cytosol fractions irreversibly decreased the abilities of both fractions to activate NADPH oxidase in the cell-free system. Translocation of p47-phox, one of the cytosolic activation factors of NADPH oxidase, from cytosol to membrane, was little affected by decavanadate. These results suggest that decavanadate inhibits the activation of NADPH oxidase in the cell-free system without affecting the phosphotyrosine phosphatase, and that decavanadate can bind to both the membrane and cytosolic activation factors when they are in a dormant state, but not to the active oxidase complex.

  17. Is spinal excitability of the triceps surae mainly affected by muscle activity or body position?

    PubMed

    Cattagni, T; Martin, A; Scaglioni, G

    2014-06-15

    The aim of this study was to determine how muscle activity and body orientation contribute to the triceps surae spinal transmission modulation, when moving from a sitting to a standing position. Maximal Hoffmann-reflex (Hmax) and motor potential (Mmax) were evoked in the soleus (SOL), medial and lateral gastrocnemius in 10 male subjects and in three conditions, passive sitting, active sitting and upright standing, with the same SOL activity in active sitting and upright standing. Moreover volitional wave (V) was evoked in the two active conditions (i.e., active sitting and upright standing). The results showed that SOL Hmax/Mmax was lower in active sitting than in passive sitting, while for the gastrocnemii it was not significantly altered. For the three plantar flexors, Hmax/Mmax was lower in upright standing than in active sitting, whereas V/Mmax was not modulated. SOL H-reflex is therefore affected by the increase in muscle activity and change in body orientation, while, in the gastrocnemii, it was only affected by a change in posture. In conclusion, passing from a sitting to a standing position affects the Hmax/Mmax of the whole triceps surae, but the mechanisms responsible for this change differ among the synergist muscles. The V/Mmax does not change when upright stance is assumed. This means that the increased inhibitory activity in orthostatic position is compensated by an increased excitatory inflow to the α-motoneurons of central and/or peripheral origin.

  18. Proteins in Intercellular Washing Fluid from Noninoculated and Rust-Affected Leaves of Wheat and Barley 1

    PubMed Central

    Holden, David W.; Rohringer, Roland

    1985-01-01

    Proteins in intercellular washing fluid (IWF) from wheat (Triticum aestivum) and barley (Hordeum vulgare) leaves were separated by two-dimensional isoelectric focusing-polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue (CBB) or silver. Intracellular protein from the cut ends of leaves accounted for only a small proportion of total protein in IWF from wheat leaves. When these were heavily infected with the stem rust fungus (Puccinia graminis f. sp. tritici) and grown at 19°C, four infection-related CBB-stainable proteins were detected in IWF. To compare IWF proteins from wheat and barley leaves infected with the same pathogen, conditions were established that permitted luxuriant growth of stem rust of wheat in barley (exposure to chloroform before inoculation and maintenance at 25°C thereafter). Under these conditions, at least 10 infection-related silver-stainable proteins were detected in IWF from infected wheat in addition to the more than 50 that were of host origin. The electrophoretic properties of 8 of the infection-related proteins were the same as those of 8 infection-related proteins in IWF from barley. IWF from wheat and barley grown under these conditions was analyzed for Concanavalin A-binding glycoproteins immobilized on nitrocellulose membrane replicas made from gels. Of the many infection-related glycoproteins that were detected in IWF from stem rust-affected wheat, approximately 20 occupied the same positions as those from stem rust-affected barley. The glycoprotein pattern of IWF prepared from wheat leaves grown at 19°C and infected with the leaf rust fungus (P. recondita f. sp. tritici) was markedly different to that of IWF from the same host infected with the stem rust fungus. We conclude that IWF from rust-affected cereal leaves may be a useful source of surface or extracellular proteins from the parasitic mycelium. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:16664314

  19. [Histidine triad protein superfamily--biological function and enzymatic activity].

    PubMed

    Krakowiak, Agnieszka; Fryc, Izabela

    2012-01-01

    The HIT superfamily consists of proteins that share the histidine triad motif, His-X-His-X-His-X-X (where X is a hydrophobic amino acid), which constitutes enzymatic catalytic center. These enzymes act as nucleotidylyl hydrolase or transferase, and the mutation of the second histidine in the triad abolishes their activity. HIT proteins were found ubiquitous in all organisms and they were classified into 5 branches, which are represented by human proteins: HINT1, FHIT, Aprataxin, GALT and DCPS. Because HINT1 orthologs, which belong to the evolutionally oldest family branch, were found from prokaryotes to eukaryotes, it is clear that HIT motif was conserved during the evolution what means that the enzymatic activity is necessary for functions of these proteins. However, in few cases, e.g. HINT1 and FHIT, the connection between the biological function and the enzymatic activity is still obscure. In this review, the relations between biology and activity for 7 HIT proteins, which were found in human, are highlighted.

  20. 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

  1. Cognition and affective style: Individual differences in brain electrical activity during spatial and verbal tasks.

    PubMed

    Bell, Martha Ann; Fox, Nathan A

    2003-12-01

    Relations between brain electrical activity and performance on two cognitive tasks were examined in a normal population selected to be high on self-reported measures of Positive or Negative Affectivity. Twenty-five right-handed women, from an original pool of 308 college undergraduates, were the participants. EEG was recorded during baseline and during psychometrically matched spatial and verbal tasks. As predicted, participants who were high in Positive Affectivity performed equally well on the verbal and spatial tasks, while participants who were high in Negative Affectivity had spatial scores that were lower than their verbal scores. There were no group differences in baseline EEG. Both groups exhibited left central activation (i.e., alpha suppression) during the verbal and spatial tasks. When EEG data were analyzed separately for the group high in Positive Affectivity, there was evidence of parietal activation for the spatial task relative to the verbal task. The EEG data for the group high in Negative Affectivity had comparable EEG power values during verbal and spatial tasks at parietal scalp locations. These data suggest that, within a selected normal population, differences in affective style may interact with cognitive performance and with the brain electrical activity associated with that performance.

  2. Phosphoproteome Profiling of SH-SY5y Neuroblastoma Cells Treated with Anesthetics: Sevoflurane and Isoflurane Affect the Phosphorylation of Proteins Involved in Cytoskeletal Regulation

    PubMed Central

    Lee, Joomin; Ahn, Eunsook; Park, Wyun Kon; Park, Seyeon

    2016-01-01

    Inhalation anesthetics are used to decrease the spinal cord transmission of painful stimuli. However, the molecular or biochemical processes within cells that regulate anesthetic-induced responses at the cellular level are largely unknown. Here, we report the phosphoproteome profile of SH-SY5y human neuroblastoma cells treated with sevoflurane, a clinically used anesthetic. Phosphoproteins were isolated from cell lysates and analyzed using two-dimensional gel electrophoresis. The phosphorylation of putative anesthetic-responsive marker proteins was validated using western blot analysis in cells treated with both sevoflurane and isoflurane. A total of 25 phosphoproteins were identified as differentially phosphorylated proteins. These included key regulators that signal cytoskeletal remodeling steps in pathways related to vesicle trafficking, axonal growth, and cell migration. These proteins included the Rho GTPase, Ras-GAP SH3 binding protein, Rho GTPase activating protein, actin-related protein, and actin. Sevoflurane and isoflurane also resulted in the dissolution of F-actin fibers in SH-SY5y cells. Our results show that anesthetics affect the phosphorylation of proteins involved in cytoskeletal remodeling pathways. PMID:27611435

  3. Effects of butyltins on mitogen-activated-protein kinase kinase kinase and Ras activity in human natural killer cells.

    PubMed

    Celada, Lindsay J; Whalen, Margaret M

    2014-09-01

    Butyltins (BTs) contaminate the environment and are found in human blood. BTs, tributyltin (TBT) and dibutyltin (DBT) diminish the cytotoxic function and levels of key proteins of human natural killer (NK) cells. NK cells are an initial immune defense against tumors, virally infected cells and antibody-coated cells and thus critical to human health. The signaling pathways that regulate NK cell functions include mitogen-activated protein kinases (MAPKs). Studies have shown that exposure to BTs leads to activation of specific MAPKs and MAPK kinases (MAP2Ks) in human NK cells. MAP2K kinases (MAP3Ks) are upstream activators of MAP2Ks, which then activate MAPKs. The current study examined if BT-induced activation of MAP3Ks was responsible for MAP2K and thus, MAPK activation. This study examines the effects of TBT and DBT on the total levels of two MAP3Ks, c-Raf and ASK1, as well as activating and inhibitory phosphorylation sites on these MAP3Ks. In addition, the immediate upstream activator of c-Raf, Ras, was examined for BT-induced alterations. Our results show significant activation of the MAP3K, c-Raf, in human NK cells within 10 min of TBT exposure and the MAP3K, ASK1, after 1 h exposures to TBT. In addition, our results suggest that both TBT and DBT affect the regulation of c-Raf.

  4. Mutations in the classical swine fever virus NS4B protein affects virulence in swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    NS4B is one of the non-structural proteins of Classical Swine Fever Virus (CSFV), the etiological agent of a severe, highly lethal disease of swine. Protein domain analysis of the predicted amino acid sequence of the NS4B protein of highly pathogenic CSFV strain Brescia (BICv) identified a Toll/Inte...

  5. Supplemental barley protein and casein similarly affect serum lipids in hypercholesterolemic women and men.

    PubMed

    Jenkins, David J A; Srichaikul, Korbua; Wong, Julia M W; Kendall, Cyril W C; Bashyam, Balachandran; Vidgen, Edward; Lamarche, Benoicirct; Rao, A Venketeshwer; Jones, Peter J H; Josse, Robert G; Jackson, Chung-Ja C; Ng, Vivian; Leong, Tracy; Leiter, Lawrence A

    2010-09-01

    High-protein diets have been advocated for weight loss and the treatment of diabetes. Yet animal protein sources are often high in saturated fat and cholesterol. Vegetable protein sources, by contrast, are low in saturated fat and without associated cholesterol. We have therefore assessed the effect on serum lipids of raising the protein intake by 5% using a cereal protein, barley protein, as part of a standard therapeutic diet. Twenty-three hypercholesterolemic men and postmenopausal women completed a randomized crossover study comparing a bread enriched with either barley protein or calcium caseinate [30 g protein, 8374 kJ (2000 kcal)] taken separately as two 1-mo treatment phases with a minimum 2-wk washout. Body weight and diet history were collected weekly during each treatment. Fasting blood samples were obtained at wk 0, 2, and 4. Palatability, satiety, and compliance were similar for both the barley protein- and casein-enriched breads, with no differences between the treatments in effects on serum LDL cholesterol or C-reactive protein, measures of oxidative stress, or blood pressure. Nevertheless, because no adverse effects were observed on cardiovascular risk factors, barley protein remains an additional option for raising the protein content of the diet.

  6. Two homologous host proteins interact with potato virus X RNAs and CPs and affect viral replication and movement.

    PubMed

    Choi, Hoseong; Cho, Won Kyong; Kim, Kook-Hyung

    2016-06-29

    Because viruses encode only a small number of proteins, all steps of virus infection rely on specific interactions between viruses and hosts. We previously screened several Nicotiana benthamiana (Nb) proteins that interact with the stem-loop 1 (SL1) RNA structure located at the 5' end of the potato virus X (PVX) genome. In this study, we characterized two of these proteins (NbCPIP2a and NbCPIP2b), which are homologous and are induced upon PVX infection. Electrophoretic mobility shift assay confirmed that both proteins bind to either SL1(+) or SL1(-) RNAs of PVX. The two proteins also interact with the PVX capsid protein (CP) in planta. Overexpression of NbCPIP2a positively regulated systemic movement of PVX in N. benthamiana, whereas NbCPIP2b overexpression did not affect systemic movement of PVX. Transient overexpression and silencing experiments demonstrated that NbCPIP2a and NbCPIP2b are positive regulators of PVX replication and that the effect on replication was greater for NbCPIP2a than for NbCPIP2b. Although these two host proteins are associated with plasma membranes, PVX infection did not affect their subcellular localization. Taken together, these results indicate that NbCPIP2a and NbCPIP2b specifically bind to PVX SL1 RNAs as well as to CP and enhance PVX replication and movement.

  7. Minor milk constituents are affected by protein concentration and forage digestibility in the feed ration.

    PubMed

    Larsen, Torben; Alstrup, Lene; Weisbjerg, Martin Riis

    2016-02-01

    The present study was conducted in order to investigate if selected minor milk components would be indicative for the nutritional situation of the cow. Forty-eight dairy cows were offered a high digestible ration vs. a lower digestible ration combined with 2 protein levels in a 4 × 4 Latin square design. Milk glucose, glucose-6-phosphate, cholesterol, triacylglycerides (TAG), uric acid and β-hydroxybutyrate (BHBA) were measured and correlated mutually and towards other milking parameters (yield, h since last milking, days in milk (DIM), urea, etc). The variation range of the suggested variables were broad, a fact that may support their utilisation as predictive parameters. The content of milk metabolites was significantly affected by the change in rations as milk glucose, glucose-6-phosphate, uric acid, and the ratio cholesterol: triacylglycerides increased with higher energy intake while BHBA and TAG decreased. The content of some of the milk metabolites changed during 24 h day/night periods: BHBA, cholesterol, uric acid and TAG increased whereas free glucose decreased in the night period. Certain associations between milk metabolites and calculated energy parameters like ECM, body condition score (BCS), and body weight gain were found, however, these associations were to some extent explained by an interaction with DIM, just as changes in milk metabolites during a 24 h period seems to interfere. It is concluded that the practical use of the suggested milk variables should be based on more than one metabolite and that stage of lactation and possibly time of the day where the milk is collected should be incorporated in predictive models.

  8. AMP-activated protein kinase--an archetypal protein kinase cascade?

    PubMed

    Hardie, D G; MacKintosh, R W

    1992-10-01

    Mammalian AMP-activated protein kinase is the central component of a protein kinase cascade which inactivates three key enzymes involved in the synthesis or release of free fatty acids and cholesterol inside the cell. The kinase cascade is activated by elevation of AMP, and perhaps also by fatty acid and cholesterol metabolites. The system may fulfil a protective function, preventing damage caused by depletion of ATP or excessive intracellular release of free lipids, a type of stress response. Recent evidence suggests that it may have been in existence for at least a billion years, since a very similar protein kinase cascade is present in higher plants. This system therefore represents an early eukaryotic protein kinase cascade, which is unique in that it is regulated by intracellular metabolites rather than extracellular signals or cell cycle events.

  9. A rapid and simple assay for growth hormone-binding protein activity in human plasma.

    PubMed

    Baumann, G; Shaw, M A; Amburn, K

    1988-12-01

    The newly discovered circulating growth hormone binding proteins dictate a re-evaluation of the state of GH in plasma in health and disease as the binding proteins are known to affect GH metabolism and action. We describe a rapid and simple GH-binding assay that allows determination of free and complexed plasma GH, as well as GH-binding protein activity as an index of GH-binding protein levels, with relative ease. The method is based on incubation of plasma with 125I-GH and separation of bound from free GH on small DEAE-cellulose columns; it can be used on a large scale for routine determinations. The results obtained by this method are comparable to those obtained with the previously used slow and more cumbersome gel filtration technique. Initial data obtained in normal subjects and certain disease states show that the bound fraction of plasma GH is similar in men, women and children, is unaffected by pregnancy or acute infection, but is marginally decreased in liver cirrhosis. In acromegaly, binding protein activity also appears normal when allowance is made for partial saturation of the binding proteins by the high prevailing GH levels. The technique we describe should facilitate investigations of normal and abnormal regulation of the GH binding proteins.

  10. Molecular crowding drives active Pin1 into nonspecific complexes with endogenous proteins prior to substrate recognition.

    PubMed

    Luh, Laura M; Hänsel, Robert; Löhr, Frank; Kirchner, Donata K; Krauskopf, Katharina; Pitzius, Susanne; Schäfer, Birgit; Tufar, Peter; Corbeski, Ivan; Güntert, Peter; Dötsch, Volker

    2013-09-18

    Proteins and nucleic acids maintain the crowded interior of a living cell and can reach concentrations in the order of 200-400 g/L which affects the physicochemical parameters of the environment, such as viscosity and hydrodynamic as well as nonspecific strong repulsive and weak attractive interactions. Dynamics, structure, and activity of macromolecules were demonstrated to be affected by these parameters. However, it remains controversially debated, which of these factors are the dominant cause for the observed alterations in vivo. In this study we investigated the globular folded peptidyl-prolyl isomerase Pin1 in Xenopus laevis oocytes and in native-like crowded oocyte extract by in-cell NMR spectroscopy. We show that active Pin1 is driven into nonspecific weak attractive interactions with intracellular proteins prior to substrate recognition. The substrate recognition site of Pin1 performs specific and nonspecific attractive interactions. Phosphorylation of the WW domain at Ser16 by PKA abrogates both substrate recognition and the nonspecific interactions with the endogenous proteins. Our results validate the hypothesis formulated by McConkey that the majority of globular folded proteins with surface charge properties close to neutral under physiological conditions reside in macromolecular complexes with other sticky proteins due to molecular crowding. In addition, we demonstrate that commonly used synthetic crowding agents like Ficoll 70 are not suitable to mimic the intracellular environment due to their incapability to simulate biologically important weak attractive interactions.

  11. STT3, a highly conserved protein required for yeast oligosaccharyl transferase activity in vivo.

    PubMed Central

    Zufferey, R; Knauer, R; Burda, P; Stagljar, I; te Heesen, S; Lehle, L; Aebi, M

    1995-01-01

    N-linked glycosylation is a ubiquitous protein modification, and is essential for viability in eukaryotic cells. A lipid-linked core-oligosaccharide is assembled at the membrane of the endoplasmic reticulum and transferred to selected asparagine residues of nascent polypeptide chains by the oligosaccharyl transferase (OTase) complex. Based on the synthetic lethal phenotype of double mutations affecting the assembly of the lipid-linked core-oligosaccharide and the OTase activity, we have performed a novel screen for mutants in Saccharomyces cerevisiae with altered N-linked glycosylation. Besides novel mutants deficient in the assembly of the lipid-linked oligosaccharide (alg mutants), we identified the STT3 locus as being required for OTase activity in vivo. The essential STT3 protein is approximately 60% identical in amino acid sequence to its human homologue. A mutation in the STT3 locus affects substrate specificity of the OTase complex in vivo and in vitro. In stt3-3 cells very little glycosyl transfer occurs from incomplete lipid-linked oligosaccharide, whereas the transfer of full-length Glc3Man9GlcNAc2 is hardly affected as compared with wild-type cells. Depletion of the STT3 protein results in loss of transferase activity in vivo and a deficiency in the assembly of OTase complex. Images PMID:7588624

  12. Selective activators of protein phosphatase 5 target the auto-inhibitory mechanism

    PubMed Central

    Haslbeck, Veronika; Drazic, Adrian; Eckl, Julia M.; Alte, Ferdinand; Helmuth, Martin; Popowicz, Grzegorz; Schmidt, Werner; Braun, Frank; Weiwad, Matthias; Fischer, Gunter; Gemmecker, Gerd; Sattler, Michael; Striggow, Frank; Groll, Michael; Richter, Klaus

    2015-01-01

    Protein phosphatase 5 (PP5) is an evolutionary conserved serine/threonine phosphatase. Its dephosphorylation activity modulates a diverse set of cellular factors including protein kinases and the microtubule-associated tau protein involved in neurodegenerative disorders. It is auto-regulated by its heat-shock protein (Hsp90)-interacting tetratricopeptide repeat (TPR) domain and its C-terminal α-helix. In the present study, we report the identification of five specific PP5 activators [PP5 small-molecule activators (P5SAs)] that enhance the phosphatase activity up to 8-fold. The compounds are allosteric modulators accelerating efficiently the turnover rate of PP5, but do barely affect substrate binding or the interaction between PP5 and the chaperone Hsp90. Enzymatic studies imply that the compounds bind to the phosphatase domain of PP5. For the most promising compound crystallographic comparisons of the apo PP5 and the PP5–P5SA-2 complex indicate a relaxation of the auto-inhibited state of PP5. Residual electron density and mutation analyses in PP5 suggest activator binding to a pocket in the phosphatase/TPR domain interface, which may exert regulatory functions. These compounds thus may expose regulatory mechanisms in the PP5 enzyme and serve to develop optimized activators based on these scaffolds. PMID:26182372

  13. Germ cell mitogenic activity is associated with nerve growth factor-like protein(s).

    PubMed

    Onoda, M; Pflug, B; Djakiew, D

    1991-12-01

    The mitogenicity of germ cell proteins released from round spermatids (RS) and pachytene spermatocytes (PS) was investigated. Germ cells were isolated by centrifugal elutriation from 90-day-old rat testes and incubated in a supplement enriched culture media that lacked exogenous proteins. The conditioned culture media of RS and PS were dialysed/concentrated and lyophilized to prepare RS protein (RSP) and PS protein (PSP). Mitogenic activity of RSP and PSP was determined by 3H-thymidine incorporation into Swiss 3T3 fibroblasts. RSP and PSP stimulated 3H-thymidine incorporation by fibroblasts in a dose-dependent manner. At a higher concentration of RSP (300 micrograms/ml), fibroblast proliferation was stimulated from 6- to 20-fold of control cultures, whereas PSP (300 micrograms/ml) stimulated fibroblast proliferation 2.5-fold of control cultures. Since RSP exhibited substantially greater mitogenic activity than PSP we further investigated the RSP mitogenic substance(s) by immunoneutralization with antibodies against several growth factors. The mitogenic activity of RSP was significantly reduced by treatment with nerve growth factor (NGF) antibody, while neither the treatment of RSP with acidic fibroblast growth factor (aFGF) antibody, nor basic fibroblast growth factor (bFGF) antibody significantly modified the mitogenic activity of RSP. Interestingly, murine NGF-beta, recombinant human NGF-beta, and bovine serum albumin (BSA) did not exhibit mitogenic activity on 3T3 fibroblasts. Nevertheless, the presence of a NGF-like protein in RS and PS was confirmed by indirect immunofluorescence staining with a murine NGF antibody. Subsequently, a Western blot analysis with the NGF antibody identified two immunoreactive bands of 41 +/- 2 kDa and 51 +/- 1 kDa in both RSP and PSP under reduced conditions. These germ cell NGF-like proteins were apparently different from similarly prepared murine and human NGFs (13 kDa) in their molecular weight. Furthermore, neurite outgrowth

  14. Heating and reduction affect the reaction with tannins of wine protein fractions differing in hydrophobicity.

    PubMed

    Marangon, Matteo; Vincenzi, Simone; Lucchetta, Marco; Curioni, Andrea

    2010-02-15

    During the storage, bottled white wines can manifest haziness due to the insolubilisation of the grape proteins that may 'survive' in the fermentation process. Although the exact mechanism of this occurrence is not fully understood, proteins and tannins are considered two of the key factors involved in wine hazing, since their aggregation leads to the formation of insoluble particles. To better understand this complex interaction, proteins and tannins from the same unfined Pinot grigio wine were separated. Wine proteins were then fractionated by hydrophobic interaction chromatography (HIC). A significant correlation between hydrophobicity of the wine protein fractions and the haze formed after reacting with wine tannins was found, with the most reactive fractions revealing (by SDS-PAGE and RP-HPLC analyses) the predominant presence of thaumatin-like proteins. Moreover, the effects of both protein heating and disulfide bonds reduction (with dithiotreithol) on haze formation in the presence of tannins were assessed. These treatments generally resulted in an improved reactivity with tannins, and this phenomenon was related to both the surface hydrophobicity and composition of the protein fractions. Therefore, haze formation in wines seems to be related to hydrophobic interactions occurring among proteins and tannins. These interactions should occur on hydrophobic tannin-binding sites, whose exposition on the proteins can depend on both protein heating and reduction.

  15. Temperature of frozen storage affects the nature and consequences of protein oxidation in beef patties.

    PubMed

    Utrera, Mariana; Morcuende, David; Estévez, Mario

    2014-03-01

    The effect of three frozen storage temperatures (-8, -18 and -80 °C) on protein oxidation in beef patties was studied through the analysis of novel oxidation markers. Additionally, the connection between lipid and protein oxidation and the impact of the latter on particular quality traits (water holding capacity, color and texture) of subsequently processed beef patties (cooking/cold-stored) were investigated. Protein oxidation was measured as the loss of tryptophan fluorescence and formation of diverse lysine oxidation products (α-aminoadipic semialdehyde, α-aminoadipic acid and Schiff bases). Lipid oxidation was assessed by levels of thiobarbituric acid reactive substances and hexanal. A significant effect of storage temperature on protein oxidation was detected. Frozen storage increased the susceptibility of meat proteins to undergo further oxidation during processing. Timely interactions were found between lipid and protein oxidation. Plausible mechanisms by which oxidative damage to proteins may have an impact in particular quality traits are thoroughly discussed.

  16. Protein Phosphorylation in Amyloplasts Regulates Starch Branching Enzyme Activity and Protein–Protein Interactions

    PubMed Central

    Tetlow, Ian J.; Wait, Robin; Lu, Zhenxiao; Akkasaeng, Rut; Bowsher, Caroline G.; Esposito, Sergio; Kosar-Hashemi, Behjat; Morell, Matthew K.; Emes, Michael J.

    2004-01-01

    Protein phosphorylation in amyloplasts and chloroplasts of Triticum aestivum (wheat) was investigated after the incubation of intact plastids with γ-32P-ATP. Among the soluble phosphoproteins detected in plastids, three forms of starch branching enzyme (SBE) were phosphorylated in amyloplasts (SBEI, SBEIIa, and SBEIIb), and both forms of SBE in chloroplasts (SBEI and SBEIIa) were shown to be phosphorylated after sequencing of the immunoprecipitated 32P-labeled phosphoproteins using quadrupole-orthogonal acceleration time of flight mass spectrometry. Phosphoamino acid analysis of the phosphorylated SBE forms indicated that the proteins are all phosphorylated on Ser residues. Analysis of starch granule–associated phosphoproteins after incubation of intact amyloplasts with γ-32P-ATP indicated that the granule-associated forms of SBEII and two granule-associated forms of starch synthase (SS) are phosphorylated, including SSIIa. Measurement of SBE activity in amyloplasts and chloroplasts showed that phosphorylation activated SBEIIa (and SBEIIb in amyloplasts), whereas dephosphorylation using alkaline phosphatase reduced the catalytic activity of both enzymes. Phosphorylation and dephosphorylation had no effect on the measurable activity of SBEI in amyloplasts and chloroplasts, and the activities of both granule-bound forms of SBEII in amyloplasts were unaffected by dephosphorylation. Immunoprecipitation experiments using peptide-specific anti-SBE antibodies showed that SBEIIb and starch phosphorylase each coimmunoprecipitated with SBEI in a phosphorylation-dependent manner, suggesting that these enzymes may form protein complexes within the amyloplast in vivo. Conversely, dephosphorylation of immunoprecipitated protein complex led to its disassembly. This article reports direct evidence that enzymes of starch metabolism (amylopectin synthesis) are regulated by protein phosphorylation and indicate a wider role for protein phosphorylation and protein–protein

  17. Interaction between transcriptional activator protein LAC9 and negative regulatory protein GAL80.

    PubMed Central

    Salmeron, J M; Langdon, S D; Johnston, S A

    1989-01-01

    In Saccharomyces cerevisiae, transcriptional activation mediated by the GAL4 regulatory protein is repressed in the absence of galactose by the binding of the GAL80 protein, an interaction that requires the carboxy-terminal 28 amino acids of GAL4. The homolog of GAL4 from Kluyveromyces lactis, LAC9, activates transcription in S. cerevisiae and is highly similar to GAL4 in its carboxyl terminus but is not repressed by wild-type levels of GAL80 protein. Here we show that GAL80 does repress LAC9-activated transcription in S. cerevisiae if overproduced. We sought to determine the molecular basis for the difference in the responses of the LAC9 and GAL4 proteins to GAL80. Our results indicate that this difference is due primarily to the fact that under wild-type conditions, the level of LAC9 protein in S. cerevisiae is much higher than that of GAL4, which suggests that LAC9 escapes GAL80-mediated repression by titration of GAL80 protein in vivo. The difference in response to GAL80 is not due to amino acid sequence differences between the LAC9 and GAL4 carboxyl termini. We discuss the implications of these results for the mechanism of galactose metabolism regulation in S. cerevisiae and K. lactis. Images PMID:2550790

  18. Seed protein, oil, fatty acids, and minerals concentration as affected by foliar K-glyphosate application in soybean cultivars

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previous studies showed that glyphosate (Gly) may chelate cation nutrients, including potassium (K), which might affect the nutritional status of soybean seed. The objective of this study was to evaluate seed composition (protein, oil, fatty acids, and minerals) as influenced by foliar applications ...

  19. 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

  20. Assessing the impact of long term frozen storage of faecal samples on protein concentration and protease activity

    PubMed Central

    Morris, Laura S.; Marchesi, Julian R.

    2016-01-01

    Background The proteome is the second axis of the microbiome:host interactome and proteases are a significant aspect in this interaction. They interact with a large variety of host proteins and structures and in many situations are implicated in pathogenesis. Furthermore faecal samples are commonly collected and stored frozen so they can be analysed at a later date. So we were interested to know whether long term storage affected the integrity of proteases and total protein and whether historical native faecal samples were still a viable option for answering research questions around the functional proteome. Methods Faecal samples were collected from 3 healthy volunteers (3 biological replicates) and processed in order to be stored at both − 20 °C and − 80 °C and in a variety of storage buffers. Protein extraction, protein content and protease activity were assessed at the time of collection, after 24 h, 1 week, 1 month, 3 months 6 months and finally 1 year. Results Beadbeating impacted the quantity of protein extracted, while sodium azide did not impact protease assays. Long term storage of extracted proteins showed that both total protein and protease activity were affected when they were stored as extracted protein. Intact faecal samples were shown to maintain both protein levels and protease activity regardless of time and temperature. Conclusions Beadbeating increases the protein and protease activity when extracting from a faecal sample, however, the extracted protein is not stable and activity is lost, even with a suitable storage buffer. The most robust solution is to store the proteins in an intact frozen native faecal matrix and extract at the time of assay or analysis, this approach was shown to be suitable for samples in which, there are low levels of protease activity and which had been frozen for a year. PMID:26853125

  1. Apelin-13 protects against apoptosis by activating AMP-activated protein kinase pathway in ischemia stroke.

    PubMed

    Yang, Yi; Zhang, Xiang-Jian; Li, Li-Tao; Cui, Hai-Ying; Zhang, Cong; Zhu, Chun-Hua; Miao, Jiang-Yong

    2016-01-01

    Apelin has been proved to be protective against apoptosis induced by ischemic reperfusion. However, mechanisms whereby apelin produces neuroprotection remain to be elucidated. AMP-activated protein kinase (AMPK) is a master energy sensor that monitors levels of key energy metabolites. It is activated via AMPKαThr172 phosphorylation during cerebral ischemia and appears to be neuroprotective. In this study, we investigated the effect of apelin on AMPKα and tested whether apelin protecting against apoptosis was associated with AMPK signals. Focal transient cerebral ischemia/reperfusion (I/R) model in male ICR mice was induced by 60 min of ischemia followed by reperfusion. Apelin-13 was injected intracerebroventricularly 15 min before reperfusion. AMPK inhibitor, compound C, was injected to mice intraperitoneally at the onset of ischemia. In experiment 1, the effect of apelin-13 on AMPKα was measured. In experiment 2, the relevance of AMPKα and apelin-13' effect on apoptosis was measured. Data showed that apelin-13 significantly increased AMPKα phosphorylation level after cerebral I/R. Apelin-13, with the co-administration of saline, reduced apoptosis cells, down-regulated Bax and cleaved-caspase3 and up-regulated Bcl2. However, with the co-administration of compound C, apelin-13 was inefficient in affecting apoptosis and Bax, Bcl2 and cleaved-caspase3. The study provided the evidence that apelin-13 up-regulated AMPKα phosphorylation level in cerebral ischemia insults and AMPK signals participated in the mechanism of apelin-mediated neuroprotection.

  2. 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.

  3. Multiple switches in G protein-coupled receptor activation.

    PubMed

    Ahuja, Shivani; Smith, Steven O

    2009-09-01

    The activation mechanism of G protein-coupled receptors has presented a puzzle that finally may be close to solution. These receptors have a relatively simple architecture consisting of seven transmembrane helices that contain just a handful of highly conserved amino acids, yet they respond to light and a range of chemically diverse ligands. Recent NMR structural studies on the active metarhodopsin II intermediate of the visual receptor rhodopsin, along with the recent crystal structure of the apoprotein opsin, have revealed multiple structural elements or 'switches' that must be simultaneously triggered to achieve full activation. The confluence of several required structural changes is an example of "coincidence counting", which is often used by nature to regulate biological processes. In ligand-activated G protein-coupled receptors, the presence of multiple switches may provide an explanation for the differences between full, partial and inverse agonists.

  4. Functional modulation of AMP-activated protein kinase by cereblon.

    PubMed

    Lee, Kwang Min; Jo, Sooyeon; Kim, Hyunyoung; Lee, Jongwon; Park, Chul-Seung

    2011-03-01

    Mutations in cereblon (CRBN), a substrate binding component of the E3 ubiquitin ligase complex, cause a form of mental retardation in humans. However, the cellular proteins that interact with CRBN remain largely unknown. Here, we report that CRBN directly interacts with the α1 subunit of AMP-activated protein kinase (AMPK α1) and inhibits the activation of AMPK activation. The ectopic expression of CRBN reduces phosphorylation of AMPK α1 and, thus, inhibits the enzyme in a nutrient-independent manner. Moreover, AMPK α1 can be potently activated by suppressing endogenous CRBN using CRBN-specific small hairpin RNAs. Thus, CRBN may act as a negative modulator of the AMPK signaling pathway in vivo.

  5. 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

  6. Sake Protein Supplementation Affects Exercise Performance and Biochemical Profiles in Power-Exercise-Trained Mice

    PubMed Central

    Chen, Yi-Ming; Lin, Che-Li; Wei, Li; Hsu, Yi-Ju; Chen, Kuan-Neng; Huang, Chi-Chang; Kao, Chin-Hsung

    2016-01-01

    Exercise and fitness training programs have attracted the public’s attention in recent years. Sports nutrition supplementation is an important issue in the global sports market. Purpose: In this study, we designed a power exercise training (PET) program with a mouse model based on a strength and conditional training protocol for humans. We tested the effect of supplementation with functional branched-chain amino acid (BCAA)-rich sake protein (SP) to determine whether the supplement had a synergistic effect during PET and enhanced athletic performance and resistance to fatigue. Methods: Male ICR mice were divided into three groups (n = 8 per group) for four-week treatment: sedentary controls with vehicle (SC), and PET and PET groups with SP supplementation (3.8 g/kg, PET + SP). Exercise performance was evaluated by forelimb grip strength and exhaustive swimming time as well as changes in body composition and anti-fatigue activity levels of serum lactate, ammonia, glucose, and creatine kinase (CK) after a 15-min swimming exercise. The biochemical parameters were measured at the end of the experiment. Results: four-week PET significantly increased grip strength and exhaustive swimming time and decreased epididymal fat pad (EFP) weight and area. Levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine, and uric acid (UA) were significantly increased. PET + SP supplementation significantly decreased serum lactate, ammonia and CK levels after the 15-min swimming exercise. The resting serum levels of AST, ALT, CREA and UA were all significantly decreased with PET + SP. Conclusion: The PET program could increase the exercise performance and modulate the body composition of mice. PET with SP conferred better anti-fatigue activity, improved biochemical profiles, and may be an effective ergogenic aid in strength training. PMID:26907336

  7. Meditation-induced neuroplastic changes in amygdala activity during negative affective processing.

    PubMed

    Leung, Mei-Kei; Lau, Way K W; Chan, Chetwyn C H; Wong, Samuel S Y; Fung, Annis L C; Lee, Tatia M C

    2017-04-10

    Recent evidence suggests that the effects of meditation practice on affective processing and resilience have the potential to induce neuroplastic changes within the amygdala. Notably, literature speculates that meditation training may reduce amygdala activity during negative affective processing. Nonetheless, studies have thus far not verified this speculation. In this longitudinal study, participants (N = 21, 9 men) were trained in awareness-based compassion meditation (ABCM) or matched relaxation training. The effects of meditation training on amygdala activity were examined during passive viewing of affective and neutral stimuli in a non-meditative state. We found that the ABCM group exhibited significantly reduced anxiety and right amygdala activity during negative emotion processing than the relaxation group. Furthermore, ABCM participants who performed more compassion practice had stronger right amygdala activity reduction during negative emotion processing. The lower right amygdala activity after ABCM training may be associated with a general reduction in reactivity and distress. As all participants performed the emotion processing task in a non-meditative state, it appears likely that the changes in right amygdala activity are carried over from the meditation practice into the non-meditative state. These findings suggest that the distress-reducing effects of meditation practice on affective processing may transfer to ordinary states, which have important implications on stress management.

  8. Single Amino Acid Polymorphisms of Pertussis Toxin Subunit S2 (PtxB) Affect Protein Function

    PubMed Central

    Millen, Scott H.; Watanabe, Mineo; Komatsu, Eiji; Yamaguchi, Fuminori; Nagasawa, Yuki; Suzuki, Eri; Monaco, Haleigh; Weiss, Alison A.

    2015-01-01

    Whooping cough due to Bordetella pertussis is increasing in incidence, in part due to accumulation of mutations which increase bacterial fitness in highly vaccinated populations. Polymorphisms in the pertussis toxin, ptxA and ptxB genes, and the pertactin, prn genes of clinical isolates of Bordetella pertussis collected in Cincinnati from 1989 through 2005 were examined. While the ptxA and prn genotypes were variable, all 48 strains had the ptxB2 genotype; ptxB1 encodes glycine at amino acid 18 of the S2 subunit of pertussis toxin, while ptxB2 encodes serine. We investigated antigenic and functional differences of PtxB1 and PtxB2. The S2 protein was not very immunogenic. Only a few vaccinated or individuals infected with B. pertussis developed antibody responses to the S2 subunit, and these sera recognized both polymorphic forms equally well. Amino acid 18 of S2 is in a glycan binding domain, and the PtxB forms displayed differences in receptor recognition and toxicity. PtxB1 bound better to the glycoprotein, fetuin, and Jurkat T cells in vitro, but the two forms were equally effective at promoting CHO cell clustering. To investigate in vivo activity of Ptx, one μg of Ptx was administered to DDY mice and blood was collected on 4 days after injection. PtxB2 was more effective at promoting lymphocytosis in mice. PMID:26375454

  9. Plakophilin 2 Affects Cell Migration by Modulating Focal Adhesion Dynamics and Integrin Protein Expression

    PubMed Central

    Koetsier, Jennifer L.; Amargo, Evangeline V.; Todorović, Viktor; Green, Kathleen J.; Godsel, Lisa M.

    2014-01-01

    Plakophilin 2 (PKP2), a desmosome component, modulates the activity and localization of the small GTPase RhoA at sites of cell–cell contact. PKP2 regulates cortical actin rearrangement during junction formation, and its loss is accompanied by an increase in actin stress fibers. We hypothesized that PKP2 may regulate focal adhesion dynamics and cell migration. Here we show that PKP2-deficient cells bind efficiently to the extracellular matrix, but upon spreading display total cell areas ~30% smaller than control cells. Focal adhesions in PKP2-deficient cells are ~2× larger and more stable than in control cells, and vinculin displays an increased time for fluorescence recovery after photobleaching. Furthermore, β4 and β1 integrin protein and mRNA expression is elevated in PKP2-silenced cells. Normal focal adhesion phenotypes can be restored in PKP2-null cells by dampening the RhoA pathway or silencing β1 integrin. However, integrin expression levels are not restored by RhoA signaling inhibition. These data uncover a potential role for PKP2 upstream of β1 integrin and RhoA in integrating cell–cell and cell–substrate contact signaling in basal keratinocytes necessary for the morphogenesis, homeostasis, and reepithelialization of the stratified epidermis. PMID:23884246

  10. 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).

  11. [Modulators of the regulatory protein activity acting at microdoses].

    PubMed

    Iamskova, V P; Krasnov, M S; Skripnikova, V S; Moliavka, A A; Il'ina, A P; Margasiuk, D V; Borisenko, A V; Berezin, B B; Iamskov, I A

    2009-01-01

    New, previously not studied bioregulators active in the ultra low doses corresponding of 10(-8) - 10(-17) mg/ml have been isolated from vitreoretinal tissue of eye. It has been shown that these bioregulators comprise some regulatory peptides-modulators represented by proteins with molecular weights 15-70 KDa one of which is bovine serum albumin. Correlation between the nanosize of bioregulators and their ability to show activity in ultra low doses is established.

  12. STAT5-Interacting Proteins: A Synopsis of Proteins that Regulate STAT5 Activity

    PubMed Central

    Able, Ashley A.; Burrell, Jasmine A.; Stephens, Jacqueline M.

    2017-01-01

    Signal Transducers and Activators of Transcription (STATs) are key components of the JAK/STAT pathway. Of the seven STATs, STAT5A and STAT5B are of particular interest for their critical roles in cellular differentiation, adipogenesis, oncogenesis, and immune function. The interactions of STAT5A and STAT5B with cytokine/hormone receptors, nuclear receptors, transcriptional regulators, proto-oncogenes, kinases, and phosphatases all contribute to modulating STAT5 activity. Among these STAT5 interacting proteins, some serve as coactivators or corepressors to regulate STAT5 transcriptional activity and some proteins can interact with STAT5 to enhance or repress STAT5 signaling. In addition, a few STAT5 interacting proteins have been identified as positive regulators of STAT5 that alter serine and tyrosine phosphorylation of STAT5 while other proteins have been identified as negative regulators of STAT5 via dephosphorylation. This review article will discuss how STAT5 activity is modulated by proteins that physically interact with STAT5. PMID:28287479

  13. Effects of prenatal protein malnutrition on the electrical cerebral activity during development.

    PubMed

    De Frías, V; Varela, O; Oropeza, J J; Bisiacchi, B; Alvarez, A

    2010-10-04

    Early protein restriction during the prenatal period has significant repercussions on the ontogeny and development of the central nervous system. The present study investigates whether early prenatal protein malnutrition could alter the electrical cerebral activity of the progeny. We used Sprague-Dawley female rats of 200 g randomly divided into three groups: a control group that received a diet with 25% of the protein content (lactalbumin), the experimental group, that received a diet with 6% of the protein content and the rehabilitated group that initially received a diet with 6% of the protein content, then switched to a diet with 25% of the protein content after the weaning period (P20D) up to 60 days of life (P60D). Reduction of the protein content from 25% to 6% of lactalbumin in the diet of pregnant rats produces impairment in the electrical cerebral activity in the progeny at P20D and at P60D. The power spectral analysis for each one of the electroencephalograms revealed that prenatal protein malnutrition in rats produced a significant reduction of the alpha (8-13 Hz) and the beta bands (13-30 Hz) and a significant increase of the theta (4-8 Hz), and delta bands (1-4 Hz), at two different stages of life (P20D and P60D). Similar results were obtained for the rehabilitated group. These results indicate that early malnutrition in life affects the ontogeny of the electrical cerebral activity. This insult probably disrupts the establishment of cortical neural circuits during the critical period of brain development. The rehabilitation period did not revert the impairment in the electrical cerebral activity produced by malnutrition. We used one-way ANOVA analysis, followed by Tukey test (*p<0.001).

  14. Protein kinase A regulates the osteogenic activity of Osterix.

    PubMed

    He, Siyuan; Choi, You Hee; Choi, Joong-Kook; Yeo, Chang-Yeol; Chun, ChangJu; Lee, Kwang Youl

    2014-10-01

    Osterix belongs to the SP gene family and is a core transcription factor responsible for osteoblast differentiation and bone formation. Activation of protein kinase A (PKA), a serine/threonine kinase, is essential for controlling bone formation and BMP-induced osteoblast differentiation. However, the relationship between Osterix and PKA is still unclear. In this report, we investigated the precise role of the PKA pathway in regulating Osterix during osteoblast differentiation. We found that PKA increased the protein level of Osterix; PKA phosphorylated Osterix, increased protein stability, and enhanced the transcriptional activity of Osterix. These results suggest that Osterix is a novel target of PKA, and PKA modulates osteoblast differentiation partially through the regulation of Osterix.

  15. 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.

  16. 5'-AMP-activated protein kinase signaling in Caenorhabditis elegans.

    PubMed

    Beale, Elmus G

    2008-01-01

    5'-AMP-activated protein kinase (AMPK) has been called "the metabolic master switch" because of its central role in regulating fuel homeostasis. AMPK, a heterotrimeric serine/threonine protein kinase composed of alpha, beta, and gamma subunits, is activated by upstream kinases and by 5'-AMP in response to various nutritional and stress signals. Downstream effects include regulation of metabolism, protein synthesis, cell growth, and mediation of the actions of a number of hormones, including leptin. However, AMPK research represents a young and growing field; hence, there are many unanswered questions regarding the control and action of AMPK. This review presents evidence for the existence of AMPK signaling pathways in Caenorhabditis elegans, a genetically tractable model organism that has yet to be fully exploited to elucidate AMPK signaling mechanisms.

  17. A designed supramolecular protein assembly with in vivo enzymatic activity.

    PubMed

    Song, Woon Ju; Tezcan, F Akif

    2014-12-19

    The generation of new enzymatic activities has mainly relied on repurposing the interiors of preexisting protein folds because of the challenge in designing functional, three-dimensional protein structures from first principles. Here we report an artificial metallo-β-lactamase, constructed via the self-assembly of a structurally and functionally unrelated, monomeric redox protein into a tetrameric assembly that possesses catalytic zinc sites in its interfaces. The designed metallo-β-lactamase is functional in the Escherichia coli periplasm and enables the bacteria to survive treatment with ampicillin. In vivo screening of libraries has yielded a variant that displays a catalytic proficiency [(k(cat)/K(m))/k(uncat)] for ampicillin hydrolysis of 2.3 × 10(6) and features the emergence of a highly mobile loop near the active site, a key component of natural β-lactamases to enable substrate interactions.

  18. Bacillus thuringiensis insecticidal crystal proteins affect lifespan and reproductive performance of Helicoverpa armigera and Spodoptera exigua adults.

    PubMed

    Zhang, Ying; Ma, Yan; Wan, Pin-Jun; Mu, Li-Li; Li, Guo-Qing

    2013-04-01

    Being delivered as sprays or expressed in plant, Bacillus thuringiensis (Bt) crystalline proteins (Cry toxins) display insecticidal activities against numerous Lepidopteran, Dipteran, and Coleopteran larvae. Comparative study of toxicities of Bt Cry toxins between larvae and adults may afford important new insights into the interactions of the toxins with receptor proteins in host insect, and represent intriguing targets for the control of insect pests. However, the effectiveness of Bt Cry toxins in insect adults has paid less attention. In the present article, the effectiveness of Cry1Ac and Cry1Ca on lifespans and reproductive performance of Helicoverpa armigera (Hübner) and Spodoptera exigua (Hübner) adults were evaluated by in vivo experiments. Considering transgenic plants express modified, truncated versions of cry genes yielding active toxin fragment, we used activated Bt toxins at the concentration of 500, 100, and 20 microg/ml in a 10% sucrose aquous solution. At the highest concentration, Cry1Ac and Cry1Ca shortened 48.1 and 48.9% of H. armigera female lifespan, and 43.5 and 38.5% of S. exigua female lifespan, and they reduced 37.8 and 40.3%, and 50.5 and 47.4% of H. armigera and S. exigua male lifespans respectively. Bt toxins negatively affected copulation. Exposure to 500 microg/ml of Cry1Ac and Cry1Ca greatly reduced 50.0 and 46.8%, and 58.7 and 57.3% spermatophore acceptance by H. armigera and S. exigua females, respectively. Similarly, Cry1Ac and Cry1Ca exposure decreased 40.0 and 50.3%, and 61.3 and 60.0% of spermatophore transfer by H. armigera and S. exigua males, respectively. Moreover, exposure females rather than males to 500 microg/ml of Cry1Ac and Cry1Ca significantly dropped 57.5 and 57.5% of the number of eggs laid by H. armigera, and 35.4 and 45.8% of the number of egg masses deposited by S. exigua. In contrast, both Cry1Ac and Cry1Ca did not negatively influence the egg hatchability. At the middle and the lowest concentrations, however

  19. Protein S as an in vivo cofactor to activated protein C in prevention of microarterial thrombosis in rabbits.

    PubMed Central

    Arnljots, B; Dahlbäck, B

    1995-01-01

    The antithrombotic effects of bovine activated protein C (APC) and protein S were investigated in a rabbit model of microarterial thrombosis. Because of the species specificity of the APC-protein S interaction, bovine APC expresses potent anticoagulant activity in rabbit plasma only when bovine protein S is also present. This provided a way to assess the contribution of bovine protein S to the antithrombotic effect of bovine APC. Rabbits were infused with boluses of activated protein C (0.1, 0.2, 0.4, or 0.8 mg/kg), protein S (0.5 mg/kg), or activated protein C (0.1 or 0.01 mg/kg) plus protein S (0.5 mg/kg). APC alone produced a dose-dependent antithrombotic effect, but only the group receiving the highest dose differed significantly from controls. While a low dose of activated protein C (0.1 mg/kg) alone had no antithrombotic effect, together with protein S (0.5 mg/kg) it produced a potent response. The presented results demonstrate the in vivo significance of protein S as a cofactor to activated protein C. The data show that a potent antithrombotic effect, without hemorrhagic side effects or significant systemic anticoagulation, may be achieved by low doses of activated protein C when combined with protein S. Images PMID:7738165

  20. 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

  1. Visible-Light-Triggered Activation of a Protein Kinase Inhibitor.

    PubMed

    Wilson, Danielle; Li, Jason W; Branda, Neil R

    2017-02-20

    A photoresponsive small molecule undergoes a ring-opening reaction when exposed to visible light and becomes an active inhibitor of the enzyme protein kinase C. This "turning on" of enzyme inhibition with light puts control into the hands of the user, creating the opportunity to regulate when and where enzyme catalysis takes place.

  2. Structure and Activity Changes of Phytohemagglutinin from Red Kidney Bean (Phaseolus vulgaris) Affected by Ultrahigh-Pressure Treatments.

    PubMed

    Lu, Yunjun; Liu, Cencen; Zhao, Mouming; Cui, Chun; Ren, Jiaoyan

    2015-11-04

    Phytohemagglutin (PHA), purified from red kidney beans (Phaseolus vulgaris) by Affi-Gel blue affinity chromatography, was subjected to ultrahigh-pressure (UHP) treatment (150, 250, 350, and 450 MPa). The purified PHA lost its hemagglutination activity after 450 MPa treatment and showed less pressure tolerance than crude PHA. However, the saccharide specificity and α-glucosidase inhibition activity of the purified PHA did not change much after UHP treatment. Electrophoresis staining by periodic acid-Schiff (PAS) manifested that the glycone structure of purified PHA remained stable even after 450 MPa pressure treatment. However, electrophoresis staining by Coomassie Blue as well as circular dichroism (CD) and differential scanning calorimetry (DSC) assay proved that the protein unit structure of purified PHA unfolded when treated at 0-250 MPa but reaggregates at 250-450 MPa. Therefore, the hemagglutination activity tends to be affected by the protein unit structure, while the stability of the glycone structure contributed to the remaining α-glucosidase inhibition activity.

  3. Quantitative protein composition and baking quality of winter wheat as affected by late sulfur fertilization.

    PubMed

    Zörb, Christian; Steinfurth, Dorothee; Seling, Simone; Langenkämper, Georg; Koehler, Peter; Wieser, Herbert; Lindhauer, Meinolf G; Mühling, Karl H

    2009-05-13

    Increasing prices for wheat products and fertilizers, as well as reduced sulfur (S) contributions from the atmosphere, call for an improvement of product quality and agricultural management. To detect the impact of a time-dependent S fertilization, the quantitative protein composition and the baking quality of two different wheat cultivars, Batis and Turkis, were evaluated. The glutathione concentration in grains serves as a reliable marker of the need for added S fertilizer. The quantitation of gliadins and glutenin subunits by reversed-phase high-performance liquid chromatography confirmed that S-rich proteins significantly increased with S fertilization, whereas the S-poor proteins significantly decreased. Proteome analysis by means of high-resolution protein profiles detected 55 and 37 proteins from Batis and Turkis changed by late S fertilization. A microscale baking test using wholemeal flour was implemented for the evaluation of baking quality, and late S fertilization was found to improve the composition of gluten proteins and baking quality.

  4. Identification of a common protein association region in the neuronal Cdk5 activator.

    PubMed

    Wang, X; Ching, Y P; Lam, W H; Qi, Z; Zhang, M; Wang, J H

    2000-10-13

    Cyclin-dependent protein kinase 5 (Cdk5) depends on the association with neuronal Cdk5 activator (Nck5a) for kinase activity. A variety of cellular proteins have been shown to undergo high affinity association with Nck5a, including three novel proteins, C42, C48, and C53 found by a yeast two-hybrid screen (Ching, Y. P., Qi, Z., and Wang, J. H. (2000) Gene 242, 285-294). The three proteins show competitive binding to Nck5a suggesting that they bind at a common site. The binding site has been mapped to a region of 26 amino acid residues (residues 145 to 170) at the N-terminal boundary of the kinase activation domain of Nck5a. This region of Nck5a contains an amphipathic alpha-helix whose hydrophobic face is involved in Cdk5 activation (Chin, K. T., Ohki, S, Tang, D., Cheng, H. C., Wang, J. H. , and Zhang, M. (1999) J. Biol. Chem. 274, 7120-7127). Several lines of evidence suggest that Nck5a interacts with the binding proteins at the hydrophilic face of the amphipathic alpha-helix. First, the Nck5a-(145-170) peptide can bind Cdk5 and Nck5a-binding proteins simultaneously. Second, the association of Nck5a-(145-170) to C48 can be markedly reduced by high ionic strength whereas the interaction between Nck5a and Cdk5 is not affected. Third, substitution of Glu(157) by glutamine in Nck5a-(145-170) abolishes the peptide's ability to bind to the three Nck5a-binding proteins without diminishing its Cdk5 binding activity.

  5. Differential range and activity of various forms of the Hedgehog protein

    PubMed Central

    Dawber, Rebecca J; Hebbes, Stephen; Herpers, Bram; Docquier, France; van den Heuvel, Marcel

    2005-01-01

    Background The Hedgehog (Hh) family of secreted proteins act as extracellular messengers to control and coordinate growth and differentiation. The mechanism by which Hh protein travels across a field of cells, and results in a range of specific effects relating to the distance from the source, has been the subject of much debate. It has been suggested that the range and activity of the pathway can be linked to modifications of the Hh protein, specifically the addition of lipid groups at N- and C-terminal sites. Results Here we have addressed the potency of different forms of Hh protein by expressing these in Drosophila, where we are able to precisely establish pathway activity and range in naïve but responsive tissues. As expected, a construct that can produce all forms of Hh recapitulates endogenous signaling potencies. In comparison, expression of a form that lacks the cholesterol moiety (HhN) leads to an extended range, but the product is less effective at inducing maximal Hh responses. Expression of a point mutant that lacks the N-terminal palmitate binding site shows that the palmitoylation of Hh is absolutely required for activity in this system. Conclusion We conclude that the addition of the cholesterol moiety limits the range of the protein and is required for maximal activity, while addition of palmitate is required for all activity. These findings have implications for understanding how Hedgehog proteins move, and thus their potential at influencing distant sites, and concomitantly, how modifications of the signaling protein can affect the efficacy of the response in exposed cells. PMID:16197551

  6. Bt proteins Cry1Ah and Cry2Ab do not affect cotton aphid Aphis gossypii and ladybeetle Propylea japonica.

    PubMed

    Zhao, Yao; Zhang, Shuai; Luo, Jun-Yu; Wang, Chun-Yi; Lv, Li-Min; Wang, Xiao-Ping; Cui, Jin-Jie; Lei, Chao-Liang

    2016-02-01

    Plant varieties expressing the Bt (Bacillus thuringiensis) insecticidal proteins Cry1Ah and Cry2Ab have potential commercialization prospects in China. However, their potential effects on non-target arthropods (NTAs) remain uncharacterized. The cotton aphid Aphis gossypii is a worldwide pest that damages various important crops. The ladybeetle Propylea japonica is a common and abundant natural enemy in many cropping systems in East Asia. In the present study, the effects of Cry1Ah and Cry2Ab proteins on A. gossypii and P. japonica were assessed from three aspects. First, neither of the Cry proteins affected the growth or developmental characteristics of the two test insects. Second, the expression levels of the detoxification-related genes of the two test insects did not change significantly in either Cry protein treatment. Third, neither of the Cry proteins had a favourable effect on the expression of genes associated with the amino acid metabolism of A. gossypii and the nutrition utilization of P. japonica. In conclusion, the Cry1Ah and Cry2Ab proteins do not appear to affect the cotton aphid A. gossypii or the ladybeetle P. japonica.

  7. Bt proteins Cry1Ah and Cry2Ab do not affect cotton aphid Aphis gossypii and ladybeetle Propylea japonica

    PubMed Central

    Zhao, Yao; Zhang, Shuai; Luo, Jun-Yu; Wang, Chun-Yi; Lv, Li-Min; Wang, Xiao-Ping; Cui, Jin-Jie; Lei, Chao-Liang

    2016-01-01

    Plant varieties expressing the Bt (Bacillus thuringiensis) insecticidal proteins Cry1Ah and Cry2Ab have potential commercialization prospects in China. However, their potential effects on non-target arthropods (NTAs) remain uncharacterized. The cotton aphid Aphis gossypii is a worldwide pest that damages various important crops. The ladybeetle Propylea japonica is a common and abundant natural enemy in many cropping systems in East Asia. In the present study, the effects of Cry1Ah and Cry2Ab proteins on A. gossypii and P. japonica were assessed from three aspects. First, neither of the Cry proteins affected the growth or developmental characteristics of the two test insects. Second, the expression levels of the detoxification-related genes of the two test insects did not change significantly in either Cry protein treatment. Third, neither of the Cry proteins had a favourable effect on the expression of genes associated with the amino acid metabolism of A. gossypii and the nutrition utilization of P. japonica. In conclusion, the Cry1Ah and Cry2Ab proteins do not appear to affect the cotton aphid A. gossypii or the ladybeetle P. japonica. PMID:26829252

  8. Autophagy activation in COL6 myopathic patients by a low-protein-diet pilot trial

    PubMed Central

    Castagnaro, Silvia; Pellegrini, Camilla; Pellegrini, Massimo; Chrisam, Martina; Sabatelli, Patrizia; Toni, Silvia; Grumati, Paolo; Ripamonti, Claudio; Pratelli, Loredana; Maraldi, Nadir M.; Cocchi, Daniela; Righi, Valeria; Faldini, Cesare; Sandri, Marco; Bonaldo, Paolo; Merlini, Luciano

    2016-01-01

    ABSTRACT A pilot clinical trial based on nutritional modulation was designed to assess the efficacy of a one-year low-protein diet in activating autophagy in skeletal muscle of patients affected by COL6/collagen VI-related myopathies. Ullrich congenital muscular dystrophy and Bethlem myopathy are rare inherited muscle disorders caused by mutations of COL6 genes and for which no cure is yet available. Studies in col6 null mice revealed that myofiber degeneration involves autophagy defects and that forced activation of autophagy results in the amelioration of muscle pathology. Seven adult patients affected by COL6 myopathies underwent a controlled low-protein diet for 12 mo and we evaluated the presence of autophagosomes and the mRNA and protein levels for BECN1/Beclin 1 and MAP1LC3B/LC3B in muscle biopsies and blood leukocytes. Safety measures were assessed, including muscle strength, motor and respiratory function, and metabolic parameters. After one y of low-protein diet, autophagic markers were increased in skeletal muscle and blood leukocytes of patients. The treatment was safe as shown by preservation of lean:fat percentage of body composition, muscle strength and function. Moreover, the decreased incidence of myofiber apoptosis indicated benefits in muscle homeostasis, and the metabolic changes pointed at improved mitochondrial function. These data provide evidence that a low-protein diet is able to activate autophagy and is safe and tolerable in patients with COL6 myopathies, pointing at autophagy activation as a potential target for therapeutic applications. In addition, our findings indicate that blood leukocytes are a promising noninvasive tool for monitoring autophagy activation in patients. PMID:27656840

  9. Proteins affecting thylakoid morphology - the key to understanding vesicle transport in chloroplasts?

    PubMed

    Lindquist, Emelie; Aronsson, Henrik

    2014-01-01

    We recently showed that a Rab protein, CPRabA5e (CP = chloroplast localized), is located in chloroplasts of Arabidopsis thaliana where it is involved in various processes, such as thylakoid biogenesis and vesicle transport. Using a yeast two-hybrid method, CPRabA5e was shown to interact with a number of chloroplast proteins, including the CURVATURE THYLAKOID 1A (CURT1A) protein and the light-harvesting chlorophyll a/b binding protein (LHCB1.5). CURT1A has recently been shown to modify thylakoid architecture by inducing membrane curvature in grana, whereas LHCB1.5 is a protein of PSII (Photosystem II) facilitating light capture. LHCB1.5 is imported to chloroplasts and transported to thylakoid membranes using the post-translational Signal Recognition Particle (SRP) pathway. With this information as starting point, we here discuss their subsequent protein-protein interactions, given by the literature and Interactome 3D. CURT1A itself and several of the proteins interacting with CURT1A and LHCB1.5 have relations to vesicle transport and thylakoid morphology, which are also characteristics of cprabA5e mutants. This highlights the previous hypothesis of an alternative thylakoid targeting pathway for LHC proteins using vesicles, in addition to the SRP pathway.

  10. 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 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.

  11. 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

  12. Drive for thinness, affect regulation and physical activity in eating disorders: a daily life study.

    PubMed

    Vansteelandt, Kristof; Rijmen, Frank; Pieters, Guido; Probst, Michel; Vanderlinden, Johan

    2007-08-01

    Using Ecological Momentary Assessment, the within patient associations between drive for thinness, emotional states, momentary urge to be physically active and physical activity were studied in 32 inpatients with an eating disorder. Participants received an electronic device and had to indicate at nine random times a day during 1 week their momentary drive for thinness, positive and negative emotional states and their urge to be physically active and physical activity. Multilevel analyses indicated that patients with higher mean levels for urge to be physically active were characterized by lower body mass index (BMI) and chronically negative affect whereas patients with higher mean levels for physical activity were characterized by lower BMI and higher dispositions for drive for thinness. In addition, within patient relations between drive for thinness and urge to be physically active were moderated by BMI and chronically negative affect whereas within patient relations between drive for thinness and physical activity were moderated by BMI. Finally, also positive emotional states were significantly associated with physical activity within patients. By using a daily process design, characteristics of physical activity were revealed that have not been identified with assessment methods that have a lower time resolution.