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Sample records for highly toxic protein

  1. Toxic proteins in plants.

    PubMed

    Dang, Liuyi; Van Damme, Els J M

    2015-09-01

    Plants have evolved to synthesize a variety of noxious compounds to cope with unfavorable circumstances, among which a large group of toxic proteins that play a critical role in plant defense against predators and microbes. Up to now, a wide range of harmful proteins have been discovered in different plants, including lectins, ribosome-inactivating proteins, protease inhibitors, ureases, arcelins, antimicrobial peptides and pore-forming toxins. To fulfill their role in plant defense, these proteins exhibit various degrees of toxicity towards animals, insects, bacteria or fungi. Numerous studies have been carried out to investigate the toxic effects and mode of action of these plant proteins in order to explore their possible applications. Indeed, because of their biological activities, toxic plant proteins are also considered as potentially useful tools in crop protection and in biomedical applications, such as cancer treatment. Genes encoding toxic plant proteins have been introduced into crop genomes using genetic engineering technology in order to increase the plant's resistance against pathogens and diseases. Despite the availability of ample information on toxic plant proteins, very few publications have attempted to summarize the research progress made during the last decades. This review focuses on the diversity of toxic plant proteins in view of their toxicity as well as their mode of action. Furthermore, an outlook towards the biological role(s) of these proteins and their potential applications is discussed.

  2. Screening of high toxic Metarhizium strain against Plutella xylostella and its marking with green fluorescent protein.

    PubMed

    Cui, Qianqian; Zhang, Yi; Zang, Yanchao; Nong, Xiangqun; Wang, Guangjun; Zhang, Zehua

    2014-10-01

    Entomopathogenic fungus is proposed to be one of the best biocontrol agents against the destructive insect pest Plutella xylostella. In this study, we tested the virulence of 11 Metarhizium strain isolates against P. xylostella using a leaf dipping method, and found one strain, named 609, which had displayed the highest pathogenicity. Bioassay results showed that the accumulated corrected mortality rate was 86.7 % on the eighth day after inoculation with a spore concentration 1 × 10(8) conidia/mL, and that the time to 50 % lethality was 5.7-day. The strain was identified as Metarhizium anisopliae var. acridum by internal transcribed spacer (ITS) region sequencing. A green fluorescent protein (GFP) marker containing vector, camben-gfp, was constructed and delivered into strain 609 by Agrobacterium tumefaciens-mediated transformation. Six positive clones expressing GFP were selected and tested for toxicity against P. xylostella, all of which displayed the same toxicity as the parental wild type strain. The survival rate of transformant T1 was investigated by monitoring GFP levels at 4-day intervals after inoculation into soil. We found that the concentration of Metarhizium spores decreased sharply from 1 × 10(7) conidia/g to 1 × 10(6) conidia/g in the first 5 days after inoculation. The decreasing trend then stabilized and the spore count declined to approximately 1 × 10(4)-10(5) conidia/g after 1 month. The results of this study indicate that the expression of gfp gene in strain 609 does not alter the virulence capability of Metarhizium. This strain will therefore be useful for the control of P. xylostella and as a tool to study molecular biology properties and monitor colonization of M. anisopliae in the field. PMID:25037866

  3. High concentrations of protein test substances may have non-toxic effects on Daphnia magna: implications for regulatory study designs and ecological risk assessments for GM crops.

    PubMed

    Raybould, Alan; Burns, Andrea; Hamer, Mick

    2014-01-01

    Laboratory testing for possible adverse effects of insecticidal proteins on non-target organisms (NTOs) is an important part of many ecological risk assessments for regulatory decision-making about the cultivation of insect-resistant genetically modified (IRGM) crops. To increase confidence in the risk assessments, regulatory guidelines for effects testing specify that representative surrogate species for NTOs are exposed to concentrations of insecticidal proteins that are in excess of worst-case predicted exposures in the field. High concentrations in effects tests are achieved by using protein test substances produced in microbes, such as Escherichia coli. In a study that exposed Daphnia magna to a single high concentration of a microbial test substance containing Vip3Aa20, the insecticidal protein in MIR162 maize, small reductions in growth were observed. These effects were surprising as many other studies strongly suggest that the activity of Vip3Aa20 is limited to Lepidoptera. A plausible explanation for the effect on growth is that high concentrations of test substance have a non-toxic effect on Daphnia, perhaps by reducing its feeding rate. A follow-up study tested that hypothesis by exposing D. magna to several concentrations of Vip3Aa20, and a high concentration of a non-toxic protein, bovine serum albumin (BSA). Vip3Aa20 and BSA had sporadic effects on the reproduction and growth of D. magna. The pattern of the effects suggests that they result from non-toxic effects of high concentrations of protein, and not from toxicity. The implications of these results for regulatory NTO effects testing and ERA of IRGM crops are discussed.

  4. High concentrations of protein test substances may have non-toxic effects on Daphnia magna: implications for regulatory study designs and ecological risk assessments for GM crops.

    PubMed

    Raybould, Alan; Burns, Andrea; Hamer, Mick

    2014-01-01

    Laboratory testing for possible adverse effects of insecticidal proteins on non-target organisms (NTOs) is an important part of many ecological risk assessments for regulatory decision-making about the cultivation of insect-resistant genetically modified (IRGM) crops. To increase confidence in the risk assessments, regulatory guidelines for effects testing specify that representative surrogate species for NTOs are exposed to concentrations of insecticidal proteins that are in excess of worst-case predicted exposures in the field. High concentrations in effects tests are achieved by using protein test substances produced in microbes, such as Escherichia coli. In a study that exposed Daphnia magna to a single high concentration of a microbial test substance containing Vip3Aa20, the insecticidal protein in MIR162 maize, small reductions in growth were observed. These effects were surprising as many other studies strongly suggest that the activity of Vip3Aa20 is limited to Lepidoptera. A plausible explanation for the effect on growth is that high concentrations of test substance have a non-toxic effect on Daphnia, perhaps by reducing its feeding rate. A follow-up study tested that hypothesis by exposing D. magna to several concentrations of Vip3Aa20, and a high concentration of a non-toxic protein, bovine serum albumin (BSA). Vip3Aa20 and BSA had sporadic effects on the reproduction and growth of D. magna. The pattern of the effects suggests that they result from non-toxic effects of high concentrations of protein, and not from toxicity. The implications of these results for regulatory NTO effects testing and ERA of IRGM crops are discussed. PMID:25523175

  5. High concentrations of protein test substances may have non-toxic effects on Daphnia magna: Implications for regulatory study designs and ecological risk assessments for GM crops

    PubMed Central

    Raybould, Alan; Burns, Andrea; Hamer, Mick

    2014-01-01

    Laboratory testing for possible adverse effects of insecticidal proteins on non-target organisms (NTOs) is an important part of many ecological risk assessments for regulatory decision-making about the cultivation of insect-resistant genetically modified (IRGM) crops. To increase confidence in the risk assessments, regulatory guidelines for effects testing specify that representative surrogate species for NTOs are exposed to concentrations of insecticidal proteins that are in excess of worst-case predicted exposures in the field. High concentrations in effects tests are achieved by using protein test substances produced in microbes, such as Escherichia coli. In a study that exposed Daphnia magna to a single high concentration of a microbial test substance containing Vip3Aa20, the insecticidal protein in MIR162 maize, small reductions in growth were observed. These effects were surprising as many other studies strongly suggest that the activity of Vip3Aa20 is limited to Lepidoptera. A plausible explanation for the effect on growth is that high concentrations of test substance have a non-toxic effect on Daphnia, perhaps by reducing its feeding rate. A follow-up study tested that hypothesis by exposing D. magna to several concentrations of Vip3Aa20, and a high concentration of a non-toxic protein, bovine serum albumin (BSA). Vip3Aa20 and BSA had sporadic effects on the reproduction and growth of D. magna. The pattern of the effects suggests that they result from non-toxic effects of high concentrations of protein, and not from toxicity. The implications of these results for regulatory NTO effects testing and ERA of IRGM crops are discussed. PMID:25523175

  6. Cyt1Aa Protein of Bacillus thuringiensis Is Toxic to the Cottonwood Leaf Beetle, Chrysomela scripta, and Suppresses High Levels of Resistance to Cry3Aa

    PubMed Central

    Federici, Brian A.; Bauer, Leah S.

    1998-01-01

    The insecticidal activity of Bacillus thuringiensis is due primarily to Cry and Cyt proteins. Cry proteins are typically toxic to lepidopterous, coleopterous, or dipterous insects, whereas the known toxicity of Cyt proteins is limited to dipterans. We report here that a Cyt protein, Cyt1Aa, is also highly toxic to the cottonwood leaf beetle, Chrysomela scripta, with a median lethal concentration of 2.5 ng/mm2 of leaf surface for second-instar larvae. Additionally, we show that Cyt1Aa suppresses resistance to Cry3Aa greater than 5,000-fold in C. scripta, a level only partially overcome by Cry1Ba due to cross-resistance. Studies of the histopathology of C. scripta larvae treated with Cyt1Aa revealed disruption and sloughing of midgut epithelial cells, indicating that its mechanism of action against C. scripta is similar to that observed in mosquito and blackfly larvae. These novel properties suggest that Cyt proteins may have an even broader spectrum of activity against insects and, owing to their different mechanism of action in comparison to Cry proteins, might be useful in managing resistance to Cry3 and possibly other Cry toxins used in microbial insecticides and transgenic plants. PMID:9797292

  7. Protein-deficient diet and DDT toxicity*

    PubMed Central

    Boyd, Eldon M.; De Castro, Elvira S.

    1968-01-01

    The objective of the study reported was to investigate the hypothesis that a diet low in protein would affect ability to detoxify pesticides. If this were so, the results would have application to use of pesticides in areas of the world where the diet is normally low in protein. To test the hypothesis, weanling albino rats were fed for 4 weeks on three types of diet: (1) a standard laboratory chow diet, (2) a synthetic diet containing 8% casein but otherwise adequate in calories, vitamins and minerals, or (3) the same diet as group (2) but containing a normal protein content of 27% casein. DDT was selected as the challenging agent and was given orally at the end of 4 weeks in a range of doses which permitted delineation of the LD50 and associated clinicopathological signs of toxicity in rats of the 3 dietary groups. The median lethal dose and the syndrome of toxicity were essentially similar in all three groups of rats. Within the parameters of toxicity measured, the results suggest that DDT toxicity is not augmented by a low-protein diet. PMID:5302290

  8. Lead-protein interactions as a basis for lead toxicity.

    PubMed

    Goering, P L

    1993-01-01

    The interaction of lead (Pb) with proteins may represent a fundamental mechanism by which Pb exerts toxicity. In this overview, various factors which influence the interaction of Pb with proteins will be discussed. Pb interacts with enzyme functional groups, and high-affinity metal-binding proteins, such as Pb-binding proteins and metallothioneins, can mediate this Pb-enzyme interaction. Many other factors influence Pb-protein interactions including ligand competition and binding affinities; protein folding and the nature of the metal-binding site; rates of protein synthesis and degradation; and intracellular localization of the ligand and metal. The remainder of this overview will focus on specific examples of important proteins known to be influenced by Pb or which hypothetically may be influenced by Pb. Gaps in knowledge and important research needs are emphasized. Many of the factors discussed play a role in the relative sensitivity of various enzymes in heme biosynthesis to Pb. Disruption of this critical pathway by Pb may result in neuropathologies and accumulation of neurotoxic heme precursors. High-affinity metal-binding proteins have been shown to play a role in mediating Pb inhibition of the octameric Zn-containing enzyme, ALA dehydratase. Knowledge of regional localization in brain and the postnatal ontogeny of the high-affinity metal-binding proteins may be pivotal in understanding Pb neurotoxicity. Other specific examples related to or potentially related to Pb toxicity which are discussed include nucleic acid binding proteins, calmodulin, protein kinase C, and carbonic anhydrase. These proteins will serve as models to understand some basic principles and differences in Pb-protein interactions.

  9. A multigrain protein enriched diet mitigates fluoride toxicity.

    PubMed

    Vasant, Rupal A; Amaravadi V R L, Narasimhacharya

    2013-06-01

    Fluorosis is a major health problem in many parts of the world. The present work focuses on investigating the utility of nutrient and antioxidant rich grains- ragi, jowar, bajra, maize in formulation of basal, high carbohydrate low protein and low carbohydrate high protein diets in mitigating fluoride toxicity. Exposure to fluoride through drinking water not only significantly increased plasma glucose and lipid profiles, but also elevated both hepatic and renal lipid peroxidation, hepatic lipid profiles and G-6-Pase activity with a reduction in plasma HDL-C, hepatic glycogen content, hexokinase activity and antioxidant status. Even though basal and high carbohydrate diets did not significantly alter plasma glucose, lipid profiles in fluoride administered animals, protein enriched multigrain diet significantly decreased plasma glucose and lipid levels. However, the multigrain basal and high carbohydrate diets influenced the hepatic glycogen, lipid profiles, hexokinase and G-6-Pase activities, hepatic and renal lipid peroxidation and antioxidant status though not as significantly as that of multigrain diet enriched with protein. Thus the results of the present study indicate that both a multigrain diet rich in nutrients and antioxidants, and fortified with protein is useful in mitigating the fluoride toxicity. PMID:24425948

  10. Ureases as multifunctional toxic proteins: A review.

    PubMed

    Carlini, Celia R; Ligabue-Braun, Rodrigo

    2016-02-01

    Ureases are metalloenzymes that hydrolyze urea into ammonia and carbon dioxide. They were the first enzymes to be crystallized and, with them, the notion that enzymes are proteins became accepted. Novel toxic properties of ureases that are independent of their enzyme activity have been discovered in the last three decades. Since our first description of the neurotoxic properties of canatoxin, an isoform of the jack bean urease, which appeared in Toxicon in 1981, about one hundred articles have been published on "new" properties of plant and microbial ureases. Here we review the present knowledge on the non-enzymatic properties of ureases. Plant ureases and microbial ureases are fungitoxic to filamentous fungi and yeasts by a mechanism involving fungal membrane permeabilization. Plant and at least some bacterial ureases have potent insecticidal effects. This entomotoxicity relies partly on an internal peptide released upon proteolysis of ingested urease by insect digestive enzymes. The intact protein and its derived peptide(s) are neurotoxic to insects and affect a number of other physiological functions, such as diuresis, muscle contraction and immunity. In mammal models some ureases are acutely neurotoxic upon injection, at least partially by enzyme-independent effects. For a long time bacterial ureases have been recognized as important virulence factors of diseases by urease-producing microorganisms. Ureases activate exocytosis in different mammalian cells recruiting eicosanoids and Ca(2+)-dependent pathways, even when their ureolytic activity is blocked by an irreversible inhibitor. Ureases are chemotactic factors recognized by neutrophils (and some bacteria), activating them and also platelets into a pro-inflammatory "status". Secretion-induction by ureases may play a role in fungal and bacterial diseases in humans and other animals. The now recognized "moonlighting" properties of these proteins have renewed interest in ureases for their biotechnological

  11. Stress protein synthesis, a potential toxicity marker in Escherichia coli.

    PubMed

    Odberg-Ferragut, C; Espigares, M; Dive, D

    1991-06-01

    Various chemicals were tested in Escherichia coli for the ability to modify the cellular growth rate and to induce the synthesis of heat shock and stress proteins. The toxicity of chemicals as observed by modification of the growth rate depended on concentration and duration of treatment, except for thiram. In this last case, no modification was observed up to a concentration of 10 micrograms.ml-1. In contrast, all toxicants tested enhanced the synthesis of heat shock and stress proteins. The stress response was similar but not identical. Heat shock proteins and stress proteins appear to be a more sensitive toxicity marker than growth inhibition. Suggestions for the use of stress proteins as a practical bioassay are made.

  12. Determination of protein markers in human serum: Analysis of protein expression in toxic oil syndrome studies.

    PubMed

    Quero, Carmen; Colomé, Nuria; Prieto, Maria Rosario; Carrascal, Montserrat; Posada, Manuel; Gelpí, Emilio; Abian, Joaquin

    2004-02-01

    Toxic oil syndrome (TOS) is a disease that appeared in Spain in 1981. It affected more than 20 000 people and produced over 300 deaths in the first 2 years. In this paper, a prospective study on the differences in gene expression in sera between a control versus a TOS-affected population, both originally exposed to the toxic oil, is presented. Differential protein expression was analyzed by two-dimensional electrophoresis (2-DE). Several problems related with serum analysis by 2-DE were addressed in order to improve protein detection in the gel images. Three new commercial systems for albumin depletion were tested to optimize the detection of minor proteins that can be obscured by the presence of a few families of high abundance proteins (albumin, immunoglobulins). Other factors, such as the use of nonionic reductants or the presence of thiourea in the gels, were also tested. From these optimized images, a group of 329 major gel spots was located, matched and compared in serum samples. Thirty-five of these protein spots were found to be under- or overexpressed in TOS patients (> three-fold increase or decrease). Proteins in the differential spots were identified by matrix-assisted laser desorption/ionization-time of flight peptide map fingerprinting and database search. Several haptoglobin isoforms were found to be differentially expressed, showing expression phenotypes that could be related with TOS affection. Haptoglobin phenotypes have been previously reported to have important biological and clinical consequences and have been described as risk factors for several diseases.

  13. Oxidative Stress, Unfolded Protein Response, and Apoptosis in Developmental Toxicity

    PubMed Central

    Kupsco, Allison; Schlenk, Daniel

    2016-01-01

    Physiological development requires precise spatiotemporal regulation of cellular and molecular processes. Disruption of these key events can generate developmental toxicity in the form of teratogenesis or mortality. The mechanism behind many developmental toxicants remains unknown. While recent work has focused on the unfolded protein response (UPR), oxidative stress, and apoptosis in the pathogenesis of disease, few studies have addressed their relationship in developmental toxicity. Redox regulation, UPR, and apoptosis are essential for physiological development and can be disturbed by a variety of endogenous and exogenous toxicants to generate lethality and diverse malformations. This review examines the current knowledge of the role of oxidative stress, UPR, and apoptosis in physiological development as well as in developmental toxicity, focusing on studies and advances in vertebrates model systems. PMID:26008783

  14. Synergistic Effects of Toxic Elements on Heat Shock Proteins

    PubMed Central

    Mahmood, Khalid; Mahmood, Qaisar; Irshad, Muhammad; Hussain, Jamshaid

    2014-01-01

    Heat shock proteins show remarkable variations in their expression levels under a variety of toxic conditions. A research span expanded over five decades has revealed their molecular characterization, gene regulation, expression patterns, vast similarity in diverse groups, and broad range of functional capabilities. Their functions include protection and tolerance against cytotoxic conditions through their molecular chaperoning activity, maintaining cytoskeleton stability, and assisting in cell signaling. However, their role as biomarkers for monitoring the environmental risk assessment is controversial due to a number of conflicting, validating, and nonvalidating reports. The current knowledge regarding the interpretation of HSPs expression levels has been discussed in the present review. The candidature of heat shock proteins as biomarkers of toxicity is thus far unreliable due to synergistic effects of toxicants and other environmental factors. The adoption of heat shock proteins as “suit of biomarkers in a set of organisms” requires further investigation. PMID:25136596

  15. Toxicity of Bacillus thuringiensis crystal proteins against eri silkworm, Samia cynthia ricini (Lepidoptera: Saturniidae).

    PubMed

    Sandeep Kumar, Donthula; Tarakeswari, Muddanuru; Lakshminarayana, Maddukuri; Sujatha, Mulpuri

    2016-07-01

    Ten purified crystal proteins of Bacillus thuringiensis (Bt) were tested at concentrations ranging from 2.93 to 3000ng/cm(2) for their toxicity to eri silkworm through protein paint bioassays using castor leaves. Based on LC50 values, Cry1Aa (2.6ng/cm(2)) was highly toxic followed by Cry1Ac (29.3ng/cm(2)) and Cry1Ab (68.7ng/cm(2)). The Cry1Ca and Cry1Ea proteins were moderately toxic to eri silkworm larvae and resulted in 23% and 28% mortality, respectively at the highest concentration tested (3000ng/cm(2)). Only reduction in larval weight was observed with Cry2Aa, Cry1Da and Cry9Aa proteins while Cry3Aa and Cry1Ba proteins were found to be nontoxic. PMID:27377590

  16. Silver nanoparticle protein corona and toxicity: a mini-review.

    PubMed

    Durán, Nelson; Silveira, Camila P; Durán, Marcela; Martinez, Diego Stéfani T

    2015-01-01

    Silver nanoparticles are one of the most important materials in the nanotechnology industry. Additionally, the protein corona is emerging as a key entity at the nanobiointerface; thus, a comprehensive understanding of the interactions between proteins and silver nanoparticles is imperative. Therefore, literature reporting studies involving both single molecule protein coronas (i.e., bovine and human serum albumin, tubulin, ubiquitin and hyaluronic-binding protein) and complex protein coronas (i.e., fetal bovine serum and yeast extract proteins) were selected to demonstrate the effects of protein coronas on silver nanoparticle cytotoxicity and antimicrobial activity. There is evidence that distinct and differential protein components may yield a "protein corona signature" that is related to the size and/or surface curvature of the silver nanoparticles. Therefore, the formation of silver nanoparticle protein coronas together with the biological response to these coronas (i.e., oxidative stress, inflammation and cytotoxicity) as well as other cellular biophysicochemical mechanisms (i.e., endocytosis, biotransformation and biodistribution) will be important for nanomedicine and nanotoxicology. Researchers may benefit from the information contained herein to improve biotechnological applications of silver nanoparticles and to address related safety concerns. In summary, the main aim of this mini-review is to highlight the relationship between the formation of silver nanoparticle protein coronas and toxicity. PMID:26337542

  17. Low toxicity high temperature PMR polyimide

    NASA Technical Reports Server (NTRS)

    Pater, Ruth H. (Inventor)

    1992-01-01

    In-situ polymerization of monomer reactants (PMR) type polyimides constitute an important class of ultra high performance composite matrix resins. PMR-15 is the best known and most widely used PMR polyimide. An object of the present invention is to provide a substantially improved high temperature PMR-15 system that exhibits better processability, toughness, and thermo-oxidative stability than PMR-15, as well as having a low toxicity. Another object is to provide new PMR polyimides that are useful as adhesives, moldings, and composite matrices. By the present invention, a new PMR polyimide comprises a mixture of the following compounds: 3,4'-oxydianiline (3,4'-ODA), NE, and BTDE which are then treated with heat. This PMR was designated LaRC-RP46 and has a broader processing window, better reproducibility of high quality composite parts, better elevated temperature mechanical properties, and higher retention of mechanical properties at an elevated temperature, particularly, at 371 C.

  18. Identification of protein adduction using mass spectrometry: Protein adducts as biomarkers and predictors of toxicity mechanisms.

    PubMed

    Yang, Xiangkun; Bartlett, Michael G

    2016-03-15

    The determination of protein-xenobiotic adducts using mass spectrometry is an emerging area which allows detailed understanding of the underlying mechanisms involved in toxicity. These approaches can also be used to reveal potential biomarkers of exposure or toxic response. The following review covers studies of protein adducts resulting from exposure to a wide variety of xenobiotics including organophosphates, polycyclic aromatic hydrocarbons, acetaminophen, alkylating agents and other related compounds. PMID:26842586

  19. Toxicological relationships between proteins obtained from protein target predictions of large toxicity databases

    SciTech Connect

    Nigsch, Florian; Mitchell, John B.O.

    2008-09-01

    The combination of models for protein target prediction with large databases containing toxicological information for individual molecules allows the derivation of 'toxiclogical' profiles, i.e., to what extent are molecules of known toxicity predicted to interact with a set of protein targets. To predict protein targets of drug-like and toxic molecules, we built a computational multiclass model using the Winnow algorithm based on a dataset of protein targets derived from the MDL Drug Data Report. A 15-fold Monte Carlo cross-validation using 50% of each class for training, and the remaining 50% for testing, provided an assessment of the accuracy of that model. We retained the 3 top-ranking predictions and found that in 82% of all cases the correct target was predicted within these three predictions. The first prediction was the correct one in almost 70% of cases. A model built on the whole protein target dataset was then used to predict the protein targets for 150 000 molecules from the MDL Toxicity Database. We analysed the frequency of the predictions across the panel of protein targets for experimentally determined toxicity classes of all molecules. This allowed us to identify clusters of proteins related by their toxicological profiles, as well as toxicities that are related. Literature-based evidence is provided for some specific clusters to show the relevance of the relationships identified.

  20. Oligomerization and toxicity of A{beta} fusion proteins

    SciTech Connect

    Caine, Joanne M.; Bharadwaj, Prashant R.; Sankovich, Sonia E.; Ciccotosto, Giuseppe D.; Streltsov, Victor A.; Varghese, Jose

    2011-06-10

    Highlights: {yields} We expressed amyloid-{beta} (A{beta}) peptide as a soluble maltose binding protein fusion (MBP-A{beta}42 and MBP-A{beta}16). {yields} The full length A{beta} peptide fusion, MBP-A{beta}42, forms oligomeric species as determined by SDS-PAGE gels, gel filtration and DLS. {yields} The MBP-A{beta}42, but not MBP-A{beta}16 or MBP alone, is toxic to both yeast and mammalian cells as determined by toxicity assays. -- Abstract: This study has found that the Maltose binding protein A{beta}42 fusion protein (MBP-A{beta}42) forms soluble oligomers while the shorter MBP-A{beta}16 fusion and control MBP did not. MBP-A{beta}42, but neither MBP-A{beta}16 nor control MBP, was toxic in a dose-dependent manner in both yeast and primary cortical neuronal cells. This study demonstrates the potential utility of MBP-A{beta}42 as a reagent for drug screening assays in yeast and neuronal cell cultures and as a candidate for further A{beta}42 characterization.

  1. Effects of cadmium on Drosophila: toxicity, proteins, and transfer RNAs

    SciTech Connect

    Jacobson, K.B.; Opresko, L.; Owenby, R.K.; Christie, N.T.

    1981-01-01

    An animal model with well-defined genetic and biochemical characteristics is needed for a detailed understanding of the mechanism of toxicity by metal ions. Drosophila melanogaster was used in the present study to demonstrate a number of responses to Cd/sup 2 +/, including lethality, age-related changes in resistance, alterations of the normal developmental changes in proteins, and alterations in specific transfer RNAs. Genotype-specific differences in resistance to Cd/sup 2 +/ were found: the v; bw strain was 5-10 times more resistant than su(s)/sup 2/v; bw for developmental exposure; upon treatment of the young adults the differences were in the same direction, but the sensitivities differed by only two- to three-fold. The adult fly became more sensitive to Cd/sup 2 +/ as it aged through 2 weeks, but changed little thereafter.The electrophoretic patterns of proteins of adult flies underwent changes during aging from 1 to 8 days; these changes were markedly altered by 0.55 mM CdCl/sub 2/ but not by 0.74 mM ZnCl/sub 2/ in the medium on which the flies were maintained. The appearance of queuosine-containing tRNA was stimulated by CdCl/sub 2/ (0.05-0.8 mM) in the growth medium, but not by ZnCl/sub 2/ (0.07-1.1 mM).Further studies involving D. melanogaster should be useful in defining specific interactions of toxic metal ions with macromolecules to enhance the understanding of the toxic effects of these and similar pollutants.

  2. Protein kinase CK2 regulates metal toxicity in neuronal cells.

    PubMed

    Zaman, Mohammad S; Johnson, Adam J; Bobek, Gabriele; Kueh, Sindy; Kersaitis, Cindy; Bailey, Trevor D; Buskila, Yossi; Wu, Ming J

    2016-01-01

    Protein kinase CK2 is a pleiotropic tetrameric enzyme, regulating numerous biological processes from cell proliferation to stress response. This study demonstrates for the first time that CK2 is involved in the regulation of metal uptake and toxicity in neuronal cells. After the determination of inhibitory concentrations (IC50) for a range of metal salts (ZnSO4, Al(mal)3, CoCl2, CrO3, NaAsO2 and CaCl2) in Neuro-2a mouse neuroblastoma cells, the effect of CK2 on metal toxicity was investigated by three lines of experiments using CK2 inhibitors, metal ion specific fluorophores and siRNA-mediated knockdown of CK2 expression. The results showed that both CK2 inhibitors, 4,5,6,7-tetrabromobenzotriazole (TBB) and quinalizarin, markedly reduced the toxicity of Zn(ii), Al(iii), Co(ii), Cr(vi) and As(iii). Confocal microscopy imaging revealed that Zn(ii) uptake was accompanied by the increase of intracellular Ca(ii) in Neuro-2a cells treated with IC50 of ZnSO4 (240 μM), and such concurrent elevation of intracellular Zn(ii) and Ca(ii) was blocked by TBB and quinalizarin. The role of CK2 in metal uptake was further characterised using specific siRNA against each of the three subunits (CK2α, α' and β) and the data demonstrate that CK2α' is the prominent subunit regulating the metal toxicity. Finally, the role of CK2 in metal toxicity was found to be conserved in the distant species-Saccharomyces cerevisiae by employing the complete deletion mutants of CK2 (cka1Δ, cka2Δ, ckb1Δ and ckb2Δ). Taken together, these findings shed light on a new facet of CK2 functionality and provide a basis for further research on the regulation of Zn(ii) and Ca(ii) homeostasis by CK2.

  3. Highly thermostable fluorescent proteins

    DOEpatents

    Bradbury, Andrew M.; Waldo, Geoffrey S.; Kiss, Csaba

    2011-03-22

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  4. Highly thermostable fluorescent proteins

    DOEpatents

    Bradbury, Andrew M.; Waldo, Geoffrey S.; Kiss, Csaba

    2012-05-01

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  5. Highly thermostable fluorescent proteins

    DOEpatents

    Bradbury, Andrew M.; Waldo, Geoffrey S.; Kiss, Csaba

    2011-11-29

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  6. Differential Toxicity of Antibodies to the Prion Protein.

    PubMed

    Reimann, Regina R; Sonati, Tiziana; Hornemann, Simone; Herrmann, Uli S; Arand, Michael; Hawke, Simon; Aguzzi, Adriano

    2016-01-01

    Antibodies against the prion protein PrPC can antagonize prion replication and neuroinvasion, and therefore hold promise as possible therapeutics against prion diseases. However, the safety profile of such antibodies is controversial. It was originally reported that the monoclonal antibody D13 exhibits strong target-related toxicity, yet a subsequent study contradicted these findings. We have reported that several antibodies against certain epitopes of PrPC, including antibody POM1, are profoundly neurotoxic, yet antibody ICSM18, with an epitope that overlaps with POM1, was reported to be innocuous when injected into mouse brains. In order to clarify this confusing situation, we assessed the neurotoxicity of antibodies D13 and ICSM18 with dose-escalation studies using diffusion-weighted magnetic resonance imaging and various histological techniques. We report that both D13 and ICSM18 induce rapid, dose-dependent, on-target neurotoxicity. We conclude that antibodies directed to this region may not be suitable as therapeutics. No such toxicity was found when antibodies against the flexible tail of PrPC were administered. Any attempt at immunotherapy or immunoprophylaxis of prion diseases should account for these potential untoward effects. PMID:26821311

  7. Differential Toxicity of Antibodies to the Prion Protein

    PubMed Central

    Hornemann, Simone; Herrmann, Uli S.; Arand, Michael; Hawke, Simon; Aguzzi, Adriano

    2016-01-01

    Antibodies against the prion protein PrPC can antagonize prion replication and neuroinvasion, and therefore hold promise as possible therapeutics against prion diseases. However, the safety profile of such antibodies is controversial. It was originally reported that the monoclonal antibody D13 exhibits strong target-related toxicity, yet a subsequent study contradicted these findings. We have reported that several antibodies against certain epitopes of PrPC, including antibody POM1, are profoundly neurotoxic, yet antibody ICSM18, with an epitope that overlaps with POM1, was reported to be innocuous when injected into mouse brains. In order to clarify this confusing situation, we assessed the neurotoxicity of antibodies D13 and ICSM18 with dose-escalation studies using diffusion-weighted magnetic resonance imaging and various histological techniques. We report that both D13 and ICSM18 induce rapid, dose-dependent, on-target neurotoxicity. We conclude that antibodies directed to this region may not be suitable as therapeutics. No such toxicity was found when antibodies against the flexible tail of PrPC were administered. Any attempt at immunotherapy or immunoprophylaxis of prion diseases should account for these potential untoward effects. PMID:26821311

  8. Essential bacterial helicases that counteract the toxicity of recombination proteins.

    PubMed

    Petit, Marie-Agnès; Ehrlich, Dusko

    2002-06-17

    PcrA, Rep and UvrD are three closely related bacterial helicases with a DExx signature. PcrA is encoded by Gram-positive bacteria and is essential for cell growth. Rep and UvrD are encoded by Gram-negative bacteria, and mutants lacking both helicases are also not viable. To understand the non-viability of the helicase mutants, we characterized spontaneous extragenic suppressors of a Bacillus subtilis pcrA null mutation. Here we report that one of these suppressors maps in recF and that previously isolated mutations in B.subtilis recF, recL, recO and recR, which belong to the same complementation group, all suppress the lethality of a pcrA mutation. Similarly, recF, recO or recR mutations suppress the lethality of the Escherichia coli rep uvrD double mutant. We conclude that RecFOR proteins are toxic in cells devoid of PcrA in Gram-positive bacteria, or Rep and UvrD in Gram-negative bacteria, and propose that the RecFOR proteins interfere with an essential cellular process, possibly replication, when DExx helicases PcrA, or Rep and UvrD are absent. PMID:12065426

  9. Nrf2 enhances myocardial clearance of toxic ubiquitinated proteins.

    PubMed

    Wang, Wenjuan; Li, Siying; Wang, Hui; Li, Bin; Shao, Lei; Lai, Yimu; Horvath, Gary; Wang, Qian; Yamamoto, Masayuki; Janicki, Joseph S; Wang, Xing Li; Tang, Dongqi; Cui, Taixing

    2014-07-01

    Nuclear factor erythroid-2 related factor 2 (Nrf2) is a master transcription factor that controls the basal and inducible expression of a battery of antioxidant genes and other cytoprotective phase II detoxifying enzymes. While knockout of Nrf2 exaggerates cardiac pathological remodeling and dysfunction in diverse pathological settings, pharmacological activation of Nrf2 protects against cardiomyocyte injury and cardiac dysfunction. In contrast, there is also a concern that the chronic activation of Nrf2 secondary to oxidative stress is a contributing mechanism for the reductive stress-mediated heart failure. However, a direct link between cardiac specific activation of Nrf2 and cardiac protection or dysfunction in vivo remains to be established. Therefore, we investigated the effect of cardiomyocyte-specific transgenic activation of Nrf2 (Nrf2(ctg)) on cardiac pathological remodeling and dysfunction. We found that the cardiomyocyte-specific activation of Nrf2 suppressed myocardial oxidative stress as well as cardiac apoptosis, fibrosis, hypertrophy, and dysfunction in a setting of sustained pressure overload induced by transverse aortic arch constriction (TAC) in mice. Notably, the constitutive activation of Nrf2 increased the steady level of autophagosomes while decreasing the ubiquitinated protein aggregates in the heart after TAC. Nrf2 gene gain- and loss-of-function approaches revealed that Nrf2 enhances autophagosome formation and autophagic flux in cardiomyocytes. Unexpectedly, while Nrf2 minimally regulated apoptosis, it suppressed significantly the proteotoxic necrosis in cardiomyocytes. In addition, Nrf2 attenuated the proteocytotoxicity presumably via enhancing autophagy-mediated clearance of ubiquitinated protein aggregates in cardiomyocytes. Taken together, we demonstrated for the first time that cardiac specific activation of Nrf2 suppresses cardiac maladaptive remodeling and dysfunction most likely by enhancing autophagic clearance of toxic protein

  10. Nrf2 enhances myocardial clearance of toxic ubiquitinated proteins

    PubMed Central

    Wang, Wenjuan; Li, Siying; Wang, Hui; Li, Bin; Shao, Lei; Lai, Yimu; Horvath, Gary; Wang, Qian; Yamamoto, Masayuki; Janicki, Joseph S.; Wang, Xing Li; Tang, Dongqi; Cui, Taixing

    2015-01-01

    Nuclear factor erythroid-2 related factor 2 (Nrf2) is a master transcription factor that controls the basal and inducible expression of a battery of antioxidant genes and other cytoprotective phase II detoxifying enzymes. While knockout of Nrf2 exaggerates cardiac pathological remodeling and dysfunction in diverse pathological settings, pharmacological activation of Nrf2 protects against cardiomyocyte injury and cardiac dysfunction. In contrast, there is also a concern that the chronic activation of Nrf2 secondary to oxidative stress is a contributing mechanism for the reductive stress-mediated heart failure. However, a direct link between cardiac specific activation of Nrf2 and cardiac protection or dysfunction in vivo remains to be established. Therefore, we investigated the effect of cardiomyocyte-specific transgenic activation of Nrf2 (Nrf2ctg) on cardiac pathological remodeling and dysfunction. We found that the cardiomyocyte-specific activation of Nrf2 suppressed myocardial oxidative stress as well as cardiac apoptosis, fibrosis, hypertrophy, and dysfunction in a setting of sustained pressure overload induced by transverse aortic arch constriction (TAC) in mice. Notably, the constitutive activation of Nrf2 increased the steady level of autophagosomes while decreasing the ubiquitinated protein aggregates in the heart after TAC. Nrf2 gene gain- and loss-of-function approaches revealed that Nrf2 enhances autophagosome formation and autophagic flux in cardiomyocytes. Unexpectedly, while Nrf2 minimally regulated apoptosis, it suppressed significantly the proteotoxic necrosis in cardiomyocytes. In addition, Nrf2 attenuated the proteocytotoxicity presumably via enhancing autophagy-mediated clearance of ubiquitinated protein aggregates in cardiomyocytes. Taken together, we demonstrated for the first time that cardiac specific activation of Nrf2 suppresses cardiac maladaptive remodeling and dysfunction most likely by enhancing autophagic clearance of toxic protein

  11. [Toxic effects of high concentrations of ammonia on Euglena gracilis].

    PubMed

    Liu, Yan; Shi, Xiao-Rong; Cui, Yi-Bin; Li, Mei

    2013-11-01

    Ammonia is among the common contaminants in aquatic environments. The present study aimed at evaluation of the toxicity of ammonia at high concentration by detecting its effects on the growth, pigment contents, antioxidant enzyme activities, and DNA damage (comet assay) of a unicellular microalga, Euglena gracilis. Ammonia restrained the growth of E. gracilis, while at higher concentrations, ammonia showed notable inhibition effect, the growth at 2 000 mg x L(-1) was restrained to 55.7% compared with that of the control; The contents of photosynthetic pigments and protein went up with increasing ammonia dosage and decreased when the ammonia concentration was above 1000 mg x L(-1); In addition, there was an obvious increase in SOD and POD activities, at higher concentration (2 000 mg x L(-1)), activities of SOD and POD increased by 30.7% and 49.4% compared with those of the control, indicating that ammonia could promote activities of antioxidant enzymes in E. gracilis; The degree of DNA damage observed in the comet assay increased with increasing ammonia concentration, which suggested that high dose of ammonia may have potential mutagenicity on E. gracilis.

  12. Treating chronic arsenic toxicity with high selenium lentil diets

    SciTech Connect

    Sah, Shweta; Vandenberg, Albert; Smits, Judit

    2013-10-01

    Arsenic (As) toxicity causes serious health problems in humans, especially in the Indo-Gangetic plains and mountainous areas of China. Selenium (Se), an essential micronutrient is a potential mitigator of As toxicity due to its antioxidant and antagonistic properties. Selenium is seriously deficient in soils world-wide but is present at high, yet non-toxic levels in the great plains of North America. We evaluate the potential of dietary Se in counteracting chronic As toxicity in rats through serum biochemistry, blood glutathione levels, immunotoxicity (antibody response), liver peroxidative stress, thyroid response and As levels in tissues and excreta. To achieve this, we compare diets based on high-Se Saskatchewan (SK) lentils versus low-Se lentils from United States. Rats drank control (0 ppm As) or As (40 ppm As) water while consuming SK lentils (0.3 ppm Se) or northwestern USA lentils (< 0.01 ppm Se) diets for 14 weeks. Rats on high Se diets had higher glutathione levels regardless of As exposure, recovered antibody responses in As-exposed group, higher fecal and urinary As excretion and lower renal As residues. Selenium deficiency caused greater hepatic peroxidative damage in the As exposed animals. Thyroid hormones, triiodothyronine (T3) and thyroxine (T4), were not different. After 14 weeks of As exposure, health indicators in rats improved in response to the high Se lentil diets. Our results indicate that high Se lentils have a potential to mitigate As toxicity in laboratory mammals, which we hope will translate into benefits for As exposed humans. - Highlights: • We reduce chronic arsenic toxicity in rats with a whole food solution. • High selenium lentils decrease liver damage and increase blood glutathione levels. • High selenium lentil diets increase urinary and fecal arsenic excretion. • High selenium lentil diets decrease arsenic levels in kidney, the storage organ. • High selenium lentil diets reverse arsenic suppression of the B cell

  13. Accelerated Hematopoietic Toxicity by High Energy 56Fe Radiation

    PubMed Central

    Datta, Kamal; Suman, Shubhankar; Trani, Daniela; Doiron, Kathryn; Rotolo, Jimmy A.; Kallakury, Bhaskar V. S.; Kolesnick, Richard; Cole, Michael F.; Fornace, Albert J.

    2013-01-01

    Purpose There is little information on the relative toxicity of highly charged (Z) high-energy (HZE) radiation in animal models compared to γ or x-rays, and the general assumption based on in vitro studies has been that acute toxicity is substantially greater. Methods C57BL/6J mice were irradiated with 56Fe ions (1 GeV/nucleon), and acute (within 30 d) toxicity compared to that of γ rays or protons (1 GeV). To assess relative hematopoietic and gastrointestinal toxicity, the effects of 56Fe ions were compared to γ rays using complete blood count (CBC), bone marrow granulocyte-macrophage colony forming unit (GM-CFU), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for apoptosis in bone marrow, and intestinal crypt survival. Results Although onset was more rapid, 56Fe ions were only slightly more toxic than γ rays or protons with lethal dose (LD)50/30 (a radiation dose at which 50% lethality occurs at 30-day) values of 5.8, 7.25, and 6.8 Gy respectively with relative biologic effectiveness for 56Fe ions of 1.25 and 1.06 for protons. Conclusions 56Fe radiation caused accelerated and more severe hematopoietic toxicity. Early mortality correlated with more profound leukopenia and subsequent sepsis. Results indicate that there is selective enhanced toxicity to bone marrow progenitor cells, which are typically resistant to γ rays, and bone marrow stem cells, because intestinal crypt cells did not show increased HZE toxicity. PMID:22077279

  14. High throughput protein production screening

    DOEpatents

    Beernink, Peter T.; Coleman, Matthew A.; Segelke, Brent W.

    2009-09-08

    Methods, compositions, and kits for the cell-free production and analysis of proteins are provided. The invention allows for the production of proteins from prokaryotic sequences or eukaryotic sequences, including human cDNAs using PCR and IVT methods and detecting the proteins through fluorescence or immunoblot techniques. This invention can be used to identify optimized PCR and WT conditions, codon usages and mutations. The methods are readily automated and can be used for high throughput analysis of protein expression levels, interactions, and functional states.

  15. Protein Corona Modulates Uptake and Toxicity of Nanoceria via Clathrin-Mediated Endocytosis.

    PubMed

    Mazzolini, Julie; Weber, Ralf J M; Chen, Hsueh-Shih; Khan, Abdullah; Guggenheim, Emily; Shaw, Robert K; Chipman, James K; Viant, Mark R; Rappoport, Joshua Z

    2016-08-01

    Particles present in diesel exhaust have been proposed as a significant contributor to the development of acute and chronic lung diseases, including respiratory infection and allergic asthma. Nanoceria (CeO2 nanoparticles) are used to increase fuel efficiency in internal combustion engines, are present in exhaust fumes, and could affect cells of the airway. Components from the environment such as biologically derived proteins, carbohydrates, and lipids can form a dynamic layer, commonly referred to as the "protein corona" which alters cellular nanoparticle interactions and internalization. Using confocal reflectance microscopy, we quantified nanoceria uptake by lung-derived cells in the presence and absence of a serum-derived protein corona. Employing mass spectrometry, we identified components of the protein corona, and demonstrated that the interaction between transferrin in the protein corona and the transferrin receptor is involved in mediating the cellular entry of nanoceria via clathrin-mediated endocytosis. Furthermore, under these conditions nanoceria does not affect cell growth, viability, or metabolism, even at high concentration. Alternatively, despite the antioxidant capacity of nanoceria, in serum-free conditions these nanoparticles induce plasma membrane disruption and cause changes in cellular metabolism. Thus, our results identify a specific receptor-mediated mechanism for nanoceria entry, and provide significant insight into the potential for nanoparticle-dependent toxicity.

  16. Protein Corona Modulates Uptake and Toxicity of Nanoceria via Clathrin-Mediated Endocytosis.

    PubMed

    Mazzolini, Julie; Weber, Ralf J M; Chen, Hsueh-Shih; Khan, Abdullah; Guggenheim, Emily; Shaw, Robert K; Chipman, James K; Viant, Mark R; Rappoport, Joshua Z

    2016-08-01

    Particles present in diesel exhaust have been proposed as a significant contributor to the development of acute and chronic lung diseases, including respiratory infection and allergic asthma. Nanoceria (CeO2 nanoparticles) are used to increase fuel efficiency in internal combustion engines, are present in exhaust fumes, and could affect cells of the airway. Components from the environment such as biologically derived proteins, carbohydrates, and lipids can form a dynamic layer, commonly referred to as the "protein corona" which alters cellular nanoparticle interactions and internalization. Using confocal reflectance microscopy, we quantified nanoceria uptake by lung-derived cells in the presence and absence of a serum-derived protein corona. Employing mass spectrometry, we identified components of the protein corona, and demonstrated that the interaction between transferrin in the protein corona and the transferrin receptor is involved in mediating the cellular entry of nanoceria via clathrin-mediated endocytosis. Furthermore, under these conditions nanoceria does not affect cell growth, viability, or metabolism, even at high concentration. Alternatively, despite the antioxidant capacity of nanoceria, in serum-free conditions these nanoparticles induce plasma membrane disruption and cause changes in cellular metabolism. Thus, our results identify a specific receptor-mediated mechanism for nanoceria entry, and provide significant insight into the potential for nanoparticle-dependent toxicity. PMID:27638694

  17. Treating chronic arsenic toxicity with high selenium lentil diets.

    PubMed

    Sah, Shweta; Vandenberg, Albert; Smits, Judit

    2013-10-01

    Arsenic (As) toxicity causes serious health problems in humans, especially in the Indo-Gangetic plains and mountainous areas of China. Selenium (Se), an essential micronutrient is a potential mitigator of As toxicity due to its antioxidant and antagonistic properties. Selenium is seriously deficient in soils world-wide but is present at high, yet non-toxic levels in the great plains of North America. We evaluate the potential of dietary Se in counteracting chronic As toxicity in rats through serum biochemistry, blood glutathione levels, immunotoxicity (antibody response), liver peroxidative stress, thyroid response and As levels in tissues and excreta. To achieve this, we compare diets based on high-Se Saskatchewan (SK) lentils versus low-Se lentils from United States. Rats drank control (0ppm As) or As (40ppm As) water while consuming SK lentils (0.3ppm Se) or northwestern USA lentils (<0.01ppm Se) diets for 14weeks. Rats on high Se diets had higher glutathione levels regardless of As exposure, recovered antibody responses in As-exposed group, higher fecal and urinary As excretion and lower renal As residues. Selenium deficiency caused greater hepatic peroxidative damage in the As exposed animals. Thyroid hormones, triiodothyronine (T3) and thyroxine (T4), were not different. After 14weeks of As exposure, health indicators in rats improved in response to the high Se lentil diets. Our results indicate that high Se lentils have a potential to mitigate As toxicity in laboratory mammals, which we hope will translate into benefits for As exposed humans. PMID:23800687

  18. Treating chronic arsenic toxicity with high selenium lentil diets.

    PubMed

    Sah, Shweta; Vandenberg, Albert; Smits, Judit

    2013-10-01

    Arsenic (As) toxicity causes serious health problems in humans, especially in the Indo-Gangetic plains and mountainous areas of China. Selenium (Se), an essential micronutrient is a potential mitigator of As toxicity due to its antioxidant and antagonistic properties. Selenium is seriously deficient in soils world-wide but is present at high, yet non-toxic levels in the great plains of North America. We evaluate the potential of dietary Se in counteracting chronic As toxicity in rats through serum biochemistry, blood glutathione levels, immunotoxicity (antibody response), liver peroxidative stress, thyroid response and As levels in tissues and excreta. To achieve this, we compare diets based on high-Se Saskatchewan (SK) lentils versus low-Se lentils from United States. Rats drank control (0ppm As) or As (40ppm As) water while consuming SK lentils (0.3ppm Se) or northwestern USA lentils (<0.01ppm Se) diets for 14weeks. Rats on high Se diets had higher glutathione levels regardless of As exposure, recovered antibody responses in As-exposed group, higher fecal and urinary As excretion and lower renal As residues. Selenium deficiency caused greater hepatic peroxidative damage in the As exposed animals. Thyroid hormones, triiodothyronine (T3) and thyroxine (T4), were not different. After 14weeks of As exposure, health indicators in rats improved in response to the high Se lentil diets. Our results indicate that high Se lentils have a potential to mitigate As toxicity in laboratory mammals, which we hope will translate into benefits for As exposed humans.

  19. Developmental toxicity assay using high content screening of zebrafish embryos

    PubMed Central

    Lantz-McPeak, Susan; Guo, Xiaoqing; Cuevas, Elvis; Dumas, Melanie; Newport, Glenn D.; Ali, Syed F.; Paule, Merle G.; Kanungo, Jyotshna

    2016-01-01

    Typically, time-consuming standard toxicological assays using the zebrafish (Danio rerio) embryo model evaluate mortality and teratogenicity after exposure during the first 2 days post-fertilization. Here we describe an automated image-based high content screening (HCS) assay to identify the teratogenic/embryotoxic potential of compounds in zebrafish embryos in vivo. Automated image acquisition was performed using a high content microscope system. Further automated analysis of embryo length, as a statistically quantifiable endpoint of toxicity, was performed on images post-acquisition. The biological effects of ethanol, nicotine, ketamine, caffeine, dimethyl sulfoxide and temperature on zebrafish embryos were assessed. This automated developmental toxicity assay, based on a growth-retardation endpoint should be suitable for evaluating the effects of potential teratogens and developmental toxicants in a high throughput manner. This approach can significantly expedite the screening of potential teratogens and developmental toxicants, thereby improving the current risk assessment process by decreasing analysis time and required resources. Published 2014. This article is a U.S. Government work and is in the public domain in the USA. PMID:24871937

  20. Legal but lethal: functional protein aggregation at the verge of toxicity

    PubMed Central

    Falsone, Angelika; Falsone, S. Fabio

    2015-01-01

    Many neurodegenerative disorders are linked to irreversible protein aggregation, a process that usually comes along with toxicity and serious cellular damage. However, it is emerging that protein aggregation can also serve for physiological purposes, as impressively shown for prions. While the aggregation of this protein family was initially considered exclusively toxic in mammalians organisms, it is now almost clear that many other proteins adopt prion-like attributes to rationally polymerize into higher order complexes with organized physiologic roles. This implies that cells can tolerate at least in some measure the accumulation of inherently dangerous protein aggregates for functional profit. This review summarizes currently known strategies that living organisms adopt to preserve beneficial aggregation, and to prevent the catastrophic accumulation of toxic aggregates that frequently accompany neurodegeneration. PMID:25741240

  1. Disease Phenotypes in a Mouse Model of RNA Toxicity Are Independent of Protein Kinase Cα and Protein Kinase Cβ

    PubMed Central

    Kim, Yun K.; Yadava, Ramesh S.; Mandal, Mahua; Mahadevan, Karunasai; Yu, Qing; Leitges, Michael; Mahadevan, Mani S.

    2016-01-01

    Myotonic dystrophy type 1(DM1) is the prototype for diseases caused by RNA toxicity. RNAs from the mutant allele contain an expanded (CUG)n tract within the 3' untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. The toxic RNAs affect the function of RNA binding proteins leading to sequestration of muscleblind-like (MBNL) proteins and increased levels of CELF1 (CUGBP, Elav-like family member 1). The mechanism for increased CELF1 is not very clear. One favored proposition is hyper-phosphorylation of CELF1 by Protein Kinase C alpha (PKCα) leading to increased CELF1 stability. However, most of the evidence supporting a role for PKC-α relies on pharmacological inhibition of PKC. To further investigate the role of PKCs in the pathogenesis of RNA toxicity, we generated transgenic mice with RNA toxicity that lacked both the PKCα and PKCβ isoforms. We find that these mice show similar disease progression as mice wildtype for the PKC isoforms. Additionally, the expression of CELF1 is also not affected by deficiency of PKCα and PKCβ in these RNA toxicity mice. These data suggest that disease phenotypes of these RNA toxicity mice are independent of PKCα and PKCβ. PMID:27657532

  2. High-throughput protein crystallization.

    PubMed

    Stevens, R C

    2000-10-01

    The combinatorial chemistry industry has made major advances in the handling and mixing of small volumes, and in the development of robust liquid-handling systems. In addition, developments have been made in the area of material handling for the high-throughput drug screening and combinatorial chemistry fields. Lastly, improvements in beamline optics at synchrotron sources have enabled the use of flash-frozen micron-sized (10-50 microm) crystals. The combination of these and other recent advances will make high-throughput protein crystallography possible. Further advances in high-throughput methods of protein crystallography will require application of the above developments and the accumulation of success/failure data in a more systematic manner. Major changes in crystallography technology will emerge based on the data collected by first-generation high-throughput systems.

  3. Toxicity and oxidative stress of different forms of organic selenium and dietary protein in mallard ducklings

    USGS Publications Warehouse

    Hoffman, D.J.; Heinz, G.H.; LeCaptain, L.J.; Eisemann, J.D.; Pendleton, G.W.

    1996-01-01

    Concentrations of over 100 ppm (mg/kg) selenium (Se) have been found in aquatic plants and insects associated with irrigation drainwater and toxicity to fish and wildlife. Composition of diet for wild ducklings may vary in selenium-contaminated environments. Earlier studies have compared toxicities and oxidative stress of Se as selenite to those of seleno-DL-methionine (DL) in mallards (Anas platyrhynchos). This study compares DL, seleno-L-methionine (L), selenized yeast (Y) and selenized wheat (W). Day-old mallard ducklings received an untreated diet (controls) containing 75% wheat (22% protein) or the same diet containing 15 or 30 ppm Se in the above forms except for 30 ppm Se as W. After 2 weeks blood and liver samples were collected for biochemical assays and Se analysis. All forms of selenium caused significant increases in plasma and hepatic glutathione peroxidase activities. Se as L at 30 ppm in the diet was the most toxic form, resulting in high mortality (64%) and impaired growth (>50%) in survivors and the greatest increase in ratio of oxidized to reduced hepatic glutathione (GSH). Se as both L and DL decreased the concentrations of hepatic GSH and total thiols. Se as Y accumulated the least in liver (approximately 50% of other forms) and had less effect on GSH and total thiols. In a second experiment, in which the basal diet was a commercial duck feed (22 % protein), survival was not affected by 30 ppm Se as DL, L, or Y and oxidative effects on GSH metabolism were less pronounced than with the wheat diet.

  4. Slight temperature changes affect protein affinity and cellular uptake/toxicity of nanoparticles.

    PubMed

    Mahmoudi, Morteza; Shokrgozar, Mohammad A; Behzadi, Shahed

    2013-04-21

    It is known that what the cell actually "sees" at the nanoscale is an outer shell formed of 'protein corona' on the surface of nanoparticles (NPs). The amount and composition of various proteins on the corona are strongly dependent on the biophysicochemical properties of NPs, which have been extensively studied. However, the effect of a small variation in temperature, due to the human circadian rhythm, on the composition of the protein corona and the affinity of various proteins to the surface of NPs, was ignored. Here, the effect of temperature on the composition of protein corona and the affinity of various proteins to the surface of NPs and, subsequently, cell responses to the protein coated NPs are probed. The results confirmed that cellular entrance, dispersion, and toxicity of NPs are strongly diverse with slight body temperature changes. This new finding can help scientists to maximise NP entrance to specific cells/organs with lower toxicity by adjusting the cellular/organ temperature.

  5. Therapeutic approaches against common structural features of toxic oligomers shared by multiple amyloidogenic proteins.

    PubMed

    Guerrero-Muñoz, Marcos J; Castillo-Carranza, Diana L; Kayed, Rakez

    2014-04-15

    Impaired proteostasis is one of the main features of all amyloid diseases, which are associated with the formation of insoluble aggregates from amyloidogenic proteins. The aggregation process can be caused by overproduction or poor clearance of these proteins. However, numerous reports suggest that amyloid oligomers are the most toxic species, rather than insoluble fibrillar material, in Alzheimer's, Parkinson's, and Prion diseases, among others. Although the exact protein that aggregates varies between amyloid disorders, they all share common structural features that can be used as therapeutic targets. In this review, we focus on therapeutic approaches against shared features of toxic oligomeric structures and future directions.

  6. In vitro bioassay for reactive toxicity towards proteins implemented for water quality monitoring.

    PubMed

    Tang, Janet Y M; Glenn, Eva; Thoen, Hanne; Escher, Beate I

    2012-03-01

    , Australia were used to evaluate the applicability of the E. coli assay for reactive toxicity in water samples. While the EC(50) values of the GSH+ strain showed similar trends as in other biological endpoints over the various treatment chains, the specific response indicative of protein damage was only observed in samples that had undergone chlorination as a disinfection process. High natural organic matter or other matrix components disturbed the bioassay so much that we recommend it for future routine testing only in tertiary treated water or drinking water. PMID:22331350

  7. In vitro bioassay for reactive toxicity towards proteins implemented for water quality monitoring.

    PubMed

    Tang, Janet Y M; Glenn, Eva; Thoen, Hanne; Escher, Beate I

    2012-03-01

    , Australia were used to evaluate the applicability of the E. coli assay for reactive toxicity in water samples. While the EC(50) values of the GSH+ strain showed similar trends as in other biological endpoints over the various treatment chains, the specific response indicative of protein damage was only observed in samples that had undergone chlorination as a disinfection process. High natural organic matter or other matrix components disturbed the bioassay so much that we recommend it for future routine testing only in tertiary treated water or drinking water.

  8. Molecular and Insecticidal Characterization of a Novel Cry-Related Protein from Bacillus Thuringiensis Toxic against Myzus persicae

    PubMed Central

    Palma, Leopoldo; Muñoz, Delia; Berry, Colin; Murillo, Jesús; Ruiz de Escudero, Iñigo; Caballero, Primitivo

    2014-01-01

    This study describes the insecticidal activity of a novel Bacillus thuringiensis Cry-related protein with a deduced 799 amino acid sequence (~89 kDa) and ~19% pairwise identity to the 95-kDa-aphidicidal protein (sequence number 204) from patent US 8318900 and ~40% pairwise identity to the cancer cell killing Cry proteins (parasporins Cry41Ab1 and Cry41Aa1), respectively. This novel Cry-related protein contained the five conserved amino acid blocks and the three conserved domains commonly found in 3-domain Cry proteins. The protein exhibited toxic activity against the green peach aphid, Myzus persicae (Sulzer) (Homoptera: Aphididae) with the lowest mean lethal concentration (LC50 = 32.7 μg/mL) reported to date for a given Cry protein and this insect species, whereas it had no lethal toxicity against the Lepidoptera of the family Noctuidae Helicoverpa armigera (Hübner), Mamestra brassicae (L.), Spodoptera exigua (Hübner), S. frugiperda (J.E. Smith) and S. littoralis (Boisduval), at concentrations as high as ~3.5 μg/cm2. This novel Cry-related protein may become a promising environmentally friendly tool for the biological control of M. persicae and possibly also for other sap sucking insect pests. PMID:25384108

  9. Acclimation and toxicity of high ammonium concentrations to unicellular algae.

    PubMed

    Collos, Yves; Harrison, Paul J

    2014-03-15

    A literature review on the effects of high ammonium concentrations on the growth of 6 classes of microalgae suggests the following rankings. Mean optimal ammonium concentrations were 7600, 2500, 1400, 340, 260, 100 μM for Chlorophyceae, Cyanophyceae, Prymnesiophyceae, Diatomophyceae, Raphidophyceae, and Dinophyceae respectively and their tolerance to high toxic ammonium levels was 39,000, 13,000, 2300, 3600, 2500, 1200 μM respectively. Field ammonium concentrations <100 μM would not likely reduce the growth rate of most microalgae. Chlorophytes were significantly more tolerant to high ammonium than diatoms, prymnesiophytes, dinoflagellates, and raphidophytes. Cyanophytes were significantly more tolerant than dinoflagellates which were the least tolerant. A smaller but more complete data set was used to estimate ammonium EC₅₀ values, and the ranking was: Chlorophyceae>Cyanophyceae, Dinophyceae, Diatomophyceae, and Raphidophyceae. Ammonia toxicity is mainly attributed to NH₃ at pHs >9 and at pHs <8, toxicity is likely associated with the ammonium ion rather than ammonia. PMID:24533997

  10. Quinone-induced protein handling changes: Implications for major protein handling systems in quinone-mediated toxicity

    SciTech Connect

    Xiong, Rui; Siegel, David; Ross, David

    2014-10-15

    Para-quinones such as 1,4-Benzoquinone (BQ) and menadione (MD) and ortho-quinones including the oxidation products of catecholamines, are derived from xenobiotics as well as endogenous molecules. The effects of quinones on major protein handling systems in cells; the 20/26S proteasome, the ER stress response, autophagy, chaperone proteins and aggresome formation, have not been investigated in a systematic manner. Both BQ and aminochrome (AC) inhibited proteasomal activity and activated the ER stress response and autophagy in rat dopaminergic N27 cells. AC also induced aggresome formation while MD had little effect on any protein handling systems in N27 cells. The effect of NQO1 on quinone induced protein handling changes and toxicity was examined using N27 cells stably transfected with NQO1 to generate an isogenic NQO1-overexpressing line. NQO1 protected against BQ–induced apoptosis but led to a potentiation of AC- and MD-induced apoptosis. Modulation of quinone-induced apoptosis in N27 and NQO1-overexpressing cells correlated only with changes in the ER stress response and not with changes in other protein handling systems. These data suggested that NQO1 modulated the ER stress response to potentiate toxicity of AC and MD, but protected against BQ toxicity. We further demonstrated that NQO1 mediated reduction to unstable hydroquinones and subsequent redox cycling was important for the activation of the ER stress response and toxicity for both AC and MD. In summary, our data demonstrate that quinone-specific changes in protein handling are evident in N27 cells and the induction of the ER stress response is associated with quinone-mediated toxicity. - Highlights: • Unstable hydroquinones contributed to quinone-induced ER stress and toxicity.

  11. Protein kinase c inhibitor attenuates cyanide toxicity in vivo

    SciTech Connect

    Maduh, E.U.; Nealley, E.W.; Song, H.; Wang, P.C.; Baskin, S.I.

    1995-12-31

    We have examined the effect of pretreatment with a potent protein kinase C (PKC) inhibitor, l-(5-isoquinoline sulfonyl)-2-methylpiperazine (H-7), against metabolic alterations induced by sodium cyanide (NaCN), 4.2 mg/kg, in brain of anesthetized male micropigs (6-10 kg). Brain high energy phosphates were analyzed using a 3/P nuclear magnetic resonance (NMR) spectroscopic surface coil in a 4.7 Tesla horizontal bore magnet. H-7, I mg/kg, was given intravenously (i.v.) 30 min before NaCN challenge (H-7 + CN). Prior to NaCN, H-7, or H-7 + CN administration, baseline 31P resonance spectra of 1-min duration were acquired for 5-10 min, and continued for an additional 60 min following i.v. NaCN injection, each animal serving as its own control. Peaks were identified as phosphomonoester (PME), inorganic phosphate (Pi), phosphodiester (PDE), phosphocreatine (PCr) and adenosine triphosphate (ATP), based on their respective chemical shifts. Without H-7 pretreatment, NaCN effects were marked by a rising Pi and a declining PCr peak 2 min after injection, with only 2/5 of the animals surviving the 60 min experiment. Through a pretreatment period of 30 min, H-7 did not affect baseline cell energy profile as reflected by the 31P-NMR spectra, but in its presence, those changes (i.e. diminishing PCr and rising Pi peaks) elicited by NaCN were markedly blunted; 4/5 of the animals in this group survived the NaCN challenge. It is proposed that H-7, a pharmacologic inhibitor of PKC, may be useful in CN antagonism, underscoring the role of PKC in cyanide intoxication.

  12. High light intensity augments mercury toxicity in cyanobacterium Nostoc muscorum.

    PubMed

    Singh, Ranjana; Dubey, Gunjan; Singh, Vijay Pratap; Srivastava, Prabhat Kumar; Kumar, Sushil; Prasad, Sheo Mohan

    2012-11-01

    The present study is aimed at investigating the role of growth irradiance in determining the extent of mercury (Hg) toxicity on various physiological parameters viz. growth, pigment contents, photosynthesis, respiration, (14)CO(2) fixation, photosynthetic electron transport, photorespiration and enzyme activity of cyanobacterium Nostoc muscorum. A general decline was observed in all these parameters with increasing concentration of Hg except for carotenoids content and respiratory activity which exhibited significant enhancement. This effect was more pronounced in high light (130 μmol photon m(-2) s(-1)) exposed cells as compared to normal (70 μmol photon m(-2) s(-1)) and low (10 μmol photon m(-2) s(-1)) light exposed cells. Among the photosynthetic electron transport activities, whole chain was found to be more sensitive than photosystem II (PSII) and photosystem I (PSI). (14)CO(2) fixation was more affected as compared to O(2) evolution when exposed to Hg and different light intensities. Photorespiratory activity, which is an index of protecting organisms from light-induced damage, also showed a similar declining trend. Enzyme assay revealed that among the carboxylating enzymes, activity of RUBISCO was more severely inhibited than PEPCase. Thus, these results suggest that Hg itself was toxic at all tested concentrations and high light intensity augmented its toxicity in N. muscorum inhibiting the growth, pigment contents and photosynthetic activity of the organism.

  13. High-throughput and multiplexed protein array technology: protein-DNA and protein-protein interactions.

    PubMed

    Sakanyan, Vehary

    2005-02-01

    Miniaturized protein arrays address protein interactions with various types of molecules in a high-throughput and multiplexed fashion. This review focuses on achievements in the analysis of protein-DNA and protein-protein interactions. The technological feasibility of protein arrays depends on the different factors that enable the arrayed proteins to recognize molecular partners and on the specificity of the interactions involved. Proteome-scale studies of molecular interactions require high-throughput approaches for both the production and purification of functionally active proteins. Various solutions have been proposed to avoid non-specific protein interactions on array supports and to monitor low-abundance molecules. The data accumulated indicate that this emerging technology is perfectly suited to resolve networks of protein interactions involved in complex physiological and pathological phenomena in different organisms and to develop sensitive tools for biomedical applications.

  14. Metalloids: essential, beneficial or toxic? Major intrinsic proteins sort it out.

    PubMed

    Bienert, Gerd P; Schüssler, Manuela D; Jahn, Thomas P

    2008-01-01

    Major intrinsic proteins (MIPs) are a family of selective membrane channels comprising water-channelling aquaporins and glycerol-channelling aquaglyceroporins. Recently, several MIPs within all domains of life were shown to facilitate the diffusion of reduced and non-charged species of the metalloids silicon, boron, arsenic and antimony. Metalloids encompass a group of biologically important elements ranging from the essential to the highly toxic. Consequently, all organisms require efficient membrane transport systems to control the exchange of metalloids with the environment. Recent genetic evidence has demonstrated a crucial role for specific MIPs in metalloid homeostasis. We propose that specific MIPs represent an ancient and indispensable transport mechanism for metalloids, which suggests that they could be potential pharmacological targets.

  15. Ubiquilin overexpression reduces GFP-polyalanine-induced protein aggregates and toxicity

    SciTech Connect

    Wang Hongmin; Monteiro, Mervyn J. . E-mail: monteiro@umbi.umd.edu

    2007-08-01

    Several human disorders are associated with an increase in a continuous stretch of alanine amino acids in proteins. These so-called polyalanine expansion diseases share many similarities with polyglutamine-related disorders, including a length-dependent reiteration of amino acid induction of protein aggregation and cytotoxicity. We previously reported that overexpression of ubiquilin reduces protein aggregates and toxicity of expanded polyglutamine proteins. Here, we demonstrate a similar role for ubiquilin toward expanded polyalanine proteins. Overexpression of ubiquilin-1 in HeLa cells reduced protein aggregates and the cytotoxicity associated with expression of a transfected nuclear-targeted GFP-fusion protein containing 37-alanine repeats (GFP-A37), in a dose dependent manner. Ubiquilin coimmunoprecipitated more with GFP proteins containing a 37-polyalanine tract compared to either 7 (GFP-A7), or no alanine tract (GFP). Moreover, overexpression of ubiquilin suppressed the increased vulnerability of HeLa cell lines stably expressing the GFP-A37 fusion protein to oxidative stress-induced cell death compared to cell lines expressing GFP or GFP-A7 proteins. By contrast, siRNA knockdown of ubiquilin expression in the GFP-A37 cell line was associated with decreased cellular proliferation, and increases in GFP protein aggregates, nuclear fragmentation, and cell death. Our results suggest that boosting ubiquilin levels in cells might provide a universal and attractive strategy to prevent toxicity of proteins containing reiterative expansions of amino acids involved in many human diseases.

  16. Effects of protein-calorie malnutrition and refeeding on fluorouracil toxicity

    SciTech Connect

    Gamelli, R.L.; Foster, R.S. Jr.

    1983-10-01

    Mice were used to study the effects of protein-calorie malnutrition and its reversal on granulocyte-macrophage production and fluorouracil's toxic effect on bone marrow. An in vitro quantitative clonal culture technique for bone marrow granulocyte-macrophage progenitor cells (GM-CFC) was used. Animals on a protein-free but otherwise complete diet for ten days had a significant contraction in total marrow cellularity and GM-CFC numbers paralleling the animal's weight loss. The acute toxic effect of fluorouracil on bone marrow was not increased in protein-deprived animals. On refeeding, there was a biphasic response in the degree of toxic effect on marrow. Animals refed for one day had significantly increased fluorouracil-related marrow abnormalities. However, animals refed for four days, when marrows were repleted, were partially protected from the drug's cytotoxic effects. The increased sensitivity in mice refed for one day was related to more GM-CFC in active DNA synthesis.

  17. Toxicity and oxidative stress of different forms of organic selenium (Se) and dietary protein in mallard (Anas platyrhynchos) ducklings

    USGS Publications Warehouse

    Hoffman, D.; Heinz, G.; Eisemann, J.; Pendleton, G.

    1994-01-01

    High concentrations of Se have been found in aquatic food chains associated with irrigation drainwater and toxicity to fish and wildlife. Earlier studies have compared toxicities of Se as selenite and as seleno-DL-methionine (DL) in mallards. This study compares DL, seleno-L-methionine (L), selenized yeast (Y) and selenized wheat (W). Day-old mallard ducklings received an untreated diet (controls) containing 75% wheat (22% protein) or the same diet containing 15 or 30 ppm Se in the above forms. After 2 weeks blood and liver samples were collected for biochemical assays and Se analysis. All forms of selenium caused significant increases in plasma and hepatic glutathione peroxidase activities. Se as L was the most toxic, resulting in high mortality (64%) and impaired growth (>50%) and the greatest increase in ratio of oxidized to reduced glutathione with 30 ppm in the diet. Se as Y accumulated the least in liver. In a subsequent experiment with 30% dietary protein Se as L was less toxic.

  18. Apolipoprotein E expression and behavioral toxicity of high charge, high energy (HZE) particle radiation

    NASA Technical Reports Server (NTRS)

    Higuchi, Yoshinori; Nelson, Gregory A.; Vazquez, Marcelo; Laskowitz, Daniel T.; Slater, James M.; Pearlstein, Robert D.

    2002-01-01

    Apolipoprotein E (apoE) is a lipid binding protein that plays an important role in tissue repair following brain injury. In the present studies, we have investigated whether apoE affects the behavioral toxicity of high charge, high energy (HZE) particle radiation. METHODS: Sixteen male apoE knockout (KO) mice and sixteen genetically matched wild-type (WT) C57BL mice were used in this experiment. Half of the KO and half of the WT animals were irradiated with 600 MeV/amu iron particles (2 Gy whole body). The effect of irradiation on motor coordination and stamina (Rotarod test), exploratory behavior (open field test), and spatial working and reference memory (Morris water maze) was assessed. ROTAROD TEST: Performance was adversely affected by radiation exposure in both KO and WT groups at 30 d after irradiation. By 60 d after radiation, the radiation effect was lost in WT, but still apparent in irradiated KO mice. OPEN FIELD TEST: Radiation reduced open field exploratory activity 14, 28, 56, 84, and 168 d after irradiation of KO mice, but had no effect on WT mice. MORRIS WATER MAZE: Radiation adversely affected spatial working memory in the KO mice, but had no discernible effect in the WT mice as assessed 180 d after irradiation. In contrast, irradiated WT mice showed marked impairment of spatial reference memory in comparison to non-irradiated mice, while no effect of radiation was observed in KO mice. CONCLUSIONS: These studies show that apoE expression influences the behavioral toxicity of HZE particle radiation and suggest that apoE plays a role in the repair/recovery from radiation injury of the CNS. ApoE deficiency may exacerbate the previously reported effects of HZE particle radiation in accelerating the brain aging process.

  19. Enhanced toxicity of silver nanoparticles in transgenic Caenorhabditis elegans expressing amyloidogenic proteins.

    PubMed

    Soria, Cristina; Coccini, Teresa; De Simone, Uliana; Marchese, Loredana; Zorzoli, Irene; Giorgetti, Sofia; Raimondi, Sara; Mangione, P Patrizia; Ramat, Stefano; Bellotti, Vittorio; Manzo, Luigi; Stoppini, Monica

    2015-01-01

    The increasing number of applications of silver nanoparticles (AgNP) prompted us to assess their toxicity in vivo. We have investigated their effects on wild type and transgenic Caenorhabditis elegans (C. elegans) strains expressing two prototypic amyloidogenic proteins: β2-microglobulin and Aβ peptide3-42. The use of C. elegans allowed us to highlight AgNP toxicity in the early phase of the worm's life cycle (LC50 survival, 0.9 µg/ml). A comparative analysis of LC50 values revealed that our nematode strains were more sensitive to assess AgNP toxicity than the cell lines, classically used in toxicity tests. Movement and superoxide production in the adult population were significantly affected by exposure to AgNP; the transgenic strains were more affected than the wild type worms. Our screening approach could be applied to other types of nanomaterials that can enter the body and express any nanostructure-related bioactivities. We propose that C. elegans reproducing the molecular events associated with protein misfolding diseases, e.g. Alzheimer's disease and systemic amyloidosis, may help to investigate the specific toxicity of a range of potentially harmful molecules. Our study suggests that transgenic C. elegans may be used to predict the effect of chemicals in a "fragile population", where an underlying pathologic state may amplify their toxicity.

  20. Toxic Compound, Anti-Nutritional Factors and Functional Properties of Protein Isolated from Detoxified Jatropha curcas Seed Cake

    PubMed Central

    Saetae, Donlaporn; Suntornsuk, Worapot

    2011-01-01

    Jatropha curcas is a multipurpose tree, which has potential as an alternative source for biodiesel. All of its parts can also be used for human food, animal feed, fertilizer, fuel and traditional medicine. J. curcas seed cake is a low-value by-product obtained from biodiesel production. The seed cake, however, has a high amount of protein, with the presence of a main toxic compound: phorbol esters as well as anti-nutritional factors: trypsin inhibitors, phytic acid, lectin and saponin. The objective of this work was to detoxify J. curcas seed cake and study the toxin, anti-nutritional factors and also functional properties of the protein isolated from the detoxified seed cake. The yield of protein isolate was approximately 70.9%. The protein isolate was obtained without a detectable level of phorbol esters. The solubility of the protein isolate was maximal at pH 12.0 and minimal at pH 4.0. The water and oil binding capacities of the protein isolate were 1.76 g water/g protein and 1.07 mL oil/g protein, respectively. The foam capacity and stability, including emulsion activity and stability of protein isolate, had higher values in a range of basic pHs, while foam and emulsion stabilities decreased with increasing time. The results suggest that the detoxified J. curcas seed cake has potential to be exploited as a novel source of functional protein for food applications. PMID:21339978

  1. Antioxidant-Mediated Modulation of Protein Reactivity for 3,4-Dihydroxyphenylacetaldehyde, a Toxic Dopamine Metabolite.

    PubMed

    Anderson, David G; Florang, Virginia R; Schamp, Josephine H; Buettner, Garry R; Doorn, Jonathan A

    2016-07-18

    3,4-Dihydroxyphenylacetaldehyde (DOPAL) is an endogenously produced toxic aldehyde. It is a bifunctional electrophile implicated in the loss of dopaminergic cells concomitant with Parkinson's disease and neurodegeneration. DOPAL is known to react with proteins and amino acids such as N-acetyl lysine (NAL); oxidation of the catechol moiety to the quinone of DOPAL increases this reactivity. Here, we demonstrate the ability of the antioxidants N-acetylcysteine, glutathione, and ascorbic acid to mitigate the reactivity of DOPAL with proteins and amino acids in a dose-dependent fashion. Conversely, Trolox did not lessen the observed reactivity with proteins. Interestingly, use of tricine, a buffer and reducing agent, in these systems also decreased the reactivity of DOPAL with amines, yielding tricine-derived free radical species. Modification of amines with aldehydes typically involves Schiff base chemistry; however, the observance of free radicals suggests that an oxidative step is involved in the reaction of DOPAL with lysine. Furthermore, while Schiff base formation is usually optimal at pH 5, the reaction rate of DOPAL with NAL is negligible at pH 5 and is enhanced under basic conditions (e.g., pH 9). Conditions of high pH are also favorable for catechol auto-oxidation, known to occur for DOPAL. The antioxidant-mediated protection demonstrated here suggests that oxidative stress may impart cellular vulnerability to protein modification by DOPAL. Therefore, depleted antioxidants and increased levels of lipid peroxidation products, known to prevent the detoxifying metabolism of DOPAL, may present a survival challenge to dopaminergic cells targeted in Parkinson's disease. PMID:27268734

  2. BH3 domain-independent apolipoprotein L1 toxicity rescued by BCL2 prosurvival proteins

    PubMed Central

    Heneghan, J. F.; Vandorpe, D. H.; Shmukler, B. E.; Giovinazzo, J. A.; Raper, J.; Friedman, D. J.; Pollak, M. R.

    2015-01-01

    The potent trypanolytic properties of human apolipoprotein L1 (APOL1) can be neutralized by the trypanosome variant surface antigen gene product known as serum resistance-associated protein. However, two common APOL1 haplotypes present uniquely in individuals of West African ancestry each encode APOL1 variants resistant to serum resistance-associated protein, and each confers substantial resistance to human African sleeping sickness. In contrast to the dominantly inherited anti-trypanosomal activity of APOL1, recessive inheritance of these two trypanoprotective APOL1 alleles predisposes to kidney disease. Proposed mechanisms of APOL1 toxicity have included BH3 domain-dependent autophagy and/or ion channel activity. We probed these potential mechanisms by expressing APOL1 in Xenopus laevis oocytes. APOL1 expression in oocytes increased ion permeability and caused profound morphological deterioration (toxicity). Coexpression of BCL2 family members rescued APOL1-associated oocyte toxicity in the order MCL1 ∼ BCLW > BCLXL ∼ BCL2A1 ≫ BCL2. Deletion of nine nominal core BH3 domain residues abolished APOL1-associated toxicity, but missense substitution of the same residues abolished neither oocyte toxicity nor its rescue by coexpressed MCL1. The APOL1 BH3 domain was similarly dispensable for the ability of APOL1 to rescue intact mice from lethal trypanosome challenge. Replacement of most extracellular Na+ by K+ also reduced APOL1-associated oocyte toxicity, allowing demonstration of APOL1-associated increases in Ca2+ and Cl− fluxes and oocyte ion currents, which were similarly reduced by MCL1 coexpression. Thus APOL1 toxicity in Xenopus oocytes is BH3-independent, but can nonetheless be rescued by some BCL2 family proteins. PMID:26108665

  3. Role of Protein–Protein Interactions in Cytochrome P450-Mediated Drug Metabolism and Toxicity

    PubMed Central

    2015-01-01

    Through their unique oxidative chemistry, cytochrome P450 monooxygenases (CYPs) catalyze the elimination of most drugs and toxins from the human body. Protein–protein interactions play a critical role in this process. Historically, the study of CYP–protein interactions has focused on their electron transfer partners and allosteric mediators, cytochrome P450 reductase and cytochrome b5. However, CYPs can bind other proteins that also affect CYP function. Some examples include the progesterone receptor membrane component 1, damage resistance protein 1, human and bovine serum albumin, and intestinal fatty acid binding protein, in addition to other CYP isoforms. Furthermore, disruption of these interactions can lead to altered paths of metabolism and the production of toxic metabolites. In this review, we summarize the available evidence for CYP protein–protein interactions from the literature and offer a discussion of the potential impact of future studies aimed at characterizing noncanonical protein–protein interactions with CYP enzymes. PMID:25133307

  4. A 13-week subchronic intravaginal toxicity study of pokeweed antiviral protein in mice.

    PubMed

    D'Cruz, O J; Waurzyniakt, B; Uckun, F M

    2004-01-01

    Pokeweed antiviral protein (PAP), a 29-kDa plant-derived protein isolated from Phytolacca americana, is a broad-spectrum antiviral agent. PAP shows unique clinical potential to become the active ingredient of a non-spermicidal microbicide because of its potent in vivo anti-HIV activity, non-interference with in vivo sperm functions, and lack of cytotoxicity to genital tract epithelial cells. Over 13 weeks the subchronic and reproductive toxicity potential of an intravaginally administered gel formulation of PAP was studied in mice to support its further development as a vaginal microbicide. Female B6C3F1 and CD-1 mice in subgroups of 20, were exposed intravaginally to a gel formulation containing 0, 0.025, 0.05, or 0.1% PAP, 5 days/week for 13 consecutive weeks. On a molar basis, these concentrations are 500- to 2000-times higher than the in vitro anti-HIV IC50 value. After 13 weeks of intravaginal treatment, B6C3F1 mice were evaluated for survival, body weight gain, and absolute and relative organ weights. Blood was analyzed for hematology and clinical chemistry profiles. Microscopic examination was performed on hematoxylin and eosin-stained tissue sections from each study animal. Placebo-control and PAP-dosed female CD-1 mice were mated with untreated males in order to evaluate if PAP has any deleterious effects on reproductive performance. There were no treatment-related mortalities. Mean body weight gain was not reduced by PAP treatment during the dosing period. The hemogram and blood chemistry profiles revealed lack of systemic toxicity following daily intravaginal instillation of PAP for 13 weeks. No clinically significant changes in absolute and relative organ weights were noted in the PAP dose groups. Extensive histopathological examination of tissues showed no increase in treatment-related microscopic lesions in any of the three PAP dose groups. Repeated intravaginal exposure of CD-1 mice to increasing concentrations of PAP for 13 weeks showed no adverse

  5. Persistence of detectable insecticidal proteins from Bacillus thuringiensis (Cry) and toxicity after adsorption on contrasting soils.

    PubMed

    Hung, T P; Truong, L V; Binh, N D; Frutos, R; Quiquampoix, H; Staunton, S

    2016-01-01

    Insecticidal Cry, or Bt, proteins are produced by the soil-endemic bacterium, Bacillus thuringiensis and some genetically modified crops. Their environmental fate depends on interactions with soil. Little is known about the toxicity of adsorbed proteins and the change in toxicity over time. We incubated Cry1Ac and Cry2A in contrasting soils subjected to different treatments to inhibit microbial activity. The toxin was chemically extracted and immunoassayed. Manduca sexta was the target insect for biotests. Extractable toxin decreased during incubation for up to four weeks. Toxicity of Cry1Ac was maintained in the adsorbed state, but lost after 2 weeks incubation at 25 °C. The decline in extractable protein and toxicity were much slower at 4 °C with no significant effect of soil sterilization. The major driving force for decline may be time-dependent fixation of adsorbed protein, leading to a decrease in the extraction yield in vitro, paralleled by decreasing solubilisation in the larval gut. PMID:26549751

  6. Persistence of detectable insecticidal proteins from Bacillus thuringiensis (Cry) and toxicity after adsorption on contrasting soils.

    PubMed

    Hung, T P; Truong, L V; Binh, N D; Frutos, R; Quiquampoix, H; Staunton, S

    2016-01-01

    Insecticidal Cry, or Bt, proteins are produced by the soil-endemic bacterium, Bacillus thuringiensis and some genetically modified crops. Their environmental fate depends on interactions with soil. Little is known about the toxicity of adsorbed proteins and the change in toxicity over time. We incubated Cry1Ac and Cry2A in contrasting soils subjected to different treatments to inhibit microbial activity. The toxin was chemically extracted and immunoassayed. Manduca sexta was the target insect for biotests. Extractable toxin decreased during incubation for up to four weeks. Toxicity of Cry1Ac was maintained in the adsorbed state, but lost after 2 weeks incubation at 25 °C. The decline in extractable protein and toxicity were much slower at 4 °C with no significant effect of soil sterilization. The major driving force for decline may be time-dependent fixation of adsorbed protein, leading to a decrease in the extraction yield in vitro, paralleled by decreasing solubilisation in the larval gut.

  7. Destabilizing protein polymorphisms in the genetic background direct phenotypic expression of mutant SOD1 toxicity.

    PubMed

    Gidalevitz, Tali; Krupinski, Thomas; Garcia, Susana; Morimoto, Richard I

    2009-03-01

    Genetic background exerts a strong modulatory effect on the toxicity of aggregation-prone proteins in conformational diseases. In addition to influencing the misfolding and aggregation behavior of the mutant proteins, polymorphisms in putative modifier genes may affect the molecular processes leading to the disease phenotype. Mutations in SOD1 in a subset of familial amyotrophic lateral sclerosis (ALS) cases confer dominant but clinically variable toxicity, thought to be mediated by misfolding and aggregation of mutant SOD1 protein. While the mechanism of toxicity remains unknown, both the nature of the SOD1 mutation and the genetic background in which it is expressed appear important. To address this, we established a Caenorhabditis elegans model to systematically examine the aggregation behavior and genetic interactions of mutant forms of SOD1. Expression of three structurally distinct SOD1 mutants in C. elegans muscle cells resulted in the appearance of heterogeneous populations of aggregates and was associated with only mild cellular dysfunction. However, introduction of destabilizing temperature-sensitive mutations into the genetic background strongly enhanced the toxicity of SOD1 mutants, resulting in exposure of several deleterious phenotypes at permissive conditions in a manner dependent on the specific SOD1 mutation. The nature of the observed phenotype was dependent on the temperature-sensitive mutation present, while its penetrance reflected the specific combination of temperature-sensitive and SOD1 mutations. Thus, the specific toxic phenotypes of conformational disease may not be simply due to misfolding/aggregation toxicity of the causative mutant proteins, but may be defined by their genetic interactions with cellular pathways harboring mildly destabilizing missense alleles. PMID:19266020

  8. Slight temperature changes affect protein affinity and cellular uptake/toxicity of nanoparticles

    NASA Astrophysics Data System (ADS)

    Mahmoudi, Morteza; Shokrgozar, Mohammad A.; Behzadi, Shahed

    2013-03-01

    It is known that what the cell actually ``sees'' at the nanoscale is an outer shell formed of `protein corona' on the surface of nanoparticles (NPs). The amount and composition of various proteins on the corona are strongly dependent on the biophysicochemical properties of NPs, which have been extensively studied. However, the effect of a small variation in temperature, due to the human circadian rhythm, on the composition of the protein corona and the affinity of various proteins to the surface of NPs, was ignored. Here, the effect of temperature on the composition of protein corona and the affinity of various proteins to the surface of NPs and, subsequently, cell responses to the protein coated NPs are probed. The results confirmed that cellular entrance, dispersion, and toxicity of NPs are strongly diverse with slight body temperature changes. This new finding can help scientists to maximise NP entrance to specific cells/organs with lower toxicity by adjusting the cellular/organ temperature.It is known that what the cell actually ``sees'' at the nanoscale is an outer shell formed of `protein corona' on the surface of nanoparticles (NPs). The amount and composition of various proteins on the corona are strongly dependent on the biophysicochemical properties of NPs, which have been extensively studied. However, the effect of a small variation in temperature, due to the human circadian rhythm, on the composition of the protein corona and the affinity of various proteins to the surface of NPs, was ignored. Here, the effect of temperature on the composition of protein corona and the affinity of various proteins to the surface of NPs and, subsequently, cell responses to the protein coated NPs are probed. The results confirmed that cellular entrance, dispersion, and toxicity of NPs are strongly diverse with slight body temperature changes. This new finding can help scientists to maximise NP entrance to specific cells/organs with lower toxicity by adjusting the cellular

  9. Modulation of diamide toxicity in thermotolerant cells by inhibition of protein synthesis.

    PubMed

    Freeman, M L; Meredith, M J

    1989-08-15

    Chinese hamster ovary cells were exposed in vitro to various concentrations of diamide for 1 h at 37 degrees C. This treatment resulted in a dose dependent increase in cytotoxicity. Cells were also heated at 43 degrees C for 15 min, incubated at 37 degrees C for 3 h, and then exposed to various concentrations of diamide. This heat shock has been shown previously to trigger the synthesis of heat shock proteins and the development of thermotolerance. Further, under these experimental conditions both were inhibited if protein synthesis was inhibited by exposure to cycloheximide (M. L. Freeman et al., Radiat. Res., 112: 195-203, 1987). Diamide toxicity was diminished in cells made thermotolerant by the 43 degrees C/15-min heat shock. For example, at the highest dose used, 0.8 mM, survival increased from 0.93% to 6.1%. However, diamide toxicity was unaffected if the cells were exposed to diamide 3 h after a 43 degrees C/60 min heat shock. This latter heat shock produced significant inhibition of protein synthesis whereas the 15-min heat shock did not (M. L. Freeman et al., Cancer Res., 48: 7033-7037, 1988). Further, a 43 degrees C/15-min heat shock did not confer protection against diamide toxicity if the cells were simultaneously exposed to cycloheximide. Exposure to 0.8 mM diamide was shown to oxidize specific cellular proteins as measured by 2-dimensional thiol blotting. However, the degree of protein thiol modification was not affected by a prior heat shock. Nor did the heat shock increase the intracellular concentration of glutathione or the activity of glutathione reductase. The diamide treatment caused specific, as opposed to general, protein thiol oxidation and heat shock did not prevent this. It is hypothesized that it was the oxidation of protein thiols which led to cellular toxicity. Protein synthesis, triggered by heat shock, protected cells from the diamide toxicity without preventing protein thiol modification. These results suggest that the proteins

  10. High-throughput human metabolism and toxicity analysis.

    PubMed

    Lee, Moo-Yeal; Dordick, Jonathan S

    2006-12-01

    Poor drug candidate safety profiles are often identified late in the drug development process, manifesting themselves in the preclinical and clinical phases and significantly contributing to the high cost and low yield of drug discovery. As a result, new tools are needed to accelerate the assessment of drug candidate toxicity and human metabolism earlier in the drug development process, from primary drug candidate screening to lead optimization. Although high-throughput screens exist for much of the discovery phase of drug development, translating such screening techniques into platforms that can accurately mimic the human in vivo response and predict the impact of drug candidates on human toxicology has proven difficult. Nevertheless, some success has been achieved in recent years, which may ultimately yield widespread acceptance in the pharmaceutical industry.

  11. Aggresomes protect cells by enhancing the degradation of toxic polyglutamine-containing protein.

    PubMed

    Taylor, J Paul; Tanaka, Fumiaki; Robitschek, Jon; Sandoval, C Miguel; Taye, Addis; Markovic-Plese, Silva; Fischbeck, Kenneth H

    2003-04-01

    Expression of misfolded protein in cultured cells frequently leads to the formation of juxtanuclear inclusions that have been termed 'aggresomes'. Aggresome formation is an active cellular response that involves trafficking of the offending protein along microtubules, reorganization of intermediate filaments and recruitment of components of the ubiquitin proteasome system. Whether aggresomes are benevolent or noxious is unknown, but they are of particular interest because of the appearance of similar inclusions in protein deposition diseases. Here we present evidence that aggresomes serve a cytoprotective function and are associated with accelerated turnover of mutant proteins. We show that mutant androgen receptor (AR), the protein responsible for X-linked spinobulbar muscular atrophy, forms insoluble aggregates and is toxic to cultured cells. Mutant AR was also found to form aggresomes in a process distinct from aggregation. Molecular and pharmacological interventions were used to disrupt aggresome formation, revealing their cytoprotective function. Aggresome-forming proteins were found to have an accelerated rate of turnover, and this turnover was slowed by inhibition of aggresome formation. Finally, we show that aggresome-forming proteins become membrane-bound and associate with lysosomal structures. Together, these findings suggest that aggresomes are cytoprotective, serving as cytoplasmic recruitment centers to facilitate degradation of toxic proteins.

  12. Encapsulation of Aconitine in Self-Assembled Licorice Protein Nanoparticles Reduces the Toxicity In Vivo

    NASA Astrophysics Data System (ADS)

    Ke, Li-jing; Gao, Guan-zhen; Shen, Yong; Zhou, Jian-wu; Rao, Ping-fan

    2015-11-01

    Many herbal medicines and compositions are clinically effective but challenged by its safety risks, i.e., aconitine (AC) from aconite species. The combined use of Radix glycyrrhizae (licorice) with Radix aconite L. effectively eliminates toxicity of the later while increasing efficacy. In this study, a boiling-stable 31-kDa protein (namely GP) was purified from licorice and self-assembled into nanoparticles (206.2 ± 2.0 nm) at pH 5.0, 25 °C. The aconitine-encapsulated GP nanoparticles (238.2 ± 1.2 nm) were prepared following the same procedure and tested for its toxicity by intraperitoneal injection on ICR mouse ( n = 8). Injection of GP-AC nanoparticles and the mixed licorice-aconite decoction, respectively, caused mild recoverable toxic effects and no death, while the aconitine, particle-free GP-AC mixture and aconite decoction induced sever toxic effects and 100 % death. Encapsulation of poisonous alkaloids into self-assembled herbal protein nanoparticles contributes to toxicity attenuation of combined use of herbs, implying a prototype nanostructure and a universal principle for the safer clinical applications of herbal medicines.

  13. Genetic toxicity of high-boiling petroleum substances.

    PubMed

    McKee, Richard H; Schreiner, Ceinwen A; Nicolich, Mark J; Gray, Thomas M

    2013-11-01

    There are several specific types of high-boiling petroleum substances (HBPS) having final boiling points >343°C), in which genetic toxicity can be related to the content of polycyclic aromatic compounds (PACs), specifically crude oils, gas oils, heavy fuel oils, lubricant base oils, waxes and aromatic extracts. Evaluation of optimized Salmonella tests covering over 250 samples from 43 types of HBPS revealed that gene mutation can be determined for these substances using a protocol optimized for the detection of mutagenic PAC. The outcomes of modified Salmonella assays can be predicted using HBPS compositional information as input to a newly developed statistical model. The general outcome of the optimized Salmonella assay can be predicted for an untested substance based on its Aromatic Ring Class (ARC) profile. Review of the results from numerous cytogenetic tests showed that although a few positive study results have been reported, most HBPS do not produce chromosomal effects when tested in rodent bone marrow assays or in in vitro chromosomal aberration assays. Results of both bacterial and cytogenetic studies can be used to satisfy genetic toxicity endpoints for the HBPS category substances. PMID:23685115

  14. Genetic toxicity of high-boiling petroleum substances.

    PubMed

    McKee, Richard H; Schreiner, Ceinwen A; Nicolich, Mark J; Gray, Thomas M

    2013-11-01

    There are several specific types of high-boiling petroleum substances (HBPS) having final boiling points >343°C), in which genetic toxicity can be related to the content of polycyclic aromatic compounds (PACs), specifically crude oils, gas oils, heavy fuel oils, lubricant base oils, waxes and aromatic extracts. Evaluation of optimized Salmonella tests covering over 250 samples from 43 types of HBPS revealed that gene mutation can be determined for these substances using a protocol optimized for the detection of mutagenic PAC. The outcomes of modified Salmonella assays can be predicted using HBPS compositional information as input to a newly developed statistical model. The general outcome of the optimized Salmonella assay can be predicted for an untested substance based on its Aromatic Ring Class (ARC) profile. Review of the results from numerous cytogenetic tests showed that although a few positive study results have been reported, most HBPS do not produce chromosomal effects when tested in rodent bone marrow assays or in in vitro chromosomal aberration assays. Results of both bacterial and cytogenetic studies can be used to satisfy genetic toxicity endpoints for the HBPS category substances.

  15. Bioactivation, protein haptenation, and toxicity of sulfamethoxazole and dapsone in normal human dermal fibroblasts

    SciTech Connect

    Bhaiya, Payal; Roychowdhury, Sanjoy; Vyas, Piyush M.; Doll, Mark A.; Hein, David W.; Svensson, Craig K. . E-mail: craig-svensson@uiowa.edu

    2006-09-01

    Cutaneous drug reactions (CDRs) associated with sulfonamides are believed to be mediated through the formation of reactive metabolites that result in cellular toxicity and protein haptenation. We evaluated the bioactivation and toxicity of sulfamethoxazole (SMX) and dapsone (DDS) in normal human dermal fibroblasts (NHDF). Incubation of cells with DDS or its metabolite (D-NOH) resulted in protein haptenation readily detected by confocal microscopy and ELISA. While the metabolite of SMX (S-NOH) haptenated intracellular proteins, adducts were not evident in incubations with SMX. Cells expressed abundant N-acetyltransferase-1 (NAT1) mRNA and activity, but little NAT2 mRNA or activity. Neither NAT1 nor NAT2 protein was detected. Incubation of NHDF with S-NOH or D-NOH increased reactive oxygen species formation and reduced glutathione content. NHDF were less susceptible to the cytotoxic effect of S-NOH and D-NOH than are keratinocytes. Our studies provide the novel observation that NHDF are able to acetylate both arylamine compounds and bioactivate the sulfone DDS, giving rise to haptenated proteins. The reactive metabolites of SMX and DDS also provoke oxidative stress in these cells in a time- and concentration-dependent fashion. Further work is needed to determine the role of the observed toxicity in mediating CDRs observed with these agents.

  16. Toxicity detection using lysosomal enzymes, glycoamylase and thioredoxin fused with fluorescent protein in Saccharomyces cerevisiae.

    PubMed

    Nguyen, Ngoc-Tu; Shin, Hwa-Yoon; Kim, Yang-Hoon; Min, Jiho

    2015-11-20

    Saccharomyces cerevisiae is the simplest and a favorite eukaryotic system that contains lysosome and thus, is a suitable organism for monitoring some toxic effects in environmental pollution. In this study, S. cerevisiae was transformed with two recombinant plasmids. Sporulation-specific glycoamylase (SGA1), which was upregulated in response to arsenic, was fused with the blue fluorescent protein (BFP) for the construction of an oxidative stress-causing chemicals sensor. Additionally, thioredoxin (TRX2), a protein overexpressed exclusively under tetracycline's influence, fused with the cyan fluorescent protein (CFP) to create a detector for this kind of chemical. In summary, we developed two recombinant S. cerevisiae that facilitate the detection of both kinds of toxic chemicals, specifically visualized by different color indicators.

  17. The epitopes in wheat proteins for defining toxic units relevant to human health.

    PubMed

    Juhász, Angéla; Gell, Gyöngyvér; Békés, Frank; Balázs, Ervin

    2012-11-01

    Wheat-related disorders are well-studied health problems. Knowledge of the composition and amounts of epitopes present in a single wheat sample represents a significant gap, and the detailed wheat proteome datasets now available can provide the necessary information to carry out an estimation of allergen prediction for a single cultivar. The combined use of genome sequence and allergen databases, prediction methodology, and cereal chemistry results in better understanding of the level of toxicity present in the end-products produced from wheat flour. The workflow presented in this review provides information about the number and distribution of epitopes at single protein, or protein fraction, levels. In addition, epitopes present in the highest frequency and harmful proteins expressed in the highest amount can be identified. The "epitope toxicity" value obtained in this way is a significant research output from the analysis of large datasets that can be applied to the food industry.

  18. Cold-inducible cloning vectors for low-temperature protein expression in Escherichia coli: application to the production of a toxic and proteolytically sensitive fusion protein.

    PubMed

    Mujacic, M; Cooper, K W; Baneyx, F

    1999-10-01

    TolAI-beta-lactamase a fusion protein consisting of the inner membrane anchoring domain of the Escherichia coli transenvelope protein TolA followed by TEM-beta-lactamase was found to be toxic and highly unstable when transcribed from the bacteriophage T7 promoter at 37 degrees C. Expression at 15 or 23 degrees C alleviated toxicity, but led to only partial stabilization of the fusion protein. To evaluate the usefulness of cold-shock promoters for the production of proteolytically sensitive proteins at low temperatures, we constructed a set of cloning vectors suitable for rapidly positioning PCR products under cspA transcriptional control. TolAI-beta-lactamase degradation was completely abolished when cspA-driven transcription was induced by temperature downshift to 15 or 23 degrees C. Our results suggest that the cspA promoter system may be a valuable tool for the production of proteins containing membrane-spanning domains or otherwise unstable gene products in E. coli.

  19. Highly specific protein-protein interactions, evolution and negative design.

    PubMed

    Sear, Richard P

    2004-12-01

    We consider highly specific protein-protein interactions in proteomes of simple model proteins. We are inspired by the work of Zarrinpar et al (2003 Nature 426 676). They took a binding domain in a signalling pathway in yeast and replaced it with domains of the same class but from different organisms. They found that the probability of a protein binding to a protein from the proteome of a different organism is rather high, around one half. We calculate the probability of a model protein from one proteome binding to the protein of a different proteome. These proteomes are obtained by sampling the space of functional proteomes uniformly. In agreement with Zarrinpar et al we find that the probability of a protein binding a protein from another proteome is rather high, of order one tenth. Our results, together with those of Zarrinpar et al, suggest that designing, say, a peptide to block or reconstitute a single signalling pathway, without affecting any other pathways, requires knowledge of all the partners of the class of binding domains the peptide is designed to mimic. This knowledge is required to use negative design to explicitly design out interactions of the peptide with proteins other than its target. We also found that patches that are required to bind with high specificity evolve more slowly than those that are required only to not bind to any other patch. This is consistent with some analysis of sequence data for proteins engaged in highly specific interactions.

  20. Transcytosis, Antitumor Activity and Toxicity of Staphylococcal Enterotoxin C2 as an Oral Administration Protein Drug

    PubMed Central

    Zhao, Wenbin; Li, Yangyang; Liu, Wenhui; Ding, Ding; Xu, Yingchun; Pan, Liqiang; Chen, Shuqing

    2016-01-01

    Staphylococcal enterotoxin C2 (SEC2) is a classical superantigen (SAg), which can tremendously activate T lymphocytes at very low dosage, thus exerting its powerful antitumor activity. As an intravenous protein drug and a bacterial toxin, SEC2 has some limitations including poor patient compliance and toxic side effects. In this research, we devoted our attention to studying the antitumor activity and toxicity of SEC2 as a potential oral administration protein drug. We proved that His-tagged SEC2 (SEC2-His) could undergo facilitated transcytosis on human colon adenocarcinoma (Caco-2) cells and SEC2-His was detected in the blood of rats after oral administration. Furthermore, oral SEC2-His caused massive cytokine release and immune cell enrichment around tumor tissue, leading to inhibition of tumor growth in vivo. Meanwhile, although SEC2-His was dosed up to 32 mg/kg in mice, no significant toxicity was observed. These data showed that SEC2 can cross the intestinal epithelium in an immunologically integral form, maintaining antitumor activity but with reduced systemic toxicity. Therefore, these results may have implications for developing SEC2 as an oral administration protein drug. PMID:27322320

  1. Small heat shock proteins protect against {alpha}-synuclein-induced toxicity and aggregation

    SciTech Connect

    Outeiro, Tiago Fleming; Klucken, Jochen; Strathearn, Katherine E.; Liu Fang; Nguyen, Paul; Rochet, Jean-Christophe; Hyman, Bradley T.; McLean, Pamela J. . E-mail: touteiro@partners.org

    2006-12-22

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

  2. Regulation of OSU-03012 toxicity by ER stress proteins and ER stress-inducing drugs.

    PubMed

    Booth, Laurence; Roberts, Jane L; Cruickshanks, Nichola; Grant, Steven; Poklepovic, Andrew; Dent, Paul

    2014-10-01

    The present studies examined the toxic interaction between the non-coxib celecoxib derivative OSU-03012 and phosphodiesterase 5 (PDE5) inhibitors, and also determined the roles of endoplasmic reticulum stress response regulators in cell survival. PDE5 inhibitors interacted in a greater than additive fashion with OSU-03012 to kill parental glioma and stem-like glioma cells. Knockdown of the endoplasmic reticulum stress response proteins IRE1 or XBP1 enhanced the lethality of OSU-03012, and of [OSU-03012 + PDE5 inhibitor] treatment. Pan-caspase and caspase-9 inhibition did not alter OSU-03012 lethality but did abolish enhanced killing in the absence of IRE1 or XBP1. Expression of the mitochondrial protective protein BCL-XL or the caspase-8 inhibitor c-FLIP-s, or knockdown of death receptor CD95 or the death receptor caspase-8 linker protein FADD, suppressed killing by [OSU-03012 + PDE5 inhibitor] treatment. CD95 activation was blocked by the nitric oxide synthase inhibitor L-NAME. Knockdown of the autophagy regulatory proteins Beclin1 or ATG5 protected the cells from OSU-03012 and from [OSU-03012 + PDE5 inhibitor] toxicity. Knockdown of IRE1 enhanced OSU-03012/[OSU-03012 + PDE5 inhibitor]-induced JNK activation, and inhibition of JNK suppressed the elevated killing caused by IRE1 knockdown. Knockdown of CD95 blunted JNK activation. Collectively, our data demonstrate that PDE5 inhibitors recruit death receptor signaling to enhance OSU-03012 toxicity in glioblastoma multiforme (GBM) cells. PMID:25103559

  3. Rational Design of a Carrier Protein for the Production of Recombinant Toxic Peptides in Escherichia coli

    PubMed Central

    Pizzo, Elio; Varcamonti, Mario; Zanfardino, Anna; Sgambati, Valeria; Di Maro, Antimo; Carpentieri, Andrea; Izzo, Viviana; Di Donato, Alberto; Cafaro, Valeria; Notomista, Eugenio

    2016-01-01

    Commercial uses of bioactive peptides require low cost, effective methods for their production. We developed a new carrier protein for high yield production of recombinant peptides in Escherichia coli very well suited for the production of toxic peptides like antimicrobial peptides. GKY20, a short antimicrobial peptide derived from the C-terminus of human thrombin, was fused to the C-terminus of Onconase, a small ribonuclease (104 amino acids), which efficiently drove the peptide into inclusion bodies with very high expression levels (about 200–250 mg/L). After purification of the fusion protein by immobilized metal ion affinity chromatography, peptide was obtained by chemical cleavage in diluted acetic acid of an acid labile Asp-Pro sequence with more than 95% efficiency. To improve peptide purification, Onconase was mutated to eliminate all acid labile sequences thus reducing the release of unwanted peptides during the acid cleavage. Mutations were chosen to preserve the differential solubility of Onconase as function of pH, which allows its selective precipitation at neutral pH after the cleavage. The improved carrier allowed the production of 15–18 mg of recombinant peptide per liter of culture with 96–98% purity without the need of further chromatographic steps after the acid cleavage. The antimicrobial activity of the recombinant peptide, with an additional proline at the N-terminus, was tested on Gram-negative and Gram-positive strains and was found to be identical to that measured for synthetic GKY20. This finding suggests that N-terminal proline residue does not change the antimicrobial properties of recombinant (P)GKY20. The improved carrier, which does not contain cysteine and methionine residues, Asp-Pro and Asn-Gly sequences, is well suited for the production of peptides using any of the most popular chemical cleavage methods. PMID:26808536

  4. Rational Design of a Carrier Protein for the Production of Recombinant Toxic Peptides in Escherichia coli.

    PubMed

    Pane, Katia; Durante, Lorenzo; Pizzo, Elio; Varcamonti, Mario; Zanfardino, Anna; Sgambati, Valeria; Di Maro, Antimo; Carpentieri, Andrea; Izzo, Viviana; Di Donato, Alberto; Cafaro, Valeria; Notomista, Eugenio

    2016-01-01

    Commercial uses of bioactive peptides require low cost, effective methods for their production. We developed a new carrier protein for high yield production of recombinant peptides in Escherichia coli very well suited for the production of toxic peptides like antimicrobial peptides. GKY20, a short antimicrobial peptide derived from the C-terminus of human thrombin, was fused to the C-terminus of Onconase, a small ribonuclease (104 amino acids), which efficiently drove the peptide into inclusion bodies with very high expression levels (about 200-250 mg/L). After purification of the fusion protein by immobilized metal ion affinity chromatography, peptide was obtained by chemical cleavage in diluted acetic acid of an acid labile Asp-Pro sequence with more than 95% efficiency. To improve peptide purification, Onconase was mutated to eliminate all acid labile sequences thus reducing the release of unwanted peptides during the acid cleavage. Mutations were chosen to preserve the differential solubility of Onconase as function of pH, which allows its selective precipitation at neutral pH after the cleavage. The improved carrier allowed the production of 15-18 mg of recombinant peptide per liter of culture with 96-98% purity without the need of further chromatographic steps after the acid cleavage. The antimicrobial activity of the recombinant peptide, with an additional proline at the N-terminus, was tested on Gram-negative and Gram-positive strains and was found to be identical to that measured for synthetic GKY20. This finding suggests that N-terminal proline residue does not change the antimicrobial properties of recombinant (P)GKY20. The improved carrier, which does not contain cysteine and methionine residues, Asp-Pro and Asn-Gly sequences, is well suited for the production of peptides using any of the most popular chemical cleavage methods. PMID:26808536

  5. Proteomic analysis of secreted proteins by human bronchial epithelial cells in response to cadmium toxicity.

    PubMed

    Chen, De-Ju; Xu, Yan-Ming; Zheng, Wei; Huang, Dong-Yang; Wong, Wing-Yan; Tai, William Chi-Shing; Cho, Yong-Yeon; Lau, Andy T Y

    2015-09-01

    For years, many studies have been conducted to investigate the intracellular response of cells challenged with toxic metal(s), yet, the corresponding secretome responses, especially in human lung cells, are largely unexplored. Here, we provide a secretome analysis of human bronchial epithelial cells (BEAS-2B) treated with cadmium chloride (CdCl2 ), with the aim of identifying secreted proteins in response to Cd toxicity. Proteins from control and spent media were separated by two-dimensional electrophoresis and visualized by silver staining. Differentially-secreted proteins were identified by MALDI-TOF-MS analysis and database searching. We characterized, for the first time, the extracellular proteome changes of BEAS-2B dosed with Cd. Our results unveiled that Cd treatment led to the marked upregulation of molecular chaperones, antioxidant enzymes, enzymes associated with glutathione metabolic process, proteins involved in cellular energy metabolism, as well as tumor-suppressors. Pretreatment of cells with the thiol antioxidant glutathione before Cd treatment effectively abrogated the secretion of these proteins and prevented cell death. Taken together, our results demonstrate that Cd causes oxidative stress-induced cytotoxicity; and the differentially-secreted protein signatures could be considered as targets for potential use as extracellular biomarkers upon Cd exposure.

  6. Dynamic development of the protein corona on silica nanoparticles: composition and role in toxicity

    NASA Astrophysics Data System (ADS)

    Mortensen, Ninell P.; Hurst, Gregory B.; Wang, Wei; Foster, Carmen M.; Nallathamby, Prakash D.; Retterer, Scott T.

    2013-06-01

    The formation and composition of the protein corona on silica (SiO2) nanoparticles (NP) with different surface chemistries was evaluated over time. Native SiO2, amine (-NH2) and carboxy (-COO-) modified NP were examined following incubation in mammalian growth media containing fetal bovine serum (FBS) for 1, 4, 24 and 48 hours. The protein corona transition from its early dynamic state to the later more stable corona was evaluated using mass spectrometry. The NP diameter was 22.4 +/- 2.2 nm measured by scanning transmission electron microscopy (STEM). Changes in hydrodynamic diameter and agglomeration kinetics were studied using dynamic light scattering (DLS). The initial surface chemistry of the NP played an important role in the development and final composition of the protein corona, impacting agglomeration kinetics and NP toxicity. Particle toxicity, indicated by changes in membrane integrity and mitochondrial activity, was measured by lactate dehydrogenase (LDH) release and tetrazolium reduction (MTT), respectively, in mouse alveolar macrophages (RAW264.7) and mouse lung epithelial cells (C10). SiO2-COO- NP had a slower agglomeration rate, formed smaller aggregates, and exhibited lower cytotoxicity compared to SiO2 and SiO2-NH2. Composition of the protein corona for each of the three NP was unique, indicating a strong dependence of corona development on NP surface chemistry. This work underscores the need to understand all aspects of NP toxicity, particularly the influence of agglomeration on effective dose and particle size. Furthermore, the interplay between materials and local biological environment is emphasized and highlights the need to conduct toxicity profiling under physiologically relevant conditions that provide an appropriate estimation of material modifications that occur during exposure in natural environments.The formation and composition of the protein corona on silica (SiO2) nanoparticles (NP) with different surface chemistries was evaluated

  7. A bacterial view of the periodic table: genes and proteins for toxic inorganic ions.

    PubMed

    Silver, Simon; Phung, Le T

    2005-12-01

    Essentially all bacteria have genes for toxic metal ion resistances and these include those for Ag+, AsO2-, AsO4(3-), Cd2+ Co2+, CrO4(2-), Cu2+, Hg2+, Ni2+, Pb2+, TeO3(2-), Tl+ and Zn2+. The largest group of resistance systems functions by energy-dependent efflux of toxic ions. Fewer involve enzymatic transformations (oxidation, reduction, methylation, and demethylation) or metal-binding proteins (for example, metallothionein SmtA, chaperone CopZ and periplasmic silver binding protein SilE). Some of the efflux resistance systems are ATPases and others are chemiosmotic ion/proton exchangers. For example, Cd2+-efflux pumps of bacteria are either inner membrane P-type ATPases or three polypeptide RND chemiosmotic complexes consisting of an inner membrane pump, a periplasmic-bridging protein and an outer membrane channel. In addition to the best studied three-polypeptide chemiosmotic system, Czc (Cd2+, Zn2+, and Co2), others are known that efflux Ag+, Cu+, Ni2+, and Zn2+. Resistance to inorganic mercury, Hg2+ (and to organomercurials, such as CH3Hg+ and phenylmercury) involve a series of metal-binding and membrane transport proteins as well as the enzymes mercuric reductase and organomercurial lyase, which overall convert more toxic to less toxic forms. Arsenic resistance and metabolizing systems occur in three patterns, the widely-found ars operon that is present in most bacterial genomes and many plasmids, the more recently recognized arr genes for the periplasmic arsenate reductase that functions in anaerobic respiration as a terminal electron acceptor, and the aso genes for the periplasmic arsenite oxidase that functions as an initial electron donor in aerobic resistance to arsenite.

  8. Mitochondrial iron accumulation exacerbates hepatic toxicity caused by hepatitis C virus core protein

    SciTech Connect

    Sekine, Shuichi; Ito, Konomi; Watanabe, Haruna; Nakano, Takafumi; Moriya, Kyoji; Shintani, Yoshizumi; Fujie, Hajime; Tsutsumi, Takeya; Miyoshi, Hideyuki; Fujinaga, Hidetake; Shinzawa, Seiko; Koike, Kazuhiko; Horie, Toshiharu

    2015-02-01

    Patients with long-lasting hepatitis C virus (HCV) infection are at major risk of hepatocellular carcinoma (HCC). Iron accumulation in the livers of these patients is thought to exacerbate conditions of oxidative stress. Transgenic mice that express the HCV core protein develop HCC after the steatosis stage and produce an excess of hepatic reactive oxygen species (ROS). The overproduction of ROS in the liver is the net result of HCV core protein-induced dysfunction of the mitochondrial respiratory chain. This study examined the impact of ferric nitrilacetic acid (Fe-NTA)-mediated iron overload on mitochondrial damage and ROS production in HCV core protein-expressing HepG2 (human HCC) cells (Hep39b cells). A decrease in mitochondrial membrane potential and ROS production were observed following Fe-NTA treatment. After continuous exposure to Fe-NTA for six days, cell toxicity was observed in Hep39b cells, but not in mock (vector-transfected) HepG2 cells. Moreover, mitochondrial iron ({sup 59}Fe) uptake was increased in the livers of HCV core protein-expressing transgenic mice. This increase in mitochondrial iron uptake was inhibited by Ru360, a mitochondrial Ca{sup 2+} uniporter inhibitor. Furthermore, the Fe-NTA-induced augmentation of mitochondrial dysfunction, ROS production, and cell toxicity were also inhibited by Ru360 in Hep39b cells. Taken together, these results indicate that Ca{sup 2+} uniporter-mediated mitochondrial accumulation of iron exacerbates hepatocyte toxicity caused by the HCV core protein. - Highlights: • Iron accumulation in the livers of patients with hepatitis C virus (HCV) infection is thought to exacerbate oxidative stress. • The impact of iron overload on mitochondrial damage and ROS production in HCV core protein-expressing cells were examined. • Mitochondrial iron uptake was increased in the livers of HCV core protein-expressing transgenic mice. • Ca{sup 2+} uniporter-mediated mitochondrial accumulation of iron exacerbates

  9. Evaluating the male and female reproductive toxicity of high-boiling petroleum substances.

    PubMed

    Murray, F Jay; Gray, Thomas M; Roberts, Linda G; Roth, Randy N; Nicolich, Mark J; Simpson, Barry J

    2013-11-01

    To meet the EPA HPV Chemical Challenge Program requirement for reproductive toxicity data on sponsored high-boiling petroleum substances (HBPS), an analysis was conducted using the results of 39 repeat-dose and 59 developmental rat dermal toxicity studies on HBPS samples spanning the boiling range of the sponsored substances, and the results of three one-generation reproductive toxicity studies on two samples spanning the concentration range of polycyclic aromatic compounds of sponsored substances. The analysis found little evidence of male or female reproductive tract toxicity based on histopathology, reproductive organ weight, and sperm parameters, and no evidence of effects on fertility, while significant developmental toxicity and/or systemic repeat-dose toxicity were frequently observed. Among 14 samples of HBPS tested in both repeat-dose toxicity and developmental toxicity studies, there were no studies in which an adverse reproductive tract finding occurred at a dose lower than that producing developmental toxicity or other adverse effects in repeat-dose toxicity studies. The current analysis supports the hypothesis that effects in developmental and/or repeat-dose toxicity studies of HBPS occur at doses lower than those that might affect fertility in rat one-generation reproductive studies. When adequate developmental and repeat-dose toxicity studies are available, a reproductive toxicity study of HBPS appears unnecessary.

  10. Prohibitin, an essential protein for Colorado potato beetle larval viability, is relevant to Bacillus thuringiensis Cry3Aa toxicity.

    PubMed

    Ochoa-Campuzano, Camila; Martínez-Ramírez, Amparo C; Contreras, Estefanía; Rausell, Carolina; Real, M Dolores

    2013-11-01

    Bacillus thuringienesis (Bt) Cry toxins constitute the most extensively used environmentally safe biopesticide and their mode of action relies on the interaction of the toxins with membrane proteins in the midgut of susceptible insects that mediate toxicity and insect specificity. Therefore, identification of Bt Cry toxin interacting proteins in the midgut of target insects and understanding their role in toxicity is of great interest to exploit their insecticidal action. Using ligand blot, we demonstrated that Bt Cry3Aa toxin bound to a 30kDa protein in Colorado potato beetle (CPB) larval midgut membrane, identified by sequence homology as prohibitin-1 protein. Prohibitins comprise a highly conserved family of proteins implicated in important cellular processes. We obtained the complete CPB prohibitin-1 DNA coding sequence of 828pb, in silico translated into a 276-amino acid protein. The analysis at the amino acid level showed that the protein contains a prohibitin-homology domain (Band7_prohibitin, cd03401) conserved among prohibitin proteins. A striking feature of the CPB identified prohibitin-1 is the predicted presence of cadherin elements, potential binding sites for Cry toxins described in other Bt susceptible insects. We also showed that CPB prohibitin-1 protein partitioned into both, detergent soluble and insoluble membrane fractions, as well as a prohibitin-2 homologous protein, previously reported to form functional complexes with prohibitin-1 in other organisms. Prohibitin complexes act as membrane scaffolds ensuring the recruitment of membrane proteases to facilitate substrate processing. Accordingly, sequestration of prohibitin-1 by an anti-prohibitin-1 antibody impaired the Cry3Aa toxin inhibition of the proteolytic cleavage of a fluorogenic synthetic substrate of an ADAM-like metalloprotease previously reported to proteolize this toxin. In this work, we also demonstrated that prohibitin-1 RNAi silencing in CPB larvae produced deleterious effects and

  11. Prohibitin, an essential protein for Colorado potato beetle larval viability, is relevant to Bacillus thuringiensis Cry3Aa toxicity.

    PubMed

    Ochoa-Campuzano, Camila; Martínez-Ramírez, Amparo C; Contreras, Estefanía; Rausell, Carolina; Real, M Dolores

    2013-11-01

    Bacillus thuringienesis (Bt) Cry toxins constitute the most extensively used environmentally safe biopesticide and their mode of action relies on the interaction of the toxins with membrane proteins in the midgut of susceptible insects that mediate toxicity and insect specificity. Therefore, identification of Bt Cry toxin interacting proteins in the midgut of target insects and understanding their role in toxicity is of great interest to exploit their insecticidal action. Using ligand blot, we demonstrated that Bt Cry3Aa toxin bound to a 30kDa protein in Colorado potato beetle (CPB) larval midgut membrane, identified by sequence homology as prohibitin-1 protein. Prohibitins comprise a highly conserved family of proteins implicated in important cellular processes. We obtained the complete CPB prohibitin-1 DNA coding sequence of 828pb, in silico translated into a 276-amino acid protein. The analysis at the amino acid level showed that the protein contains a prohibitin-homology domain (Band7_prohibitin, cd03401) conserved among prohibitin proteins. A striking feature of the CPB identified prohibitin-1 is the predicted presence of cadherin elements, potential binding sites for Cry toxins described in other Bt susceptible insects. We also showed that CPB prohibitin-1 protein partitioned into both, detergent soluble and insoluble membrane fractions, as well as a prohibitin-2 homologous protein, previously reported to form functional complexes with prohibitin-1 in other organisms. Prohibitin complexes act as membrane scaffolds ensuring the recruitment of membrane proteases to facilitate substrate processing. Accordingly, sequestration of prohibitin-1 by an anti-prohibitin-1 antibody impaired the Cry3Aa toxin inhibition of the proteolytic cleavage of a fluorogenic synthetic substrate of an ADAM-like metalloprotease previously reported to proteolize this toxin. In this work, we also demonstrated that prohibitin-1 RNAi silencing in CPB larvae produced deleterious effects and

  12. Recombinant murine toxin from Yersinia pestis shows high toxicity and β-adrenergic blocking activity in mice.

    PubMed

    Fan, Yanxiao; Zhou, Yazhou; Feng, Na; Wang, Qiong; Tian, Guang; Wu, Xiaohong; Liu, Zizhong; Bi, Yujing; Yang, Ruifu; Wang, Xiaoyi

    2016-05-01

    Yersinia pestis murine toxin (Ymt) encoded on pMT1 is a 61-kDa protein, a member of the phospholipase D superfamily, which is found in all the domains of life. It is considered to be an intracellular protein required for the survival of Y. pestis in the midgut of the flea, but the exact role of Ymt in the pathogenesis of Y. pestis has not been clarified. Purified Ymt is highly toxic to mice and rats, but the exact mechanism of the animals' death is unclear. Here, we prepared a recombinant Ymt in Escherichia coli BL21 cells, and determined its toxicity and activity. We demonstrated that recombinant Ymt was as toxic to mice as the native protein when administered via the intraperitoneal or intravenous route, and inhibited the elevation of blood sugar caused by adrenaline. We also demonstrated that recombinant Ymt was highly toxic to mice when administered via the muscular or subcutaneous route. We also show that the multiple organ congestion or hemorrhage caused by Ymt poisoning may explain the death of the mice. PMID:26774329

  13. High-Content Screening for Assessing Nanomaterial Toxicity.

    PubMed

    Huo, Lingling; Chen, Rui; Shi, Xiaofei; Bai, Ru; Wang, Peng; Chang, Yanzhong; Chen, Chunying

    2015-02-01

    With rapid development of novel nanomaterials (NMs), the state of the art technologies with high efficiency and high-throughput characteristics had been applied for nanosafety evaluation. High-content screening (HCS), a cell-based multi-parametric image analysis technique, was adopted in the evaluation of eight different NMs in this study. A set of different endpoints including reactive oxygen species (ROS) production, Ca2+ transient, mitochondrial membrane potential (MMP) and cellular pH levels were checked in human bronchial epithelial (16HBE) cells after incubating with NMs for 24 hours. All NMs induced significant increase of intracellular ROS levels in 16HBE cells, although the decrease of cell viability was only found in Ag and ZnO NMs-treated cells. MMP level had a dose-response decrease in Ag, ZnO and CeO2 NMs-treated cells, while showed a significant increase in TiO2 NMs-treated cells. All tested NMs showed significant up-regulation of cellular lysosomal pH levels. However, none of NMs caused significant changes in cellular Ca2+ level at 24-hour time point. HCS allows for efficient and reliable screening of multiple responses of cells simultaneously within one screen test, which can avoid the problematic interpretation of investigations when carried on a single toxicological endpoint. Therefore, the present data provide insight and inspiration that HCS is an effective and powerful method for image-based assessments with a broad set of biological endpoints in toxicity evaluation of nanomaterials.

  14. Expression and purification of toxic anti-breast cancer p28-NRC chimeric protein

    PubMed Central

    Soleimani, Meysam; Mirmohammad-Sadeghi, Hamid; Sadeghi-Aliabadi, Hojjat; Jahanian-Najafabadi, Ali

    2016-01-01

    Background: Chimeric proteins consisting of a targeting moiety and a cytotoxic moiety are now under intense research focus for targeted therapy of cancer. Here, we report cloning, expression, and purification of such a targeted chimeric protein made up of p28 peptide as both targeting and anticancer moiety fused to NRC peptide as a cytotoxic moiety. However, since the antimicrobial activity of the NRC peptide would intervene expression of the chimeric protein in Escherichia coli, we evaluated the effects of two fusion tags, that is, thioredoxin (Trx) and 6x-His tags, and various expression conditions, on the expression of p28-NRC chimeric protein. Materials and Methods: In order to express the chimeric protein with only 6x-His tag, pET28 expression plasmid was used. Cloning in pET32 expression plasmid was performed to add both Trx and 6x-His tags to the chimeric protein. Expression of the chimeric protein with both plasmids was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis following optimization of expression conditions and host strains. Results: Expression of the chimeric protein in pET28a was performed. However, expression yield of the chimeric protein was low. Optimization of culture conditions and host strains led to reasonable expression yield of the toxic chimeric protein in pET32a vector. In cases of both plasmids, approximately 10 kDa deviation of the apparent molecular weight from the theoretical one was seen in SDS-PAGE of purified chimeric proteins. Conclusions: The study leads to proper expression and purification yield of p28-NRC chimeric protein with Trx tag following optimizing culture conditions and host strains. PMID:27169101

  15. Non-toxic invert analog glass compositions of high modulus

    NASA Technical Reports Server (NTRS)

    Bacon, J. F. (Inventor)

    1974-01-01

    Glass compositions having a Young's modulus of at least 15 million psi are described. They and a specific modulus of at least 110 million inches consist essentially of, in mols, 15 to 40% SiO2, 6 to 15% Li2O, 24 to 45% of at least two bivalent oxides selected from the group consisting of Ca, NzO, MgO and CuO; 13 to 39% of at least two trivalent oxides selected from the group consisting of Al2O3, Fe2O3, B2O3, La2O3, and Y2O3 and up to 15% of one or more tetravelent oxides selected from the group consisting of ZrO2, TiO2 and CeO2. The high modulus, low density glass compositions contain no toxic elements. The composition, glass density, Young's modulus, and specific modulus for 28 representative glasses are presented. The fiber modulus of five glasses are given.

  16. Cytoprotective role of astaxanthin against glycated protein/iron chelate-induced toxicity in human umbilical vein endothelial cells.

    PubMed

    Nishigaki, Ikuo; Rajendran, Peramaiyan; Venugopal, Ramachandran; Ekambaram, Gnapathy; Sakthisekaran, Dhanapal; Nishigaki, Yutaka

    2010-01-01

    Astaxanthin (ASX), a red carotenoid pigment with no pro-vitamin A activity, is a biological antioxidant that occurs naturally in a wide variety of plants, algae and seafoods. This study investigated whether ASX could inhibit glycated protein/iron chelate-induced toxicity in human umbilical-vein endothelial cells (HUVEC) by interfering with ROS generation in these cells. Glycated fetal bovine serum (GFBS) was prepared by incubating fetal bovine serum (FBS) with high-concentration glucose. Stimulation of cultured HUVECs with 50 mm 1 mL of GFBS significantly enhanced lipid peroxidation and decreased antioxidant enzyme activities and levels of phase II enzymes. However, preincubation of the cultures with ASX resulted in a marked decrease in the level of lipid peroxide (LPO) and an increase in the levels of antioxidant enzymes in an ASX concentration-dependent manner. These results demonstrate that ASX could inhibit LPO formation and enhance the antioxidant enzyme status in GFBS/iron chelate-exposed endothelial cells by suppressing ROS generation, thereby limiting the effects of the AGE-RAGE interaction. The results indicate that ASX could have a beneficial role against glycated protein/iron chelate-induced toxicity by preventing lipid and protein oxidation and increasing the activity of antioxidant enzymes. PMID:19548280

  17. Identifying Toxicity Pathways with ToxCast High-Throughput Screening and Applications to Predicting Developmental Toxicity

    EPA Science Inventory

    Results from rodent and non-rodent prenatal developmental toxicity tests for over 300 chemicals have been curated into the relational database ToxRefDB. These same chemicals have been run in concentration-response format through over 500 high-throughput screening assays assessin...

  18. Protein aggregation in bacteria: the thin boundary between functionality and toxicity.

    PubMed

    Bednarska, Natalia G; Schymkowitz, Joost; Rousseau, Frederic; Van Eldere, Johan

    2013-09-01

    Misfolding and aggregation of proteins have a negative impact on all living organisms. In recent years, aggregation has been studied in detail due to its involvement in neurodegenerative diseases, including Alzheimer's, Parkinson's and Huntington's diseases, and type II diabetes--all associated with accumulation of amyloid fibrils. This research highlighted the central importance of protein homeostasis, or proteostasis for short, defined as the cellular state in which the proteome is both stable and functional. It implicates an equilibrium between synthesis, folding, trafficking, aggregation, disaggregation and degradation. In accordance with the eukaryotic systems, it has been documented that protein aggregation also reduces fitness of bacterial cells, but although our understanding of the cellular protein quality control systems is perhaps most detailed in bacteria, the use of bacterial proteostasis as a drug target remains little explored. Here we describe protein aggregation as a normal physiological process and its role in bacterial virulence and we shed light on how bacteria defend themselves against the toxic threat of aggregates. We review the impact of aggregates on bacterial viability and look at the ways that bacteria use to maintain a balance between aggregation and functionality. The proteostasis in bacteria can be interrupted via overexpression of proteins, certain antibiotics such as aminoglycosides, as well as antimicrobial peptides--all leading to loss of cell viability. Therefore intracellular protein aggregation and disruption of proteostatic balance in bacteria open up another strategy that should be explored towards the discovery of new antimicrobials.

  19. Regulation of OSU-03012 toxicity by ER stress proteins and ER stress inducing drugs

    PubMed Central

    Booth, Laurence; Roberts, Jane L.; Cruickshanks, Nichola; Grant, Steven; Poklepovic, Andrew; Dent, Paul

    2014-01-01

    The present studies examined the toxic interaction between the non-coxib celecoxib derivative OSU-03012 and phosphodiesterase 5 (PDE5) inhibitors, and to determine the roles of endoplasmic reticulum stress response regulators in cell survival. PDE5 inhibitors interacted in a greater than additive fashion with OSU-03012 to kill parental glioma and stem-like glioma cells. Knock down of the endoplasmic reticulum stress response proteins IRE1 or XBP1 enhanced the lethality of OSU-03012, and of [OSU-03012 + PDE5 inhibitor] treatment. Pan-caspase and caspase 9 inhibition did not alter OSU-03012 lethality but did abolish enhanced killing in the absence of IRE1 or XBP1. Expression of the mitochondrial protective protein BCL-XL or the caspase 8 inhibitor c-FLIP-s, or knock down of death receptor CD95 or the death receptor – caspase 8 linker protein FADD, suppressed killing by [OSU-03012 + PDE5 inhibitor] treatment. CD95 activation was blocked by the nitric oxide synthase inhibitor L-NAME. Knock down of the autophagy regulatory proteins Beclin1 or ATG5 protected cells from OSU-03012 and of [OSU-03012 + PDE5 inhibitor] toxicity. Knock down of IRE1 enhanced OSU-03012/[OSU-03012 + PDE5 inhibitor] –induced JNK activation and inhibition of JNK suppressed the elevated killing caused by IRE1 knock down. Knock down of CD95 blunted JNK activation. Collectively our data demonstrates that PDE5 inhibitors recruit death receptor signaling to enhance OSU-03012 toxicity in GBM cells. PMID:25103559

  20. High density protein crystal growth

    NASA Technical Reports Server (NTRS)

    Rouleau, Robyn (Inventor); Delucas, Lawrence (Inventor); Hedden, Douglas Keith (Inventor)

    2004-01-01

    A protein crystal growth assembly including a crystal growth cell and further including a cell body having a top side and a bottom side and a first aperture defined therethrough, the cell body having opposing first and second sides and a second aperture defined therethrough. A cell barrel is disposed within the cell body, the cell barrel defining a cavity alignable with the first aperture of the cell body, the cell barrel being rotatable within the second aperture. A reservoir is coupled to the bottom side of the cell body and a cap having a top side is disposed on the top side of the cell body. The protein crystal growth assembly may be employed in methods including vapor diffusion crystallization, liquid to liquid crystallization, batch crystallization, and temperature induction batch mode crystallization.

  1. Protein kinase A signaling and calcium ions are major players in PAF mediated toxicity against Aspergillus niger

    PubMed Central

    Binder, Ulrike; Benčina, Mojca; Fizil, Ádám; Batta, Gyula; Chhillar, Anil K.; Marx, Florentine

    2015-01-01

    The Penicillium chrysogenum antifungal protein PAF is toxic against potentially pathogenic Ascomycetes. We used the highly sensitive aequorin-expressing model Aspergillus niger to identify a defined change in cytoplasmic free Ca2+ dynamics in response to PAF. This Ca2+ signature depended on an intact positively charged lysine-rich PAF motif. By combining Ca2+ measurements in A. niger mutants with deregulated cAMP/protein kinase A (PKA) signaling, we proved the interconnection of Ca2+ perturbation and cAMP/PKA signaling in the mechanistic function of PAF. A deep understanding of the mode of action of PAF is an invaluable prerequisite for its future application as new antifungal drug. PMID:25882631

  2. Polyglutamine protein aggregation and toxicity are linked to the cellular stress response.

    PubMed

    Cowan, K J; Diamond, M I; Welch, W J

    2003-06-15

    Chronic exposure of cells to expanded polyglutamine proteins results in eventual cell demise. We constructed mouse cell lines expressing either the full-length androgen receptor (AR), or truncated forms of AR containing 25 or 65 glutamines to study the cellular consequences of chronic low-level exposure to these proteins. Expression of the polyglutamine-expanded truncated AR protein, but not the full-length expanded protein, resulted in the formation of cytoplasmic and nuclear aggregates and eventual cell death. Nuclear aggregates preferentially stained positive for heat shock protein (hsp)72, a sensitive indicator of a cellular stress response. Biochemical studies revealed that the presence of nuclear aggregates correlated with activation of the c-jun NH2-terminal kinase (JNK). Different metabolic insults, including heat shock treatment, and exposure to sodium arsenite or menadione, proved more toxic to those cells expressing the polyglutamine-expanded truncated protein than to cells expressing the non-expanded form. Cells containing cytoplasmic polyglutamine-protein aggregates exhibited a delayed expression of hsp72 after heat shock. Once expressed, hsp72 failed to localize normally and instead was sequestered within the protein aggregates. This was accompanied by an inability of the aggregate-containing cells to cease their stress response as evidenced by the continued presence of activated JNK. Finally, activation of the cellular stress response increased the overall extent of polyglutamine protein aggregation, especially within the nucleus. Inclusion of a JNK inhibitor reduced this stress-dependent increase in nuclear aggregates. Abnormal stress responses may contribute to enhanced cell vulnerability in cells expressing polyglutamine-expanded proteins and may increase the propensity of such cells to form cytoplasmic and nuclear inclusions. PMID:12783846

  3. Subacute toxicity of Dietary T-2 toxin in mice: influence of protein nutrition.

    PubMed Central

    Hayes, M A; Schiefer, H B

    1980-01-01

    The subacute toxic effects of dietary T-2 toxin (20 ppm) incorporated in semipurified diets of 8%, 12% or 16% protein, were examined in young Swiss mice after one, two, three and four weeks. Dietary T-2 toxin caused substantial reductions in growth and food consumptaion, the degrees of which were greatest in mice fed the diets of reduced protein content. T-2 toxin consistently caused similar degrees of nonregenerative anemia, lymphopenia, thymic atrophy and gastric hyperkeratosis irrespective of the dietary protein level. However, erythroid hypoplasia was temporary in mice fed T-2 toxin in the 16%-protein diet such that erythroid precursors regenerated in splenic and bone marrow and were hyperplastic after four weeks. Liver to body weight ratios of mice fed T-2 toxin in the 16%-and 12%-protein diets increased during the four week trial in comparison to control mice fed at a similar rate. These observations indicated that suppression of erythropoiesis in mice by dietary T-2 toxin was temporarty and that the interval before regeneration was prolonged by diets of reduced protein content. PMID:7407693

  4. Studies on Bacterial Proteins Corona Interaction with Saponin Imprinted ZnO Nanohoneycombs and Their Toxic Responses.

    PubMed

    Sharma, Deepali; Ashaduzzaman, Md; Golabi, Mohsen; Shriwastav, Amritanshu; Bisetty, Krishna; Tiwari, Ashutosh

    2015-11-01

    Molecular imprinting generates robust, efficient, and highly mesoporous surfaces for biointeractions. Mechanistic interfacial interaction between the surface of core substrate and protein corona is crucial to understand the substantial microbial toxic responses at a nanoscale. In this study, we have focused on the mechanistic interactions between synthesized saponin imprinted zinc oxide nanohoneycombs (SIZnO NHs), average size 80-125 nm, surface area 20.27 m(2)/g, average pore density 0.23 pore/nm and number-average pore size 3.74 nm and proteins corona of bacteria. The produced SIZnO NHs as potential antifungal and antibacterial agents have been studied on Sclerotium rolfsii (S. rolfsii), Pythium debarynum (P. debarynum) and Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), respectively. SIZnO NHs exhibited the highest antibacterial (∼50%) and antifungal (∼40%) activity against Gram-negative bacteria (E. coli) and fungus (P. debarynum), respectively at concentration of 0.1 mol. Scanning electron spectroscopy (SEM) observation showed that the ZnO NHs ruptured the cell wall of bacteria and internalized into the cell. The molecular docking studies were carried out using binding proteins present in the gram negative bacteria (lipopolysaccharide and lipocalin Blc) and gram positive bacteria (Staphylococcal Protein A, SpA). It was envisaged that the proteins present in the bacterial cell wall were found to interact and adsorb on the surface of SIZnO NHs thereby blocking the active sites of the proteins used for cell wall synthesis. The binding affinity and interaction energies were higher in the case of binding proteins present in gram negative bacteria as compared to that of gram positive bacteria. In addition, a kinetic mathematical model (KMM) was developed in MATLAB to predict the internalization in the bacterial cellular uptake of the ZnO NHs for better understanding of their controlled toxicity. The results obtained from KMM exhibited a good

  5. ULTRA HIGH EFFICIENCY ESP DEVELOPMENT FOR AIR TOXICS CONTROL

    SciTech Connect

    David K. Anderson

    1999-11-01

    Because more than 90 percent of U.S. coal-fired utility boilers are equipped with electrostatic precipitators (ESPs), retrofitable ESP technologies represent a logical approach towards achieving the Department of Energy's (DOE) goal of a major reduction in fine particulate and mercury emissions (air toxics) from coal based power systems. EPA's recent issuance of significantly tightened ambient air standards for particles smaller than 2.5 {micro}m (PM{sub 2.5}) creates a new urgency for developing cost-effective means to control fine particulate emissions. This challenge is compounded by the on-going switch in the utility industry to low-sulfur Powder River Basin (PRB) coals, that generate higher resistivity and difficult-to-collect fly ash. Particulate emissions can increase by a factor of ten when a utility switches to a low-sulfur coal. Numerous power plants are presently limited in operation by the inability of their ESPs to control opacity at high loads. In Phase I of this program, ABB investigated five technologies to improve the collection of fine particulate and trace metals in ESPs. These included: (1) flue-gas cooling, (2) flue-gas humidification, (3) pulsed energization, (4) wet ESP and precharger modules, and (5) sorbent injection for mercury control. Tests were conducted with an Eastern bituminous coal and a Powder River Basin sub-bituminous low-sulfur coal in an integrated pilot-scale combustor and ESP test facility. The impacts of the different retrofit technologies on ESP performance, individually and in combination, were evaluated indepth through advanced sampling and measurement techniques. In Phase II, the most promising concepts identified from Phase I testing, flue-gas cooling and humidification, pulsed energization, and sorbent injection at low flue-gas temperatures for mercury control, were integrated into a commercially oriented sub-scale system for field testing at Commonwealth Edison's Waukegan Unit No. 8. The main objective of the proposed

  6. Toxicity of hemoglobin solutions: hemoglobin is a lipopolysaccharide (LPS) binding protein which enhances LPS biological activity.

    PubMed

    Roth, R I; Kaca, W

    1994-01-01

    Administration of alpha alpha-crosslinked stroma-free hemoglobin (SFH) as a cell-free resuscitation fluid is associated with multiple organ toxicities. Many of these toxicities are characteristic of the pathophysiological effects of bacterial endotoxins (lipopolysaccharide, LPS). To better understand the potential role of LPS in the observed in vivo toxicities of SFH, we examined mixtures of SFH and E. coli LPS for evidence of LPS-SFH complex formation. LPS-SFH complexes were demonstrated by three techniques: ultrafiltration through 300 kDa cut-off membranes, which distinguished LPS in complexes (87-89% < 300 kDa) from LPS alone (90% > 300 kDa); density centrifugation through 5% sucrose, which distinguished denser LPS alone from LPS-SFH complexes; and precipitation by 67% ethanol, which demonstrated 2-3 fold increased precipitability of complexes compared to SFH alone. Interaction of LPS with SFH was also associated with markedly increased biological activity of LPS, as manifested by enhancement of LPS activation of Limulus amebocyte lysate (LAL), increased release of human mononuclear cell tissue factor, and enhanced production of cultured human endothelial cell tissue factor. These results demonstrated that hemoglobin can serve as an endotoxin binding protein, and that this interaction results in the alteration of several LPS physical characteristics and enhancement of LPS biological activities.

  7. Amyloid β-sheet mimics that antagonize protein aggregation and reduce amyloid toxicity

    NASA Astrophysics Data System (ADS)

    Cheng, Pin-Nan; Liu, Cong; Zhao, Minglei; Eisenberg, David; Nowick, James S.

    2012-11-01

    The amyloid protein aggregation associated with diseases such as Alzheimer's, Parkinson's and type II diabetes (among many others) features a bewildering variety of β-sheet-rich structures in transition from native proteins to ordered oligomers and fibres. The variation in the amino-acid sequences of the β-structures presents a challenge to developing a model system of β-sheets for the study of various amyloid aggregates. Here, we introduce a family of robust β-sheet macrocycles that can serve as a platform to display a variety of heptapeptide sequences from different amyloid proteins. We have tailored these amyloid β-sheet mimics (ABSMs) to antagonize the aggregation of various amyloid proteins, thereby reducing the toxicity of amyloid aggregates. We describe the structures and inhibitory properties of ABSMs containing amyloidogenic peptides from the amyloid-β peptide associated with Alzheimer's disease, β2-microglobulin associated with dialysis-related amyloidosis, α-synuclein associated with Parkinson's disease, islet amyloid polypeptide associated with type II diabetes, human and yeast prion proteins, and Tau, which forms neurofibrillary tangles.

  8. Arabidopsis thaliana atrab28: a nuclear targeted protein related to germination and toxic cation tolerance.

    PubMed

    Borrell, Antonio; Cutanda, M Cruz; Lumbreras, Victoria; Pujal, Judit; Goday, Adela; Culiáñez-Macià, Francisco A; Pagès, Montserrat

    2002-09-01

    The Arabidopsis gene Atrab28 has been shown to be expressed during late embryogenesis. The pattern of expression of Atrab28 mRNA and protein during embryo development is largely restricted to provascular tissues of mature embryos, and in contrast to the maize Rab28 homologue it cannot be induced by ABA and dehydration in vegetative tissues. Here, we have studied the subcellular location of Atrab28 protein and the effect of its over-expression in transgenic Arabidopsis plants. The Atrab28 protein was mainly detected in the nucleus and nucleolus of cells from mature embryos. In frame fusion of Atrab28 to the reporter green fluorescent protein (GFP) directed the GFP to the nucleus in transgenic Arabidopsis and in transiently transformed onion cells. Analysis of chimeric constructs identified an N-terminal region of 60 amino acids containing a five amino acid motif QPKRP that was necessary for targeting GFP to the nucleus. These results indicate that Atrab28 protein is targeted to the nuclear compartments by a new nuclear localization signal (NLS). Transgenic Arabidopsis plants, with gain of Atrab28 function, showed faster germination rates under either standard or salt and osmotic stress conditions. Moreover, improved cation toxicity tolerance was also observed not only during germination but also in seedlings. These results suggest a role of Atrab28 in the ion cell balance during late embryogenesis and germination. PMID:12175017

  9. Synthesis of highly fluorescent gold nanoclusters using egg white proteins.

    PubMed

    Joseph, Dickson; Geckeler, Kurt E

    2014-03-01

    Gold nanoclusters (AuNCs) have gained interest during the recent years because of their low toxicity and finer size for the bioimaging and biolabeling applications in comparison to the semiconductor quantum dot analogues. Diverse materials such as sulfur compounds, peptides, dendrimers, proteins, etc., are exploited for the preparation of AuNCs. Henceforth, highly fluorescent, water-soluble, and few atom-containing gold nanoclusters are created using a rapid, straightforward, and green method. In this regard for the first time chicken egg white (CEW), one of the most unique materials, is utilized in an aqueous solution under basic conditions at physiological temperature for the preparation of AuNCs. Tyrosine and tryptophan amino acid residues are responsible for the conversion of Au ions to Au(0) under alkaline condtions. CEW contains four major proteins of which the main constituent protein, ovalbumin also leads to the formation of the AuNCs with a higher fluorescence emission compared to the CEW. The ratios between the different reaction partners are very crucial, along with temperature and time for the preparation of AuNCs with high photoluminescence emission. The limited vibrational motion of the proteins under alkaline condition and the bulkiness of the proteins help in the formation of AuNCs.

  10. Mucosal toxicity studies of a gel formulation of native pokeweed antiviral protein.

    PubMed

    D'Cruz, Osmond J; Waurzyniak, Barbara; Uckun, Fatih M

    2004-01-01

    Pokeweed antiviral protein (PAP), a 29-kDa plant-derived protein isolated from Phytolacca americana, is a promising nonspermicidal broad-spectrum antiviral microbicide. This study evaluated the mucosal toxicity potential of native PAP in the in vivo rabbit vaginal irritation model as well as the in vitro reconstituted human vaginal epithelial tissue model. Twenty-two New Zealand white rabbits in 4 subgroups were exposed intravaginally to a gel with and without 0.01, 0.1, or 1.0% native PAP for 10 consecutive days. The dose of PAP used represented nearly 200- to 20,000 times its in vitro anti-HIV IC50 value. Animals were euthanized on day 11 and vaginal tissues were evaluated for histologic and immunohistochemical evidence of mucosal toxicity, cellular inflammation, and hyperplasia. Blood was analyzed for changes in hematology and clinical chemistry profiles. Reconstituted human vaginal epithelial tissue grown on membrane filters was exposed to 0.01, 0.1, or 1.0% native PAP in medium or topically via a gel for 24 hours and tissue damage was evaluated by histological assessment. In the in vivo rabbit vaginal irritation model, half of all PAP-treated rabbits (8/16) exhibited an acceptable range of vaginal mucosal irritation (total score <8 out of a possible 16), whereas nearly a third of PAP-treated rabbits (5/16) developed moderate to marked vaginal mucosal irritation (total score >11). However, no treatment-related adverse effects were seen in hematological or clinical chemistry measurements. Furthermore, in vitro exposure of a 3-dimensional human vaginal tissue grown on polycarbonate membrane filters to identical concentrations of PAP either added to culture medium or applied topically via gel formulation did not result in direct toxicity as determined by histologic evaluation. These findings indicate careful monitoring of vaginal irritation will be required in the clinical development of PAP as a nonspermicidal microbicide.

  11. High pressure effects on allergen food proteins.

    PubMed

    Somkuti, Judit; Smeller, László

    2013-12-15

    There are several proteins, which can cause allergic reaction if they are inhaled or ingested. Our everyday food can also contain such proteins. Food allergy is an IgE-mediated immune disorder, a growing health problem of great public concern. High pressure is known to affect the structure of proteins; typically few hundred MPa pressure can lead to denaturation. That is why several trials have been performed to alter the structure of the allergen proteins by high pressure, in order to reduce its allergenicity. Studies have been performed both on simple protein solutions and on complex food systems. Here we review those allergens which have been investigated under or after high pressure treatment by methods capable of detecting changes in the secondary and tertiary structure of the proteins. We focus on those allergenic proteins, whose structural changes were investigated by spectroscopic methods under pressure in correlation with the observed allergenicity (IgE binding) changes. According to this criterion we selected the following allergen proteins: Mal d 1 and Mal d 3 (apple), Bos d 5 (milk), Dau c 1 (carrot), Gal d 2 (egg), Ara h 2 and Ara h 6 (peanut), and Gad m 1 (cod).

  12. Estimating Toxicity Pathway Activating Doses for High Throughput Chemical Risk Assessments

    EPA Science Inventory

    Estimating a Toxicity Pathway Activating Dose (TPAD) from in vitro assays as an analog to a reference dose (RfD) derived from in vivo toxicity tests would facilitate high throughput risk assessments of thousands of data-poor environmental chemicals. Estimating a TPAD requires def...

  13. The protein transportation pathway from Golgi to vacuoles via endosomes plays a role in enhancement of methylmercury toxicity

    NASA Astrophysics Data System (ADS)

    Hwang, Gi-Wook; Murai, Yasutaka; Takahashi, Tsutomu; Naganuma, Akira

    2014-07-01

    Methylmercury causes serious damage to the central nervous system, but the molecular mechanisms of methylmercury toxicity are only marginally understood. In this study, we used a gene-deletion mutant library of budding yeast to conduct genome-wide screening for gene knockouts affecting the sensitivity of methylmercury toxicity. We successfully identified 31 genes whose deletions confer resistance to methylmercury in yeast, and 18 genes whose deletions confer hypersensitivity to methylmercury. Yeast genes whose deletions conferred resistance to methylmercury included many gene encoding factors involved in protein transport to vacuoles. Detailed examination of the relationship between the factors involved in this transport system and methylmercury toxicity revealed that mutants with loss of the factors involved in the transportation pathway from the trans-Golgi network (TGN) to the endosome, protein uptake into the endosome, and endosome-vacuole fusion showed higher methylmercury resistance than did wild-type yeast. The results of our genetic engineering study suggest that this vesicle transport system (proteins moving from the TGN to vacuole via endosome) is responsible for enhancing methylmercury toxicity due to the interrelationship between the pathways. There is a possibility that there may be proteins in the cell that enhance methylmercury toxicity through the protein transport system.

  14. The Mitochondrial Chaperone Protein TRAP1 Mitigates α-Synuclein Toxicity

    PubMed Central

    Lutz, A. Kathrin; Toegel, Jane P.; Gerhardt, Ellen; Karsten, Peter; Falkenburger, Björn; Reinartz, Andrea; Winklhofer, Konstanze F.; Schulz, Jörg B.

    2012-01-01

    Overexpression or mutation of α-Synuclein is associated with protein aggregation and interferes with a number of cellular processes, including mitochondrial integrity and function. We used a whole-genome screen in the fruit fly Drosophila melanogaster to search for novel genetic modifiers of human [A53T]α-Synuclein–induced neurotoxicity. Decreased expression of the mitochondrial chaperone protein tumor necrosis factor receptor associated protein-1 (TRAP1) was found to enhance age-dependent loss of fly head dopamine (DA) and DA neuron number resulting from [A53T]α-Synuclein expression. In addition, decreased TRAP1 expression in [A53T]α-Synuclein–expressing flies resulted in enhanced loss of climbing ability and sensitivity to oxidative stress. Overexpression of human TRAP1 was able to rescue these phenotypes. Similarly, human TRAP1 overexpression in rat primary cortical neurons rescued [A53T]α-Synuclein–induced sensitivity to rotenone treatment. In human (non)neuronal cell lines, small interfering RNA directed against TRAP1 enhanced [A53T]α-Synuclein–induced sensitivity to oxidative stress treatment. [A53T]α-Synuclein directly interfered with mitochondrial function, as its expression reduced Complex I activity in HEK293 cells. These effects were blocked by TRAP1 overexpression. Moreover, TRAP1 was able to prevent alteration in mitochondrial morphology caused by [A53T]α-Synuclein overexpression in human SH-SY5Y cells. These results indicate that [A53T]α-Synuclein toxicity is intimately connected to mitochondrial dysfunction and that toxicity reduction in fly and rat primary neurons and human cell lines can be achieved using overexpression of the mitochondrial chaperone TRAP1. Interestingly, TRAP1 has previously been shown to be phosphorylated by the serine/threonine kinase PINK1, thus providing a potential link of PINK1 via TRAP1 to α-Synuclein. PMID:22319455

  15. Esophageal Toxicity From High-Dose, Single-Fraction Paraspinal Stereotactic Radiosurgery

    SciTech Connect

    Cox, Brett W.; Jackson, Andrew; Hunt, Margie; Bilsky, Mark; Yamada, Yoshiya

    2012-08-01

    Purpose: To report the esophageal toxicity from single-fraction paraspinal stereotactic radiosurgery (SRS) and identify dosimetric and clinical risk factors for toxicity. Methods and Materials: A total of 204 spinal metastases abutting the esophagus (182 patients) were treated with high-dose single-fraction SRS during 2003-2010. Toxicity was scored using the National Cancer Institute Common Toxicity Criteria for Adverse Events, version 4.0. Dose-volume histograms were combined to generate a comprehensive atlas of complication incidence that identifies risk factors for toxicity. Correlation of dose-volume factors with esophageal toxicity was assessed using Fisher's exact test and logistic regression. Clinical factors were correlated with toxicity. Results: The median dose to the planning treatment volume was 24 Gy. Median follow-up was 12 months (range, 3-81). There were 31 (15%) acute and 24 (12%) late esophageal toxicities. The rate of grade {>=}3 acute or late toxicity was 6.8% (14 patients). Fisher's exact test resulted in significant median splits for grade {>=}3 toxicity at V12 = 3.78 cm{sup 3} (relative risk [RR] 3.7, P=.05), V15 = 1.87 cm{sup 3} (RR 13, P=.0013), V20 = 0.11 cm{sup 3} (RR 6, P=0.01), and V22 = 0.0 cm{sup 3} (RR 13, P=.0013). The median split for D2.5 cm{sup 3} (14.02 Gy) was also a significant predictor of toxicity (RR 6; P=.01). A highly significant logistic regression model was generated on the basis of D2.5 cm{sup 3}. One hundred percent (n = 7) of grade {>=}4 toxicities were associated with radiation recall reactions after doxorubicin or gemcitabine chemotherapy or iatrogenic manipulation of the irradiated esophagus. Conclusions: High-dose, single-fraction paraspinal SRS has a low rate of grade {>=}3 esophageal toxicity. Severe esophageal toxicity is minimized with careful attention to esophageal doses during treatment planning. Iatrogenic manipulation of the irradiated esophagus and systemic agents classically associated with radiation

  16. Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions.

    PubMed

    Fasolo, Joseph; Im, Hogune; Snyder, Michael P

    2015-01-01

    High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis to determine a high confidence set of protein interactions for downstream validation in vivo. PMID:26274875

  17. High Protein Diet and Huntington's Disease

    PubMed Central

    Wu, Yih-Ru; Chen, Pei; Tsai, Fuu-Jen; Yang, Chueh-Lien; Tsao, Ya-Tzu; Chang, Wen; Hsieh, I-Shan; Chern, Yijuang; Soong, Bing-Wen

    2015-01-01

    Huntington’s disease (HD) is a neurodegenerative disorder caused by the huntingtin (HTT) gene with expanded CAG repeats. In addition to the apparent brain abnormalities, impairments also occur in peripheral tissues. We previously reported that mutant Huntingtin (mHTT) exists in the liver and causes urea cycle deficiency. A low protein diet (17%) restores urea cycle activity and ameliorates symptoms in HD model mice. It remains unknown whether the dietary protein content should be monitored closely in HD patients because the normal protein consumption is lower in humans (~15% of total calories) than in mice (~22%). We assessed whether dietary protein content affects the urea cycle in HD patients. Thirty HD patients were hospitalized and received a standard protein diet (13.7% protein) for 5 days, followed by a high protein diet (HPD, 26.3% protein) for another 5 days. Urea cycle deficiency was monitored by the blood levels of citrulline and ammonia. HD progression was determined by the Unified Huntington’s Disease Rating Scale (UHDRS). The HPD increased blood citrulline concentration from 15.19 μmol/l to 16.30 μmol/l (p = 0.0378) in HD patients but did not change blood ammonia concentration. A 2-year pilot study of 14 HD patients found no significant correlation between blood citrulline concentration and HD progression. Our results indicated a short period of the HPD did not markedly compromise urea cycle function. Blood citrulline concentration is not a reliable biomarker of HD progression. PMID:25992839

  18. Comparison of Bile Acids and Acetaminophen Protein Adducts in Children and Adolescents with Acetaminophen Toxicity.

    PubMed

    James, Laura; Yan, Ke; Pence, Lisa; Simpson, Pippa; Bhattacharyya, Sudeepa; Gill, Pritmohinder; Letzig, Lynda; Kearns, Gregory; Beger, Richard

    2015-01-01

    Metabolomics approaches have enabled the study of new mechanisms of liver injury in experimental models of drug toxicity. Disruption of bile acid homeostasis is a known mechanism of drug induced liver injury. The relationship of individual bile acids to indicators of oxidative drug metabolism (acetaminophen protein adducts) and liver injury was examined in children with acetaminophen overdose, hospitalized children with low dose exposure to acetaminophen, and children with no recent exposure to acetaminophen. Nine bile acids were quantified through targeted metabolomic analysis in the serum samples of the three groups. Bile acids were compared to serum levels of acetaminophen protein adducts and alanine aminotransferase. Glycodeoxycholic acid, taurodeoxycholic acid, and glycochenodeoxycholic acid were significantly increased in children with acetaminophen overdose compared to healthy controls. Among patients with acetaminophen overdose, bile acids were higher in subjects with acetaminophen protein adduct values > 1.0 nmol/mL and modest correlations were noted for three bile acids and acetaminophen protein adducts as follows: taurodeoxycholic acid (R=0.604; p<0.001), glycodeoxycholic acid (R=0.581; p<0.001), and glycochenodeoxycholic acid (R=0.571; p<0.001). Variability in bile acids was greater among hospitalized children receiving low doses of acetaminophen than in healthy children with no recent acetaminophen exposure. Compared to bile acids, acetaminophen protein adducts more accurately discriminated among children with acetaminophen overdose, children with low dose exposure to acetaminophen, and healthy control subjects. In children with acetaminophen overdose, elevations of conjugated bile acids were associated with specific indicators of acetaminophen metabolism and non-specific indicators of liver injury. PMID:26208104

  19. Comparison of Bile Acids and Acetaminophen Protein Adducts in Children and Adolescents with Acetaminophen Toxicity.

    PubMed

    James, Laura; Yan, Ke; Pence, Lisa; Simpson, Pippa; Bhattacharyya, Sudeepa; Gill, Pritmohinder; Letzig, Lynda; Kearns, Gregory; Beger, Richard

    2015-01-01

    Metabolomics approaches have enabled the study of new mechanisms of liver injury in experimental models of drug toxicity. Disruption of bile acid homeostasis is a known mechanism of drug induced liver injury. The relationship of individual bile acids to indicators of oxidative drug metabolism (acetaminophen protein adducts) and liver injury was examined in children with acetaminophen overdose, hospitalized children with low dose exposure to acetaminophen, and children with no recent exposure to acetaminophen. Nine bile acids were quantified through targeted metabolomic analysis in the serum samples of the three groups. Bile acids were compared to serum levels of acetaminophen protein adducts and alanine aminotransferase. Glycodeoxycholic acid, taurodeoxycholic acid, and glycochenodeoxycholic acid were significantly increased in children with acetaminophen overdose compared to healthy controls. Among patients with acetaminophen overdose, bile acids were higher in subjects with acetaminophen protein adduct values > 1.0 nmol/mL and modest correlations were noted for three bile acids and acetaminophen protein adducts as follows: taurodeoxycholic acid (R=0.604; p<0.001), glycodeoxycholic acid (R=0.581; p<0.001), and glycochenodeoxycholic acid (R=0.571; p<0.001). Variability in bile acids was greater among hospitalized children receiving low doses of acetaminophen than in healthy children with no recent acetaminophen exposure. Compared to bile acids, acetaminophen protein adducts more accurately discriminated among children with acetaminophen overdose, children with low dose exposure to acetaminophen, and healthy control subjects. In children with acetaminophen overdose, elevations of conjugated bile acids were associated with specific indicators of acetaminophen metabolism and non-specific indicators of liver injury.

  20. Proteomics approach on microcystin binding proteins in mouse liver for investigation of microcystin toxicity.

    PubMed

    Imanishi, Susumu; Harada, Ken-ichi

    2004-05-01

    Microcystins (MC) produced by freshwater cyanobacteria are potent hepatotoxins. MC inhibit protein phosphatases (PP) 1 and 2A. MC and okadaic acid (OA), which is a similar PP inhibitor whereas it has a less affinity to PP1 than PP2A, behave similarly to primary culture hepatocytes, with inducements of phosphorylations of cytoskeleton, morphological changes and apoptosis. Although the distribution of OA in mouse liver was observed immunohistochemically, no OA injury was found. The purpose of this study was therefore to determine why only MC has specific toxicities on the liver. A systematic process of MC affinity chromatography and proteomics, using two-dimensional gel electrophoresis and MALDI-TOFMS, indicated the existence of some MC-binding proteins including the complexes of PP1, PP2A, and PP4 with their own regulatory subunits in mouse liver extracts. The competitive inhibition experiments using affinity chromatography with OA showed that two of the three protein complexes strongly interacted with OA, whereas only the complex of PP1 with the inhibitory subunit NIPP1 did not strongly interacted with OA. These results suggest that the PP1 complex is not related to the common behavior of MC and OA of primary culture hepatocytes, and is related to the specific hepatotoxicities of MC.

  1. Integration of Dosimetry, Exposure and High-Throughput Screening Data in Chemical Toxicity Assessment

    EPA Science Inventory

    High-throughput in vitro toxicity screening can provide an efficient way to identify potential biological targets for chemicals. However, relying on nominal assay concentrations may misrepresent potential in vivo effects of these chemicals due to differences in bioavailability, c...

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

    PubMed Central

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

    2016-01-01

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

  3. Categorizing biases in high-confidence high-throughput protein-protein interaction data sets.

    PubMed

    Yu, Xueping; Ivanic, Joseph; Memisević, Vesna; Wallqvist, Anders; Reifman, Jaques

    2011-12-01

    We characterized and evaluated the functional attributes of three yeast high-confidence protein-protein interaction data sets derived from affinity purification/mass spectrometry, protein-fragment complementation assay, and yeast two-hybrid experiments. The interacting proteins retrieved from these data sets formed distinct, partially overlapping sets with different protein-protein interaction characteristics. These differences were primarily a function of the deployed experimental technologies used to recover these interactions. This affected the total coverage of interactions and was especially evident in the recovery of interactions among different functional classes of proteins. We found that the interaction data obtained by the yeast two-hybrid method was the least biased toward any particular functional characterization. In contrast, interacting proteins in the affinity purification/mass spectrometry and protein-fragment complementation assay data sets were over- and under-represented among distinct and different functional categories. We delineated how these differences affected protein complex organization in the network of interactions, in particular for strongly interacting complexes (e.g. RNA and protein synthesis) versus weak and transient interacting complexes (e.g. protein transport). We quantified methodological differences in detecting protein interactions from larger protein complexes, in the correlation of protein abundance among interacting proteins, and in their connectivity of essential proteins. In the latter case, we showed that minimizing inherent methodology biases removed many of the ambiguous conclusions about protein essentiality and protein connectivity. We used these findings to rationalize how biological insights obtained by analyzing data sets originating from different sources sometimes do not agree or may even contradict each other. An important corollary of this work was that discrepancies in biological insights did not

  4. Mitochondrial Toxicity Studied with the PBMC of Children from the Chinese National Pediatric Highly Active Antiretroviral Therapy Cohort

    PubMed Central

    Liu, Daojie; Yin, Jiming; Qiao, Luxin; Shi, Ying; Dong, Yaowu; Li, Ning; Zhang, Fujie; Chen, Dexi

    2013-01-01

    As the backbone of highly active antiretroviral therapy (HAART), nucleoside reverse transcriptase inhibitors (NRTIs) have effectively improved outcomes for HIV-infected patients. However, long-term treatment with NRTIs can cause a series of pathologies associated with mitochondrial toxicity. To date, the status and mechanism of mitochondrial toxicity induced by NRTIs are still not clear, especially in HIV-infected children. As part of the national pediatric HAART program in China, our study focused on mitochondrial toxicity and its potential mechanism in HIV-1-infected children who were divided into two groups based on their duration of treatment with NRTIs: one group received treatment for less than 36 months and one group was treated for 36 to 72 months. The control group comprised age-matched non-HIV-infected children. Blood lactic acid and ATP levels in peripheral blood mononuclear cells (PBMCs) were measured to evaluate mitochondrial function, and mtDNA copies and mutations in PBMCs were determined for detecting mtDNA lesions. Simultaneously, TK2 and P53R2 gene expression in PBMC was measured. As compared with the control group, blood lactic acid levels in both NRTI treatment groups were significantly higher, whereas ATP levels and mtDNA mutation rates in PBMCs did not differ between the control and the two NRTI treatment groups. Both NRTI treatment groups exhibited significant mtDNA loss. N Moreover, we found that P53R2 mRNA expression and protein levels were significantly reduced in both treatment groups and that TK2 mRNA expression and protein levels were induced in the long-term NRTI treatment group. These results suggest that mitochondrial toxicity occurs in long-term HAART patients and that P53R2 and TK2 levels in PBMCs are useful biomarkers for detecting mitochondrial toxicity in patients on long-term treatment with NRTIs. PMID:23468942

  5. Integrated process for high conversion and high yield protein PEGylation.

    PubMed

    Pfister, David; Morbidelli, Massimo

    2016-08-01

    Over the past decades, PEGylation has become a powerful technique to increase the in vivo circulation half-life of therapeutic proteins while maintaining their activity. The development of new therapeutic proteins is likely to require further improvement of the PEGylation methods to reach even better selectivity and yield for reduced costs. The intensification of the PEGylation process was investigated through the integration of a chromatographic step in order to increase yield and conversion for the production of mono-PEGylated protein. Lysozyme was used as a model protein to demonstrate the feasibility of such approach. In the integrated reaction/separation process, chromatography was used as fractionation technique in order to isolate and recycle the unreacted protein from the PEGylated products. This allows operating the reactor with short reaction times so as to minimize the production of multi-PEGylated proteins (i.e., conjugated to more than one polymer). That is, the reaction is stopped before the desired product (i.e., the mono-PEGylated protein) can further react, thus leading to limited conversion but high yield. The recycling of the unreacted protein was then considered to drive the protein overall conversion to completion. This approach has great potential to improve processes whose yield is limited by the further reaction of the product leading to undesirable by-products. Biotechnol. Bioeng. 2016;113: 1711-1718. © 2016 Wiley Periodicals, Inc.

  6. Highly toxic ribbon worm Cephalothrix simula containing tetrodotoxin in Hiroshima Bay, Hiroshima Prefecture, Japan.

    PubMed

    Asakawa, Manabu; Ito, Katsutoshi; Kajihara, Hiroshi

    2013-02-20

    In 1998, during a toxicological surveillance of various marine fouling organisms in Hiroshima Bay, Japan, specimens of the ribbon worm, Cephalothrix simula (Nemertea: Palaeonemertea) were found. These ribbon worms contained toxins with extremely strong paralytic activity. The maximum toxicity in terms of tetrodotoxin (TTX) was 25,590 mouse units (MU) per gram for the whole worm throughout the monitoring period. The main toxic component was isolated and recrystallized from an acidified methanolic solution. The crystalline with a specific toxicity of 3520 MU/mg was obtained and identified as TTX by high performance liquid chromatography (HPLC)-fluorescent detection (FLD) (HPLC-FLD), electrospray ionization-mass spectrometry (ESI-MS), infrared (IR), nuclear magnetic resonance (NMR) and gas chromatography-mass spectrometry (GC-MS). The highest toxicity of C. simula exceeded the human lethal dose per a single worm. A toxicological surveillance of C. simula from 1998 to 2005 indicated approximately 80% of the individuals were ranked as "strongly toxic" (≥1000 MU/g). Forty-eight percent of the specimens possessed toxicity scores of more than 2000 MU/g. Seasonal variations were observed in the lethal potency of C. simula. Specimens collected on January 13, 2000 to December 26, 2000 showed mean toxicities of 665-5300 MU/g (n = 10). These data prompted a toxicological surveillance of ribbon worms from other localities with different habitats in Japan, including Akkeshi Bay (Hokkaido) under stones on rocky intertidal beaches, as well as Otsuchi (Iwate) among calcareous tubes of serpulid polychaetes on rocky shores. Within twelve species of ribbon worms examined, only C. simula possessed extremely high toxicity. Therefore, C. simula appears to show generally high toxicity irrespective of their locality and habitat.

  7. Relative Impact of Incorporating Pharmacokinetics on Predicting In Vivo Hazard and Mode of Action from High-Throughput In Vitro Toxicity Assays

    EPA Science Inventory

    The use of high-throughput in vitro assays has been proposed to play a significant role in the future of toxicity testing. In this study, rat hepatic metabolic clearance and plasma protein binding were measured for 59 ToxCast phase I chemicals. Computational in vitro-to-in vivo e...

  8. Manganese toxicity and Saccharomyces cerevisiae Mam3p, a member of the ACDP (ancient conserved domain protein) family.

    PubMed

    Yang, Mei; Jensen, Laran T; Gardner, Allison J; Culotta, Valeria C

    2005-03-15

    Manganese is an essential, but potentially toxic, trace metal in biological systems. Overexposure to manganese is known to cause neurological deficits in humans, but the pathways that lead to manganese toxicity are largely unknown. We have employed the bakers' yeast Saccharomyces cerevisiae as a model system to identify genes that contribute to manganese-related damage. In a genetic screen for yeast manganese-resistance mutants, we identified S. cerevisiae MAM3 as a gene which, when deleted, would increase cellular tolerance to toxic levels of manganese and also increased the cell's resistance towards cobalt and zinc. By sequence analysis, Mam3p shares strong similarity with the mammalian ACDP (ancient conserved domain protein) family of polypeptides. Mutations in human ACDP1 have been associated with urofacial (Ochoa) syndrome. However, the functions of eukaryotic ACDPs remain unknown. We show here that S. cerevisiae MAM3 encodes an integral membrane protein of the yeast vacuole whose expression levels directly correlate with the degree of manganese toxicity. Surprisingly, Mam3p contributes to manganese toxicity without any obvious changes in vacuolar accumulation of metals. Furthermore, through genetic epistasis studies, we demonstrate that MAM3 operates independently of the well-established manganese-trafficking pathways in yeast, involving the manganese transporters Pmr1p, Smf2p and Pho84p. This is the first report of a eukaryotic ACDP family protein involved in metal homoeostasis. PMID:15498024

  9. Role of oxidative stress in methamphetamine-induced dopaminergic toxicity mediated by protein kinase Cδ.

    PubMed

    Shin, Eun-Joo; Duong, Chu Xuan; Nguyen, Xuan-Khanh Thi; Li, Zhengyi; Bing, Guoying; Bach, Jae-Hyung; Park, Dae Hun; Nakayama, Keiichi; Ali, Syed F; Kanthasamy, Anumantha G; Cadet, Jean Lud; Nabeshima, Toshitaka; Kim, Hyoung-Chun

    2012-06-15

    This study examined the role of protein kinase C (PKC) isozymes in methamphetamine (MA)-induced dopaminergic toxicity. Multiple-dose administration of MA did not significantly alter PKCα, PKCβI, PKCβII, or PKCζ expression in the striatum, but did significantly increase PKCδ expression. Gö6976 (a co-inhibitor of PKCα and -β), hispidin (PKCβ inhibitor), and PKCζ pseudosubstrate inhibitor (PKCζ inhibitor) did not significantly alter MA-induced behavioral impairments. However, rottlerin (PKCδ inhibitor) significantly attenuated behavioral impairments in a dose-dependent manner. In addition, MA-induced behavioral impairments were not apparent in PKCδ knockout (-/-) mice. MA-induced oxidative stress (i.e., lipid peroxidation and protein oxidation) was significantly attenuated in rottlerin-treated mice and was not apparent in PKCδ (-/-) mice. Consistent with this, MA-induced apoptosis (i.e., terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive apoptotic cells) was significantly attenuated in rottlerin-treated mice. Furthermore, MA-induced increases in the dopamine (DA) turnover rate and decreases in tyrosine hydroxylase (TH) activity and the expression of TH, dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) were not significantly observed in rottlerin-treated or PKCδ (-/-) mice. Our results suggest that PKCδ gene expression is a key mediator of oxidative stress and dopaminergic damage induced by MA. Thus, inhibition of PKCδ may be a useful target for protection against MA-induced neurotoxicity.

  10. The Protein Oxidation Repair Enzyme Methionine Sulfoxide Reductase A Modulates Aβ Aggregation and Toxicity In Vivo

    PubMed Central

    Minniti, Alicia N.; Arrazola, Macarena S.; Bravo-Zehnder, Marcela; Ramos, Francisca; Inestrosa, Nibaldo C.

    2015-01-01

    Abstract Aims: To examine the role of the enzyme methionine sulfoxide reductase A-1 (MSRA-1) in amyloid-β peptide (Aβ)-peptide aggregation and toxicity in vivo, using a Caenorhabditis elegans model of the human amyloidogenic disease inclusion body myositis. Results: MSRA-1 specifically reduces oxidized methionines in proteins. Therefore, a deletion of the msra-1 gene was introduced into transgenic C. elegans worms that express the Aβ-peptide in muscle cells to prevent the reduction of oxidized methionines in proteins. In a constitutive transgenic Aβ strain that lacks MSRA-1, the number of amyloid aggregates decreases while the number of oligomeric Aβ species increases. These results correlate with enhanced synaptic dysfunction and mislocalization of the nicotinic acetylcholine receptor ACR-16 at the neuromuscular junction (NMJ). Innovation: This approach aims at modulating the oxidation of Aβ in vivo indirectly by dismantling the methionine sulfoxide repair system. The evidence presented here shows that the absence of MSRA-1 influences Aβ aggregation and aggravates locomotor behavior and NMJ dysfunction. The results suggest that therapies which boost the activity of the Msr system could have a beneficial effect in managing amyloidogenic pathologies. Conclusion: The absence of MSRA-1 modulates Aβ-peptide aggregation and increments its deleterious effects in vivo. Antioxid. Redox Signal. 22, 48–62. PMID:24988428

  11. Role of oxidative stress in methamphetamine-induced dopaminergic toxicity mediated by protein kinase Cδ

    PubMed Central

    Nguyen, Xuan-Khanh Thi; Li, Zhengyi; Bing, Guoying; Bach, Jae-Hyung; Park, Dae Hun; Nakayama, Keiichi; Ali, Syed F.; Kanthasamy, Anumantha G.; Cadet, Jean Lud; Nabeshima, Toshitaka; Kim, Hyoung-Chun

    2014-01-01

    This study examined the role of protein kinase C (PKC) isozymes in methamphetamine (MA)-induced dopaminergic toxicity. Multiple-dose administration of MA did not significantly alter PKCα, PKCβI, PKCβII, or PKCζ expression in the striatum, but did significantly increase PKCδ expression. Gö6976 (a co-inhibitor of PKCα and -β), hispidin (PKCβ inhibitor), and PKCζ pseudosubstrate inhibitor (PKCζ inhibitor) did not significantly alter MA-induced behavioral impairments. However, rottlerin (PKCδ inhibitor) significantly attenuated behavioral impairments in a dose-dependent manner. In addition, MA-induced behavioral impairments were not apparent in PKCδ knockout (–/–) mice. MA-induced oxidative stress (i.e., lipid peroxidation and protein oxidation) was significantly attenuated in rottlerin-treated mice and was not apparent in PKCδ (–/–) mice. Consistent with this, MA-induced apoptosis (i.e., terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive apoptotic cells) was significantly attenuated in rottlerin-treated mice. Furthermore, MA-induced increases in the dopamine (DA) turnover rate and decreases in tyrosine hydroxylase (TH) activity and the expression of TH, dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) were not significantly observed in rottlerin-treated or PKCδ (–/–) mice. Our results suggest that PKCδ gene expression is a key mediator of oxidative stress and dopaminergic damage induced by MA. Thus, inhibition of PKCδ may be a useful target for protection against MA-induced neurotoxicity. PMID:22512859

  12. Molecular-receptor-specific, non-toxic, near-infrared-emitting Au cluster-protein nanoconjugates for targeted cancer imaging

    NASA Astrophysics Data System (ADS)

    Retnakumari, Archana; Setua, Sonali; Menon, Deepthy; Ravindran, Prasanth; Muhammed, Habeeb; Pradeep, Thalappil; Nair, Shantikumar; Koyakutty, Manzoor

    2010-02-01

    Molecular-receptor-targeted imaging of folate receptor positive oral carcinoma cells using folic-acid-conjugated fluorescent Au25 nanoclusters (Au NCs) is reported. Highly fluorescent Au25 clusters were synthesized by controlled reduction of Au+ ions, stabilized in bovine serum albumin (BSA), using a green-chemical reducing agent, ascorbic acid (vitamin-C). For targeted-imaging-based detection of cancer cells, the clusters were conjugated with folic acid (FA) through amide linkage with the BSA shell. The bioconjugated clusters show excellent stability over a wide range of pH from 4 to 14 and fluorescence efficiency of ~5.7% at pH 7.4 in phosphate buffer saline (PBS), indicating effective protection of nanoclusters by serum albumin during the bioconjugation reaction and cell-cluster interaction. The nanoclusters were characterized for their physico-chemical properties, toxicity and cancer targeting efficacy in vitro. X-ray photoelectron spectroscopy (XPS) suggests binding energies correlating to metal Au 4f7/2~83.97 eV and Au 4f5/2~87.768 eV. Transmission electron microscopy and atomic force microscopy revealed the formation of individual nanoclusters of size ~1 nm and protein cluster aggregates of size ~8 nm. Photoluminescence studies show bright fluorescence with peak maximum at ~674 nm with the spectral profile covering the near-infrared (NIR) region, making it possible to image clusters at the 700-800 nm emission window where the tissue absorption of light is minimum. The cell viability and reactive oxygen toxicity studies indicate the non-toxic nature of the Au clusters up to relatively higher concentrations of 500 µg ml-1. Receptor-targeted cancer detection using Au clusters is demonstrated on FR+ve oral squamous cell carcinoma (KB) and breast adenocarcinoma cell MCF-7, where the FA-conjugated Au25 clusters were found internalized in significantly higher concentrations compared to the negative control cell lines. This study demonstrates the potential of using

  13. Toxicity of Bacillus thuringiensis (L.) Cry proteins against summer fruit tortrix (Adoxophyes orana - Fischer von Rösslerstamm).

    PubMed

    Radosavljevic, Jelena; Naimov, Samir

    2016-07-01

    The activity of seven Cry1, one Cry9 and one hybrid Cry1 protoxins against neonate larvae of summer fruit tortrix (Adoxophyes orana - Fischer von Rösslerstamm) has been investigated. Cry1Ia is identified as the most toxic protein, followed by Cry1Aa and Cry1Ac. Cry1Ca, Cry1Cb, Cry1Da and Cry1Fa were less active, while SN19 (Cry1 hybrid protein with domain composition 1Ba/1Ia/1Ba) and Cry9Aa exhibited negligible toxicity against A. orana. In vitro trypsin-activated Cry1Ac is still less active than Cry1Ia protoxin, suggesting that toxicity of Cry1Ia is most probably due to more complex differences in further downstream processing, toxin-receptor interactions and pore formation in A. orana's midgut epithelium. PMID:27311897

  14. Bridging high-throughput genetic and transcriptional data reveals cellular responses to alpha-synuclein toxicity.

    PubMed

    Yeger-Lotem, Esti; Riva, Laura; Su, Linhui Julie; Gitler, Aaron D; Cashikar, Anil G; King, Oliver D; Auluck, Pavan K; Geddie, Melissa L; Valastyan, Julie S; Karger, David R; Lindquist, Susan; Fraenkel, Ernest

    2009-03-01

    Cells respond to stimuli by changes in various processes, including signaling pathways and gene expression. Efforts to identify components of these responses increasingly depend on mRNA profiling and genetic library screens. By comparing the results of these two assays across various stimuli, we found that genetic screens tend to identify response regulators, whereas mRNA profiling frequently detects metabolic responses. We developed an integrative approach that bridges the gap between these data using known molecular interactions, thus highlighting major response pathways. We used this approach to reveal cellular pathways responding to the toxicity of alpha-synuclein, a protein implicated in several neurodegenerative disorders including Parkinson's disease. For this we screened an established yeast model to identify genes that when overexpressed alter alpha-synuclein toxicity. Bridging these data and data from mRNA profiling provided functional explanations for many of these genes and identified previously unknown relations between alpha-synuclein toxicity and basic cellular pathways. PMID:19234470

  15. Highly Toxic Ribbon Worm Cephalothrix simula Containing Tetrodotoxin in Hiroshima Bay, Hiroshima Prefecture, Japan

    PubMed Central

    Asakawa, Manabu; Ito, Katsutoshi; Kajihara, Hiroshi

    2013-01-01

    In 1998, during a toxicological surveillance of various marine fouling organisms in Hiroshima Bay, Japan, specimens of the ribbon worm, Cephalothrix simula (Nemertea: Palaeonemertea) were found. These ribbon worms contained toxins with extremely strong paralytic activity. The maximum toxicity in terms of tetrodotoxin (TTX) was 25,590 mouse units (MU) per gram for the whole worm throughout the monitoring period. The main toxic component was isolated and recrystallized from an acidified methanolic solution. The crystalline with a specific toxicity of 3520 MU/mg was obtained and identified as TTX by high performance liquid chromatography (HPLC)-fluorescent detection (FLD) (HPLC-FLD), electrospray ionization-mass spectrometry (ESI-MS), infrared (IR), nuclear magnetic resonance (NMR) and gas chromatography–mass spectrometry (GC-MS). The highest toxicity of C. simula exceeded the human lethal dose per a single worm. A toxicological surveillance of C. simula from 1998 to 2005 indicated approximately 80% of the individuals were ranked as “strongly toxic” (≥1000 MU/g). Forty-eight percent of the specimens possessed toxicity scores of more than 2000 MU/g. Seasonal variations were observed in the lethal potency of C. simula. Specimens collected on January 13, 2000 to December 26, 2000 showed mean toxicities of 665–5300 MU/g (n = 10). These data prompted a toxicological surveillance of ribbon worms from other localities with different habitats in Japan, including Akkeshi Bay (Hokkaido) under stones on rocky intertidal beaches, as well as Otsuchi (Iwate) among calcareous tubes of serpulid polychaetes on rocky shores. Within twelve species of ribbon worms examined, only C. simula possessed extremely high toxicity. Therefore, C. simula appears to show generally high toxicity irrespective of their locality and habitat. PMID:23430577

  16. Environmental toxicants perturb human Sertoli cell adhesive function via changes in F-actin organization mediated by actin regulatory proteins

    PubMed Central

    Xiao, Xiang; Mruk, Dolores D.; Tang, Elizabeth I.; Wong, Chris K.C.; Lee, Will M.; John, Constance M.; Turek, Paul J.; Silvestrini, Bruno; Cheng, C. Yan

    2014-01-01

    STUDY QUESTION Can human Sertoli cells cultured in vitro and that have formed an epithelium be used as a model to monitor toxicant-induced junction disruption and to better understand the mechanism(s) by which toxicants disrupt cell adhesion at the Sertoli cell blood–testis barrier (BTB)? SUMMARY ANSWER Our findings illustrate that human Sertoli cells cultured in vitro serve as a reliable system to monitor the impact of environmental toxicants on the BTB function. WHAT IS KNOWN ALREADY Suspicions of a declining trend in semen quality and a concomitant increase in exposures to environmental toxicants over the past decades reveal the need of an in vitro system that efficiently and reliably monitors the impact of toxicants on male reproductive function. Furthermore, studies in rodents have confirmed that environmental toxicants impede Sertoli cell BTB function in vitro and in vivo. STUDY DESIGN, SIZE AND DURATION We examined the effects of two environmental toxicants: cadmium chloride (0.5–20 µM) and bisphenol A (0.4–200 µM) on human Sertoli cell function. Cultured Sertoli cells from three men were used in this study, which spanned an 18-month period. PARTICIPANTS/MATERIALS, SETTING, METHODS Human Sertoli cells from three subjects were cultured in F12/DMEM containing 5% fetal bovine serum. Changes in protein expression were monitored by immunoblotting using specific antibodies. Immunofluorescence analyses were used to assess changes in the distribution of adhesion proteins, F-actin and actin regulatory proteins following exposure to two toxicants: cadmium chloride and bisphenol A (BPA). MAIN RESULTS AND THE ROLE OF CHANCE Human Sertoli cells were sensitive to cadmium and BPA toxicity. Changes in the localization of cell adhesion proteins were mediated by an alteration of the actin-based cytoskeleton. This alteration of F-actin network in Sertoli cells as manifested by truncation and depolymerization of actin microfilaments at the Sertoli cell BTB was caused by

  17. Surfactant protein A (SP-A) inhibits agglomeration and macrophage uptake of toxic amine modified nanoparticles

    PubMed Central

    McKenzie, Zofi; Kendall, Michaela; Mackay, Rose-Marie; Whitwell, Harry; Elgy, Christine; Ding, Ping; Mahajan, Sumeet; Morgan, Cliff; Griffiths, Mark; Clark, Howard; Madsen, Jens

    2015-01-01

    Abstract The lung provides the main route for nanomaterial exposure. Surfactant protein A (SP-A) is an important respiratory innate immune molecule with the ability to bind or opsonise pathogens to enhance phagocytic removal from the airways. We hypothesised that SP-A, like surfactant protein D, may interact with inhaled nanoparticulates, and that this interaction will be affected by nanoparticle (NP) surface characteristics. In this study, we characterise the interaction of SP-A with unmodified (U-PS) and amine-modified (A-PS) polystyrene particles of varying size and zeta potential using dynamic light scatter analysis. SP-A associated with both 100 nm U-PS and A-PS in a calcium-independent manner. SP-A induced significant calcium-dependent agglomeration of 100 nm U-PS NPs but resulted in calcium-independent inhibition of A-PS self agglomeration. SP-A enhanced uptake of 100 nm U-PS into macrophage-like RAW264.7 cells in a dose-dependent manner but in contrast inhibited A-PS uptake. Reduced association of A-PS particles in RAW264.7 cells following pre-incubation of SP-A was also observed with coherent anti-Stokes Raman spectroscopy. Consistent with these findings, alveolar macrophages (AMs) from SP-A−/− mice were more efficient at uptake of 100 nm A-PS compared with wild type C57Bl/6 macrophages. No difference in uptake was observed with 500 nm U-PS or A-PS particles. Pre-incubation with SP-A resulted in a significant decrease in uptake of 100 nm A-PS in macrophages isolated from both groups of mice. In contrast, increased uptake by AMs of U-PS was observed after pre-incubation with SP-A. Thus we have demonstrated that SP-A promotes uptake of non-toxic U-PS particles but inhibits the clearance of potentially toxic A-PS particles by blocking uptake into macrophages. PMID:25676620

  18. Eukaryotic resistance to fluoride toxicity mediated by a widespread family of fluoride export proteins.

    PubMed

    Li, Sanshu; Smith, Kathryn D; Davis, Jared H; Gordon, Patricia B; Breaker, Ronald R; Strobel, Scott A

    2013-11-19

    Fluorine is an abundant element and is toxic to organisms from bacteria to humans, but the mechanisms by which eukaryotes resist fluoride toxicity are unknown. The Escherichia coli gene crcB was recently shown to be regulated by a fluoride-responsive riboswitch, implicating it in fluoride response. There are >8,000 crcB homologs across all domains of life, indicating that it has an important role in biology. Here we demonstrate that eukaryotic homologs [renamed FEX (fluoride exporter)] function in fluoride export. FEX KOs in three eukaryotic model organisms, Neurospora crassa, Saccharomyces cerevisiae, and Candida albicans, are highly sensitized to fluoride (>200-fold) but not to other halides. Some of these KO strains are unable to grow in fluoride concentrations found in tap water. Using the radioactive isotope of fluoride, (18)F, we developed an assay to measure the intracellular fluoride concentration and show that the FEX deletion strains accumulate fluoride in excess of the external concentration, providing direct evidence of FEX function in fluoride efflux. In addition, they are more sensitive to lower pH in the presence of fluoride. These results demonstrate that eukaryotic FEX genes encode a previously unrecognized class of fluoride exporter necessary for survival in standard environmental conditions. PMID:24173035

  19. Multi-endpoint, high-throughput study of nanomaterial toxicity in Caenorhabditis elegans.

    PubMed

    Jung, Sang-Kyu; Qu, Xiaolei; Aleman-Meza, Boanerges; Wang, Tianxiao; Riepe, Celeste; Liu, Zheng; Li, Qilin; Zhong, Weiwei

    2015-02-17

    The booming nanotechnology industry has raised public concerns about the environmental health and safety impact of engineered nanomaterials (ENMs). High-throughput assays are needed to obtain toxicity data for the rapidly increasing number of ENMs. Here we present a suite of high-throughput methods to study nanotoxicity in intact animals using Caenorhabditis elegans as a model. At the population level, our system measures food consumption of thousands of animals to evaluate population fitness. At the organism level, our automated system analyzes hundreds of individual animals for body length, locomotion speed, and lifespan. To demonstrate the utility of our system, we applied this technology to test the toxicity of 20 nanomaterials at four concentrations. Only fullerene nanoparticles (nC60), fullerol, TiO2, and CeO2 showed little or no toxicity. Various degrees of toxicity were detected from different forms of carbon nanotubes, graphene, carbon black, Ag, and fumed SiO2 nanoparticles. Aminofullerene and ultraviolet-irradiated nC60 also showed small but significant toxicity. We further investigated the effects of nanomaterial size, shape, surface chemistry, and exposure conditions on toxicity. Our data are publicly available at the open-access nanotoxicity database www.QuantWorm.org/nano. PMID:25611253

  20. A multi-endpoint, high-throughput study of nanomaterial toxicity in Caenorhabditis elegans

    PubMed Central

    Jung, Sang-Kyu; Qu, Xiaolei; Aleman-Meza, Boanerges; Wang, Tianxiao; Riepe, Celeste; Liu, Zheng; Li, Qilin; Zhong, Weiwei

    2015-01-01

    The booming nanotech industry has raised public concerns about the environmental health and safety impact of engineered nanomaterials (ENMs). High-throughput assays are needed to obtain toxicity data for the rapidly increasing number of ENMs. Here we present a suite of high-throughput methods to study nanotoxicity in intact animals using Caenorhabditis elegans as a model. At the population level, our system measures food consumption of thousands of animals to evaluate population fitness. At the organism level, our automated system analyzes hundreds of individual animals for body length, locomotion speed, and lifespan. To demonstrate the utility of our system, we applied this technology to test the toxicity of 20 nanomaterials under four concentrations. Only fullerene nanoparticles (nC60), fullerol, TiO2, and CeO2 showed little or no toxicity. Various degrees of toxicity were detected from different forms of carbon nanotubes, graphene, carbon black, Ag, and fumed SiO2 nanoparticles. Aminofullerene and UV irradiated nC60 also showed small but significant toxicity. We further investigated the effects of nanomaterial size, shape, surface chemistry, and exposure conditions on toxicity. Our data are publicly available at the open-access nanotoxicity database www.QuantWorm.org/nano. PMID:25611253

  1. Ocular toxicity following high dose chemotherapy and autologous transplant.

    PubMed

    Rubin, P; Hulette, C; Khawly, J A; Elkordy, M; Hussein, A; Vredenburgh, J J; Jaffe, G J; Peters, W P

    1996-07-01

    A 49-year-old woman received an autologous transplant for breast cancer. Six weeks later she noticed visual disturbance of the left eye which correlated with a visual field abnormality. There was a milder degree of visual disturbance in the right eye. Treatment with high-dose steroids partially stabilized the problem, which was felt to be an ischemic optic neuropathy. She ultimately died of respiratory failure. Pathology of the optic nerves revealed demyelination. Visual disturbances following high-dose chemotherapy are uncommon; the pathology to date has not been elucidated. Steroid therapy may be useful. PMID:8832031

  2. Acrolein, a highly toxic aldehyde generated under oxidative stress in vivo, aggravates the mouse liver damage after acetaminophen overdose.

    PubMed

    Arai, Tomoya; Koyama, Ryo; Yuasa, Makoto; Kitamura, Daisuke; Mizuta, Ryushin

    2014-01-01

    Although acetaminophen-induced liver injury in mice has been extensively studied as a model of human acute drug-induced hepatitis, the mechanism of liver injury remains unclear. Liver injury is believed to be initiated by metabolic conversion of acetaminophen to the highly reactive intermediate N-acetyl p-benzoquinoneimine, and is aggravated by subsequent oxidative stress via reactive oxygen species (ROS), including hydrogen peroxide (H2O2) and the hydroxyl radical (•OH). In this study, we found that a highly toxic unsaturated aldehyde acrolein, a byproduct of oxidative stress, has a major role in acetaminophen-induced liver injury. Acetaminophen administration in mice resulted in liver damage and increased acrolein-protein adduct formation. However, both of them were decreased by treatment with N-acetyl-L-cysteine (NAC) or sodium 2-mercaptoethanesulfonate (MESNA), two known acrolein scavengers. The specificity of NAC and MESNA was confirmed in cell culture, because acrolein toxicity, but not H2O2 or •OH toxicity, was inhibited by NAC and MESNA. These results suggest that acrolein may be more strongly correlated with acetaminophen-induced liver injury than ROS, and that acrolein produced by acetaminophen-induced oxidative stress can spread from dying cells at the primary injury site, causing damage to the adjacent cells and aggravating liver injury.

  3. Optimization of high-throughput nanomaterial developmental toxicity testing in zebrafish embryos

    EPA Science Inventory

    Nanomaterial (NM) developmental toxicities are largely unknown. With an extensive variety of NMs available, high-throughput screening methods may be of value for initial characterization of potential hazard. We optimized a zebrafish embryo test as an in vivo high-throughput assay...

  4. Toxic effects of zinc from trout farm sediments on ATP, protein, and hemoglobin concentrations of Limnodrilus hoffmeisteri.

    PubMed

    Martinez-Tabche, L; Gutiérrez Cabrera, I; Gómez Oliván, L; Galar Martinez, M; Germán Faz, C

    2000-04-14

    Zinc (Zn) is a nutritionally essential metal, and deficiency results in severe health consequences to aquatic organisms. In this study toxicity data for Limnodrilus hoffmeisteri produced by Zn in systems using three natural sediments (trout farms: El Oyamel, El Truchón, and El Potrero) are presented. Hemoglobin, adenosine triphosphate (ATP), and protein concentrations were measured in L. hoffmeisteri exposed to spiked sediments, as indicators of exposure. Physicochemical characteristics of water and sediments were also considered. Zn concentrations were measured in water and sediment. El Oyamel, El Truchón, and El Potrero pond sediments did not have similar physicochemical characteristics. Zn concentrations of water obtained from the rustic ponds were near 0.4575 mg/L; however, this metal was always found to be higher in the sediments (0.0271-0.9754 mg/kg). The bioassay with worms demonstrated that pond sediments from El Oyamel, El Potrero, and El Truchón produced toxicity since ATP and protein concentrations were low compared to controls (organisms without metal). All spiked sediments had a significant reduction effect on ATP, protein, and hemoglobin concentrations. This investigation clearly shows that sediments of El Truchón, El Oyamel, and El Potrero possess toxicity potential. These results suggest the usefulness of these bioassays to evaluate the toxicity of sediments polluted with heavy metals. PMID:10777248

  5. Complete genome sequence of Bacillus thuringiensis YBT-1518, a typical strain with high toxicity to nematodes.

    PubMed

    Wang, Pengxia; Zhang, Chunyi; Guo, Mengmeng; Guo, Suxia; Zhu, Yiguang; Zheng, Jinshui; Zhu, Lei; Ruan, Lifang; Peng, Donghai; Sun, Ming

    2014-02-10

    Bacillus thuringiensis is a ubiquitous spore-forming bacterium and has been widely used as a biopesticide for controlling agricultural insects by the production of insecticidal crystal proteins (ICPs). B. thuringiensis YBT-1518 displays effective toxicity to nematodes. This strain harbors three nematicidal crystal protein genes, including cry55Aa1, cry6Aa2 and cry5Ba2, and also contains multiple potential virulence factors. Here, we report the complete genome sequence of B. thuringiensis YBT-1518, which consists of one circular chromosome and six circular plasmids.

  6. “Non-Toxic” Proteins of the Botulinum Toxin Complex Exert In-vivo Toxicity

    PubMed Central

    Miyashita, Shin-Ichiro; Sagane, Yoshimasa; Suzuki, Tomonori; Matsumoto, Takashi; Niwa, Koichi; Watanabe, Toshihiro

    2016-01-01

    The botulinum neurotoxin (BoNT) causes muscle paralysis and is the most potent toxin in nature. BoNT is associated with a complex of auxiliary “Non-Toxic” proteins, which constitute a large-sized toxin complex (L-TC). However, here we report that the “Non-Toxic” complex of serotype D botulinum L-TC, when administered to rats, exerts in-vivo toxicity on small-intestinal villi. Moreover, Serotype C and D of the “Non-Toxic” complex, but not BoNT, induced vacuole-formation in a rat intestinal epithelial cell line (IEC-6), resulting in cell death. Our results suggest that the vacuole was formed in a manner distinct from the mechanism by which Helicobacter pylori vacuolating toxin (VacA) and Vibrio cholerae haemolysin induce vacuolation. We therefore hypothesise that the serotype C and D botulinum toxin complex is a functional hybrid of the neurotoxin and vacuolating toxin (VT) which arose from horizontal gene transfer from an ancestral BoNT-producing bacterium to a hypothetical VT-producing bacterium. PMID:27507612

  7. Multidrug Resistance-Associated Protein 3 Plays an Important Role in Protection against Acute Toxicity of Diclofenac.

    PubMed

    Scialis, Renato J; Csanaky, Iván L; Goedken, Michael J; Manautou, José E

    2015-07-01

    Diclofenac (DCF) is a nonsteroidal anti-inflammatory drug commonly prescribed to reduce pain in acute and chronic inflammatory diseases. One of the main DCF metabolites is a reactive diclofenac acyl glucuronide (DCF-AG) that covalently binds to biologic targets and may contribute to adverse drug reactions arising from DCF use. Cellular efflux of DCF-AG is partially mediated by multidrug resistance-associated proteins (Mrp). The importance of Mrp2 during DCF-induced toxicity has been established, yet the role of Mrp3 remains largely unexplored. In the present work, Mrp3-null (KO) mice were used to study the toxicokinetics and toxicodynamics of DCF and its metabolites. DCF-AG plasma concentrations were 90% lower in KO mice than in wild-type (WT) mice, indicating that Mrp3 mediates DCF-AG basolateral efflux. In contrast, there were no differences in DCF-AG biliary excretion between WT and KO, suggesting that only DCF-AG basolateral efflux is compromised by Mrp3 deletion. Susceptibility to toxicity was also evaluated after a single high DCF dose. No signs of injury were detected in livers and kidneys; however, ulcers were found in the small intestines. Furthermore, the observed intestinal injuries were consistently more severe in KO compared with WT. DCF covalent adducts were observed in liver and small intestines; however, staining intensity did not correlate with the severity of injuries, implying that tissues respond differently to covalent modification. Overall, the data provide strong evidence that (1) in vivo Mrp3 plays an important role in DCF-AG disposition and (2) compromised Mrp3 function can enhance injury in the gastrointestinal tract after DCF treatment.

  8. A single amino acid (Asp159) from the dog prion protein suppresses the toxicity of the mouse prion protein in Drosophila.

    PubMed

    Sanchez-Garcia, J; Jensen, K; Zhang, Y; Rincon-Limas, D E; Fernandez-Funez, P

    2016-11-01

    Misfolding of the prion protein (PrP) is the key step in the transmission of spongiform pathologies in humans and several animals. Although PrP is highly conserved in mammals, a few changes in the sequence of endogenous PrP are proposed to confer protection to dogs, which were highly exposed to prion during the mad-cow epidemics. D159 is a unique amino acid found in PrP from dogs and other canines that was shown to alter surface charge, but its functional relevance has never been tested in vivo. Here, we show in transgenic Drosophila that introducing the N159D substitution on mouse PrP decreases its turnover. Additionally, mouse PrP-N159D demonstrates no toxicity and accumulates no pathogenic conformations, suggesting that a single D159 substitution is sufficient to prevent PrP conformational change and pathogenesis. Understanding the mechanisms mediating the protective activity of D159 is likely to lessen the burden of prion diseases in humans and domestic animals. PMID:27477054

  9. Characterization of a highly toxic strain of Bacillus thuringiensis serovar kurstaki very similar to the HD-73 strain.

    PubMed

    Reinoso-Pozo, Yaritza; Del Rincón-Castro, Ma Cristina; Ibarra, Jorge E

    2016-09-01

    The LBIT-1200 strain of Bacillus thuringiensis was recently isolated from soil, and showed a 6.4 and 9.5 increase in toxicity, against Manduca sexta and Trichoplusia ni, respectively, compared to HD-73. However, LBIT-1200 was still highly similar to HD-73, including the production of bipyramidal crystals containing only one protein of ∼130 000 kDa, its flagellin gene sequence related to the kurstaki serotype, plasmid and RepPCR patterns similar to HD-73, no production of β-exotoxin and no presence of VIP genes. Sequencing of its cry gene showed the presence of a cry1Ac-type gene with four amino acid differences, including two amino acid replacements in domain III, compared to Cry1Ac1, which may explain its higher toxicity. In conclusion, the LBIT-1200 strain is a variant of the HD-73 strain but shows a much higher toxicity, which makes this new strain an important candidate to be developed as a bioinsecticide, once it passes other tests, throughout its biotechnological development. PMID:27535648

  10. Characterization of a highly toxic strain of Bacillus thuringiensis serovar kurstaki very similar to the HD-73 strain.

    PubMed

    Reinoso-Pozo, Yaritza; Del Rincón-Castro, Ma Cristina; Ibarra, Jorge E

    2016-09-01

    The LBIT-1200 strain of Bacillus thuringiensis was recently isolated from soil, and showed a 6.4 and 9.5 increase in toxicity, against Manduca sexta and Trichoplusia ni, respectively, compared to HD-73. However, LBIT-1200 was still highly similar to HD-73, including the production of bipyramidal crystals containing only one protein of ∼130 000 kDa, its flagellin gene sequence related to the kurstaki serotype, plasmid and RepPCR patterns similar to HD-73, no production of β-exotoxin and no presence of VIP genes. Sequencing of its cry gene showed the presence of a cry1Ac-type gene with four amino acid differences, including two amino acid replacements in domain III, compared to Cry1Ac1, which may explain its higher toxicity. In conclusion, the LBIT-1200 strain is a variant of the HD-73 strain but shows a much higher toxicity, which makes this new strain an important candidate to be developed as a bioinsecticide, once it passes other tests, throughout its biotechnological development.

  11. Incorporating human dosimetry and exposure into high-throughput in vitro toxicity screening.

    PubMed

    Rotroff, Daniel M; Wetmore, Barbara A; Dix, David J; Ferguson, Stephen S; Clewell, Harvey J; Houck, Keith A; Lecluyse, Edward L; Andersen, Melvin E; Judson, Richard S; Smith, Cornelia M; Sochaski, Mark A; Kavlock, Robert J; Boellmann, Frank; Martin, Matthew T; Reif, David M; Wambaugh, John F; Thomas, Russell S

    2010-10-01

    Many chemicals in commerce today have undergone limited or no safety testing. To reduce the number of untested chemicals and prioritize limited testing resources, several governmental programs are using high-throughput in vitro screens for assessing chemical effects across multiple cellular pathways. In this study, metabolic clearance and plasma protein binding were experimentally measured for 35 ToxCast phase I chemicals. The experimental data were used to parameterize a population-based in vitro-to-in vivo extrapolation model for estimating the human oral equivalent dose necessary to produce a steady-state in vivo concentration equivalent to in vitro AC(50) (concentration at 50% of maximum activity) and LEC (lowest effective concentration) values from the ToxCast data. For 23 of the 35 chemicals, the range of oral equivalent doses for up to 398 ToxCast assays was compared with chronic aggregate human oral exposure estimates in order to assess whether significant in vitro bioactivity occurred within the range of maximum expected human oral exposure. Only 2 of the 35 chemicals, triclosan and pyrithiobac-sodium, had overlapping oral equivalent doses and estimated human oral exposures. Ranking by the potencies of the AC(50) and LEC values, these two chemicals would not have been at the top of a prioritization list. Integrating both dosimetry and human exposure information with the high-throughput toxicity screening efforts provides a better basis for making informed decisions on chemical testing priorities and regulatory attention. Importantly, these tools are necessary to move beyond hazard rankings to estimates of possible in vivo responses based on in vitro screens. PMID:20639261

  12. Quantitative GFP fluorescence as an indicator of arsenite developmental toxicity in mosaic heat shock protein 70 transgenic zebrafish

    SciTech Connect

    Seok, Seung-Hyeok; Baek, Min-Won; Lee, Hui-Young; Kim, Dong-Jae; Na, Yi-Rang; Noh, Kyoung-Jin; Park, Sung-Hoon; Lee, Hyun-Kyoung; Lee, Byoung-Hee; Ryu, Doug-Young; Park, Jae-Hak

    2007-12-01

    In transgenic zebrafish (Danio rerio), green fluorescent protein (GFP) is a promising marker for environmental pollutants. In using GFP, one of the obstacles which we faced was how to compare toxicity among different toxicants or among a specific toxicant in different model species with the intensity of GFP expression. Using a fluorescence detection method, we first validated our method for estimating the amount of GFP fluorescence present in transgenic fish, which we used as an indicator of developmental toxicity caused by the well-known toxicant, arsenite. To this end, we developed mosaic transgenic zebrafish with the human heat shock response element (HSE) fused to the enhanced GFP (EGFP) reporter gene to indicate exposure to arsenite. We confirmed that EGFP expression sites correlate with gross morphological disruption caused by arsenite exposure. Arsenite (300.0 {mu}M) caused stronger EGFP fluorescence intensity and quantity than 50.0 {mu}M and 10.0 {mu}M arsenite in our transgenic zebrafish. Furthermore, arsenite-induced apoptosis was demonstrated by TUNEL assay. Apoptosis was inhibited by the antioxidant, N-acetyl-cystein (NAC) in this transgenic zebrafish. The distribution of TUNEL-positive cells in embryonic tissues was correlated with the sites of arsenite toxicity and EGFP expression. The EGFP values quantified using the standard curve equation from the known GFP quantity were consistent with the arsenite-induced EGFP expression pattern and arsenite concentration, indicating that this technique can be a reliable and applicable measurement. In conclusion, we propose that fluorescence-based EGFP quantification in transgenic fish containing the hsp70 promoter-EGFP reporter-gene construct is a useful indicator of development toxicity caused by arsenite.

  13. High Throughput Prioritization for Integrated Toxicity Testing Based on ToxCast Chemical Profiling

    EPA Science Inventory

    The rational prioritization of chemicals for integrated toxicity testing is a central goal of the U.S. EPA’s ToxCast™ program (http://epa.gov/ncct/toxcast/). ToxCast includes a wide-ranging battery of over 500 in vitro high-throughput screening assays which in Phase I was used to...

  14. Perspectives on Validation of High-Throughput Assays Supporting 21st Century Toxicity Testing

    EPA Science Inventory

    In vitro high-throughput screening (HTS) assays are seeing increasing use in toxicity testing. HTS assays can simultaneously test many chemicals but have seen limited use in the regulatory arena, in part because of the need to undergo rigorous, time-consuming formal validation. ...

  15. How Much is Too Much? Toxic Chemicals in High School Labs.

    ERIC Educational Resources Information Center

    Nagel, Miriam C.

    1982-01-01

    Lists 37 chemicals classified as suspected carcinogens and suspected teratogens (chemicals capable of producing malformations in an embryo). Offers suggestions to high school chemistry teachers for conducting safe laboratory investigations by avoiding use of these potentially toxic materials. (Author/JN)

  16. Predictive Model of Rat Reproductive Toxicity from ToxCast High Throughput Screening

    EPA Science Inventory

    The EPA ToxCast research program uses high throughput screening for bioactivity profiling and predicting the toxicity of large numbers of chemicals. ToxCast Phase‐I tested 309 well‐characterized chemicals in over 500 assays for a wide range of molecular targets and cellular respo...

  17. pH controlled gating of toxic protein pores by dendrimers

    NASA Astrophysics Data System (ADS)

    Mandal, Taraknath; Kanchi, Subbarao; Ayappa, K. G.; Maiti, Prabal K.

    2016-06-01

    Designing effective nanoscale blockers for membrane inserted pores formed by pore forming toxins, which are expressed by several virulent bacterial strains, on a target cell membrane is a challenging and active area of research. Here we demonstrate that PAMAM dendrimers can act as effective pH controlled gating devices once the pore has been formed. We have used fully atomistic molecular dynamics (MD) simulations to characterize the cytolysin A (ClyA) protein pores modified with fifth generation (G5) PAMAM dendrimers. Our results show that the PAMAM dendrimer, in either its protonated (P) or non-protonated (NP) states can spontaneously enter the protein lumen. Protonated dendrimers interact strongly with the negatively charged protein pore lumen. As a consequence, P dendrimers assume a more expanded configuration efficiently blocking the pore when compared with the more compact configuration adopted by the neutral NP dendrimers creating a greater void space for the passage of water and ions. To quantify the effective blockage of the protein pore, we have calculated the pore conductance as well as the residence times by applying a weak force on the ions/water. Ionic currents are reduced by 91% for the P dendrimers and 31% for the NP dendrimers. The preferential binding of Cl- counter ions to the P dendrimer creates a zone of high Cl- concentration in the vicinity of the internalized dendrimer and a high concentration of K+ ions in the transmembrane region of the pore lumen. In addition to steric effects, this induced charge segregation for the P dendrimer effectively blocks ionic transport through the pore. Our investigation shows that the bio-compatible PAMAM dendrimers can potentially be used to develop therapeutic protocols based on the pH sensitive gating of pores formed by pore forming toxins to mitigate bacterial infections.Designing effective nanoscale blockers for membrane inserted pores formed by pore forming toxins, which are expressed by several virulent

  18. The toxic effect of lead on seed germination, growth, chlorophyll and protein contents of wheat and lens.

    PubMed

    Mesmar, M N; Jaber, K

    1991-01-01

    Lead is a heavy metal which is believed to be toxic when present in excessive amount. Excess Pb in Triticum sativum and Lens esculanta alters several physiological and biochemical processes in both species. Seed germination of both species grown on soaked filter paper with Pb (NO3)2 was highly inhibited (about 60% at 20 mM Pb (NO3)2). Results obtained from measurement of lead content in the roots and shoots of both species indicated that most of the lead accumulated in the roots of both species with a lower degree within the shoots. Lead uptake by both species whether grown in perlite medium or on filter paper soaked with Pb (NO3)2, was correlated with lead concentration. These results indicate a passive process of lead translocation. These results also show that lead inhibits the growth of both plant species, but root growth inhibition was more pronounced than shoot growth inhibition at different lead concentration. Total chlorophyll content was found to be decreased in both species after treatment with Pb (NO3)2. Total protein content in the seedlings, as our results have indicated, was found to be increased with increasing lead concentration in both species. PMID:1841484

  19. pH controlled gating of toxic protein pores by dendrimers.

    PubMed

    Mandal, Taraknath; Kanchi, Subbarao; Ayappa, K G; Maiti, Prabal K

    2016-07-14

    Designing effective nanoscale blockers for membrane inserted pores formed by pore forming toxins, which are expressed by several virulent bacterial strains, on a target cell membrane is a challenging and active area of research. Here we demonstrate that PAMAM dendrimers can act as effective pH controlled gating devices once the pore has been formed. We have used fully atomistic molecular dynamics (MD) simulations to characterize the cytolysin A (ClyA) protein pores modified with fifth generation (G5) PAMAM dendrimers. Our results show that the PAMAM dendrimer, in either its protonated (P) or non-protonated (NP) states can spontaneously enter the protein lumen. Protonated dendrimers interact strongly with the negatively charged protein pore lumen. As a consequence, P dendrimers assume a more expanded configuration efficiently blocking the pore when compared with the more compact configuration adopted by the neutral NP dendrimers creating a greater void space for the passage of water and ions. To quantify the effective blockage of the protein pore, we have calculated the pore conductance as well as the residence times by applying a weak force on the ions/water. Ionic currents are reduced by 91% for the P dendrimers and 31% for the NP dendrimers. The preferential binding of Cl(-) counter ions to the P dendrimer creates a zone of high Cl(-) concentration in the vicinity of the internalized dendrimer and a high concentration of K(+) ions in the transmembrane region of the pore lumen. In addition to steric effects, this induced charge segregation for the P dendrimer effectively blocks ionic transport through the pore. Our investigation shows that the bio-compatible PAMAM dendrimers can potentially be used to develop therapeutic protocols based on the pH sensitive gating of pores formed by pore forming toxins to mitigate bacterial infections. PMID:27328315

  20. Label-free detection of protein molecules secreted from an organ-on-a-chip model for drug toxicity assays

    NASA Astrophysics Data System (ADS)

    Morales, Andres W.; Zhang, Yu S.; Aleman, Julio; Alerasool, Parissa; Dokmeci, Mehmet R.; Khademhosseini, Ali; Ye, Jing Yong

    2016-03-01

    Clinical attrition is about 30% from failure of drug candidates due to toxic side effects, increasing the drug development costs significantly and slowing down the drug discovery process. This partly originates from the fact that the animal models do not accurately represent human physiology. Hence there is a clear unmet need for developing drug toxicity assays using human-based models that are complementary to traditional animal models before starting expensive clinical trials. Organ-on-a-chip techniques developed in recent years have generated a variety of human organ models mimicking different human physiological conditions. However, it is extremely challenging to monitor the transient and long-term response of the organ models to drug treatments during drug toxicity tests. First, when an organ-on-a-chip model interacts with drugs, a certain amount of protein molecules may be released into the medium due to certain drug effects, but the amount of the protein molecules is limited, since the organ tissue grown inside microfluidic bioreactors have minimum volume. Second, traditional fluorescence techniques cannot be utilized for real-time monitoring of the concentration of the protein molecules, because the protein molecules are continuously secreted from the tissue and it is practically impossible to achieve fluorescence labeling in the dynamically changing environment. Therefore, direct measurements of the secreted protein molecules with a label-free approach is strongly desired for organs-on-a-chip applications. In this paper, we report the development of a photonic crystal-based biosensor for label-free assays of secreted protein molecules from a liver-on-a-chip model. Ultrahigh detection sensitivity and specificity have been demonstrated.

  1. Water-protein interactions from high-resolution protein crystallography.

    PubMed Central

    Nakasako, Masayoshi

    2004-01-01

    To understand the role of water in life at molecular and atomic levels, structures and interactions at the protein-water interface have been investigated by cryogenic X-ray crystallography. The method enabled a much clearer visualization of definite hydration sites on the protein surface than at ambient temperature. Using the structural models of proteins, including several hydration water molecules, the characteristics in hydration structures were systematically analysed for the amount, the interaction geometries between water molecules and proteins, and the local and global distribution of water molecules on the surface of proteins. The tetrahedral hydrogen-bond geometry of water molecules in bulk solvent was retained at the interface and enabled the extension of a three-dimensional chain connection of a hydrogen-bond network among hydration water molecules and polar protein atoms over the entire surface of proteins. Networks of hydrogen bonds were quite flexible to accommodate and/or to regulate the conformational changes of proteins such as domain motions. The present experimental results may have profound implications in the understanding of the physico-chemical principles governing the dynamics of proteins in an aqueous environment and a discussion of why water is essential to life at a molecular level. PMID:15306376

  2. Screening of the toxic effects of a high melamine dose on the biochemical hematological and histopathological investigations in male rats.

    PubMed

    El Rabey, Haddad A; Al-Sieni, Abdulbasit I; Majami, Abdullah A

    2014-11-01

    Screening of the toxic effect of a high oral melamine dose (30,000 ppm supplemented in the diet) was performed for 28 days on male rats. The morphology, anatomy, complete blood count (CBC), serum electrolytes, kidney function, serum proteins, serum bilirubin, serum liver enzymes, catalase, glutathion-S-transferase, lipid peroxide, serum melamine concentration, total body weight, food intake, food efficiency ratio (FER), body weight gain percentage (BWG%), body weight gain, water consumption, and histopathological examinations of kidney, urinary bladder, testis, liver, heart, and spleen were investigated. The melamine-supplemented rats turned yellow and showed different degrees of hypertrophy and congestion, particularly the kidney and the ureter as a result of melamine toxicity. The CBC showed minimal changes in the melamine-supplemented groups. Na and Cl were decreased, whereas K, P, and Ca were increased. Serum creatinine, uric acid, and urea were elevated. Liver function enzymes were nonsignificantly affected. Catalase and glutathion-S-transferase were decreased, whereas lipid peroxide was increased in the kidney tissue homogenate. It was also noted that serum protein was decreased and serum bilirubin was increased. Histopathologically, most examined organs were severely injured specially the kidneys, liver, and testes. PMID:24253415

  3. Toxicity assessment of polycyclic aromatic hydrocarbons in sediments from European high mountain lakes.

    PubMed

    Quiroz, Roberto; Grimalt, Joan O; Fernández, Pilar

    2010-05-01

    Sediment quality guidelines and toxic equivalent factors have been used for assessment of the toxicity of sedimentary long-range atmospherically transported polycyclic aromatic hydrocarbons (PAHs) to the organisms living in high mountain European lakes. This method has provided indices that are consistent with experimental studies evaluating in situ sedimentary estrogenic activity or physiological response to AhR binding in fish from the same lakes. All examined lakes in north, central, west, northeast and southeast European mountains have shown sedimentary PAH concentrations that are above thresholds of no effect but only those situated in the southeast lakes district exhibited concentrations above the indices of probable effects. These mountains, Tatras, are also those having PAH concentrations of highest activity for AhR binding. Chrysene+triphenylene, dibenz[a]anthracene, benzo[k]fluoranthene and indeno[1,2,3-cd]pyrene are the main compounds responsible for the observed toxic effects.

  4. The Drosophila melanogaster seminal fluid protein Acp62F is a protease inhibitor that is toxic upon ectopic expression.

    PubMed

    Lung, Oliver; Tram, Uyen; Finnerty, Casey M; Eipper-Mains, Marcie A; Kalb, John M; Wolfner, Mariana F

    2002-01-01

    Drosophila melanogaster seminal fluid proteins stimulate sperm storage and egg laying in the mated female but also cause a reduction in her life span. We report here that of eight Drosophila seminal fluid proteins (Acps) and one non-Acp tested, only Acp62F is toxic when ectopically expressed. Toxicity to preadult male or female Drosophila occurs upon one exposure, whereas multiple exposures are needed for toxicity to adult female flies. Of the Acp62F received by females during mating, approximately 10% enters the circulatory system while approximately 90% remains in the reproductive tract. We show that in the reproductive tract, Acp62F localizes to the lumen of the uterus and the female's sperm storage organs. Analysis of Acp62F's sequence, and biochemical assays, reveals that it encodes a trypsin inhibitor with sequence and structural similarities to extracellular serine protease inhibitors from the nematode Ascaris. In light of previous results demonstrating entry of Acp62F into the mated female's hemolymph, we propose that Acp62F is a candidate for a molecule to contribute to the Acp-dependent decrease in female life span. We propose that Acp62F's protease inhibitor activity exerts positive protective functions in the mated female's reproductive tract but that entry of a small amount of this protein into the female's hemolymph could contribute to the cost of mating. PMID:11805057

  5. Sources of toxicity and exposure information for identifying chemicals of high concern to children

    SciTech Connect

    Stone, Alex; Delistraty, Damon

    2010-11-15

    Due to the large number of chemicals in commerce without adequate toxicity characterization data, coupled with an ineffective federal policy for chemical management in the United States, many states are grappling with the challenge to identify toxic chemicals that may pose a risk to human health and the environment. Specific populations (e.g., children, elderly) are particularly sensitive to these toxic chemicals. In 2008, the Children's Safe Product Act (CSPA) was passed in Washington State. The CSPA included specific requirements to identify High Priority Chemicals (HPCs) and Chemicals of High Concern to Children (CHCCs). To implement this legislation, a methodology was developed to identify HPCs from authoritative scientific and regulatory sources on the basis of toxicity criteria. Another set of chemicals of concern was then identified from authoritative sources, based on their potential exposure to children. Exposure potential was evaluated by identifying chemicals detected in biomonitoring studies (i.e., human tissues), as well as those present in residential exposure media (e.g., indoor air, house dust, drinking water, consumer products). Accordingly, CHCCs were defined as HPCs that also appear in biomonitoring studies or relevant exposure media. For chemicals with unique Chemical Abstracts Service (CAS) numbers, we identified 2044 HPCs and 2219 chemicals with potential exposure to children, resulting in 476 CHCCs. The process of chemical identification is dynamic, so that chemicals may be added or subtracted as new information becomes available. Although beyond the scope of this paper, the 476 CHCCs will be prioritized in a more detailed assessment, based on the strength and weight of evidence of toxicity and exposure data. Our approach was developed to be flexible which allows the addition or removal of specific sources of toxicity or exposure information, as well as transparent to allow clear identification of inputs. Although the methodology was

  6. O-GlcNAc modification blocks the aggregation and toxicity of the protein α-synuclein associated with Parkinson's disease

    NASA Astrophysics Data System (ADS)

    Marotta, Nicholas P.; Lin, Yu Hsuan; Lewis, Yuka E.; Ambroso, Mark R.; Zaro, Balyn W.; Roth, Maxwell T.; Arnold, Don B.; Langen, Ralf; Pratt, Matthew R.

    2015-11-01

    Several aggregation-prone proteins associated with neurodegenerative diseases can be modified by O-linked N-acetyl-glucosamine (O-GlcNAc) in vivo. One of these proteins, α-synuclein, is a toxic aggregating protein associated with synucleinopathies, including Parkinson's disease. However, the effect of O-GlcNAcylation on α-synuclein is not clear. Here, we use synthetic protein chemistry to generate both unmodified α-synuclein and α-synuclein bearing a site-specific O-GlcNAc modification at the physiologically relevant threonine residue 72. We show that this single modification has a notable and substoichiometric inhibitory effect on α-synuclein aggregation, while not affecting the membrane binding or bending properties of α-synuclein. O-GlcNAcylation is also shown to affect the phosphorylation of α-synuclein in vitro and block the toxicity of α-synuclein that was exogenously added to cells in culture. These results suggest that increasing O-GlcNAcylation may slow the progression of synucleinopathies and further support a general function for O-GlcNAc in preventing protein aggregation.

  7. Pyrrolizidine Alkaloid-Protein Adducts: Potential Non-invasive Biomarkers of Pyrrolizidine Alkaloid-Induced Liver Toxicity and Exposure.

    PubMed

    Xia, Qingsu; Zhao, Yuewei; Lin, Ge; Beland, Frederick A; Cai, Lining; Fu, Peter P

    2016-08-15

    Pyrrolizidine alkaloids (PAs) are phytochemicals present in hundreds of plant species from different families widely distributed in many geographical regions around the world. PA-containing plants are probably the most common type of poisonous plants affecting livestock, wildlife, and humans. There have been many large-scale human poisonings caused by the consumption of food contaminated with toxic PAs. PAs require metabolic activation to generate pyrrolic metabolites to exert their toxicity. In this study, we developed a novel method to quantify pyrrole-protein adducts present in the blood. This method involves the use of AgNO3 in acidic ethanol to cleave the thiol linkage of pyrrole-protein (DHP-protein) adducts, and the resulting 7,9-di-C2H5O-DHP is quantified by HPLC-ES-MS/MS multiple reaction monitoring analysis in the presence of a known quantity of isotopically labeled 7,9-di-C2D5O-DHP internal standard. Using this method, we determined that diester-type PAs administered to rats produced higher levels of DHP-protein adducts than other types of PAs. The results suggest that DHP-protein adducts can potentially serve as minimally invasive biomarkers of PA exposure. PMID:27388689

  8. Predictors of Toxicity After Image-guided High-dose-rate Interstitial Brachytherapy for Gynecologic Cancer

    SciTech Connect

    Lee, Larissa J.; Viswanathan, Akila N.

    2012-12-01

    Purpose: To identify predictors of grade 3-4 complications and grade 2-4 rectal toxicity after three-dimensional image-guided high-dose-rate (HDR) interstitial brachytherapy for gynecologic cancer. Methods and Materials: Records were reviewed for 51 women (22 with primary disease and 29 with recurrence) treated with HDR interstitial brachytherapy. A single interstitial insertion was performed with image guidance by computed tomography (n = 43) or magnetic resonance imaging (n = 8). The median delivered dose in equivalent 2-Gy fractions was 72.0 Gy (45 Gy for external-beam radiation therapy and 24 Gy for brachytherapy). Toxicity was reported according to the Common Toxicity Criteria for Adverse Events. Actuarial toxicity estimates were calculated by the Kaplan-Meier method. Results: At diagnosis, the median patient age was 62 years and the median tumor size was 3.8 cm. The median D90 and V100 were 71.4 Gy and 89.5%; the median D2cc for the bladder, rectum, and sigmoid were 64.6 Gy, 61.0 Gy, and 52.7 Gy, respectively. The actuarial rates of all grade 3-4 complications at 2 years were 20% gastrointestinal, 9% vaginal, 6% skin, 3% musculoskeletal, and 2% lymphatic. There were no grade 3-4 genitourinary complications and no grade 5 toxicities. Grade 2-4 rectal toxicity was observed in 10 patients, and grade 3-4 complications in 4; all cases were proctitis with the exception of 1 rectal fistula. D2cc for rectum was higher for patients with grade 2-4 (68 Gy vs 57 Gy for grade 0-1, P=.03) and grade 3-4 (73 Gy vs 58 Gy for grade 0-2, P=.02) rectal toxicity. The estimated dose that resulted in a 10% risk of grade 2-4 rectal toxicity was 61.8 Gy (95% confidence interval, 51.5-72.2 Gy). Discussion: Image-guided HDR interstitial brachytherapy results in acceptable toxicity for women with primary or recurrent gynecologic cancer. D2cc for the rectum is a reliable predictor of late rectal complications. Three-dimensional-based treatment planning should be performed to ensure

  9. Highly Charged Proteins: The Achilles' Heel of Aging Proteomes.

    PubMed

    de Graff, Adam M R; Hazoglou, Michael J; Dill, Ken A

    2016-02-01

    As cells and organisms age, their proteins sustain increasing amounts of oxidative damage. It is estimated that half of all proteins are damaged in old organisms, yet the dominant mechanisms by which damage affects proteins and cellular phenotypes are not known. Here, we show that random modification of side chain charge induced by oxidative damage is likely to be a dominant source of protein stability loss in aging cells. Using an established model of protein electrostatics, we find that short, highly charged proteins are particularly susceptible to large destabilization from even a single side chain oxidation event. This mechanism identifies 20 proteins previously established to be important in aging that are at particularly high risk for oxidative destabilization, including transcription factors, histone and histone-modifying proteins, ribosomal and telomeric proteins, and proteins essential for homeostasis. Cellular processes enriched in high-risk proteins are shown to be particularly abundant in the aggregates of old organisms. PMID:26724998

  10. Printing Proteins as Microarrays for High-Throughput Function Determination

    NASA Astrophysics Data System (ADS)

    MacBeath, Gavin; Schreiber, Stuart L.

    2000-09-01

    Systematic efforts are currently under way to construct defined sets of cloned genes for high-throughput expression and purification of recombinant proteins. To facilitate subsequent studies of protein function, we have developed miniaturized assays that accommodate extremely low sample volumes and enable the rapid, simultaneous processing of thousands of proteins. A high-precision robot designed to manufacture complementary DNA microarrays was used to spot proteins onto chemically derivatized glass slides at extremely high spatial densities. The proteins attached covalently to the slide surface yet retained their ability to interact specifically with other proteins, or with small molecules, in solution. Three applications for protein microarrays were demonstrated: screening for protein-protein interactions, identifying the substrates of protein kinases, and identifying the protein targets of small molecules.

  11. Highly efficient and lowly toxic docetaxel nanoemulsions for intravenous injection to animals.

    PubMed

    Li, Xin; Du, Lina; Wang, Chenyun; Liu, Yan; Mei, Xingguo; Jin, Yiguang

    2011-07-01

    Hypersensitivity many occur with commercial docetaxel injections containing Tween 80 and ethanol. An alternative formulation of docetaxel, an oil-in-water nanoemulsion was prepared using the high-pressure homogenization method. It was composed of medium-chain triglyceride, oleic acid, egg lecithin, and poloxamer. These ingredients are known as safe agents for intravenous (i.v.) injection. The nanoemulsion had a small size of 169 nm, and a high surface charge with the zeta potential of -33.9 mV. It maintained well stable even under high centrifugation. Acute toxicity of i.v. injection, erythrocyte hemolysis experiment, and rabbit ear vein irritation test showed no toxicity for the docetaxel nanoemulsion. The docetaxel nanoemulsion led to a larger apparent distribution volume and area under curve than the docetaxel injection after i.v. administration to rats. The histopathological test of tumor further demonstrated the highly anticancer efficiency of the docetaxel nanoemulsion. Thus, the nanoemulsion is a promising delivery system for docetaxel with highly anticancer efficiency and low toxicity. PMID:21812321

  12. Mammalian ribosomal and chaperone protein RPS3A counteracts α-synuclein aggregation and toxicity in a yeast model system.

    PubMed

    De Graeve, Stijn; Marinelli, Sarah; Stolz, Frank; Hendrix, Jelle; Vandamme, Jurgen; Engelborghs, Yves; Van Dijck, Patrick; Thevelein, Johan M

    2013-11-01

    Accumulation of aggregated forms of αSyn (α-synuclein) into Lewy bodies is a known hallmark associated with neuronal cell death in Parkinson's disease. When expressed in the yeast Saccharomyces cerevisiae, αSyn interacts with the plasma membrane, forms inclusions and causes a concentration-dependent growth defect. We have used a yeast mutant, cog6Δ, which is particularly sensitive to moderate αSyn expression, for screening a mouse brain-specific cDNA library in order to identify mammalian proteins that counteract αSyn toxicity. The mouse ribosomal and chaperone protein RPS3A was identified as a suppressor of αSyn [WT (wild-type) and A53T] toxicity in yeast. We demonstrated that the 50 N-terminal amino acids are essential for this function. The yeast homologues of RPS3A were not effective in suppressing the αSyn-induced growth defect, illustrating the potential of our screening system to identify modifiers that would be missed using yeast gene overexpression as the first screening step. Co-expression of mouse RPS3A delayed the formation of αSyn-GFP inclusions in the yeast cells. The results of the present study suggest that the recently identified extraribosomal chaperonin function of RPS3A also acts on the neurodegeneration-related protein αSyn and reveal a new avenue for identifying promising candidate mammalian proteins involved in αSyn functioning.

  13. Reduced toxicity and broad spectrum resistance to viral and fungal infection in transgenic plants expressing pokeweed antiviral protein II.

    PubMed

    Wang, P; Zoubenko, O; Tumer, N E

    1998-12-01

    Pokeweed antiviral protein II (PAPII), a 30 kDa protein isolated from leaves of Phytolacca americana, inhibits translation by catalytically removing a specific adenine residue from the large rRNA of the 60S subunit of eukaryotic ribosomes. The protein sequence of PAPII shows only 41% identity to PAP and PAP-S, two other antiviral proteins isolated from pokeweed. We isolated a cDNA corresponding to PAPII and introduced it into tobacco plants. PAPII expressed in transgenic tobacco was correctly processed to the mature form as in pokeweed and accumulated to at least 10-fold higher levels than wild-type PAP. We had previously observed a significant decrease in transformation frequency with PAP and recovered only two transgenic lines expressing 1-2 ng per mg protein. In contrast, eight different transgenic lines expressing up to 250 ng/mg PAPII were recovered, indicating that PAPII is less toxic than PAP. Two symptomless transgenic lines expressing PAPII were resistant to tobacco mosaic virus, potato virus X and the fungal pathogen Rhizoctonia solani. The level of viral and fungal resistance observed correlated well with the amount of PAPII protein accumulated. Pathogenesis-related protein PR1 was constitutively expressed in transgenic lines expressing PAPII. Although PR1 was constitutively expressed, no increase in salicylic acid levels was detected, indicating that PAPII may elicit a salicylic acid-independent signal transduction pathway.

  14. Acetaminophen-induced liver injury in rats and mice: Comparison of protein adducts, mitochondrial dysfunction, and oxidative stress in the mechanism of toxicity

    SciTech Connect

    McGill, Mitchell R.; Williams, C. David; Xie, Yuchao; Ramachandran, Anup; Jaeschke, Hartmut

    2012-11-01

    Acetaminophen (APAP) overdose is the most common cause of acute liver failure in the West. In mice, APAP hepatotoxicity can be rapidly induced with a single dose. Because it is both clinically relevant and experimentally convenient, APAP intoxication has become a popular model of liver injury. Early data demonstrated that rats are resistant to APAP toxicity. As a result, mice are the preferred species for mechanistic studies. Furthermore, recent work has shown that the mechanisms of APAP toxicity in humans are similar to mice. Nevertheless, some investigators still use rats. New mechanistic information from the last forty years invites a reevaluation of the differences between these species. Comparison may provide interesting insights and confirm or exclude the rat as an option for APAP studies. To this end, we treated rats and mice with APAP and measured parameters of liver injury, APAP metabolism, oxidative stress, and activation of the c-Jun N-terminal kinase (JNK). Consistent with earlier data, we found that rats were highly resistant to APAP toxicity. Although overall APAP metabolism was similar in both species, mitochondrial protein adducts were significantly lower in rats. Accordingly, rats also had less oxidative stress. Finally, while mice showed extensive activation and mitochondrial translocation of JNK, this could not be detected in rat livers. These data support the hypothesis that mitochondrial dysfunction is critical for the development of necrosis after APAP treatment. Because mitochondrial damage also occurs in humans, rats are not a clinically relevant species for studies of APAP hepatotoxicity. Highlights: ► Acetaminophen overdose causes severe liver injury only in mice but not in rats. ► APAP causes hepatic GSH depletion and protein adduct formation in rats and mice. ► Less protein adducts were measured in rat liver mitochondria compared to mouse. ► No oxidant stress, peroxynitrite formation or JNK activation was present in rats. ► The

  15. Inhalation threshold of toxicological concern (TTC) - Structural alerts discriminate high from low repeated-dose inhalation toxicity.

    PubMed

    Schüürmann, Gerrit; Ebert, Ralf-Uwe; Tluczkiewicz, Inga; Escher, Sylvia E; Kühne, Ralph

    2016-03-01

    The threshold of toxicological concern (TTC) of a compound represents an exposure value below which the associated human health risk is considered negligible. As such, this approach offers assessing the risk of potential toxicants when little or no toxicological information is available. For the inhalation repeated-dose TTC, the goal was to derive structural alerts that discriminate between high- and low-toxic compounds. A further aim was to identify physicochemical parameters related to the inhalation-specific bioavailability of the compounds, and to explore their use as predictors of high vs low toxicity. 296 compounds with subacute, subchronic and chronic inhalation toxicity NOEC (no-observed effect concentration) values were subdivided into three almost equal-sized high-, medium- and low-toxic (HTox, MTox, LTox) potency classes. Whereas the derived 14 HTox and 7 LTox structural alerts yield an only moderate discrimination between these three groups, the high-toxic vs low-toxic mis-classification is very low: LTox-predicted compounds are not HTox to 97.5%, and HTox-predicted compounds not LTox to 88.6%. The probability of a compound being HTox vs LTox is triggered further by physicochemical properties encoding the tendency to evaporate from blood. The new structural alerts may aid in the predictive inhalation toxicity assessment of compounds as well as in designing low-toxicity chemicals, and provide a rationale for the chemistry underlying the toxicological outcome that can also be used for scoping targeted experimental studies.

  16. Inhalation threshold of toxicological concern (TTC) - Structural alerts discriminate high from low repeated-dose inhalation toxicity.

    PubMed

    Schüürmann, Gerrit; Ebert, Ralf-Uwe; Tluczkiewicz, Inga; Escher, Sylvia E; Kühne, Ralph

    2016-03-01

    The threshold of toxicological concern (TTC) of a compound represents an exposure value below which the associated human health risk is considered negligible. As such, this approach offers assessing the risk of potential toxicants when little or no toxicological information is available. For the inhalation repeated-dose TTC, the goal was to derive structural alerts that discriminate between high- and low-toxic compounds. A further aim was to identify physicochemical parameters related to the inhalation-specific bioavailability of the compounds, and to explore their use as predictors of high vs low toxicity. 296 compounds with subacute, subchronic and chronic inhalation toxicity NOEC (no-observed effect concentration) values were subdivided into three almost equal-sized high-, medium- and low-toxic (HTox, MTox, LTox) potency classes. Whereas the derived 14 HTox and 7 LTox structural alerts yield an only moderate discrimination between these three groups, the high-toxic vs low-toxic mis-classification is very low: LTox-predicted compounds are not HTox to 97.5%, and HTox-predicted compounds not LTox to 88.6%. The probability of a compound being HTox vs LTox is triggered further by physicochemical properties encoding the tendency to evaporate from blood. The new structural alerts may aid in the predictive inhalation toxicity assessment of compounds as well as in designing low-toxicity chemicals, and provide a rationale for the chemistry underlying the toxicological outcome that can also be used for scoping targeted experimental studies. PMID:26735350

  17. Targeting Protein-Protein Interactions with Trimeric Ligands: High Affinity Inhibitors of the MAGUK Protein Family

    PubMed Central

    Nissen, Klaus B.; Haugaard-Kedström, Linda M.; Wilbek, Theis S.; Nielsen, Line S.; Åberg, Emma; Kristensen, Anders S.; Bach, Anders; Jemth, Per; Strømgaard, Kristian

    2015-01-01

    PDZ domains in general, and those of PSD-95 in particular, are emerging as promising drug targets for diseases such as ischemic stroke. We have previously shown that dimeric ligands that simultaneously target PDZ1 and PDZ2 of PSD-95 are highly potent inhibitors of PSD-95. However, PSD-95 and the related MAGUK proteins contain three consecutive PDZ domains, hence we envisioned that targeting all three PDZ domains simultaneously would lead to more potent and potentially more specific interactions with the MAGUK proteins. Here we describe the design, synthesis and characterization of a series of trimeric ligands targeting all three PDZ domains of PSD-95 and the related MAGUK proteins, PSD-93, SAP-97 and SAP-102. Using our dimeric ligands targeting the PDZ1-2 tandem as starting point, we designed novel trimeric ligands by introducing a PDZ3-binding peptide moiety via a cysteine-derivatized NPEG linker. The trimeric ligands generally displayed increased affinities compared to the dimeric ligands in fluorescence polarization binding experiments and optimized trimeric ligands showed low nanomolar inhibition towards the four MAGUK proteins, thus being the most potent inhibitors described. Kinetic experiments using stopped-flow spectrometry showed that the increase in affinity is caused by a decrease in the dissociation rate of the trimeric ligand as compared to the dimeric ligands, likely reflecting the lower probability of simultaneous dissociation of all three PDZ ligands. Thus, we have provided novel inhibitors of the MAGUK proteins with exceptionally high affinity, which can be used to further elucidate the therapeutic potential of these proteins. PMID:25658767

  18. Targeting protein-protein interactions with trimeric ligands: high affinity inhibitors of the MAGUK protein family.

    PubMed

    Nissen, Klaus B; Haugaard-Kedström, Linda M; Wilbek, Theis S; Nielsen, Line S; Åberg, Emma; Kristensen, Anders S; Bach, Anders; Jemth, Per; Strømgaard, Kristian

    2015-01-01

    PDZ domains in general, and those of PSD-95 in particular, are emerging as promising drug targets for diseases such as ischemic stroke. We have previously shown that dimeric ligands that simultaneously target PDZ1 and PDZ2 of PSD-95 are highly potent inhibitors of PSD-95. However, PSD-95 and the related MAGUK proteins contain three consecutive PDZ domains, hence we envisioned that targeting all three PDZ domains simultaneously would lead to more potent and potentially more specific interactions with the MAGUK proteins. Here we describe the design, synthesis and characterization of a series of trimeric ligands targeting all three PDZ domains of PSD-95 and the related MAGUK proteins, PSD-93, SAP-97 and SAP-102. Using our dimeric ligands targeting the PDZ1-2 tandem as starting point, we designed novel trimeric ligands by introducing a PDZ3-binding peptide moiety via a cysteine-derivatized NPEG linker. The trimeric ligands generally displayed increased affinities compared to the dimeric ligands in fluorescence polarization binding experiments and optimized trimeric ligands showed low nanomolar inhibition towards the four MAGUK proteins, thus being the most potent inhibitors described. Kinetic experiments using stopped-flow spectrometry showed that the increase in affinity is caused by a decrease in the dissociation rate of the trimeric ligand as compared to the dimeric ligands, likely reflecting the lower probability of simultaneous dissociation of all three PDZ ligands. Thus, we have provided novel inhibitors of the MAGUK proteins with exceptionally high affinity, which can be used to further elucidate the therapeutic potential of these proteins.

  19. High Efficiency InP Solar Cells from Low Toxicity Tertiarybutylphosphine

    NASA Technical Reports Server (NTRS)

    Hoffman, Richard W., Jr.; Fatemi, Navid S.; Wilt, David M.; Jenkins, Phillip P.; Brinker, David J.; Scheiman, David A.

    1994-01-01

    Large scale manufacture of phosphide based semiconductor devices by organo-metallic vapor phase epitaxy (OMVPE) typically requires the use of highly toxic phosphine. Advancements in phosphine substitutes have identified tertiarybutylphosphine (TBP) as an excellent precursor for OMVPE of InP. High quality undoped and doped InP films were grown using TBP and trimethylindium. Impurity doped InP films were achieved utilizing diethylzinc and silane for p and n type respectively. 16 percent efficient solar cells under air mass zero, one sun intensity were demonstrated with Voc of 871 mV and fill factor of 82.6 percent. It was shown that TBP could replace phosphine, without adversely affecting device quality, in OMVPE deposition of InP thus significantly reducing toxic gas exposure risk.

  20. Microstructural Changes in High-Protein Nutrition Bars Formulated with Extruded or Toasted Milk Protein Concentrate.

    PubMed

    Banach, J C; Clark, S; Lamsal, B P

    2016-02-01

    Milk protein concentrates with more than 80% protein (that is, MPC80) are underutilized as the primary protein source in high-protein nutrition bars as they impart crumbliness and cause hardening during storage. High-protein nutrition bar texture changes are often associated with internal protein aggregations and macronutrient phase separation. These changes were investigated in model high-protein nutrition bars formulated with MPC80 and physically modified MPC80s. High-protein nutrition bars formulated with extruded MPC80s hardened slower than those formulated with toasted or unmodified MPC80. Extruded MPC80 had reduced free sulfhydryl group exposure, whereas measurable increases were seen in the toasted MPC80. High-protein nutrition bar textural performance may be related to the number of exposed free sulfhydryl groups in MPC80. Protein aggregations resulting from ingredient modification and high-protein nutrition bar storage were studied with sodium dodecyl sulfate polyacrylamide gel electrophoresis. Disulfide-based protein aggregations and changes in free sulfhydryl concentration were not consistently relatable to high-protein nutrition bar texture change. However, the high-protein nutrition bars formulated with extruded MPC80 were less prone to phase separations, as depicted by confocal laser scanning microscopy, and underwent less texture change during storage than those formulated with toasted or unmodified MPC80.

  1. Conversion of high and low pollen protein diets into protein in worker honey bees (Hymenoptera: Apidae).

    PubMed

    Basualdo, M; Barragán, S; Vanagas, L; García, C; Solana, H; Rodríguez, E; Bedascarrasbure, E

    2013-08-01

    Adequate protein levels are necessary to maintain strong honey bee [Apis mellifera (L.)] colonies. The aim of this study was to quantify how pollens with different crude protein contents influence protein stores within individual honey bees. Caged bees were fed one of three diets, consisting of high-protein-content pollen, low-protein-content pollen, or protein-free diet as control; measurements were made based on protein content in hemolymph and fat body, fat body weight, and body weight. Vitellogenin in hemolymph was also measured. Bees fed with high crude protein diet had significantly higher levels of protein in hemolymph and fat bodies. Caged bees did not increase pollen consumption to compensate for the lower protein in the diet, and ingesting approximately 4 mg of protein per bee could achieve levels of 20 microg/microl protein in hemolymph. Worker bees fed with low crude protein diet took more time in reaching similar protein content of the bees that were fed with high crude protein diet. The data showed that fat bodies and body weight were not efficient methods of measuring the protein status of bees. The determination of total protein or vitellogenin concentration in the hemolymph from 13-d-old bees and protein concentration of fat bodies from 9-d-old bees could be good indicators of nutritional status of honey bees. PMID:24020265

  2. Conversion of high and low pollen protein diets into protein in worker honey bees (Hymenoptera: Apidae).

    PubMed

    Basualdo, M; Barragán, S; Vanagas, L; García, C; Solana, H; Rodríguez, E; Bedascarrasbure, E

    2013-08-01

    Adequate protein levels are necessary to maintain strong honey bee [Apis mellifera (L.)] colonies. The aim of this study was to quantify how pollens with different crude protein contents influence protein stores within individual honey bees. Caged bees were fed one of three diets, consisting of high-protein-content pollen, low-protein-content pollen, or protein-free diet as control; measurements were made based on protein content in hemolymph and fat body, fat body weight, and body weight. Vitellogenin in hemolymph was also measured. Bees fed with high crude protein diet had significantly higher levels of protein in hemolymph and fat bodies. Caged bees did not increase pollen consumption to compensate for the lower protein in the diet, and ingesting approximately 4 mg of protein per bee could achieve levels of 20 microg/microl protein in hemolymph. Worker bees fed with low crude protein diet took more time in reaching similar protein content of the bees that were fed with high crude protein diet. The data showed that fat bodies and body weight were not efficient methods of measuring the protein status of bees. The determination of total protein or vitellogenin concentration in the hemolymph from 13-d-old bees and protein concentration of fat bodies from 9-d-old bees could be good indicators of nutritional status of honey bees.

  3. The neuronal nitric oxide synthase inhibitor NANT blocks acetaminophen toxicity and protein nitration in freshly isolated hepatocytes.

    PubMed

    Banerjee, Sudip; Melnyk, Stepan B; Krager, Kimberly J; Aykin-Burns, Nukhet; Letzig, Lynda G; James, Laura P; Hinson, Jack A

    2015-12-01

    3-Nitrotyrosine (3NT) in liver proteins of mice treated with hepatotoxic doses of acetaminophen (APAP) has been postulated to be causative in toxicity. Nitration is by a reactive nitrogen species formed from nitric oxide (NO). The source of the NO is unclear. iNOS knockout mice were previously found to be equally susceptible to APAP toxicity as wildtype mice and iNOS inhibitors did not decrease toxicity in mice or in hepatocytes. In this work we examined the potential role of nNOS in APAP toxicity in hepatocytes using the specific nNOS inhibitor NANT (10 µM)(N-[(4S)-4-amino-5-[(2-aminoethyl)amino]pentyl]-N'-nitroguanidinetris (trifluoroacetate)). Primary hepatocytes (1 million/ml) from male B6C3F1 mice were incubated with APAP (1mM). Cells were removed and assayed spectrofluorometrically for reactive nitrogen and oxygen species using diaminofluorescein (DAF) and Mitosox red, respectively. Cytotoxicity was determined by LDH release into media. Glutathione (GSH, GSSG), 3NT, GSNO, acetaminophen-cysteine adducts, NAD, and NADH were measured by HPLC. APAP significantly increased cytotoxicity at 1.5-3.0 h. The increase was blocked by NANT. NANT did not alter APAP mediated GSH depletion or acetaminophen-cysteine adducts in proteins which indicated that NANT did not inhibit metabolism. APAP significantly increased spectroflurometric evidence of reactive nitrogen and oxygen formation at 0.5 and 1.0 h, respectively, and increased 3NT and GSNO at 1.5-3.0 h. These increases were blocked by NANT. APAP dramatically increased NADH from 0.5-3.0 h and this increase was blocked by NANT. Also, APAP decreased the Oxygen Consumption Rate (OCR), decreased ATP production, and caused a loss of mitochondrial membrane potential, which were all blocked by NANT. PMID:26454079

  4. The neuronal nitric oxide synthase inhibitor NANT blocks acetaminophen toxicity and protein nitration in freshly isolated hepatocytes.

    PubMed

    Banerjee, Sudip; Melnyk, Stepan B; Krager, Kimberly J; Aykin-Burns, Nukhet; Letzig, Lynda G; James, Laura P; Hinson, Jack A

    2015-12-01

    3-Nitrotyrosine (3NT) in liver proteins of mice treated with hepatotoxic doses of acetaminophen (APAP) has been postulated to be causative in toxicity. Nitration is by a reactive nitrogen species formed from nitric oxide (NO). The source of the NO is unclear. iNOS knockout mice were previously found to be equally susceptible to APAP toxicity as wildtype mice and iNOS inhibitors did not decrease toxicity in mice or in hepatocytes. In this work we examined the potential role of nNOS in APAP toxicity in hepatocytes using the specific nNOS inhibitor NANT (10 µM)(N-[(4S)-4-amino-5-[(2-aminoethyl)amino]pentyl]-N'-nitroguanidinetris (trifluoroacetate)). Primary hepatocytes (1 million/ml) from male B6C3F1 mice were incubated with APAP (1mM). Cells were removed and assayed spectrofluorometrically for reactive nitrogen and oxygen species using diaminofluorescein (DAF) and Mitosox red, respectively. Cytotoxicity was determined by LDH release into media. Glutathione (GSH, GSSG), 3NT, GSNO, acetaminophen-cysteine adducts, NAD, and NADH were measured by HPLC. APAP significantly increased cytotoxicity at 1.5-3.0 h. The increase was blocked by NANT. NANT did not alter APAP mediated GSH depletion or acetaminophen-cysteine adducts in proteins which indicated that NANT did not inhibit metabolism. APAP significantly increased spectroflurometric evidence of reactive nitrogen and oxygen formation at 0.5 and 1.0 h, respectively, and increased 3NT and GSNO at 1.5-3.0 h. These increases were blocked by NANT. APAP dramatically increased NADH from 0.5-3.0 h and this increase was blocked by NANT. Also, APAP decreased the Oxygen Consumption Rate (OCR), decreased ATP production, and caused a loss of mitochondrial membrane potential, which were all blocked by NANT.

  5. Acute Toxicity in High-Risk Prostate Cancer Patients Treated With Androgen Suppression and Hypofractionated Intensity-Modulated Radiotherapy

    SciTech Connect

    Pervez, Nadeem; Small, Cormac; MacKenzie, Marc; Yee, Don; Parliament, Matthew; Ghosh, Sunita; Mihai, Alina; Amanie, John; Murtha, Albert; Field, Colin; Murray, David; Fallone, Gino; Pearcey, Robert

    2010-01-15

    Purpose: To report acute toxicity resulting from radiotherapy (RT) dose escalation and hypofractionation using intensity-modulated RT (IMRT) treatment combined with androgen suppression in high-risk prostate cancer patients. Methods and Materials: Sixty patients with a histological diagnosis of high-risk prostatic adenocarcinoma (having either a clinical Stage of >=T3a or an initial prostate-specific antigen [PSA] level of >=20 ng/ml or a Gleason score of 8 to 10 or a combination of a PSA concentration of >15 ng/ml and a Gleason score of 7) were enrolled. RT prescription was 68 Gy in 25 fractions (2.72 Gy/fraction) over 5 weeks to the prostate and proximal seminal vesicles. The pelvic lymph nodes and distal seminal vesicles concurrently received 45 Gy in 25 fractions. The patients were treated with helical TomoTherapy-based IMRT and underwent daily megavoltage CT image-guided verification prior to each treatment. Acute toxicity scores were recorded weekly during RT and at 3 months post-RT, using Radiation Therapy Oncology Group acute toxicity scales. Results: All patients completed RT and follow up for 3 months. The maximum acute toxicity scores were as follows: 21 (35%) patients had Grade 2 gastrointestinal (GI) toxicity; 4 (6.67%) patients had Grade 3 genitourinary (GU) toxicity; and 30 (33.33%) patients had Grade 2 GU toxicity. These toxicity scores were reduced after RT; there were only 8 (13.6%) patients with Grade 1 GI toxicity, 11 (18.97%) with Grade 1 GU toxicity, and 5 (8.62%) with Grade 2 GU toxicity at 3 months follow up. Only the V60 to the rectum correlated with the GI toxicity. Conclusion: Dose escalation using a hypofractionated schedule to the prostate with concurrent pelvic lymph node RT and long-term androgen suppression therapy is well tolerated acutely. Longer follow up for outcome and late toxicity is required.

  6. Acetaminophen-induced liver injury in rats and mice: comparison of protein adducts, mitochondrial dysfunction, and oxidative stress in the mechanism of toxicity.

    PubMed

    McGill, Mitchell R; Williams, C David; Xie, Yuchao; Ramachandran, Anup; Jaeschke, Hartmut

    2012-11-01

    Acetaminophen (APAP) overdose is the most common cause of acute liver failure in the West. In mice, APAP hepatotoxicity can be rapidly induced with a single dose. Because it is both clinically relevant and experimentally convenient, APAP intoxication has become a popular model of liver injury. Early data demonstrated that rats are resistant to APAP toxicity. As a result, mice are the preferred species for mechanistic studies. Furthermore, recent work has shown that the mechanisms of APAP toxicity in humans are similar to mice. Nevertheless, some investigators still use rats. New mechanistic information from the last forty years invites a reevaluation of the differences between these species. Comparison may provide interesting insights and confirm or exclude the rat as an option for APAP studies. To this end, we treated rats and mice with APAP and measured parameters of liver injury, APAP metabolism, oxidative stress, and activation of the c-Jun N-terminal kinase (JNK). Consistent with earlier data, we found that rats were highly resistant to APAP toxicity. Although overall APAP metabolism was similar in both species, mitochondrial protein adducts were significantly lower in rats. Accordingly, rats also had less oxidative stress. Finally, while mice showed extensive activation and mitochondrial translocation of JNK, this could not be detected in rat livers. These data support the hypothesis that mitochondrial dysfunction is critical for the development of necrosis after APAP treatment. Because mitochondrial damage also occurs in humans, rats are not a clinically relevant species for studies of APAP hepatotoxicity.

  7. THE ROLE OF PROTEIN BINDING OF TRIVALENT ARSENICALS IN ARSENIC CARCINOGENESIS AND TOXICITY

    EPA Science Inventory

    Three of the most plausible biological theories of arsenic carcinogenesis are protein binding, oxidative stress and altered DNA methylation. This review presents the role of trivalent arsenicals binding to proteins in arsenic carcinogenesis. Using vacuum filtration based receptor...

  8. A high throughput passive dosing format for the Fish Embryo Acute Toxicity test.

    PubMed

    Vergauwen, Lucia; Schmidt, Stine N; Stinckens, Evelyn; Maho, Walid; Blust, Ronny; Mayer, Philipp; Covaci, Adrian; Knapen, Dries

    2015-11-01

    High throughput testing according to the Fish Embryo Acute Toxicity (FET) test (OECD Testing Guideline 236) is usually conducted in well plates. In the case of hydrophobic test substances, sorptive and evaporative losses often result in declining and poorly controlled exposure conditions. Therefore, our objective was to improve exposure conditions in FET tests by evaluating a passive dosing format using silicone O-rings in standard 24-well polystyrene plates. We exposed zebrafish embryos to a series of phenanthrene concentrations until 120h post fertilization (hpf), and obtained a linear dilution series. We report effect values for both mortality and sublethal morphological effects based on (1) measured exposure concentrations, (2) (lipid normalized) body residues and (3) chemical activity. The LC50 for 120hpf was 310μg/L, CBR50 (critical body residue) was 2.72mmol/kg fresh wt and La50 (lethal chemical activity) was 0.047. All values were within ranges expected for baseline toxicity. Impaired swim bladder inflation was the most pronounced morphological effect and swimming activity was reduced in all exposure concentrations. Further analysis showed that the effect on swimming activity was not attributed to impaired swim bladder inflation, but rather to baseline toxicity. We conclude that silicone O-rings (1) produce a linear dilution series of phenanthrene in the 120hpf FET test, (2) generate and maintain aqueous concentrations for reliable determination of effect concentrations, and allow for obtaining mechanistic toxicity information, and (3) cause no toxicity, demonstrating its potential as an extension of the FET test when testing hydrophobic chemicals. PMID:26026258

  9. Nutritional study of two Brazilian soybean (Glycine max) cultivars differing in the contents of antinutritional and toxic proteins.

    PubMed

    Vasconcelos, I M.; Maia, A A.B.; Siebra, E A.; Oliveira, J T.A.; Carvalho, A d.F.F.U.; Melo, V M.M.; Carlini, C R.; Castelar, L I.d.M.

    2001-01-01

    The research was conducted with two different recently released Brazilian soybean cultivars (Rio Balsas and Bays) to evaluate whether there is any correlation between the different levels of antinutritional and/or toxic proteins in the cultivars and their nutritive value as sources of protein for monogastric animals (rats). Furthermore, it is discussed, for the first time, the role of the dietary soyatoxin on the performance of rats fed on diets containing soyatoxin-rich (cv. Bays) and soyatoxin-free (cv. Rio Balsas) soybean cultivars. Feeding rats with diets containing raw soybean cultivars showed a lower growth rate, net protein utilization and digestibility, a much higher dry matter and nitrogen excretion and macroscopic alterations in internal organs when compared to rats fed on egg-white protein. The nutritional parameters measured for the diet based on raw Bays cultivar were poorer than those of the diet prepared with Rio Balsas. In the raw soybeans, trypsin inhibitor and lectin, and urease to a lesser extent, significantly affected at different fashion the soybean protein utilization. Heating treatment of the Bays seeds increased the growth rate, NPU, in vivo protein digestibility and practically eliminated or attenuated all the organ alterations observed. This study might be helpful in the choice of safe and nutritious soybean cultivars. PMID:11179862

  10. [Elimination of toxic compounds, biological evaluation and partial characterization of the protein from jojoba meal (Simmondsia chinensis [Link] Schneider].

    PubMed

    Medina Juárez, L A; Trejo González, A

    1989-12-01

    The purpose of this study was to establish a new methodology to remove the toxic compounds present in jojoba meal and flour. Also, to perform the biological evaluation of the detoxified products and to chemically characterize the protein fractions. Jojoba meal and seed without testa were deffated with hexane and detoxified with a 7:3 isopropanol-water mixture which removed 86% of total phenolic compounds and 100% of simmondsins originally present, the resulting products had reduced bitterness and caused no deaths on experimental animals. NPR values obtained for diets containing such products were significantly different from those obtained with the casein control (p less than 0.05). Total protein was made up of three different fractions: the water-soluble fraction was the most abundant (61.8%), followed by the salt-soluble (23.6%), and the alkaline soluble fraction (14.6%). The nitrogen solubility curves showed that the isoelectric point for the water-soluble and salt-soluble fractions was pH 3.0, while that of the alkaline fraction fell in the range of 4.5-5.0. All fractions had a maximum solubility at pH 7.0. The methodology reported here, offers a viable solution to eliminate toxic compounds from jojoba meal or seeds, and upgrades the potential use of products such as animal feed or raw material for the production of protein isolates.

  11. Tier-1 assays for assessing the toxicity of insecticidal proteins produced by genetically engineered plants to non-target arthropods.

    PubMed

    Li, Yun-He; Romeis, Jörg; Wu, Kong-Ming; Peng, Yu-Fa

    2014-04-01

    In assessing an insect-resistant genetically engineered (IRGE) crop before its commercialization, researchers normally use so-called "Tier-1 assays" as the initial step to determine the effects of the crop on non-target organisms. In these tests, the insecticidal proteins (IPs) produced by the IRGEs are added to the diets of test organisms in the laboratory. Test organisms in such assays can be directly exposed to much higher concentrations of the test IPs than they would encounter in the field. The results of Tier-1 assays are thus more conservative than those generated in studies in which the organisms are exposed to the IPs by feeding on IRGE plant tissue or in the case of predators or parasites, by feeding on invertebrate prey or hosts that have fed on IRGE plant tissue. In this report, we consider three important factors that must be considered in Tier-1 assays: (i) methods for delivery of the IP to the test organisms; (ii) the need for and selection of compounds used as positive controls; and (iii) methods for monitoring the concentration, stability and bioactivity of the IP during the assay. We also analyze the existing data from Tier-1 assays regarding the toxicity of Bt Cry proteins to non-target arthropod species. The data indicate that the widely used Bt proteins have no direct toxicity to non-target organisms.

  12. High-Yield Secretion of Multiple Client Proteins in Aspergillus

    SciTech Connect

    Segato, F.; Damasio, A. R. L.; Goncalves, T. A.; de Lucas, R. C.; Squina, F. M.; Decker, S. R.; Prade, R. A.

    2012-07-15

    Production of pure and high-yield client proteins is an important technology that addresses the need for industrial applications of enzymes as well as scientific experiments in protein chemistry and crystallization. Fungi are utilized in industrial protein production because of their ability to secrete large quantities of proteins. In this study, we engineered a high-expression-secretion vector, pEXPYR that directs proteins towards the extracellular medium in two Aspergillii host strains, examine the effect of maltose-induced over-expression and protein secretion as well as time and pH-dependent protein stability in the medium. We describe five client proteins representing a core set of hemicellulose degrading enzymes that accumulated up to 50-100 mg/L of protein. Using a recyclable genetic marker that allows serial insertion of multiple genes, simultaneous hyper-secretion of three client proteins in a single host strain was accomplished.

  13. A high-throughput method for assessing chemical toxicity using a Caenorhabditis elegans reproduction assay

    SciTech Connect

    Boyd, Windy A.; McBride, Sandra J.; Rice, Julie R.; Snyder, Daniel W.; Freedman, Jonathan H.

    2010-06-01

    The National Research Council has outlined the need for non-mammalian toxicological models to test the potential health effects of a large number of chemicals while also reducing the use of traditional animal models. The nematode Caenorhabditis elegans is an attractive alternative model because of its well-characterized and evolutionarily conserved biology, low cost, and ability to be used in high-throughput screening. A high-throughput method is described for quantifying the reproductive capacity of C. elegans exposed to chemicals for 48 h from the last larval stage (L4) to adulthood using a COPAS Biosort. Initially, the effects of exposure conditions that could influence reproduction were defined. Concentrations of DMSO vehicle {<=} 1% did not affect reproduction. Previous studies indicated that C. elegans may be influenced by exposure to low pH conditions. At pHs greater than 4.5, C. elegans reproduction was not affected; however below this pH there was a significant decrease in the number of offspring. Cadmium chloride was chosen as a model toxicant to verify that automated measurements were comparable to those of traditional observational studies. EC{sub 50} values for cadmium for automated measurements (176-192 {mu}M) were comparable to those previously reported for a 72-h exposure using manual counting (151 {mu}M). The toxicity of seven test toxicants on C. elegans reproduction was highly correlative with rodent lethality suggesting that this assay may be useful in predicting the potential toxicity of chemicals in other organisms.

  14. 19-Substituted Benzoquinone Ansamycin Heat Shock Protein-90 Inhibitors: Biological Activity and Decreased Off-Target Toxicity

    PubMed Central

    Chang, Chuan-Hsin; Drechsel, Derek A.; Kitson, Russell R. A.; Siegel, David; You, Qiang; Backos, Donald S.; Ju, Cynthia; Moody, Christopher J.

    2014-01-01

    The benzoquinone ansamycins (BQAs) are a valuable class of antitumor agents that serve as inhibitors of heat shock protein (Hsp)-90. However, clinical use of BQAs has resulted in off-target toxicities, including concerns of hepatotoxicity. Mechanisms underlying the toxicity of quinones include their ability to redox cycle and/or arylate cellular nucleophiles. We have therefore designed 19-substituted BQAs to prevent glutathione conjugation and nonspecific interactions with protein thiols to minimize off-target effects and reduce hepatotoxicity. 19-Phenyl– and 19-methyl–substituted versions of geldanamycin and its derivatives, 17-allylamino-17-demethoxygeldanamycin and 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG), did not react with glutathione, whereas marked reactivity was observed using parent BQAs. Importantly, although 17-DMAG induced cell death in primary and cultured mouse hepatocytes, 19-phenyl and 19-methyl DMAG showed reduced toxicity, validating the overall approach. Furthermore, our data suggest that arylation reactions, rather than redox cycling, are a major mechanism contributing to BQA hepatotoxicity. 19-Phenyl BQAs inhibited purified Hsp90 in a NAD(P)H:quinone oxidoreductase 1 (NQO1)–dependent manner, demonstrating increased efficacy of the hydroquinone ansamycin relative to its parent quinone. Molecular modeling supported increased stability of the hydroquinone form of 19-phenyl-DMAG in the active site of human Hsp90. In human breast cancer cells, 19-phenyl BQAs induced growth inhibition also dependent upon metabolism via NQO1 with decreased expression of client proteins and compensatory induction of Hsp70. These data demonstrate that 19-substituted BQAs are unreactive with thiols, display reduced hepatotoxicity, and retain Hsp90 and growth-inhibitory activity in human breast cancer cells, although with diminished potency relative to parent BQAs. PMID:24682466

  15. High mobility group box 1 protein as a late-acting mediator of acute lung inflammation.

    PubMed

    Lutz, Waldemar; Stetkiewicz, Jan

    2004-01-01

    Acute inflammatory lung injury is often a delayed complication of critical illness and is associated with increased mortality. High mobility group box 1 (HMGB1) protein, in addition to its role as a transcriptional regulator factor, has been identified as a late mediator of endotoxin lethality and might be also involved in the development and progression of acute lung injury. HMGB1 protein itself can cause an acute inflammatory response manifested by increased production of proinflammatory cytokines and neutrophil accumulation. The delayed kinetics of HMGB1 protein release indicate that this protein is a distal mediator of acute inflamatory lung injury. Anti-HMGB1 protein antibodies attenuated endotoxin-induced lung injury, but not the early release of TNF-alpha and IL-1beta, indicating that HMGB1 protein is a late mediator of endotoxin-induced acute lung injury. HMGB1 protein is not released by apoptotic cells but is passively released by necrotic or damaged somatic and immune cells and it functions as a major stimulus of necrosis-induced inflammation. HMGB1 protein is also released by activated monocytes/macrophages and induces delayed and biphasic release of proinflammatory mediators from these cells. HMGB1 protein failed to stimulate cytokines release in lymphocytes, indicating that cellular stimulation is specific. We would like to suggest that HMGB1 protein may be also a primary mediator of the inflammatory responses to lung cells injury caused by toxic environmental chemicals.

  16. The Toxicity of a Novel Antifungal Compound Is Modulated by Endoplasmic Reticulum-Associated Protein Degradation Components

    PubMed Central

    Raj, Shriya; Krishnan, Karthik; Askew, David S.; Helynck, Olivier; Suzanne, Peggy; Lesnard, Aurélien; Rault, Sylvain; Zeidler, Ute; d'Enfert, Christophe

    2015-01-01

    In a search for new antifungal compounds, we screened a library of 4,454 chemicals for toxicity against the human fungal pathogen Aspergillus fumigatus. We identified sr7575, a molecule that inhibits growth of the evolutionary distant fungi A. fumigatus, Cryptococcus neoformans, Candida albicans, and Saccharomyces cerevisiae but lacks acute toxicity for mammalian cells. To gain insight into the mode of inhibition, sr7575 was screened against 4,885 S. cerevisiae mutants from the systematic collection of haploid deletion strains and 977 barcoded haploid DAmP (decreased abundance by mRNA perturbation) strains in which the function of essential genes was perturbed by the introduction of a drug resistance cassette downstream of the coding sequence region. Comparisons with previously published chemogenomic screens revealed that the set of mutants conferring sensitivity to sr7575 was strikingly narrow, affecting components of the endoplasmic reticulum-associated protein degradation (ERAD) stress response and the ER membrane protein complex (EMC). ERAD-deficient mutants were hypersensitive to sr7575 in both S. cerevisiae and A. fumigatus, indicating a conserved mechanism of growth inhibition between yeast and filamentous fungi. Although the unfolded protein response (UPR) is linked to ERAD regulation, sr7575 did not trigger the UPR in A. fumigatus and UPR mutants showed no enhanced sensitivity to the compound. The data from this chemogenomic analysis demonstrate that sr7575 exerts its antifungal activity by disrupting ER protein quality control in a manner that requires ERAD intervention but bypasses the need for the canonical UPR. ER protein quality control is thus a specific vulnerability of fungal organisms that might be exploited for antifungal drug development. PMID:26666917

  17. Carbaryl toxicity prediction to soil organisms under high and low temperature regimes.

    PubMed

    Lima, Maria P R; Cardoso, Diogo N; Soares, Amadeu M V M; Loureiro, Susana

    2015-04-01

    Many studies on risk assessment of pesticides on non-target organisms have been performed based on standardized protocols that reflect conditions in temperate climates. However, the responses of organisms to chemical compounds may differ according to latitude and thus predicting the toxicity of chemicals at different temperatures is an important factor to consider in risk assessment. The toxic effects of the pesticide carbaryl were evaluated at different temperature regimes, which are indicative of temperate and tropical climates and are relevant to climate change predictions or seasonal temperature fluctuations. Four standard organisms were used (Folsomia candida, Eisenia andrei; Triticum aestivum and Brassica rapa) and the effects were assessed using synergistic ratios, calculated from EC/LC50 values. When possible, the MIXTOX tool was used based on the reference model of independent action (IA) and possible deviations. A decrease on carbaryl toxicity at higher temperatures was found in F. candida reproduction, but when the mixtox tool was used no interactions between these stressors (Independent Action) was observed, so an additive response was suggested. Synergistic ratios showed a tendency to synergism at high temperatures for E. andrei and B. rapa and antagonism at low temperatures for both species. T. aestivum showed to be less affected than expected (antagonism), when exposed to both low and high temperatures. The results showed that temperature may increase the deleterious effects of carbaryl to non-target organisms, which is important considering both seasonal and latitude related differences, as well as the global climate change context.

  18. Carbaryl toxicity prediction to soil organisms under high and low temperature regimes.

    PubMed

    Lima, Maria P R; Cardoso, Diogo N; Soares, Amadeu M V M; Loureiro, Susana

    2015-04-01

    Many studies on risk assessment of pesticides on non-target organisms have been performed based on standardized protocols that reflect conditions in temperate climates. However, the responses of organisms to chemical compounds may differ according to latitude and thus predicting the toxicity of chemicals at different temperatures is an important factor to consider in risk assessment. The toxic effects of the pesticide carbaryl were evaluated at different temperature regimes, which are indicative of temperate and tropical climates and are relevant to climate change predictions or seasonal temperature fluctuations. Four standard organisms were used (Folsomia candida, Eisenia andrei; Triticum aestivum and Brassica rapa) and the effects were assessed using synergistic ratios, calculated from EC/LC50 values. When possible, the MIXTOX tool was used based on the reference model of independent action (IA) and possible deviations. A decrease on carbaryl toxicity at higher temperatures was found in F. candida reproduction, but when the mixtox tool was used no interactions between these stressors (Independent Action) was observed, so an additive response was suggested. Synergistic ratios showed a tendency to synergism at high temperatures for E. andrei and B. rapa and antagonism at low temperatures for both species. T. aestivum showed to be less affected than expected (antagonism), when exposed to both low and high temperatures. The results showed that temperature may increase the deleterious effects of carbaryl to non-target organisms, which is important considering both seasonal and latitude related differences, as well as the global climate change context. PMID:24836932

  19. Copper oxide nanoparticles are highly toxic: a comparison between metal oxide nanoparticles and carbon nanotubes.

    PubMed

    Karlsson, Hanna L; Cronholm, Pontus; Gustafsson, Johanna; Möller, Lennart

    2008-09-01

    Since the manufacture and use of nanoparticles are increasing, humans are more likely to be exposed occupationally or via consumer products and the environment. However, so far toxicity data for most manufactured nanoparticles are limited. The aim of this study was to investigate and compare different nanoparticles and nanotubes regarding cytotoxicity and ability to cause DNA damage and oxidative stress. The study was focused on different metal oxide particles (CuO, TiO2, ZnO, CuZnFe2O4, Fe3O4, Fe2O3), and the toxicity was compared to that of carbon nanoparticles and multiwalled carbon nanotubes (MWCNT). The human lung epithelial cell line A549 was exposed to the particles, and cytotoxicity was analyzed using trypan blue staining. DNA damage and oxidative lesions were determined using the comet assay, and intracellular production of reactive oxygen species (ROS) was measured using the oxidation-sensitive fluoroprobe 2',7'-dichlorofluorescin diacetate (DCFH-DA). The results showed that there was a high variation among different nanoparticles concerning their ability to cause toxic effects. CuO nanoparticles were most potent regarding cytotoxicity and DNA damage. The toxicity was likely not explained by Cu ions released to the cell medium. These particles also caused oxidative lesions and were the only particles that induced an almost significant increase (p = 0.058) in intracellular ROS. ZnO showed effects on cell viability as well as DNA damage, whereas the TiO2 particles (a mix of rutile and anatase) only caused DNA damage. For iron oxide particles (Fe3O4, Fe2O3), no or low toxicity was observed, but CuZnFe2O4 particles were rather potent in inducing DNA lesions. Finally, the carbon nanotubes showed cytotoxic effects and caused DNA damage in the lowest dose tested. The effects were not explained by soluble metal impurities. In conclusion, this study highlights the in vitro toxicity of CuO nanoparticles.

  20. Cerebrospinal fluid proteins and free amino acids in patients with solvent induced chronic toxic encephalopathy and healthy controls.

    PubMed Central

    Moen, B E; Kyvik, K R; Engelsen, B A; Riise, T

    1990-01-01

    The concentrations of protein, albumin, IgG, and free amino acids in the cerebrospinal fluid of 16 patients with chronic toxic encephalopathy due to organic solvents were measured. The patient group consisted of all patients with this diagnosis in a neurological department in 1985. The diagnosis was based on neuraesthenic symptoms, pathological psychometric performance, and verified exposure to neurotoxic organic solvents. A control group of 16 patients with myalgias or backache, or both, and no signs of disease was used for comparison. The purpose was to study possible changes in the cerebrospinal fluid that might contribute to understanding the aetiology of solvent induced chronic toxic encephalopathy. A rise in protein, albumin, and IgG was found in the patient group compared with the control group, as well as reduced concentrations of phosphoethanolamine, taurine, homocarnosine, ethanolamine, alpha-aminobutyric acid, and leucine. Using a stepwise multiple regression analysis, taurine was negatively correlated to exposure to solvents. These findings may indicate membrane alterations in the central nervous system related to exposure to organic solvents. PMID:2337535

  1. Development of protein based bioremediation and drugs for heavy metal toxicity

    SciTech Connect

    Opella, Stanley J.

    2001-09-18

    Structural studies were performed on several proteins of the bacterial detoxification system. These proteins are responsible for binding (MerP) and transport of heavy metals, including mercury, across membranes. The structural information obtained from NMR experiments provides insight into the selectivity and sequestration processes towards heavy metal toxins.

  2. Dietary high vanadium causes oxidative damage-induced renal and hepatic toxicity in broilers.

    PubMed

    Liu, Juan; Cui, Hengmin; Liu, Xiaodong; Peng, Xi; Deng, Junliang; Zuo, Zhicai; Cui, Wei; Deng, Yuanxin; Wang, Kangping

    2012-02-01

    The purpose of this study was to investigate the renal and hepatic oxidative damage and toxicity caused by dietary high vanadium in broilers. A total of 420 one-day-old avian broilers were divided into six groups and fed on a corn-soybean basal diet as control diet (vanadium 0.073 mg/kg), and five high vanadium diets (vanadium 5 mg/kg, high vanadium group I; 15 mg/kg, high vanadium group II; 30 mg/kg, high vanadium group III; 45 mg/kg, high vanadium group IV; and 60 mg/kg, high vanadium group V) throughout the experimental period of 42 days. The results showed that the renal and hepatic superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities, ability to inhibit hydroxy radical, and malondialdehyde (MDA), glutathione, and vanadium contents were not significantly changed in high vanadium group I and II when compared with those of the control groups. However, the SOD and GSH-Px activities, ability to inhibit hydroxy radical, and GSH content were significantly decreased, and the MDA and vanadium contents were markedly increased in high vanadium groups III, IV, and V. At the same time, the lesions were also observed in the kidney and liver of high vanadium groups III, IV, and V. The renal tubular epithelial cells showed granular degeneration and vacuolar degeneration, and hepatocytes showed granular degeneration, vacuolar degeneration, and fatty degeneration. It was concluded that dietary vanadium in the range of 30-60 mg/kg could cause oxidative damage and vanadium accumulation, which induced renal and hepatic toxicity and lesions. The renal and hepatic function was finally impaired in boilers.

  3. Gender-specific reduction of hepatic Mrp2 expression by high-fat diet protects female mice from ANIT toxicity

    SciTech Connect

    Kong, Bo; Csanaky, Iván L.; Aleksunes, Lauren M.; Patni, Meghan; Chen, Qi; Ma, Xiaochao; Jaeschke, Hartmut; Weir, Scott; Broward, Melinda; Klaassen, Curtis D.; Guo, Grace L.

    2012-06-01

    Emerging evidence suggests that feeding a high-fat diet (HFD) to rodents affects the expression of genes involved in drug transport. However, gender-specific effects of HFD on drug transport are not known. The multidrug resistance-associated protein 2 (Mrp2, Abcc2) is a transporter highly expressed in the hepatocyte canalicular membrane and is important for biliary excretion of glutathione-conjugated chemicals. The current study showed that hepatic Mrp2 expression was reduced by HFD feeding only in female, but not male, C57BL/6J mice. In order to determine whether down-regulation of Mrp2 in female mice altered chemical disposition and toxicity, the biliary excretion and hepatotoxicity of the Mrp2 substrate, α-naphthylisothiocyanate (ANIT), were assessed in male and female mice fed control diet or HFD for 4 weeks. ANIT-induced biliary injury is a commonly used model of experimental cholestasis and has been shown to be dependent upon Mrp2-mediated efflux of an ANIT glutathione conjugate that selectively injures biliary epithelial cells. Interestingly, HFD feeding significantly reduced early-phase biliary ANIT excretion in female mice and largely protected against ANIT-induced liver injury. In summary, the current study showed that, at least in mice, HFD feeding can differentially regulate Mrp2 expression and function and depending upon the chemical exposure may enhance or reduce susceptibility to toxicity. Taken together, these data provide a novel interaction between diet and gender in regulating hepatobiliary excretion and susceptibility to injury. -- Highlights: ► High-fat diet decreases hepatic Mrp2 expression only in female but not in male mice. ► HFD significantly reduces early-phase biliary ANIT excretion in female mice. ► HFD protects female mice against ANIT-induced liver injury.

  4. ELEVATED LEVELS OF INDUCIBLE HEAT SHOCK PROTEIN (HSP70-1) PROTECT MCF-7 CELLS FROM ARSENITE TOXICITY

    EPA Science Inventory

    Heat shock proteins (HSPs) belong to the highly conserved family of stress proteins and are induced following exposure to arsenic. Elevated HSPs protect against cellular damage from heat but it is unclear whether HSP induction alters the damaging effects of environmental chemical...

  5. Adapting the medaka embryo assay to a high-throughput approach for developmental toxicity testing.

    PubMed

    Oxendine, Sharon L; Cowden, John; Hinton, David E; Padilla, Stephanie

    2006-09-01

    Chemical exposure during embryonic development may cause persistent effects, yet developmental toxicity data exist for very few chemicals. Current testing procedures are time consuming and costly, underlining the need for rapid and low cost screening strategies. While in vitro methods are useful for screening, these methods do not replicate all the intricacies of embryonic development and should ideally be complemented by an in vivo screening strategy. In this study, we modify a medaka fish embryo assay to meet the requirements of high-throughput, developmental toxicant testing in vivo. The Japanese medaka (Oryzias latipes) offers several advantages over traditional mammalian model systems, including economic husbandry, high fecundity, and rapid ex utero development. In most studies where fish eggs are exposed to a chemical, the exposure takes place in a common vessel, with many embryos being exposed to the same solution. This type of design is not amenable to high-throughput methodology, does not allow the investigator to follow the same embryo throughout gestation, and may confound statistical analysis of the results. Therefore, we developed a 96-well microtiter plate method to facilitate exposure of individual medaka embryos in single wells and compared this approach to the common vessel method using the industrial solvent dimethyl sulfoxide (DMSO) as the test compound. At lower DMSO concentrations (0% or 1%), the 96-well microtiter plate assay replicated results obtained using the common vessel exposure method. There was, however, increased lethality and decreased hatching rate in the bottle-reared embryos treated with the higher DMSO concentrations (5% or 10%). Because the embryos reared in the 96-well microtiter plates never showed increased adverse effects (as compared to the bottle-reared embryos) at any DMSO concentration, we conclude that the 96-well microtiter plate assay provides a rapid and efficient alternative for developmental toxicity screens that

  6. Toxicity Analysis of N- and C-Terminus-Deleted Vegetative Insecticidal Protein from Bacillus thuringiensis

    PubMed Central

    Selvapandiyan, A.; Arora, N.; Rajagopal, R.; Jalali, S. K.; Venkatesan, T.; Singh, S. P.; Bhatnagar, Raj K.

    2001-01-01

    A vegetative insecticidal protein (VIP)-encoding gene from a local isolate of Bacillus thuringiensis has been cloned, sequenced, and expressed in Escherichia coli. The expressed protein shows insecticidal activity against several lepidopteran pests but is ineffective against Agrotis ipsilon. Comparison of the amino acid sequence with those of reported VIPs revealed a few differences. Analysis of insecticidal activity with N- and C-terminus deletion mutants suggests a differential mode of action of VIP against different pests. PMID:11722946

  7. A cadherin-like protein influences Bacillus thuringiensis Cry1Ab toxicity in the oriental armyworm, Mythimna separata.

    PubMed

    Wang, Ling; Jiang, Xingfu; Luo, Lizhi; Stanley, David; Sappington, Thomas W; Zhang, Lei

    2013-06-01

    Cadherins comprise a family of calcium-dependent cell adhesion proteins that act in cell-cell interactions. Cadherin-like proteins (CADs) in midguts of some insects act as receptors that bind some of the toxins produced by the Bacillus thuringiensis (Bt). We cloned a CAD gene associated with larval midguts prepared from Mythimna separata. The full-length cDNA (MsCAD1, GenBank Accession No. JF951432) is 5642 bp, with an open reading frame encoding a 1757 amino acid and characteristics typical of insect CADs. Expression of MsCAD1 is predominantly in midgut tissue, with highest expression in the 3rd- to 6th-instars and lowest in newly hatched larvae. Knocking-down MsCAD1 decreased Cry1Ab susceptibility, indicated by reduced developmental time, increased larval weight and reduced larval mortality. We expressed MsCAD1 in E. coli and recovered the recombinant protein, rMsCAD1, which binds Cry1Ab toxin. Truncation analysis and binding experiments revealed that a contiguous 209-aa, located in CR11 and CR12, is the minimal Cry1Ab binding region. These results demonstrate that MsCAD1 is associated with Cry1Ab toxicity and is one of the Cry1Ab receptors in this insect. The significance of this work lies in identifying MsCAD1 as a Cry1Ab receptor, which helps understand the mechanism of Cry1Ab toxicity and of potential resistance to Bt in M. separata. PMID:23754724

  8. Role of uptake of (14C)valine into protein in the development of tolerance to diisopropylphosphorofluoridate (DFP) toxicity

    SciTech Connect

    Gupta, R.C.; Dettbarn, W.D.

    1986-07-01

    In a subchronic toxicity study male Sprague-Dawley rats were daily treated with diisopropylphosphorofluoridate (DFP) (0.5 mg/kg, sc) for 14 days. Maximum signs of anticholinesterase toxicity were observed during Days 4 and 5 comparable to those seen 10-15 min following a single sublethal dosage (1.5 mg DFP/kg, sc). Signs disappeared after Days 6-7 of exposure and rats became apparently normal during the remainder of the treatment period. Significant hypothermia was seen following the second to fifth doses with maximum effect after the fifth injection. Subsequent injections of DFP did not cause any reduction in temperature. Incorporation of (/sup 14/C)valine was measured 24 hr after the 5th and 14th injections of DFP, at a time when body temperature had recovered to control values. The rate of in vivo incorporation of (/sup 14/C)valine was measured 0.5, 1.0, and 2.0 hr after a subcutaneous injection of L-(1-/sup 14/C)valine at a dose of 5 microCi/mmol/100 g body wt. After five injections the rate of L-(1-/sup 14/C)valine uptake into the free amino acid pool and the incorporation into the protein bound pool was significantly (p less than 0.01) reduced in discrete brain regions, liver, kidney, and skeletal muscles. At the end of the 14-day treatment, protein synthesis in all the skeletal muscles tested had recovered completely (p greater than 0.01) to the values of nontreated control animals. In brain, liver, and kidney, however, no recovery was seen during this period. The recovery of protein synthesis in skeletal muscle may be one of the mechanisms that lead to tolerance development during prolonged administration of subacute concentrations of DFP.

  9. Soy-Protein-Based Nanofabrics for Highly Efficient and Multifunctional Air Filtration.

    PubMed

    Souzandeh, Hamid; Johnson, Kyle S; Wang, Yu; Bhamidipaty, Keshava; Zhong, Wei-Hong

    2016-08-10

    Proteins are well-known by their numerous active functional groups along the polypeptide chain. The variety of functional groups of proteins provides a great potential for proteins to interact with airborne pollutants with varying surface properties. However, to our knowledge, a successful demonstration of this potential has not been reported before. In this work, soy protein, a type of abundant plant protein, has been employed for the first time to fabricate multifunctional air-filtration materials. To take advantage of the functional groups of soy protein for air filtration, the soy protein was first well denatured to unfold the polypeptide chains and then fabricated into nanofibers with the help of poly(vinyl alcohol). It was found that the resultant nanofabrics showed high filtration efficiency not only for airborne particulates with a broad range of size but also for various toxic gaseous chemicals (e.g., formaldehyde and carbon monoxide), a capability that has not been realized by conventional air-filtering materials. This study indicates that protein-based nanofabrics are promising nanomaterials for multifunctional air-filtration applications.

  10. Synthetic Biological Protein Nanowires with High Conductivity.

    PubMed

    Tan, Yang; Adhikari, Ramesh Y; Malvankar, Nikhil S; Pi, Shuang; Ward, Joy E; Woodard, Trevor L; Nevin, Kelly P; Xia, Qiangfei; Tuominen, Mark T; Lovley, Derek R

    2016-09-01

    Genetic modification to add tryptophan to PilA, the monomer for the electrically conductive pili of Geobacter sulfurreducens, yields conductive protein filaments 2000-fold more conductive than the wild-type pili while cutting the diameter in half to 1.5 nm. PMID:27409066

  11. A systematic study of mitochondrial toxicity of environmental chemicals using quantitative high throughput screening

    PubMed Central

    Attene-Ramos, Matias S.; Huang, Ruili; Sakamuru, Srilatha; Witt, Kristine L.; Beeson, Gyda C.; Shou, Louie; Schnellmann, Rick G.; Beeson, Craig C.; Tice, Raymond R.; Austin, Christopher P.; Xia, Menghang

    2014-01-01

    A goal of the Tox21 program is to transit toxicity testing from traditional in vivo models to in vitro assays that assess how chemicals affect cellular responses and toxicity pathways. A critical contribution of the NIH Chemical Genomics center (NCGC) to the Tox21 program is the implementation of a quantitative high throughput screening (qHTS) approach, using cell- and biochemical-based assays to generate toxicological profiles for thousands of environmental compounds. Here, we evaluated the effect of chemical compounds on mitochondrial membrane potential in HepG2 cells by screening a library of 1,408 compounds provided by the National Toxicology Program (NTP) in a qHTS platform. Compounds were screened over 14 concentrations, and results showed that 91 and 88 compounds disrupted mitochondrial membrane potential after treatment for one or five h, respectively. Seventy-six compounds active at both time points were clustered by structural similarity, producing 11 clusters and 23 singletons. Thirty-eight compounds covering most of the active chemical space were more extensively evaluated. Thirty-six of the 38 compounds were confirmed to disrupt mitochondrial membrane potential using a fluorescence plate reader and 35 were confirmed using a high content imaging approach. Among the 38 compounds, 4 and 6 induced LDH release, a measure of cytotoxicity, at 1 or 5 h, respectively. Compounds were further assessed for mechanism of action (MOA) by measuring changes in oxygen consumption rate, which enabled identification of 20 compounds as uncouplers. This comprehensive approach allows for evaluation of thousands of environmental chemicals for mitochondrial toxicity and identification of possible MOAs. PMID:23895456

  12. Toxic and nontoxic components of botulinum neurotoxin complex are evolved from a common ancestral zinc protein

    SciTech Connect

    Inui, Ken; Sagane, Yoshimasa; Miyata, Keita; Miyashita, Shin-Ichiro; Suzuki, Tomonori; Shikamori, Yasuyuki; Ohyama, Tohru; Niwa, Koichi; Watanabe, Toshihiro

    2012-03-16

    Highlights: Black-Right-Pointing-Pointer BoNT and NTNHA proteins share a similar protein architecture. Black-Right-Pointing-Pointer NTNHA and BoNT were both identified as zinc-binding proteins. Black-Right-Pointing-Pointer NTNHA does not have a classical HEXXH zinc-coordinating motif similar to that found in all serotypes of BoNT. Black-Right-Pointing-Pointer Homology modeling implied probable key residues involved in zinc coordination. -- Abstract: Zinc atoms play an essential role in a number of enzymes. Botulinum neurotoxin (BoNT), the most potent toxin known in nature, is a zinc-dependent endopeptidase. Here we identify the nontoxic nonhemagglutinin (NTNHA), one of the BoNT-complex constituents, as a zinc-binding protein, along with BoNT. A protein structure classification database search indicated that BoNT and NTNHA share a similar domain architecture, comprising a zinc-dependent metalloproteinase-like, BoNT coiled-coil motif and concanavalin A-like domains. Inductively coupled plasma-mass spectrometry analysis demonstrated that every single NTNHA molecule contains a single zinc atom. This is the first demonstration of a zinc atom in this protein, as far as we know. However, the NTNHA molecule does not possess any known zinc-coordinating motif, whereas all BoNT serotypes possess the classical HEXXH motif. Homology modeling of the NTNHA structure implied that a consensus K-C-L-I-K-X{sub 35}-D sequence common among all NTNHA serotype molecules appears to coordinate a single zinc atom. These findings lead us to propose that NTNHA and BoNT may have evolved distinct functional specializations following their branching out from a common ancestral zinc protein.

  13. The novel hydroxylamine derivative NG-094 suppresses polyglutamine protein toxicity in Caenorhabditis elegans.

    PubMed

    Haldimann, Pierre; Muriset, Maude; Vígh, László; Goloubinoff, Pierre

    2011-05-27

    Aggregation-prone polyglutamine (polyQ) expansion proteins cause several neurodegenerative disorders, including Huntington disease. The pharmacological activation of cellular stress responses could be a new strategy to combat protein conformational diseases. Hydroxylamine derivatives act as co-inducers of heat-shock proteins (HSPs) and can enhance HSP expression in diseased cells, without significant adverse effects. Here, we used Caenorhabditis elegans expressing polyQ expansions with 35 glutamines fused to the yellow fluorescent protein (Q35-YFP) in body wall muscle cells as a model system to investigate the effects of treatment with a novel hydroxylamine derivative, NG-094, on the progression of polyQ diseases. NG-094 significantly ameliorated polyQ-mediated animal paralysis, reduced the number of Q35-YFP aggregates and delayed polyQ-dependent acceleration of aging. Micromolar concentrations of NG-094 in animal tissues with only marginal effects on the nematode fitness sufficed to confer protection against polyQ proteotoxicity, even when the drug was administered after disease onset. NG-094 did not reduce insulin/insulin-like growth factor 1-like signaling, but conferred cytoprotection by a mechanism involving the heat-shock transcription factor HSF-1 that potentiated the expression of stress-inducible HSPs. NG-094 is thus a promising candidate for tests on mammalian models of polyQ and other protein conformational diseases. PMID:21471208

  14. High-throughput identification of proteins with AMPylation using self-assembled human protein (NAPPA) microarrays

    PubMed Central

    Yu, Xiaobo; LaBaer, Joshua

    2015-01-01

    Summary AMPylation (adenylylation) has been recognized as an important post translational modification employed by pathogens to regulate host cellular proteins and their associated signaling pathways. AMPylation has potential functions in various cellular processes and is widely conserved across both prokaryotes and eukaryotes. However, despite the identification of many AMPylators, relatively few candidate substrates of AMPylation are known. This is changing with the recent development of a robust and reliable method to identify new substrates using protein microarrays, which can significantly expand the list of potential substrates. Here, we describe procedures to detect AMPylated and auto-AMPylated proteins in a sensitive, high throughput, and non-radioactive manner. The approach employs high-density protein microarrays fabricated using NAPPA (Nucleic Acid Programmable Protein Arrays) technology, which enables the highly successful display of fresh recombinant human proteins in situ. The modification of target proteins is determined via copper-catalyzed azide–alkyne cycloaddition. The assay can be accomplished within 11 hours. PMID:25881200

  15. High Ph, Ammonia Toxicity, and the Search for Life on the Jovian Planets

    NASA Technical Reports Server (NTRS)

    Deal, P. H.; Souza, K. A.; Mack, H. M.

    1975-01-01

    The effects of pH and ammonia concentration were studied separately, where possible, on a variety of organisms, including some isolated from natural environments of high pH and/or ammonia concentration. Escherichia coli and Bacillus subtilis are both extremely sensitive to ammonia. An aerobic organism (growth up to pH 11.4) from an alkaline spring is more resistant, but exhibits a toxic response to ammonia at a pH much lower than its maximum for growth. The greatest ammonia resistance has been found in an unidentified organism growing at near neutral pH. Even in this case, however, urvival at ammonia concentrations reasonably expected on the Jovian planets is measured in hours. This is two to three orders of magnitude longer than for E. coli. Results support the tentative conclusion that contamination of the Jovian planets with terrestrial organisms that can grow is unlikely. However, the range of toxic response noted, coupled with the observation that terrestrial life has not been exposed to high ammonia concentrations for millions of years, suggests that adaptation to greater ammonia tolerance may be possible.

  16. Full-scale bioreactor pretreatment of highly toxic wastewater from styrene and propylene oxide production.

    PubMed

    Dao, Linh; Grigoryeva, Tatiana; Laikov, Alexander; Devjatijarov, Ruslan; Ilinskaya, Olga

    2014-10-01

    The wastewater originating from simultaneous production of styrene and propylene oxide (SPO) is classified as highly polluted with chemical oxygen demand level in the range 5965 to 9137mgL(-1)-as well as highly toxic. The dilution factor providing for a 10 percent toxic effect of wastewater samples in a test with Paramecium caudatum was 8.0-9.5. Biological approach for pretreatment and detoxification of the wastewater under full-scale bioreactor conditions was investigated. The number of suspended microorganisms and the clean up efficiency were increased up to 5.5-6.58×10(8)CFUmL(-1) and 88 percent, respectively during the bioreactor's operation. Isolates in the Citrobacter, Burkholderia, Pseudomonas, and Paracoccus genera were dominant in the mature suspended, as well as the immobilized microbial community of the bioreactor. The most dominant representatives were tested for their ability to biodegrade the major components of the SPO wastewater and evidence of their role in the treatment process was demonstrated. The investigated pretreatment process allowed the wastewater to be detoxified for conventional treatment with activated sludge and was closely related to the maturation of the bioreactor's microbial community. PMID:25086231

  17. High-Throughput Baculovirus Expression System for Membrane Protein Production.

    PubMed

    Kalathur, Ravi C; Panganiban, Marinela; Bruni, Renato

    2016-01-01

    The ease of use, robustness, cost-effectiveness, and posttranslational machinery make baculovirus expression system a popular choice for production of eukaryotic membrane proteins. This system can be readily adapted for high-throughput operations. This chapter outlines the techniques and procedures for cloning, transfection, small-scale production, and purification of membrane protein samples in a high-throughput manner. PMID:27485337

  18. [Investigating mechanism of toxicity reduction by combination of Glycyrrhizae Radix et Rhizoma and Aconiti Lateralis Radix Preparata on terms of proteins self-assembly].

    PubMed

    Li, Bing-jie; Shen, Yong; Liao, Ri-tao; Gao, Guan-zhen; Ke, Li-jing; Zhou, Jian-wu; Rao, Ping-fan

    2015-02-01

    The combination of Glycyrrhizae Radix et Rhizoma and Aconiti Lateralis Radix Preparata can increase efficacy and decrease toxicity. This study started from the phenomena of protein self-assembly in the mixed decoction of Glycyrrhizae Radix et Rhizoma with Aconiti Lateralis Radix Preparata. The attenuated mechanism was explored between the combination of Glycyrrhizae Radix et Rhizoma and Aconiti Lateralis Radix Preparata by using the protein of Glycyrrhizae Radix et Rhizoma and aconitine which was the major toxic component of Aconiti Lateralis Radix Preparata. Glycyrrhizae Radix et Rhizoma protein with aconitine could form stable particles which particle mean diameter was (206.2 ± 2.02) nm and (238.20 ± 1.23) nm at pH 5.0 in normal temperature. Through the mouse acute toxicity experiment found that injection of aconitine monomer all mice were killed, and injection of Glycyrrhizae Radix et Rhizoma protein-aconitine particles with the same content of aconitine all mice survived. Survey the stability of Glycyrrhizae Radix et Rhizoma protein-aconitine shows that the colloid particles is stable at room temperature, and it has the possibility to candidate drug carrier. Glycyrrhizae Radix et Rhizoma protein can reduce the toxicity of aconitine through self-assembly.

  19. Controlled delivery of hollow corn protein nanoparticles via non-toxic crosslinking: in vivo and drug loading study.

    PubMed

    Xu, Helan; Shen, Li; Xu, Lan; Yang, Yiqi

    2015-02-01

    In this research, controlled delivery of hollow nanoparticles from zein, the corn storage protein, to different organs of mice was achieved via crosslinking using citric acid, a non-toxic polycarboxylic acid derived from starch. Besides, crosslinking significantly enhanced water stability of nanoparticles while preserving their drug loading efficiency. Protein nanoparticles have been widely investigated as vehicles for delivery of therapeutics. However, protein nanoparticles were not stable in physiological conditions, easily cleared by mononuclear phagocyte system (MPS), and thus mainly accumulated and degraded in spleen and liver, the major MPS organs. Effective delivery to major non-MPS organs, such as kidney, was usually difficult to achieve, as well as long resident time of nanoparticles. In this research, hollow zein nanoparticles were chemically crosslinked with citric acid. Controlled delivery and prolonged accumulation of the nanoparticles in kidney, one major non-MPS organ, were achieved. The nanoparticles showed improved stability in aqueous environment at pH 7.4 without affecting the adsorption of 5-FU, a common anticancer drug. In summary, citric acid crosslinked hollow zein nanoparticles could be potential vehicles for controllable delivery of anticancer therapeutics.

  20. Polychaete richness and abundance enhanced in anthropogenically modified estuaries despite high concentrations of toxic contaminants.

    PubMed

    Dafforn, Katherine A; Kelaher, Brendan P; Simpson, Stuart L; Coleman, Melinda A; Hutchings, Pat A; Clark, Graeme F; Knott, Nathan A; Doblin, Martina A; Johnston, Emma L

    2013-01-01

    Ecological communities are increasingly exposed to multiple chemical and physical stressors, but distinguishing anthropogenic impacts from other environmental drivers remains challenging. Rarely are multiple stressors investigated in replicated studies over large spatial scales (>1000 kms) or supported with manipulations that are necessary to interpret ecological patterns. We measured the composition of sediment infaunal communities in relation to anthropogenic and natural stressors at multiple sites within seven estuaries. We observed increases in the richness and abundance of polychaete worms in heavily modified estuaries with severe metal contamination, but no changes in the diversity or abundance of other taxa. Estuaries in which toxic contaminants were elevated also showed evidence of organic enrichment. We hypothesised that the observed response of polychaetes was not a 'positive' response to toxic contamination or a reduction in biotic competition, but due to high levels of nutrients in heavily modified estuaries driving productivity in the water column and enriching the sediment over large spatial scales. We deployed defaunated field-collected sediments from the surveyed estuaries in a small scale experiment, but observed no effects of sediment characteristics (toxic or enriching). Furthermore, invertebrate recruitment instead reflected the low diversity and abundance observed during field surveys of this relatively 'pristine' estuary. This suggests that differences observed in the survey are not a direct consequence of sediment characteristics (even severe metal contamination) but are related to parameters that covary with estuary modification such as enhanced productivity from nutrient inputs and the diversity of the local species pool. This has implications for the interpretation of diversity measures in large-scale monitoring studies in which the observed patterns may be strongly influenced by many factors that covary with anthropogenic modification.

  1. Polychaete Richness and Abundance Enhanced in Anthropogenically Modified Estuaries Despite High Concentrations of Toxic Contaminants

    PubMed Central

    Dafforn, Katherine A.; Kelaher, Brendan P.; Simpson, Stuart L.; Coleman, Melinda A.; Hutchings, Pat A.; Clark, Graeme F.; Knott, Nathan A.; Doblin, Martina A.; Johnston, Emma L.

    2013-01-01

    Ecological communities are increasingly exposed to multiple chemical and physical stressors, but distinguishing anthropogenic impacts from other environmental drivers remains challenging. Rarely are multiple stressors investigated in replicated studies over large spatial scales (>1000 kms) or supported with manipulations that are necessary to interpret ecological patterns. We measured the composition of sediment infaunal communities in relation to anthropogenic and natural stressors at multiple sites within seven estuaries. We observed increases in the richness and abundance of polychaete worms in heavily modified estuaries with severe metal contamination, but no changes in the diversity or abundance of other taxa. Estuaries in which toxic contaminants were elevated also showed evidence of organic enrichment. We hypothesised that the observed response of polychaetes was not a ‘positive’ response to toxic contamination or a reduction in biotic competition, but due to high levels of nutrients in heavily modified estuaries driving productivity in the water column and enriching the sediment over large spatial scales. We deployed defaunated field-collected sediments from the surveyed estuaries in a small scale experiment, but observed no effects of sediment characteristics (toxic or enriching). Furthermore, invertebrate recruitment instead reflected the low diversity and abundance observed during field surveys of this relatively ‘pristine’ estuary. This suggests that differences observed in the survey are not a direct consequence of sediment characteristics (even severe metal contamination) but are related to parameters that covary with estuary modification such as enhanced productivity from nutrient inputs and the diversity of the local species pool. This has implications for the interpretation of diversity measures in large-scale monitoring studies in which the observed patterns may be strongly influenced by many factors that covary with anthropogenic

  2. A PLGA-encapsulated chimeric protein protects against adherence and toxicity of enterotoxigenic Escherichia coli.

    PubMed

    Nazarian, Shahram; Gargari, Seyed Latif Mousavi; Rasooli, Iraj; Hasannia, Sadegh; Pirooznia, Nazanin

    2014-01-01

    Enterotoxigenic Escherichia coli (ETEC) are the most common cause of diarrhea among children. Colonization factors and enterotoxins are the major ETEC candidate vaccines. Since protection against ETEC mostly occurs by induction of IgA antibodies, much effort is focused on the development of oral vaccines. In this study oral immunogenicity of a poly(lactic-co-glycolic acid) (PLGA) encapsulated chimeric protein containing CfaB, CstH, CotA and LTB (Heat-labile B subunit) was investigated. The protein was encapsulated in PLGA by double emulsion method and nanoparticles were characterized physicochemically. Immunogenicity was assessed by evaluating IgG1, IgG2 and IgA titers after BALB/c mice vaccination. Non aggregated nanoparticles had a spherical shape with an average particle size of 252.7±23 nm and 91.96±4.4% of encapsulation efficiency. Western blotting showed maintenance of the molecular weight and antigenicity of the released protein. Oral immunization of mice induced serum IgG and fecal IgA antibody responses. Immunization induced protection against ETEC binding to Caco-2 cells. The effect of LT toxin on fluid accumulation in ileal loops was neutralized by inhibition of enterotoxin binding to GM1-ganglosides. Delivery of the chimeric protein in PLGA elicited both systemic and mucosal immune responses. The findings could be exploited to development of oral multi-component ETEC prophylactic measures. PMID:23906742

  3. A SURROGATE SUBCHRONIC TOXICITY TEST METHOD FOR WATERS WITH HIGH TOTAL DISSOLVED SOLIDS

    EPA Science Inventory

    Total dissolved solids (TDS) are often identified as a toxicant in whole-effluent toxicity (WET) testing. The primary test organism used in WET testing, Ceriodaphnia dubia, is very sensitive to TDS ions, which can be problematic when differentiating the toxicity of TDS from those...

  4. Polysaccharide protein complex isolated from mushroom Phellinus rimosus (berk.) Pilat alleviates γ radiation-induced toxicity in mice.

    PubMed

    Joseph, Jini; Smina, Thozhuthum Parambil Pathmanabhan; Janardhanan, Kainoor Krishnankutty

    2011-06-01

    Ionizing radiations generate reactive oxygen species in irradiated tissue that induces several pathophysiological changes in the body. Radiotherapy induced toxicity is a major dose-limiting factor in anticancer treatments. Radioprotective agents are of significant importance in medical, industrial, environmental, military, and space applications. Radioprotective effect of polysaccharide protein complex (PPC-Pr) isolated from mushroom, Phellinus rimosus, was evaluated in Swiss albino mice. PPC-Pr (5 and 10 mg/kg bwt, i.p.) significantly increased leukocyte count, bone marrow cellularity, glutathione content, and activities of antioxidant enzymes such as catalase, superoxide dismutase, and glutathione peroxidase in blood as well as intestinal mucosa when compared with the irradiated control group. Histopathological observation of intestinal jejunal mucosa revealed the tissue protective effects of PPC-Pr. Further radioprotective activity of PPC-Pr was in a dose-dependent manner. The findings suggest potential radioprotective efficacy of PPC-Pr.

  5. Calculation of injection forces for highly concentrated protein solutions.

    PubMed

    Fischer, Ingo; Schmidt, Astrid; Bryant, Andrew; Besheer, Ahmed

    2015-09-30

    Protein solutions often manifest a high viscosity at high solution concentrations, thus impairing injectability. Accordingly, accurate prediction of the injection force based on solution viscosity can greatly support protein formulation and device development. In this study, the shear-dependent viscosity of three concentrated protein solutions is reported, and calculated injection forces obtained by two different mathematical models are compared against measured values. The results show that accurate determination of the needle dimensions and the shear-thinning behavior of the protein solutions is vital for injection force prediction. Additionally, one model delivered more accurate results, particularly for solutions with prominent shear-thinning behavior.

  6. Radiation chemistry of amino acids, peptides and proteins in relation to the radiation sterilization of high-protein foods

    SciTech Connect

    Garrison, W. M.

    1981-12-01

    An important source of information on the question of whether or not toxic or other deleterious substances are formed in the radiation sterilization of foods is the chemical study of reaction products and reaction mechanisms in the radiolysis of individual food components. The present evaluation of the radiation chemistry of amino acids, peptides, and proteins outlines the various radiation-induced processes which lead to amino acid degradation and to the synthesis of amino acid derivatives of higher molecular weight. Among the latter are the ..cap alpha..,..cap alpha..'-diamino dicarboxylic acids which are formed as major products in the radiolysis of peptides both in aqueous solution and in the solid state. The ..cap alpha..,..cap alpha..'-diamino acids are of particular interest as irradiation products because they represent a class of compounds not normally encountered in plant and animal protein sources. Such compounds have, however, been isolated from certain types of bacteria and bacterial products. All of the available data strongly suggest that the ..cap alpha..,..cap alpha..'-diamino acids are produced in significant yield in the radiation sterilization of high protein foods. The importance of initiating extensive chemical and biological studies of these and of other high molecular weight products in irradiated food is emphasized.

  7. CHARACTERIZATION OF DRUG INTERACTIONS WITH SERUM PROTEINS BY USING HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY

    PubMed Central

    Hage, David S.; Anguizola, Jeanethe; Barnaby, Omar; Jackson, Abby; Yoo, Michelle J.; Papastavros, Efthimia; Pfaunmiller, Erika; Sobansky, Matt; Tong, Zenghan

    2011-01-01

    The binding of drugs with serum proteins can affect the activity, distribution, rate of excretion, and toxicity of pharmaceutical agents in the body. One tool that can be used to quickly analyze and characterize these interactions is high-performance affinity chromatography (HPAC). This review shows how HPAC can be used to study drug-protein binding and describes the various applications of this approach when examining drug interactions with serum proteins. Methods for determining binding constants, characterizing binding sites, examining drug-drug interactions, and studying drug-protein dissociation rates will be discussed. Applications that illustrate the use of HPAC with serum binding agents such as human serum albumin, α1-acid glycoprotein, and lipoproteins will be presented. Recent developments will also be examined, such as new methods for immobilizing serum proteins in HPAC columns, the utilization of HPAC as a tool in personalized medicine, and HPAC methods for the high-throughput screening and characterization of drug-protein binding. PMID:21395530

  8. Incorporating High-Throughput Exposure Predictions With Dosimetry-Adjusted In Vitro Bioactivity to Inform Chemical Toxicity Testing.

    PubMed

    Wetmore, Barbara A; Wambaugh, John F; Allen, Brittany; Ferguson, Stephen S; Sochaski, Mark A; Setzer, R Woodrow; Houck, Keith A; Strope, Cory L; Cantwell, Katherine; Judson, Richard S; LeCluyse, Edward; Clewell, Harvey J; Thomas, Russell S; Andersen, Melvin E

    2015-11-01

    We previously integrated dosimetry and exposure with high-throughput screening (HTS) to enhance the utility of ToxCast HTS data by translating in vitro bioactivity concentrations to oral equivalent doses (OEDs) required to achieve these levels internally. These OEDs were compared against regulatory exposure estimates, providing an activity-to-exposure ratio (AER) useful for a risk-based ranking strategy. As ToxCast efforts expand (ie, Phase II) beyond food-use pesticides toward a wider chemical domain that lacks exposure and toxicity information, prediction tools become increasingly important. In this study, in vitro hepatic clearance and plasma protein binding were measured to estimate OEDs for a subset of Phase II chemicals. OEDs were compared against high-throughput (HT) exposure predictions generated using probabilistic modeling and Bayesian approaches generated by the U.S. Environmental Protection Agency (EPA) ExpoCast program. This approach incorporated chemical-specific use and national production volume data with biomonitoring data to inform the exposure predictions. This HT exposure modeling approach provided predictions for all Phase II chemicals assessed in this study whereas estimates from regulatory sources were available for only 7% of chemicals. Of the 163 chemicals assessed in this study, 3 or 13 chemicals possessed AERs < 1 or < 100, respectively. Diverse bioactivities across a range of assays and concentrations were also noted across the wider chemical space surveyed. The availability of HT exposure estimation and bioactivity screening tools provides an opportunity to incorporate a risk-based strategy for use in testing prioritization.

  9. High-affinity FRβ-specific CAR T cells eradicate AML and normal myeloid lineage without HSC toxicity.

    PubMed

    Lynn, R C; Feng, Y; Schutsky, K; Poussin, M; Kalota, A; Dimitrov, D S; Powell, D J

    2016-06-01

    Acute myeloid leukemia (AML) is an aggressive malignancy, and development of new treatments to prolong remissions is warranted. Chimeric antigen receptor (CAR) T-cell therapies appear promising but on-target, off-tumor recognition of antigen in healthy tissues remains a concern. Here we isolated a high-affinity (HA) folate receptor beta (FRβ)-specific single-chain variable fragment (2.48 nm KD) for optimization of FRβ-redirected CAR T-cell therapy for AML. T cells stably expressing the HA-FRβ CAR exhibited greatly enhanced antitumor activity against FRβ(+) AML in vitro and in vivo compared with a low-affinity FRβ CAR (54.3 nm KD). Using the HA-FRβ immunoglobulin G, FRβ expression was detectable in myeloid-lineage hematopoietic cells; however, expression in CD34(+) hematopoietic stem cells (HSCs) was nearly undetectable. Accordingly, HA-FRβ CAR T cells lysed mature CD14(+) monocytes, while HSC colony formation was unaffected. Because of the potential for elimination of mature myeloid lineage, mRNA CAR electroporation for transient CAR expression was evaluated. mRNA-electroporated HA-FRβ CAR T cells retained effective antitumor activity in vitro and in vivo. Together, our results highlight the importance of antibody affinity in target protein detection and CAR development and suggest that transient delivery of potent HA-FRβ CAR T cells is highly effective against AML and reduces the risk for long-term myeloid toxicity.

  10. Toxicity of extracellular proteins from Diplodia seriata and Neofusicoccum parvum involved in grapevine Botryosphaeria dieback.

    PubMed

    Bénard-Gellon, M; Farine, S; Goddard, M L; Schmitt, M; Stempien, E; Pensec, F; Laloue, H; Mazet-Kieffer, F; Fontaine, F; Larignon, P; Chong, J; Tarnus, C; Bertsch, C

    2015-03-01

    Botryosphaeria dieback, esca and Eutypa dieback are three economic major grapevine trunk diseases that cause severe yield reduction in vineyards worldwide. The frequency of disease symptoms has increased considerably over the past decade, and no efficient treatment is currently available to control these diseases. The different fungi associated with grapevine trunk diseases mainly induce necrotic wood and characteristic foliar symptoms. In this context, fungi virulence factors and host invasion are not well understood. We hypothesise that extracellular proteins produced by Diplodia seriata and Neofusicoccum parvum, two causal agents associated with Botryosphaeria dieback, are virulence factors responsible for the pathogenicity. In our previous work, we demonstrated that the total extracellular compounds produced by N. parvum induced more necrosis on Chardonnay calli and triggered a different defence gene expression pattern than those produced by D. seriata. Furthermore, this aggressiveness was not clearly correlated with the production of mellein, a characteristic phytotoxin of Botryosphaeriaceae, in our in vitro calli model. To characterise other potential virulence factors and to understand the mechanisms of host invasion by the fungus, we evaluated the profile, quantity and the impact of extracellular proteins produced by these fungi on Vitis vinifera calli necrosis and defence gene expression. Our results reveal that, under the same conditions, N. parvum produces more extracellular proteins and in higher concentrations than D. seriata. With Vitis vinifera cv. Chardonnay cells, we showed that equivalent concentrations of proteins secreted by N. parvum were more aggressive than those of D. seriata in producing necrosis and that they clearly induced more grapevine defence genes. PMID:25323623

  11. Myelin Basic Protein Induces Neuron-Specific Toxicity by Directly Damaging the Neuronal Plasma Membrane

    PubMed Central

    Zheng, Sixin; Liu, Xiao; Jin, Jinghua; Ren, Yi; Luo, Jianhong

    2014-01-01

    The central nervous system (CNS) insults may cause massive demyelination and lead to the release of myelin-associated proteins including its major component myelin basic protein (MBP). MBP is reported to induce glial activation but its effect on neurons is still little known. Here we found that MBP specifically bound to the extracellular surface of the neuronal plasma membrane and induced neurotoxicity in vitro. This effect of MBP on neurons was basicity-dependent because the binding was blocked by acidic lipids and competed by other basic proteins. Further studies revealed that MBP induced damage to neuronal membrane integrity and function by depolarizing the resting membrane potential, increasing the permeability to cations and other molecules, and decreasing the membrane fluidity. At last, artificial liposome vesicle assay showed that MBP directly disturbed acidic lipid bilayer and resulted in increased membrane permeability. These results revealed that MBP induces neurotoxicity through its direct interaction with acidic components on the extracellular surface of neuronal membrane, which may suggest a possible contribution of MBP to the pathogenesis in the CNS disorders with myelin damage. PMID:25255088

  12. [Target proteins and mechanisms of ochratoxin toxicity. A contribution to the identification of potential ochratoxin antagonists

    PubMed

    Vedani, Angelo; Zbinden, Peter

    1997-01-01

    Ochratoxins are mycotoxins released by moulds on grain, peanuts and vegetables. Toxicological investigations have shown that ochratoxin A displays nephrotoxic, genotoxic, teratogenic, cancerogenic and immunosuppressive effects. Increased blood levels observed in humans would seem to suggest a link to a kidney desease (Balcan Endemic Nephropaty) frequently observed in the Balkan countries. The adverse effects of ochratoxin A are mainly associated with its impact on phenylalanine-metabolizing enzymes. Based on the three-dimensional structure of phenylalanine-t-RNA-synthetase, its interactions with ochratoxins are analyzed as well as with Aspartam. In animal models, Aspartam has been shown to almost fully prevent toxic effects of ochratoxin A. The topology of the binding site of phenylalanine-t-RNA-synthetase would seem to be favorable towards a few affinity-enhancing modifications of the Aspartame molecule. A known molecular mechanism is a prerequisite for a systematic search of antagonizing substances for toxins. Based on a receptor structure, binding properties of such drugs can be identified and optimized using computer-aided drug design. Susequently, only the most potent candidate structures must be subjected to a determination of their biological activity, which can lead to a significant reduction of substances to be tested in vivo. Such experiments are particularly stressful as the animals must be intoxicated beforehand. The extent of an antagonistic impact on humans suffering from a chronical ochratoxin A intoxication must be subject of clinical studies. PMID:11178501

  13. [Target proteins and mechanisms of ochratoxin toxicity. A contribution to the identification of potential ochratoxin antagonists

    PubMed

    Vedani, Angelo; Zbinden, Peter

    1997-01-01

    Ochratoxins are mycotoxins released by moulds on grain, peanuts and vegetables. Toxicological investigations have shown that ochratoxin A displays nephrotoxic, genotoxic, teratogenic, cancerogenic and immunosuppressive effects. Increased blood levels observed in humans would seem to suggest a link to a kidney desease (Balcan Endemic Nephropaty) frequently observed in the Balkan countries. The adverse effects of ochratoxin A are mainly associated with its impact on phenylalanine-metabolizing enzymes. Based on the three-dimensional structure of phenylalanine-t-RNA-synthetase, its interactions with ochratoxins are analyzed as well as with Aspartam. In animal models, Aspartam has been shown to almost fully prevent toxic effects of ochratoxin A. The topology of the binding site of phenylalanine-t-RNA-synthetase would seem to be favorable towards a few affinity-enhancing modifications of the Aspartame molecule. A known molecular mechanism is a prerequisite for a systematic search of antagonizing substances for toxins. Based on a receptor structure, binding properties of such drugs can be identified and optimized using computer-aided drug design. Susequently, only the most potent candidate structures must be subjected to a determination of their biological activity, which can lead to a significant reduction of substances to be tested in vivo. Such experiments are particularly stressful as the animals must be intoxicated beforehand. The extent of an antagonistic impact on humans suffering from a chronical ochratoxin A intoxication must be subject of clinical studies.

  14. The relationship between developmental toxicity and aromatic-ring class profile of high-boiling petroleum substances.

    PubMed

    Murray, F Jay; Roth, Randy N; Nicolich, Mark J; Gray, Thomas M; Simpson, Barry J

    2013-11-01

    In response to the US EPA HPV Challenge Program, this study was conducted to: (1) evaluate the relationship between PAC content and the developmental toxicity of high-boiling petroleum substances (HBPS) and (2) develop mathematical models to predict the developmental toxicity of similar untested substances based on their aromatic ring class (ARC) profiles. For this investigation, 68 developmental toxicity studies were reviewed. The ARC models relied on data from 21 rat dermal developmental toxicity studies conducted with similar experimental designs to ensure a consistent data set for comparison. The most sensitive general endpoints of developmental toxicity (i.e., decreased fetal survival and growth) were chosen for modeling. The ARC models demonstrated a strong correlation between the predicted vs. observed values for specific sensitive endpoints of these developmental toxicities (percent resorptions, r=0.99; live fetuses per litter, r=0.98; fetal body weight, r=0.94). Such associations provide a promising approach for predicting the developmental toxicity of untested HBPS. Efforts to corroborate the ARC models using test substances that were not used to build the ARC models produced mixed results, and further development and refinement of the ARC models is recommended before they can be reliably applied to all HBPS.

  15. Applications in high-content functional protein microarrays.

    PubMed

    Moore, Cedric D; Ajala, Olutobi Z; Zhu, Heng

    2016-02-01

    Protein microarray technology provides a versatile platform for characterization of hundreds to thousands of proteins in a parallel and high-throughput manner. Over the last decade, applications of functional protein microarrays in particular have flourished in studying protein function at a systems level and have led to the construction of networks and pathways describing these functions. Relevant areas of research include the detection of various binding properties of proteins, the study of enzyme-substrate relationships, the analysis of host-microbe interactions, and profiling antibody specificity. In addition, discovery of novel biomarkers in autoimmune diseases and cancers is emerging as a major clinical application of functional protein microarrays. In this review, we will summarize the recent advances of functional protein microarrays in both basic and clinical applications. PMID:26599287

  16. Optimizing high performance computing workflow for protein functional annotation.

    PubMed

    Stanberry, Larissa; Rekepalli, Bhanu; Liu, Yuan; Giblock, Paul; Higdon, Roger; Montague, Elizabeth; Broomall, William; Kolker, Natali; Kolker, Eugene

    2014-09-10

    Functional annotation of newly sequenced genomes is one of the major challenges in modern biology. With modern sequencing technologies, the protein sequence universe is rapidly expanding. Newly sequenced bacterial genomes alone contain over 7.5 million proteins. The rate of data generation has far surpassed that of protein annotation. The volume of protein data makes manual curation infeasible, whereas a high compute cost limits the utility of existing automated approaches. In this work, we present an improved and optmized automated workflow to enable large-scale protein annotation. The workflow uses high performance computing architectures and a low complexity classification algorithm to assign proteins into existing clusters of orthologous groups of proteins. On the basis of the Position-Specific Iterative Basic Local Alignment Search Tool the algorithm ensures at least 80% specificity and sensitivity of the resulting classifications. The workflow utilizes highly scalable parallel applications for classification and sequence alignment. Using Extreme Science and Engineering Discovery Environment supercomputers, the workflow processed 1,200,000 newly sequenced bacterial proteins. With the rapid expansion of the protein sequence universe, the proposed workflow will enable scientists to annotate big genome data. PMID:25313296

  17. Optimizing high performance computing workflow for protein functional annotation.

    PubMed

    Stanberry, Larissa; Rekepalli, Bhanu; Liu, Yuan; Giblock, Paul; Higdon, Roger; Montague, Elizabeth; Broomall, William; Kolker, Natali; Kolker, Eugene

    2014-09-10

    Functional annotation of newly sequenced genomes is one of the major challenges in modern biology. With modern sequencing technologies, the protein sequence universe is rapidly expanding. Newly sequenced bacterial genomes alone contain over 7.5 million proteins. The rate of data generation has far surpassed that of protein annotation. The volume of protein data makes manual curation infeasible, whereas a high compute cost limits the utility of existing automated approaches. In this work, we present an improved and optmized automated workflow to enable large-scale protein annotation. The workflow uses high performance computing architectures and a low complexity classification algorithm to assign proteins into existing clusters of orthologous groups of proteins. On the basis of the Position-Specific Iterative Basic Local Alignment Search Tool the algorithm ensures at least 80% specificity and sensitivity of the resulting classifications. The workflow utilizes highly scalable parallel applications for classification and sequence alignment. Using Extreme Science and Engineering Discovery Environment supercomputers, the workflow processed 1,200,000 newly sequenced bacterial proteins. With the rapid expansion of the protein sequence universe, the proposed workflow will enable scientists to annotate big genome data.

  18. The skinny on high-protein, low-carbohydrate diets.

    PubMed

    Cunningham, Wendy; Hyson, Dianne

    2006-01-01

    Short-term studies of high-protein, low-carbohydrate diets have shown weight loss and improvements in plasma lipid profiles. Studies of greater than 6 months' duration, however, have failed to show continued benefit of high-protein, low-carbohydrate diets on weight loss and cardiovascular risk factors compared with conventional diets. Without concurrent weight loss and caloric restriction, these diets offer no additional benefit to lipids or body weight over other weight-loss regimens. In fact, high-protein, low-carbohydrate diets may add additional risk to individuals with cardiovascular disease due to their high fat and cholesterol content combined with decreased intake of fruits, vegetables, whole grains, and other nutrients related to cardiovascular risk. In addition, high-protein, low-carbohydrate diets have been implicated in other risks, including impaired renal, bone, and gastrointestinal health.

  19. Antitarget Interaction, Acute Toxicity and Protein Binding Studies of Quinazolinedione Sulphonamides as GABA1 Antagonists

    PubMed Central

    Ajeet; Verma, Mansi; Rani, Sangeeta; Kumar, A.

    2016-01-01

    Diseases characterized by recurrent seizures are known as epilepsy. One of the most important mechanisms for handling it is GABA1 receptor mediated inhibition. In the same context while studying the treatment of epilepsy we observed significant effects by derivatives of sulfonamides, which prompted us to design novel derivatives by means of in silico resources with antiepileptic effects. Molecular docking approaches are routinely used in modern drug design to help understand drug–receptor interaction. This study has been performed with the help of Chemdraw Ultra 7.0, GUSAR online tool for IC50 and LD50 predictions, AutoDock Vina (Python Prescription 0.8), and PaDEL software. Results revealed that ligand-protein interaction affinity of all 10 designed molecules ranges from -5.7 Kcal/mol to -5.2 Kcal/mol, which is approximately comparable to pre-existing GABA1 inhibitor i.e. phenytoin (CID: 1775, ligand-protein interaction affinity is -6.5 Kcal/mol). PMID:27168681

  20. Automated High Throughput Protein Crystallization Screening at Nanoliter Scale and Protein Structural Study on Lactate Dehydrogenase

    SciTech Connect

    Li, Fenglei

    2006-08-09

    The purposes of our research were: (1) To develop an economical, easy to use, automated, high throughput system for large scale protein crystallization screening. (2) To develop a new protein crystallization method with high screening efficiency, low protein consumption and complete compatibility with high throughput screening system. (3) To determine the structure of lactate dehydrogenase complexed with NADH by x-ray protein crystallography to study its inherent structural properties. Firstly, we demonstrated large scale protein crystallization screening can be performed in a high throughput manner with low cost, easy operation. The overall system integrates liquid dispensing, crystallization and detection and serves as a whole solution to protein crystallization screening. The system can dispense protein and multiple different precipitants in nanoliter scale and in parallel. A new detection scheme, native fluorescence, has been developed in this system to form a two-detector system with a visible light detector for detecting protein crystallization screening results. This detection scheme has capability of eliminating common false positives by distinguishing protein crystals from inorganic crystals in a high throughput and non-destructive manner. The entire system from liquid dispensing, crystallization to crystal detection is essentially parallel, high throughput and compatible with automation. The system was successfully demonstrated by lysozyme crystallization screening. Secondly, we developed a new crystallization method with high screening efficiency, low protein consumption and compatibility with automation and high throughput. In this crystallization method, a gas permeable membrane is employed to achieve the gentle evaporation required by protein crystallization. Protein consumption is significantly reduced to nanoliter scale for each condition and thus permits exploring more conditions in a phase diagram for given amount of protein. In addition

  1. High-molecular weight protein toxins of marine invertebrates and their elaborate modes of action.

    PubMed

    Butzke, Daniel; Luch, Andreas

    2010-01-01

    High-molecular weight protein toxins significantly contribute to envenomations by certain marine invertebrates, e.g., jellyfish and fire corals. Toxic proteins frequently evolved from enzymes meant to be employed primarily for digestive purposes. The cellular intermediates produced by such enzymatic activity, e.g., reactive oxygen species or lysophospholipids, rapidly and effectively mediate cell death by disrupting cellular integrity. Membrane integrity may also be disrupted by pore-forming toxins that do not exert inherent enzymatic activity. When targeted to specific pharmacologically relevant sites in tissues or cells of the natural enemy or prey, toxic enzymes or pore-forming toxins even may provoke fast and severe systemic reactions. Since toxin-encoding genes constitute "hot spots" of molecular evolution, continuous variation and acquirement of new pharmacological properties are guaranteed. This also makes individual properties and specificities of complex proteinaceous venoms highly diverse and inconstant. In the present chapter we portray high-molecular weight constituents of venoms present in box jellyfish, sea anemones, sea hares, fire corals and the crown-of-thorns starfish. The focus lies on the latest achievements in the attempt to elucidate their molecular modes of action. PMID:20358685

  2. High-Content Assay Multiplexing for Toxicity Screening in Induced Pluripotent Stem Cell-Derived Cardiomyocytes and Hepatocytes.

    PubMed

    Grimm, Fabian Alexander; Iwata, Yasuhiro; Sirenko, Oksana; Bittner, Michael; Rusyn, Ivan

    2015-11-01

    Cell-based high-content screening (HCS) assays have become an increasingly attractive alternative to traditional in vitro and in vivo testing in pharmaceutical drug development and toxicological safety assessment. The time- and cost-effectiveness of HCS assays, combined with the organotypic nature of human induced pluripotent stem cell (iPSC)-derived cells, open new opportunities to employ physiologically relevant in vitro model systems to improve screening for potential chemical hazards. In this study, we used two human iPSC types, cardiomyocytes and hepatocytes, to test various high-content and molecular assay combinations for their applicability in a multiparametric screening format. Effects on cardiomyocyte beat frequency were characterized by calcium flux measurements for up to 90 min. Subsequent correlation with intracellular cAMP levels was used to determine if the effects on cardiac physiology were G-protein-coupled receptor dependent. In addition, we utilized high-content cell imaging to simultaneously determine cell viability, mitochondrial integrity, and reactive oxygen species (ROS) formation in both cell types. Kinetic analysis indicated that ROS formation is best detectable 30 min following initial treatment, whereas cytotoxic effects were most stable after 24 h. For hepatocytes, high-content imaging was also used to evaluate cytotoxicity and cytoskeletal integrity, as well as mitochondrial integrity and the potential for lipid accumulation. Lipid accumulation, a marker for hepatic steatosis, was most reliably detected 48 h following treatment with test compounds. Overall, our results demonstrate how a compendium of assays can be utilized for quantitative screening of chemical effects in iPSC cardiomyocytes and hepatocytes and enable rapid and cost-efficient multidimensional biological profiling of toxicity. PMID:26539751

  3. High-Content Assay Multiplexing for Toxicity Screening in Induced Pluripotent Stem Cell-Derived Cardiomyocytes and Hepatocytes

    PubMed Central

    Grimm, Fabian Alexander; Iwata, Yasuhiro; Sirenko, Oksana; Bittner, Michael

    2015-01-01

    Abstract Cell-based high-content screening (HCS) assays have become an increasingly attractive alternative to traditional in vitro and in vivo testing in pharmaceutical drug development and toxicological safety assessment. The time- and cost-effectiveness of HCS assays, combined with the organotypic nature of human induced pluripotent stem cell (iPSC)-derived cells, open new opportunities to employ physiologically relevant in vitro model systems to improve screening for potential chemical hazards. In this study, we used two human iPSC types, cardiomyocytes and hepatocytes, to test various high-content and molecular assay combinations for their applicability in a multiparametric screening format. Effects on cardiomyocyte beat frequency were characterized by calcium flux measurements for up to 90 min. Subsequent correlation with intracellular cAMP levels was used to determine if the effects on cardiac physiology were G-protein-coupled receptor dependent. In addition, we utilized high-content cell imaging to simultaneously determine cell viability, mitochondrial integrity, and reactive oxygen species (ROS) formation in both cell types. Kinetic analysis indicated that ROS formation is best detectable 30 min following initial treatment, whereas cytotoxic effects were most stable after 24 h. For hepatocytes, high-content imaging was also used to evaluate cytotoxicity and cytoskeletal integrity, as well as mitochondrial integrity and the potential for lipid accumulation. Lipid accumulation, a marker for hepatic steatosis, was most reliably detected 48 h following treatment with test compounds. Overall, our results demonstrate how a compendium of assays can be utilized for quantitative screening of chemical effects in iPSC cardiomyocytes and hepatocytes and enable rapid and cost-efficient multidimensional biological profiling of toxicity. PMID:26539751

  4. Highly Efficient Protein Misfolding Cyclic Amplification

    PubMed Central

    Ostapchenko, Valeriy G.; Savtchenk, Regina; Alexeeva, Irina; Rohwer, Robert G.; Baskakov, Ilia V.

    2011-01-01

    Protein misfolding cyclic amplification (PMCA) provides faithful replication of mammalian prions in vitro and has numerous applications in prion research. However, the low efficiency of conversion of PrPC into PrPSc in PMCA limits the applicability of PMCA for many uses including structural studies of infectious prions. It also implies that only a small sub-fraction of PrPC may be available for conversion. Here we show that the yield, rate, and robustness of prion conversion and the sensitivity of prion detection are significantly improved by a simple modification of the PMCA format. Conducting PMCA reactions in the presence of Teflon beads (PMCAb) increased the conversion of PrPC into PrPSc from ∼10% to up to 100%. In PMCAb, a single 24-hour round consistently amplified PrPSc by 600-700-fold. Furthermore, the sensitivity of prion detection in one round (24 hours) increased by 2-3 orders of magnitude. Using serial PMCAb, a 1012-fold dilution of scrapie brain material could be amplified to the level detectible by Western blotting in 3 rounds (72 hours). The improvements in amplification efficiency were observed for the commonly used hamster 263K strain and for the synthetic strain SSLOW that otherwise amplifies poorly in PMCA. The increase in the amplification efficiency did not come at the expense of prion replication specificity. The current study demonstrates that poor conversion efficiencies observed previously have not been due to the scarcity of a sub-fraction of PrPC susceptible to conversion nor due to limited concentrations of essential cellular cofactors required for conversion. The new PMCAb format offers immediate practical benefits and opens new avenues for developing fast ultrasensitive assays and for producing abundant quantities of PrPSc in vitro. PMID:21347353

  5. High-throughput Protein Purification and Quality Assessment for Crystallization

    PubMed Central

    Kim, Youngchang; Babnigg, Gyorgy; Jedrzejczak, Robert; Eschenfeldt, William H.; Li, Hui; Maltseva, Natalia; Hatzos-Skintges, Catherine; Gu, Minyi; Makowska-Grzyska, Magdalena; Wu, Ruiying; An, Hao; Chhor, Gekleng; Joachimiak, Andrzej

    2012-01-01

    The ultimate goal of structural biology is to understand the structural basis of proteins in cellular processes. In structural biology, the most critical issue is the availability of high-quality samples. “Structural biology-grade” proteins must be generated in the quantity and quality suitable for structure determination using X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy. The purification procedures must reproducibly yield homogeneous proteins or their derivatives containing marker atom(s) in milligram quantities. The choice of protein purification and handling procedures plays a critical role in obtaining high-quality protein samples. With structural genomics emphasizing a genome-based approach in understanding protein structure and function, a number of unique structures covering most of the protein folding space have been determined and new technologies with high efficiency have been developed. At the Midwest Center for Structural Genomics (MCSG), we have developed semi-automated protocols for high-throughput parallel protein expression and purification. A protein, expressed as a fusion with a cleavable affinity tag, is purified in two consecutive immobilized metal affinity chromatography (IMAC) steps: (i) the first step is an IMAC coupled with buffer-exchange, or size exclusion chromatography (IMAC-I), followed by the cleavage of the affinity tag using the highly specific Tobacco Etch Virus (TEV) protease; [1] the second step is IMAC and buffer exchange (IMAC-II) to remove the cleaved tag and tagged TEV protease. These protocols have been implemented on multidimensional chromatography workstations and, as we have shown, many proteins can be successfully produced in large-scale. All methods and protocols used for purification, some developed by MCSG, others adopted and integrated into the MCSG purification pipeline and more recently the Center for Structural Genomics of Infectious Diseases (CSGID) purification pipeline, are

  6. Comparative metalloproteomic approaches for the investigation proteins involved in the toxicity of inorganic and organic forms of mercury in rice (Oryza sativa L.) roots.

    PubMed

    Li, Yunyun; Zhao, Jiating; Li, Yu-Feng; Xu, Xiaohan; Zhang, Bowen; Liu, Yongjie; Cui, Liwei; Li, Bai; Gao, Yuxi; Chai, Zhifang

    2016-07-13

    The toxicity mechanisms of rice roots under inorganic mercury (IHg) or methylmercury (MeHg) stress were investigated using metalloproteomic approaches. Rice seedlings were cultivated in nutrient solutions with IHg or MeHg for three weeks. Proteins were extracted from the roots and separated by two-dimensional electrophoresis (2-DE). Differentially expressed proteins were analyzed using ESI-MS/MS and identified by PMF. 26 and 29 protein spots were differentially expressed in the IHg- and MeHg-exposed roots, respectively. The proteins responsive to Hg exposure are involved in antioxidative defense, sulfur and glutathione metabolism, carbohydrate and energy metabolism, programmed cell death, and pathogen defense. Chitinase and salt stress-induced proteins exhibited a greater differentially expression in response to MeHg stress compared to IHg stress. Hg-binding proteins were detected by the combined use of 1-DE, SRXRF, and ESI-MS/MS. The results showed that Hg was bound to proteins of 15-25 kDa in rice roots under Hg stress. The Hg contents in the band under IHg stress were remarkably higher than those under MeHg. Hg binds to proteins, which leads to irreversible damage of root growth. Rice roots changed the related protein expression levels in response to Hg stress. These results may provide new insights into the mechanism of toxicity of IHg and MeHg in rice.

  7. Assessing a peptidylic inhibitor-based therapeutic approach that simultaneously suppresses polyglutamine RNA- and protein-mediated toxicities in patient cells and Drosophila

    PubMed Central

    Zhang, Qian; Tsoi, Ho; Peng, Shaohong; Li, Pan P.; Lau, Kwok-Fai; Rudnicki, Dobrila D.; Ngo, Jacky Chi-Ki; Chan, Ho Yin Edwin

    2016-01-01

    ABSTRACT Polyglutamine (polyQ) diseases represent a group of progressive neurodegenerative disorders that are caused by abnormal expansion of CAG triplet nucleotides in disease genes. Recent evidence indicates that not only mutant polyQ proteins, but also their corresponding mutant RNAs, contribute to the pathogenesis of polyQ diseases. Here, we describe the identification of a 13-amino-acid peptide, P3, which binds directly and preferentially to long-CAG RNA within the pathogenic range. When administered to cell and Drosophila disease models, as well as to patient-derived fibroblasts, P3 inhibited expanded-CAG-RNA-induced nucleolar stress and suppressed neurotoxicity. We further examined the combined therapeutic effect of P3 and polyQ-binding peptide 1 (QBP1), a well-characterized polyQ protein toxicity inhibitor, on neurodegeneration. When P3 and QBP1 were co-administered to disease models, both RNA and protein toxicities were effectively mitigated, resulting in a notable improvement of neurotoxicity suppression compared with the P3 and QBP1 single-treatment controls. Our findings indicate that targeting toxic RNAs and/or simultaneous targeting of toxic RNAs and their corresponding proteins could open up a new therapeutic strategy for treating polyQ degeneration. PMID:26839389

  8. Brain Responses to High-Protein Diets12

    PubMed Central

    Journel, Marion; Chaumontet, Catherine; Darcel, Nicolas; Fromentin, Gilles; Tomé, Daniel

    2012-01-01

    Proteins are suspected to have a greater satiating effect than the other 2 macronutrients. After protein consumption, peptide hormones released from the gastrointestinal tract (mainly anorexigenic gut peptides such as cholecystokinin, glucagon peptide 1, and peptide YY) communicate information about the energy status to the brain. These hormones and vagal afferents control food intake by acting on brain regions involved in energy homeostasis such as the brainstem and the hypothalamus. In fact, a high-protein diet leads to greater activation than a normal-protein diet in the nucleus tractus solitarius and in the arcuate nucleus. More specifically, neural mechanisms triggered particularly by leucine consumption involve 2 cellular energy sensors: the mammalian target of rapamycin and AMP-activated protein kinase. In addition, reward and motivation aspects of eating behavior, controlled mainly by neurons present in limbic regions, play an important role in the reduced hedonic response of a high-protein diet. This review examines how metabolic signals emanating from the gastrointestinal tract after protein ingestion target the brain to control feeding, energy expenditure, and hormones. Understanding the functional roles of brain areas involved in the satiating effect of proteins and their interactions will demonstrate how homeostasis and reward are integrated with the signals from peripheral organs after protein consumption. PMID:22585905

  9. Nrf2 and Nrf2-Related Proteins in Development and Developmental Toxicity: Insights from studies in Zebrafish (Danio rerio)

    PubMed Central

    Hahn, Mark E.; Timme-Laragy, Alicia R.; Karchner, Sibel I.; Stegeman, John J.

    2015-01-01

    Oxidative stress is an important mechanism of chemical toxicity, contributing to developmental toxicity and teratogenesis as well as to cardiovascular and neurodegenerative diseases and diabetic embryopathy. Developing animals are especially sensitive to effects of chemicals that disrupt the balance of processes generating reactive species and oxidative stress, and those anti-oxidant defenses that protect against oxidative stress. The expression and inducibility of anti-oxidant defenses through activation of NFE2-related factor 2 (Nrf2) and related proteins is an essential process affecting the susceptibility to oxidants, but the complex interactions of Nrf2 in determining embryonic response to oxidants and oxidative stress are only beginning to be understood. The zebrafish (Danio rerio) is an established model in developmental biology and now also in developmental toxicology and redox signaling. Here we review the regulation of genes involved in protection against oxidative stress in developing vertebrates, with a focus on Nrf2 and related cap’n’collar (CNC)-basic-leucine zipper (bZIP) transcription factors. Vertebrate animals including zebrafish share Nfe2, Nrf1, Nrf2, and Nrf3 as well as a core set of genes that respond to oxidative stress, contributing to the value of zebrafish as a model system with which to investigate the mechanisms involved in regulation of redox signaling and the response to oxidative stress during embryolarval development. Moreover, studies in zebrafish have revealed nrf and keap1 gene duplications that provide an opportunity to dissect multiple functions of vertebrate NRF genes, including multiple sensing mechanisms involved in chemical-specific effects. PMID:26130508

  10. Air Toxics under the Big Sky: A Real-World Investigation to Engage High School Science Students

    ERIC Educational Resources Information Center

    Adams, Earle; Smith, Garon; Ward, Tony J.; Vanek, Diana; Marra, Nancy; Jones, David; Henthorn, Melissa; Striebel, Jim

    2008-01-01

    This paper describes a problem-based chemistry education model in which students perform scientific research on a local environmentally relevant problem. The project is a collaboration among The University of Montana and local high schools centered around Missoula, Montana. "Air Toxics under the Big Sky" involves high school students in collecting…

  11. Protein-DNA binding in high-resolution

    PubMed Central

    Mahony, Shaun; Pugh, B. Franklin

    2015-01-01

    Recent advances in experimental and computational methodologies are enabling ultra-high resolution genome-wide profiles of protein-DNA binding events. For example, the ChIP-exo protocol precisely characterizes protein-DNA crosslinking patterns by combining chromatin immunoprecipitation (ChIP) with 5′ → 3′ exonuclease digestion. Similarly, deeply sequenced chromatin accessibility assays (e.g. DNase-seq and ATACseq) enable the detection of protected footprints at protein-DNA binding sites. With these techniques and others, we have the potential to characterize the individual nucleotides that interact with transcription factors, nucleosomes, RNA polymerases, and other regulatory proteins in a particular cellular context. In this review, we explain the experimental assays and computational analysis methods that enable high-resolution profiling of protein-DNA binding events. We discuss the challenges and opportunities associated with such approaches. PMID:26038153

  12. Influence of Electromagnetic Fields on Lead Toxicity: A Study of Conformational Changes in Human Blood Proteins

    PubMed Central

    Ansarihadipour, Hadi; Bayatiani, Mohamadreza

    2016-01-01

    Background Electromagnetic fields (EMF) are associated with oxidative stress, which is in turn associated with reactive oxygen species (ROS), anemia, and hypoxia. Objectives This study focused on the synergistic effects of lead ions and EMF on oxidative modifications in hemoglobin (Hb) and plasma proteins. Patients and Methods In this experimental study, the blood samples were obtained from age- and sex-matched healthy subjects at Arak University of Medical Sciences, Arak, Iran. The collected bloods were prepared as 55 samples and then divided into different groups for incubating with 0 to 100 uM of lead ions in 2 mT and 50 Hz of EMF for 120 minutes. The carbonyl group was determined to be an oxidative biomarker in plasma proteins. The ferric reducing ability of plasma (FRAP) was considered to be an antioxidant power of human plasma. The conformational changes in hemoglobin, met-Hb, and hemichrome were considered to be oxidative markers in red blood cells. To predict the factors affecting the oxyHb, the artificial neural network (MLP: 11,2,2,1) in SPSS software was applied. Results The test subjects showed increased concentrations of metHb (1.8 ± 0.19 vs. 1.36 ± 0.25) and hemichrome (6.01 ± 0.57) in relation to the control subjects. The decreased absorbance at 340 nm (0.88 ± 0.09 vs. 1.07 ± 0.08) demonstrated the reduced interaction between the globin chain and the heme ring. The decreased absorbance at 420 nm (Soret band) (2.96 ± 0.13) and the increased absorbance at 630 nm (0.07 ± 0.002 vs. 0.064 ± 0.005) indicated the conversion of oxyHb to metHb, which confirmed the oxidative damage to the erythrocytes. The linear regression analysis showed significant positive correlations between lead concentration and the percentage of plasma carbonyl content (R2 = 0.96), the relation of plasma carbonyl content to Hb absorbance at 630 nm (R2 = 0.97), and the relation of plasma carbonyl content to metHb concentration (R2 = 0.95) after 120 minutes incubation with lead

  13. Influence of Electromagnetic Fields on Lead Toxicity: A Study of Conformational Changes in Human Blood Proteins

    PubMed Central

    Ansarihadipour, Hadi; Bayatiani, Mohamadreza

    2016-01-01

    Background Electromagnetic fields (EMF) are associated with oxidative stress, which is in turn associated with reactive oxygen species (ROS), anemia, and hypoxia. Objectives This study focused on the synergistic effects of lead ions and EMF on oxidative modifications in hemoglobin (Hb) and plasma proteins. Patients and Methods In this experimental study, the blood samples were obtained from age- and sex-matched healthy subjects at Arak University of Medical Sciences, Arak, Iran. The collected bloods were prepared as 55 samples and then divided into different groups for incubating with 0 to 100 uM of lead ions in 2 mT and 50 Hz of EMF for 120 minutes. The carbonyl group was determined to be an oxidative biomarker in plasma proteins. The ferric reducing ability of plasma (FRAP) was considered to be an antioxidant power of human plasma. The conformational changes in hemoglobin, met-Hb, and hemichrome were considered to be oxidative markers in red blood cells. To predict the factors affecting the oxyHb, the artificial neural network (MLP: 11,2,2,1) in SPSS software was applied. Results The test subjects showed increased concentrations of metHb (1.8 ± 0.19 vs. 1.36 ± 0.25) and hemichrome (6.01 ± 0.57) in relation to the control subjects. The decreased absorbance at 340 nm (0.88 ± 0.09 vs. 1.07 ± 0.08) demonstrated the reduced interaction between the globin chain and the heme ring. The decreased absorbance at 420 nm (Soret band) (2.96 ± 0.13) and the increased absorbance at 630 nm (0.07 ± 0.002 vs. 0.064 ± 0.005) indicated the conversion of oxyHb to metHb, which confirmed the oxidative damage to the erythrocytes. The linear regression analysis showed significant positive correlations between lead concentration and the percentage of plasma carbonyl content (R2 = 0.96), the relation of plasma carbonyl content to Hb absorbance at 630 nm (R2 = 0.97), and the relation of plasma carbonyl content to metHb concentration (R2 = 0.95) after 120 minutes incubation with lead

  14. Serum Creatinine Versus Plasma Methotrexate Levels to Predict Toxicities in Children Receiving High-dose Methotrexate.

    PubMed

    Tiwari, Priya; Thomas, M K; Pathania, Subha; Dhawan, Deepa; Gupta, Y K; Vishnubhatla, Sreenivas; Bakhshi, Sameer

    2015-01-01

    Facilities for measuring methotrexate (MTX) levels are not available everywhere, potentially limiting administration of high-dose methotrexate (HDMTX). We hypothesized that serum creatinine alteration after HDMTX administration predicts MTX clearance. Overall, 122 cycles in 50 patients of non-Hodgkin lymphoma or acute lymphoblastic leukemia aged ≤18 years receiving HDMTX were enrolled prospectively. Plasma MTX levels were measured at 12, 24, 36, 48, 60, and 72 hours; serum creatinine was measured at baseline, 24, 48, and 72 hours. Correlation of plasma MTX levels with creatinine levels and changes in creatinine from baseline (Δ creatinine) were evaluated. Plasma MTX levels at 72 hours showed positive correlation with serum creatinine at 48 hours (P = .011) and 72 hours (P = .013) as also Δ creatinine at 48 hours (P = .042) and 72 hours (P = .045). However, cut-off value of either creatinine or Δ creatinine could not be established to reliably predict delayed MTX clearance. Greater than 50% Δ creatinine at 48 and 72 hours significantly predicted grade 3/4 leucopenia (P = .036 and P = .001, respectively) and thrombocytopenia (P = .012 and P = .009, respectively) but not mucositis (P = .827 and P = .910, respectively). Delayed MTX elimination did not predict any grade 3/4 toxicity. In spite of demonstration of significant correlation between serum creatinine and Δ creatinine with plasma MTX levels at 72 hours, cut-off value of either variable to predict MTX delay could not be established. Thus, either of these cannot be used as a surrogate for plasma MTX estimation. Interestingly, Δ creatinine effectively predicted hematological toxicities, which were not predicted by delayed MTX clearance.

  15. Perspectives on Validation of High-Throughput Assays Supporting 21st Century Toxicity Testing1

    PubMed Central

    Judson, Richard; Kavlock, Robert; Martin, Matt; Reif, David; Houck, Keith; Knudsen, Thomas; Richard, Ann; Tice, Raymond R.; Whelan, Maurice; Xia, Menghang; Huang, Ruili; Austin, Christopher; Daston, George; Hartung, Thomas; Fowle, John R.; Wooge, William; Tong, Weida; Dix, David

    2014-01-01

    Summary In vitro, high-throughput screening (HTS) assays are seeing increasing use in toxicity testing. HTS assays can simultaneously test many chemicals, but have seen limited use in the regulatory arena, in part because of the need to undergo rigorous, time-consuming formal validation. Here we discuss streamlining the validation process, specifically for prioritization applications in which HTS assays are used to identify a high-concern subset of a collection of chemicals. The high-concern chemicals could then be tested sooner rather than later in standard guideline bioassays. The streamlined validation process would continue to ensure the reliability and relevance of assays for this application. We discuss the following practical guidelines: (1) follow current validation practice to the extent possible and practical; (2) make increased use of reference compounds to better demonstrate assay reliability and relevance; (3) deemphasize the need for cross-laboratory testing, and; (4) implement a web-based, transparent and expedited peer review process. PMID:23338806

  16. Humidified microcontact printing of proteins: universal patterning of proteins on both low and high energy surfaces.

    PubMed

    Ricoult, Sébastien G; Nezhad, Amir Sanati; Knapp-Mohammady, Michaela; Kennedy, Timothy E; Juncker, David

    2014-10-14

    Microcontact printing (μCP) of proteins is widely used for biosensors and cell biology but is constrained to printing proteins adsorbed to a low free energy, hydrophobic surface to a high free energy, hydrophilic surface. This strongly limits μCP as harsh chemical treatments are required to form a high energy surface. Here, we introduce humidified μCP (HμCP) of proteins which enables universal printing of protein on any smooth surface. We found that by flowing water in proximity to proteins adsorbed on a hydrophilized stamp, the water vapor diffusing through the stamp enables the printing of proteins on both low and high energy surfaces. Indeed, when proteins are printed using stamps with increasing spacing between water-filled microchannels, only proteins adjacent to the channels are transferred. The vapor transport through the stamp was modeled, and by comparing the humidity profiles with the protein patterns, 88% relative humidity in the stamp was identified as the threshold for HμCP. The molecular forces occurring between PDMS, peptides, and glass during printing were modeled ab initio to confirm the critical role water plays in the transfer. Using HμCP, we introduce straightforward protocols to pattern multiple proteins side-by-side down to nanometer resolution without the need for expensive mask aligners, but instead exploiting self-alignment effects derived from the stamp geometry. Finally, we introduce vascularized HμCP stamps with embedded microchannels that allow printing proteins as arbitrary, large areas patterns with nanometer resolution. This work introduces the general concept of water-assisted μCP and opens new possibilities for "solvent-assisted" printing of proteins and of other nanoparticles. PMID:25222734

  17. Humidified microcontact printing of proteins: universal patterning of proteins on both low and high energy surfaces.

    PubMed

    Ricoult, Sébastien G; Nezhad, Amir Sanati; Knapp-Mohammady, Michaela; Kennedy, Timothy E; Juncker, David

    2014-10-14

    Microcontact printing (μCP) of proteins is widely used for biosensors and cell biology but is constrained to printing proteins adsorbed to a low free energy, hydrophobic surface to a high free energy, hydrophilic surface. This strongly limits μCP as harsh chemical treatments are required to form a high energy surface. Here, we introduce humidified μCP (HμCP) of proteins which enables universal printing of protein on any smooth surface. We found that by flowing water in proximity to proteins adsorbed on a hydrophilized stamp, the water vapor diffusing through the stamp enables the printing of proteins on both low and high energy surfaces. Indeed, when proteins are printed using stamps with increasing spacing between water-filled microchannels, only proteins adjacent to the channels are transferred. The vapor transport through the stamp was modeled, and by comparing the humidity profiles with the protein patterns, 88% relative humidity in the stamp was identified as the threshold for HμCP. The molecular forces occurring between PDMS, peptides, and glass during printing were modeled ab initio to confirm the critical role water plays in the transfer. Using HμCP, we introduce straightforward protocols to pattern multiple proteins side-by-side down to nanometer resolution without the need for expensive mask aligners, but instead exploiting self-alignment effects derived from the stamp geometry. Finally, we introduce vascularized HμCP stamps with embedded microchannels that allow printing proteins as arbitrary, large areas patterns with nanometer resolution. This work introduces the general concept of water-assisted μCP and opens new possibilities for "solvent-assisted" printing of proteins and of other nanoparticles.

  18. Lipid rafts: linking prion protein to zinc transport and amyloid-β toxicity in Alzheimer's disease

    PubMed Central

    Watt, Nicole T.; Griffiths, Heledd H.; Hooper, Nigel M.

    2014-01-01

    Dysregulation of neuronal zinc homeostasis plays a major role in many processes related to brain aging and neurodegenerative diseases, including Alzheimer's disease (AD). Yet, despite the critical role of zinc in neuronal function, the cellular mechanisms underpinning its homeostatic control are far from clear. We reported that the cellular prion protein (PrPC) is involved in the uptake of zinc into neurons. This PrPC-mediated zinc influx required the metal-binding octapeptide repeats in PrPC and the presence of the zinc permeable AMPA channel with which PrPC directly interacted. Together with the observation that PrPC is evolutionarily related to the ZIP family of zinc transporters, these studies indicate that PrPC plays a key role in neuronal zinc homeostasis. Therefore, PrPC could contribute to cognitive health and protect against age-related zinc dyshomeostasis but PrPC has also been identified as a receptor for amyloid-β oligomers which accumulate in the brains of those with AD. We propose that the different roles that PrPC has are due to its interaction with different ligands and/or co-receptors in lipid raft-based signaling/transport complexes. PMID:25364748

  19. The effect of zinc on amyloid β-protein assembly and toxicity: A mechanistic investigation

    NASA Astrophysics Data System (ADS)

    Solomonov, Inna; Sagi, Irit

    2014-10-01

    Neurotoxic assemblies of amyloid β-protein (Aβ) are widely believed to be the cause for Alzheimer's disease (AD). Therefore, understanding the factors and mechanisms that control, modulate, and inhibit formation of these assemblies is crucial for the development of therapeutic intervention of AD. This information also can contribute significantly to our understanding of the mechanisms of other amyloidosis diseases, such as Parkinson's disease, Huntington's disease, type 2 diabetes, amyotrophic lateral sclerosis (Lou Gehrig's disease) and prion diseases (e.g. Mad Cow disease). We have developed a multidisciplinary experimental strategy to study structural and dynamic mechanistic aspects that underlie the Aβ assembly process. Utilizing this strategy, we explored the molecular basis leading to the perturbation of the Aβ assembly process by divalent metal ions, mainly Zn2+ ions. Using Zn2+ as reaction physiological relevant probes, it was demonstrated that Zn2+ rapidly (milliseconds) induce self-assembly of Aβ aggregates and stabilize them in a manner that prevents formation of Aβ fibrils. Importantly, the early-formed intermediates are substantially more neurotoxic than fibrils. Our results suggest that relevant Aβ modulators should be targeted against the rapidly evolved intermediate states of Aβ assembly. The design of such modulators is challenging, as they have to compete with different natural mediators (such as Zn2+) of Aβ aggregation, which diverse Aβ assemblies in both specific and nonspecific manners.

  20. Treatment of proteins with dietary polyphenols lowers the formation of AGEs and AGE-induced toxicity.

    PubMed

    Zhang, Xinchen; Hu, Shuting; Chen, Feng; Wang, Mingfu

    2014-10-01

    Advanced glycation endproducts (AGEs) are a group of harmful compounds produced either endogenously or during thermal food processing. Once absorbed by humans via food intake, AGEs can cause oxidative cell damage and contribute to pathological development of various diseases. The AGE-inhibitory activity of dietary polyphenols in vitro has been extensively reported before, but the current study is pioneering in examining the antiglycation activity of five selected dietary polyphenols (phloretin, naringenin, epicatechin, chlorogenic acid, and rosmarinic acid) during the thermal protein glycation process. When added into the glucose-casein glycation model heated at 120 °C for 2 h, these polyphenols were capable of inhibiting the formation of both total fluorescent AGEs and nonfluorescent carboxymethyllysine (CML). The thermal stability and transformation of polyphenols are likely important factors affecting their antioxidant activity and inhibitory efficacy of reactive carbonyl species formation. Treatment with epicatechin would lower not only AGE formation but also AGE-induced cytotoxicity and oxidative stress to human retinal pigment epithelial (ARPE-19) cells. PMID:25208810

  1. High-dose mitoxantrone with peripheral blood progenitor cell rescue: toxicity, pharmacokinetics and implications for dosage and schedule.

    PubMed Central

    Ballestrero, A.; Ferrando, F.; Garuti, A.; Basta, P.; Gonella, R.; Esposito, M.; Vannozzi, M. O.; Sorice, G.; Friedman, D.; Puglisi, M.; Brema, F.; Mela, G. S.; Sessarego, M.; Patrone, F.

    1997-01-01

    The optimal use of mitoxantrone (NOV) in the high-dose range requires elucidation of its maximum tolerated dose with peripheral blood progenitor cell (PBPC) support and the time interval needed between drug administration and PBPC reinfusion in order to avoid graft toxicity. The aims of this study were: (1) to verify the feasibility and haematological toxicity of escalating NOV up to 90 mg m(-2) with PBPC support; and (2) to verify the safeness of a short (96 h) interval between NOV administration and PBPC reinfusion. Three cohorts of ten patients with breast cancer (BC) or non-Hodgkin's lymphoma (NHL) received escalating doses of NOV, 60, 75 and 90 mg m(-2) plus melphalan (L-PAM), 140-180 mg m(-2), with PBPC rescue 96 h after NOV. Haematological toxicity was evaluated daily (WHO criteria). NOV plasma pharmacokinetics was also evaluated, as well as NOV cytotoxicity against PBPCs. Haematological recovery was rapid and complete at each NOV dose level without statistically significant differences, and there were no major toxicities. NOV plasma concentrations at the time of PBPC reinfusion were below the toxicity threshold against haemopoietic progenitors. It is concluded that, when adequately supported with PBPCs, NOV can be escalated up to 90 mg m(-2) with acceptable haematological toxicity. PBPCs can be safely reinfused as early as 96 h after NOV administration. PMID:9310249

  2. Strategies for integrating transcriptional profiling into high throughput toxicity testing (SOT Symposium Workshop presentation)

    EPA Science Inventory

    Presentation Description: The release of the National Research Council’s Report “Toxicity Testing in the 21st Century: A Vision and a Strategy” in 2007 initiated a broad-based movement in the toxicology community to re-think how toxicity testing and risk assessment are performed....

  3. A Call for Nominations of Quantitative High-Throughput Screening Assays from Relevant Human Toxicity Pathways

    EPA Science Inventory

    The National Research Council of the United States National Academies of Science has recently released a document outlining a long-range vision and strategy for transforming toxicity testing from largely whole animal-based testing to one based on in vitro assays. “Toxicity Testin...

  4. Predictive models of prenatal developmental toxicity from ToxCast high-throughput screening data

    EPA Science Inventory

    EPA's ToxCast™ project is profiling the in vitro bioactivity of chemicals to assess pathway-level and cell-based signatures that correlate with observed in vivo toxicity. We hypothesized that developmental toxicity in guideline animal studies captured in the ToxRefDB database wou...

  5. High Pressure NMR Methods for Characterizing Functional Substates of Proteins.

    PubMed

    Kalbitzer, Hans Robert

    2015-01-01

    Proteins usually exist in multiple conformational states in solution. High pressure NMR spectroscopy is a well-suited method to identify these states. In addition, these states can be characterized by their thermodynamic parameters, the free enthalpies at ambient pressure, the partial molar volumes, and the partial molar compressibility that can be obtained from the analysis of the high pressure NMR data. Two main types of states of proteins exist, functional states and folding states. There is a strong link between these two types, the functional states represent essential folding states (intermediates), other folding states may have no functional meaning (optional folding states). In this chapter, this concept is tested on the Ras protein, an important proto-oncogen in humans where all substates required by theory can be identified experimentally by high pressure NMR spectroscopy. Finally, we show how these data can be used to develop allosteric inhibitors of proteins. PMID:26174382

  6. Fluorescent Protein Nanowire-Mediated Protein Microarrays for Multiplexed and Highly Sensitive Pathogen Detection.

    PubMed

    Men, Dong; Zhou, Juan; Li, Wei; Leng, Yan; Chen, Xinwen; Tao, Shengce; Zhang, Xian-En

    2016-07-13

    Protein microarrays are powerful tools for high-throughput and simultaneous detection of different target molecules in complex biological samples. However, the sensitivity of conventional fluorescence-labeling protein detection methods is limited by the availability of signal molecules for binding to the target molecule. Here, we built a multifunctional fluorescent protein nanowire (FNw) by harnessing self-assembly of yeast amyloid protein. The FNw integrated a large number of fluorescent molecules, thereby enhancing the fluorescent signal output in target detection. The FNw was then combined with protein microarray technology to detect proteins derived from two pathogens, including influenza virus (hemagglutinin 1, HA1) and human immunodeficiency virus (p24 and gp120). The resulting detection sensitivity achieved a 100-fold improvement over a commercially available detection reagent. PMID:27315221

  7. Preorganized Peptide Scaffolds as Mimics of Phosphorylated Proteins Binding Sites with a High Affinity for Uranyl.

    PubMed

    Starck, Matthieu; Sisommay, Nathalie; Laporte, Fanny A; Oros, Stéphane; Lebrun, Colette; Delangle, Pascale

    2015-12-01

    Cyclic peptides with two phosphoserines and two glutamic acids were developed to mimic high-affinity binding sites for uranyl found in proteins such as osteopontin, which is believed to be a privileged target of this ion in vivo. These peptides adopt a β-sheet structure that allows the coordination of the latter amino acid side chains in the equatorial plane of the dioxo uranyl cation. Complementary spectroscopic and analytical methods revealed that these cyclic peptides are efficient uranyl chelating peptides with a large contribution from the phosphorylated residues. The conditional affinity constants were measured by following fluorescence tryptophan quenching and are larger than 10(10) at physiological pH. These compounds are therefore promising models for understanding uranyl chelation by proteins, which is relevant to this actinide ion toxicity. PMID:26583259

  8. High-bandwidth protein analysis using solid-state nanopores.

    PubMed

    Larkin, Joseph; Henley, Robert Y; Muthukumar, Murugappan; Rosenstein, Jacob K; Wanunu, Meni

    2014-02-01

    High-bandwidth measurements of the ion current through hafnium oxide and silicon nitride nanopores allow the analysis of sub-30 kD protein molecules with unprecedented time resolution and detection efficiency. Measured capture rates suggest that at moderate transmembrane bias values, a substantial fraction of protein translocation events are detected. Our dwell-time resolution of 2.5 μs enables translocation time distributions to be fit to a first-passage time distribution derived from a 1D diffusion-drift model. The fits yield drift velocities that scale linearly with voltage, consistent with an electrophoretic process. Further, protein diffusion constants (D) are lower than the bulk diffusion constants (D0) by a factor of ~50, and are voltage-independent in the regime tested. We reason that deviations of D from D0 are a result of confinement-driven pore/protein interactions, previously observed in porous systems. A straightforward Kramers model for this inhibited diffusion points to 9- to 12-kJ/mol interactions of the proteins with the nanopore. Reduction of μ and D are found to be material-dependent. Comparison of current-blockage levels of each protein yields volumetric information for the two proteins that is in good agreement with dynamic light scattering measurements. Finally, detection of a protein-protein complex is achieved.

  9. In Vivo Biodistribution and Toxicity of Highly Soluble PEG-Coated Boron Nitride in Mice

    NASA Astrophysics Data System (ADS)

    Liu, Bo; Qi, Wei; Tian, Longlong; Li, Zhan; Miao, Guoying; An, Wenzhen; Liu, Dan; Lin, Jing; Zhang, Xiaoyong; Wu, Wangsuo

    2015-12-01

    The boron nitride (BN) nanoparticles, as the structural analogues of graphene, are the potential biomedicine materials because of the excellent biocompatibility, but their solubility and biosafety are the biggest obstacle for the clinic application. Here, we first synthesized the highly soluble BN nanoparticles coated by PEG (BN-PEG) with smaller size (~10 nm), then studied their biodistribution in vivo through radioisotope (Tc99mO4 -) labeling, and the results showed that BN-PEG nanoparticles mainly accumulated in the liver, lung, and spleen with the less uptake by the brain. Moreover, the pathological changes induced by BN-PEG could be significantly observed in the sections of the liver, lung, spleen, and heart, which can be also supported by the test of biochemical indexes in serum. More importantly, we first observed the biodistribution of BN-PEG in the heart tissues with high toxicity, which would give a warning about the cardiovascular disease, and provide some opportunities for the drug delivery and treatment.

  10. Fast removal of high quantities of toxic arsenate via cationic p(APTMACl) microgels.

    PubMed

    Rehman, Saif Ur; Siddiq, Mohammed; Al-Lohedan, Hamad; Aktas, Nahit; Sahiner, Mehtap; Demirci, Sahin; Sahiner, Nurettin

    2016-01-15

    Hydrogels are resourceful materials and can be prepared in different morphology, size, surface charge and porosity adopting different polymerization techniques and reaction conditions. The cationic poly(3-acrylamidopropyl)trimethylammonium chloride (p(APTMACl)) microgels were synthesized by photo-initiated inverse suspension polymerization technique. These microgels were utilized as absorbents for the removal of toxic arsenate (As) from different aqueous environments. The experimental parameters affecting absorption efficiency were investigated, and it was demonstrated that these types of microgels are highly efficient in removing arsenate anions from different aqueous environments compared to the previously reported bulk hydrogel, and cryogel of the same material. A removal efficiency of approximately 97.25% was obtained by immersing 0.5 g microgel in 250 ppm 100 mL solution of arsenate anions for 60 min. Both Langmuir and Freundlich adsorption isotherms were applied to adsorption of arsenate anions by p(APTMACl) microgels, and the Langmuir isotherm was a better representation of the adsorption of arsenate with a high value of R(2) (0.9982). Furthermore, mag-p(APTMACl) microgels were synthesized for the adsorption of arsenate anions to provide easy removal of the microgel composite by using an externally applied magnetic field. Furthermore, re-usability of the p(APTMACl) microgels was also investigated for the adsorption of arsenate anions.

  11. A bioinspired peptide scaffold with high antibiotic activity and low in vivo toxicity

    PubMed Central

    Rabanal, Francesc; Grau-Campistany, Ariadna; Vila-Farrés, Xavier; Gonzalez-Linares, Javier; Borràs, Miquel; Vila, Jordi; Manresa, Angeles; Cajal, Yolanda

    2015-01-01

    Bacterial resistance to almost all available antibiotics is an important public health issue. A major goal in antimicrobial drug discovery is the generation of new chemicals capable of killing pathogens with high selectivity, particularly multi-drug-resistant ones. Here we report the design, preparation and activity of new compounds based on a tunable, chemically accessible and upscalable lipopeptide scaffold amenable to suitable hit-to-lead development. Such compounds could become therapeutic candidates and future antibiotics available on the market. The compounds are cyclic, contain two D-amino acids for in vivo stability and their structures are reminiscent of other cyclic disulfide-containing peptides available on the market. The optimized compounds prove to be highly active against clinically relevant Gram-negative and Gram-positive bacteria. In vitro and in vivo tests show the low toxicity of the compounds. Their antimicrobial activity against resistant and multidrug-resistant bacteria is at the membrane level, although other targets may also be involved depending on the bacterial strain. PMID:26024044

  12. The toxic mechanism of high lethality of herbicide butachlor in marine flatfish flounder, Paralichthys olivaceus

    NASA Astrophysics Data System (ADS)

    Guo, Huarong; Yin, Licheng; Zhang, Shicui; Feng, Wenrong

    2010-09-01

    The toxic mechanism of herbicide butachlor to induce extremely high lethality in marine flatfish flounder, Paralichthys Olivaceus, was analyzed by histopathological examination, antioxidant enzymes activities and ATP content assay. Histopathological examination of gill, liver and kidney of exposed fishes showed that gill was a target organ of butachlor. The butachlor seriously impaired the respiration of gills by a series of lesions such as edema, lifting and detachment of lamellar epithelium, breakdown of pillar cells, and blood congestion. The dysfunction of gill respiration caused suffocation to the exposed flounder with extremely high acute lethality. Antioxidant enzyme activity assay of the in vitro cultured flounder gill (FG) cells exposed to butachlor indicated that butachlor markedly inhibited the antioxidant enzyme activities of Superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX). Furthermore, along with the decline of antioxidant enzyme activities, ATP content in the exposed FG cells decreased, too. This infers that the oxidative stress induced by butachlor can inhibit the production of cellular ATP. Similar decrease of ATP content was also observed in the exposed flounder gill tissues. Taken together, as in FG cells, butachlor possibly induced a short supply of ATP in pillar cells by inhibiting the antioxidant enzyme activities and then affecting the contractibility of the pillar cells, which in turn resulted in the blood congestion and suffocation of exposed flounder.

  13. High doses of pseudoephedrine hydrochloride accelerate onset of CNS oxygen toxicity seizures in unanesthetized rats.

    PubMed

    Pilla, R; Held, H E; Landon, C S; Dean, J B

    2013-08-29

    Pseudoephedrine (PSE) salts (hydrochloride and sulfate) are commonly used as nasal and paranasal decongestants by scuba divers. Anecdotal reports from the Divers Alert Network suggest that taking PSE prior to diving while breathing pure O₂ increases the risk for CNS oxygen toxicity (CNS-OT), which manifests as seizures. We hypothesized that high doses of PSE reduce the latency time to seizure (LS) in unanesthetized rats breathing 5 atmospheres absolute (ATA) of hyperbaric oxygen. Sixty-three male rats were implanted with radio-transmitters that recorded electroencephalogram activity and body temperature. After ≥7-day recovery, and 2 h before "diving", each rat was administered either saline solution (control) or PSE hydrochloride intragastrically at the following doses (mg PSE/kg): 0, 40, 80, 100, 120, 160, and 320. Rats breathed pure O₂ and were dived to 5ATA until the onset of behavioral seizures coincident with neurological seizures. LS was the time elapsed between reaching 5ATA and exhibiting seizures. We observed a significant dose-dependent decrease in the LS at doses of 100-320 mg/kg, whereas no significant differences in LS from control value were observed at doses ≤80 mg/kg. Our findings showed that high doses of PSE accelerate the onset of CNS-OT seizures in unanesthetized rats breathing 5ATA of poikilocapnic hyperoxia. Extrapolating our findings to humans, we conclude that the recommended daily dose of PSE should not be abused prior to diving with oxygen-enriched gas mixes or pure O₂.

  14. Disrupting Self-Assembly and Toxicity of Amyloidogenic Protein Oligomers by “Molecular Tweezers” - from the Test Tube to Animal Models

    PubMed Central

    Attar, Aida; Bitan, Gal

    2014-01-01

    Despite decades of research, therapy for diseases caused by abnormal protein folding and aggregation (amyloidoses) is limited to treatment of symptoms and provides only temporary and moderate relief to sufferers. The failure in developing successful disease-modifying drugs for amyloidoses stems from the nature of the targets for such drugs – primarily oligomers of amyloidogenic proteins, which are distinct from traditional targets, such as enzymes or receptors. The oligomers are metastable, do not have well-defined structures, and exist in dynamically changing mixtures. Therefore, inhibiting the formation and toxicity of these oligomers likely will require out-of-the-box thinking and novel strategies. We review here the development of a strategy based on targeting the combination of hydrophobic and electrostatic interactions that are key to the assembly and toxicity of amyloidogenic proteins using lysine (K)-specific “molecular tweezers” (MTs). Our discussion includes a survey of the literature demonstrating the important role of K residues in the assembly and toxicity of amyloidogenic proteins and the development of a lead MT derivative called CLR01, from an inhibitor of protein aggregation in vitro to a drug candidate showing effective amelioration of disease symptoms in animal models of Alzheimer’s and Parkinson’s diseases. PMID:23859557

  15. Relevance Rank Platform (RRP) for Functional Filtering of High Content Protein-Protein Interaction Data.

    PubMed

    Pokharel, Yuba Raj; Saarela, Jani; Szwajda, Agnieszka; Rupp, Christian; Rokka, Anne; Lal Kumar Karna, Shibendra; Teittinen, Kaisa; Corthals, Garry; Kallioniemi, Olli; Wennerberg, Krister; Aittokallio, Tero; Westermarck, Jukka

    2015-12-01

    High content protein interaction screens have revolutionized our understanding of protein complex assembly. However, one of the major challenges in translation of high content protein interaction data is identification of those interactions that are functionally relevant for a particular biological question. To address this challenge, we developed a relevance ranking platform (RRP), which consist of modular functional and bioinformatic filters to provide relevance rank among the interactome proteins. We demonstrate the versatility of RRP to enable a systematic prioritization of the most relevant interaction partners from high content data, highlighted by the analysis of cancer relevant protein interactions for oncoproteins Pin1 and PME-1. We validated the importance of selected interactions by demonstration of PTOV1 and CSKN2B as novel regulators of Pin1 target c-Jun phosphorylation and reveal previously unknown interacting proteins that may mediate PME-1 effects via PP2A-inhibition. The RRP framework is modular and can be modified to answer versatile research problems depending on the nature of the biological question under study. Based on comparison of RRP to other existing filtering tools, the presented data indicate that RRP offers added value especially for the analysis of interacting proteins for which there is no sufficient prior knowledge available. Finally, we encourage the use of RRP in combination with either SAINT or CRAPome computational tools for selecting the candidate interactors that fulfill the both important requirements, functional relevance, and high confidence interaction detection.

  16. The relationship between repeat-dose toxicity and aromatic-ring class profile of high-boiling petroleum substances.

    PubMed

    Roth, Randy N; Simpson, Barry J; Nicolich, Mark J; Murray, F Jay; Gray, Thomas M

    2013-11-01

    A study was undertaken within the context of the U.S. EPA HPV Chemical Challenge Program to (1) characterize relationships between PAC content and repeat-dose toxicities of high-boiling petroleum substances (HBPS) and (2) develop statistical models that could be used to predict the repeat-dose toxicity of similar untested substances. The study evaluated 47 repeat-dose dermal toxicity and 157 chemical compositional studies. The four most sensitive endpoints of repeat-dose toxicity were platelet count, hemoglobin concentration, relative liver weight and thymus weight. Predictive models were developed for the dose-response relationships between the wt.% concentration of each of seven ring classes of aromatic compounds (the "ARC profile") and specific effects, with high correlations (r=0.91-0.94) between the observed and model-predicted data. The development of the mathematical models used to generate the results reported in this study is described by Nicolich et al. (2013). Model-generated dose-response curves permit the prediction of either the effect at a given dose or the dose that causes a given effect. The models generate values that are consistent with other standard measures. The models, using compositional data, can be used for predicting the repeat-dose toxicity of untested HBPS.

  17. Integrated and comparative proteomics of high-oil and high-protein soybean seeds.

    PubMed

    Xu, Xiu Ping; Liu, Hui; Tian, Lihong; Dong, Xiang Bai; Shen, Shi Hua; Qu, Le Qing

    2015-04-01

    We analysed the global protein expression in seeds of a high-oil soybean cultivar (Jiyu 73, JY73) by proteomics. More than 700 protein spots were detected and 363 protein spots were successfully identified. Comparison of the protein profile of JY73 with that of a high-protein cultivar (Zhonghuang 13, ZH13) revealed 40 differentially expressed proteins, including oil synthesis, redox/stress, hydrolysis and storage-related proteins. All redox/stress proteins were less or not expressed in JY73, whereas the expression of the major storage proteins, nitrogen and carbon metabolism-related proteins was higher in ZH13. Biochemical analysis of JY73 revealed that it was in a low oxidation state, with a high content of polyunsaturated fatty acids and vitamin E. Vitamin E was more active than antioxidant enzymes and protected the soybean seed in a lower oxidation state. The characteristics of high oil and high protein in soybean, we revealed, might provide a reference for soybean nutrition and soybean breeding.

  18. A high-throughput, in-vitro assay for Bacillus thuringiensis insecticidal proteins.

    PubMed

    Izumi Willcoxon, Michi; Dennis, Jaclyn R; Lau, Sabina I; Xie, Weiping; You, You; Leng, Song; Fong, Ryan C; Yamamoto, Takashi

    2016-01-10

    A high-throughput, in-vitro assay for Bacillus thuringiensis (Bt) insecticidal proteins designated as Cry was developed and evaluated for screening a large number of Cry protein variants produced by DNA shuffling. This automation-amenable assay exploits an insect cell line expressing a single receptor of Bt Cry proteins. The Cry toxin used to develop this assay is a variant of the Cry1Ab protein called IP1-88, which was produced previously by DNA shuffling. Cell mortality caused by the activated Bt Cry toxin was determined by chemical cell viability assay in 96/384-well microtiter plates utilizing CellTiter 96(®) obtained from Promega. A widely-accepted mode-of-action theory of certain Bt Cry proteins suggests that the activated toxin binds to one or more receptors and forms a pore through the insect gut epithelial cell apical membrane. A number of insect proteins such as cadherin-like protein (Cad), aminopeptidase-N (APN), alkaline phosphatase (ALP) and ABC transporter (ABCC) have been identified as the receptors of Bt Cry toxins. In this study, Bt Cry toxin receptors Ostrinia nubilalis (European corn borer) cadherin-like protein (On-Cad) and aminopeptidase-N 1 and 3 (On-APN1, On-APN3) and Spodoptera frugiperda (fall armyworm) cadherin-like protein (Sf-Cad) were cloned in an insect cell line, Sf21, and a mammalian cell line, Expi293F. It was observed by ligand blotting and immunofluorescence microscopy that trypsin-activated IP1-88 bound to On-Cad and On-APN1, but not Sf-Cad or On-APN3. In contrast, IP1-88 bound only to APN1 in BBMV (Brush Border Membrane Vesicles) prepared from the third and fourth-instar O. nubilalis larval midgut. The sensitivity of the recombinant cells to the toxin was then tested. IP1-88 showed no toxicity to non-recombinant Sf21 and Expi293F. Toxicity was observed only when the On-Cad gene was cloned and expressed. Sf-Cad and On-APN1 were not able to make those cells sensitive to the toxin. Since the expression of On-Cad alone was

  19. A high-throughput, in-vitro assay for Bacillus thuringiensis insecticidal proteins.

    PubMed

    Izumi Willcoxon, Michi; Dennis, Jaclyn R; Lau, Sabina I; Xie, Weiping; You, You; Leng, Song; Fong, Ryan C; Yamamoto, Takashi

    2016-01-10

    A high-throughput, in-vitro assay for Bacillus thuringiensis (Bt) insecticidal proteins designated as Cry was developed and evaluated for screening a large number of Cry protein variants produced by DNA shuffling. This automation-amenable assay exploits an insect cell line expressing a single receptor of Bt Cry proteins. The Cry toxin used to develop this assay is a variant of the Cry1Ab protein called IP1-88, which was produced previously by DNA shuffling. Cell mortality caused by the activated Bt Cry toxin was determined by chemical cell viability assay in 96/384-well microtiter plates utilizing CellTiter 96(®) obtained from Promega. A widely-accepted mode-of-action theory of certain Bt Cry proteins suggests that the activated toxin binds to one or more receptors and forms a pore through the insect gut epithelial cell apical membrane. A number of insect proteins such as cadherin-like protein (Cad), aminopeptidase-N (APN), alkaline phosphatase (ALP) and ABC transporter (ABCC) have been identified as the receptors of Bt Cry toxins. In this study, Bt Cry toxin receptors Ostrinia nubilalis (European corn borer) cadherin-like protein (On-Cad) and aminopeptidase-N 1 and 3 (On-APN1, On-APN3) and Spodoptera frugiperda (fall armyworm) cadherin-like protein (Sf-Cad) were cloned in an insect cell line, Sf21, and a mammalian cell line, Expi293F. It was observed by ligand blotting and immunofluorescence microscopy that trypsin-activated IP1-88 bound to On-Cad and On-APN1, but not Sf-Cad or On-APN3. In contrast, IP1-88 bound only to APN1 in BBMV (Brush Border Membrane Vesicles) prepared from the third and fourth-instar O. nubilalis larval midgut. The sensitivity of the recombinant cells to the toxin was then tested. IP1-88 showed no toxicity to non-recombinant Sf21 and Expi293F. Toxicity was observed only when the On-Cad gene was cloned and expressed. Sf-Cad and On-APN1 were not able to make those cells sensitive to the toxin. Since the expression of On-Cad alone was

  20. Separation of macromolecular proteins and rejection of toxic heavy metal ions by PEI/cSMM blend UF membranes.

    PubMed

    Kanagaraj, P; Nagendran, A; Rana, D; Matsuura, T; Neelakandan, S

    2015-01-01

    The charged surface modifying macromolecule (cSMM) was blended into the casting solution of poly(ether imide) (PEI) to prepare surface modified ultrafiltration membranes by phase inversion technique. The separation of proteins including bovine serum albumin, egg albumin, pepsin and trypsin was investigated by the fabricated membranes. On increasing cSMM content, solute rejection decreases whereas membrane flux increases. The pore size and surface porosity of the 5 wt% cSMM blend PEI membranes increases to 41.4 Å and 14.8%, respectively. Similarly, the molecular weight cut-off of the membranes ranged from 20 to 45 kDa, depending on the various compositions of the prepared membranes. The toxic heavy metal ions Cu(II), Cr(III), Zn(II) and Pb(II) from aqueous solutions were subjected to rejection by the prepared blended membrane with various concentration of polyethyleneimine (PETIM) as water soluble polymeric ligand. It was found that the rejection behavior of metal ion depends on the PETIM concentration and the stability complexation of metal ion with ligand.

  1. Downregulation of super oxide dismutase level in protein might be due to sulfur mustard induced toxicity in lung.

    PubMed

    Mirbagheri, Leila; Habibi Roudkenar, Mehryar; Imani Fooladi, Abbas Ali; Ghanei, Mostafa; Nourani, Mohammad Reza

    2013-05-15

    Sulfur mustard (SM) has been identified as an important chemical weapon. During the Iran-Iraq war of 1980-88, the extensive usage of SM against Iranian civilians and military forces was proven. This agent has been shown to cause severe damage mainly in the skin, eyes, lungs, and respiratory tract in Iranian veterans. The most common disease is bronchiolitis obliterans (BO)). SM increases the endogenous production of reactive oxygen species (ROS). Superoxide dismutases (SODs) are known as protective antioxidants against the harmful effects of ROS. Twenty exposed SM individuals (43.2±6.4 years), and 10 normal controls (41.3±2.5 years) were enrolled in this study. Evaluation of SODs was performed by semiquantitative RT-PCR and immunohistochemistry. Our results demonstrated that CuZnSOD and MnSOD mRNA were up-regulated 2.79±1.09 and 2.49±1.11 folds, respectively in SM-injured patients in comparison with control levels. In contrast, Immunohistochemistry results showed downregulation of CuZnSOD protein expression in SM injured patients. Our results revealed that SODs may play an important role in cellular protection against oxidative stress due to mustard gas toxicity in airway wall of SM exposed patients.

  2. Accuracy of Protein-Protein Binding Sites in High-Throughput Template-Based Modeling

    PubMed Central

    Kundrotas, Petras J.; Vakser, Ilya A.

    2010-01-01

    The accuracy of protein structures, particularly their binding sites, is essential for the success of modeling protein complexes. Computationally inexpensive methodology is required for genome-wide modeling of such structures. For systematic evaluation of potential accuracy in high-throughput modeling of binding sites, a statistical analysis of target-template sequence alignments was performed for a representative set of protein complexes. For most of the complexes, alignments containing all residues of the interface were found. The full interface alignments were obtained even in the case of poor alignments where a relatively small part of the target sequence (as low as 40%) aligned to the template sequence, with a low overall alignment identity (<30%). Although such poor overall alignments might be considered inadequate for modeling of whole proteins, the alignment of the interfaces was strong enough for docking. In the set of homology models built on these alignments, one third of those ranked 1 by a simple sequence identity criteria had RMSD<5 Å, the accuracy suitable for low-resolution template free docking. Such models corresponded to multi-domain target proteins, whereas for single-domain proteins the best models had 5 Åhigh-throughput techniques had accuracy suitable for meaningful docking experiments. This percentage will grow with the increasing availability of co-crystallized protein-protein complexes. PMID:20369011

  3. High affinity FRβ-specific CAR T cells eradicate AML and normal yeloid lineage without HSC toxicity

    PubMed Central

    Lynn, Rachel C; Feng, Yang; Schutsky, Keith; Poussin, Mathilde; Kalota, Anna; Dimitrov, Dimiter S; Powell, Daniel J

    2016-01-01

    Acute myeloid leukemia (AML) is an aggressive malignancy, and development of new treatments to prolong remissions is warranted. Chimeric antigen receptor (CAR) T-cell therapies appear promising but on-target, off-tumor recognition of antigen in healthy tissues remains a concern. Here, we isolated a high affinity (HA) folate receptor beta (FRβ)-specific scFv (2.48nM KD) for optimization of FRβ-redirected CAR T-cell therapy for AML. T-cells stably expressing the HA-FRβ CAR exhibited greatly enhanced antitumor activity against FRβ+ AML in vitro and in vivo compared to a low affinity (LA) FRβ CAR (54.3nM KD). Using the HA-FRβ IgG, FRβ expression was detectable in myeloid-lineage hematopoietic cells; however, expression in CD34+ hematopoietic stem cells (HSCs) was nearly undetectable. Accordingly, HA-FRβ CAR T-cells lysed mature CD14+ monocytes, while HSC colony formation was unaffected. Because of the potential for elimination of mature myeloid lineage, mRNA CAR electroporation for transient CAR expression was evaluated. mRNA-electroporated HA-FRβ CAR T-cells retained effective anti-tumor activity in vitro and in vivo. Together, our results highlight the importance of antibody affinity in target protein detection and CAR development and suggest that transient delivery of potent HA-FRβ CAR T-cells is highly effective against AML and reduces the risk for long-term myeloid toxicity. PMID:26898190

  4. Incorporating High-Throughput Exposure Predictions With Dosimetry-Adjusted In Vitro Bioactivity to Inform Chemical Toxicity Testing

    PubMed Central

    Wetmore, Barbara A.; Wambaugh, John F.; Allen, Brittany; Ferguson, Stephen S.; Sochaski, Mark A.; Setzer, R. Woodrow; Houck, Keith A.; Strope, Cory L.; Cantwell, Katherine; Judson, Richard S.; LeCluyse, Edward; Clewell, Harvey J.; Thomas, Russell S.; Andersen, Melvin E.

    2015-01-01

    We previously integrated dosimetry and exposure with high-throughput screening (HTS) to enhance the utility of ToxCast HTS data by translating in vitro bioactivity concentrations to oral equivalent doses (OEDs) required to achieve these levels internally. These OEDs were compared against regulatory exposure estimates, providing an activity-to-exposure ratio (AER) useful for a risk-based ranking strategy. As ToxCast efforts expand (ie, Phase II) beyond food-use pesticides toward a wider chemical domain that lacks exposure and toxicity information, prediction tools become increasingly important. In this study, in vitro hepatic clearance and plasma protein binding were measured to estimate OEDs for a subset of Phase II chemicals. OEDs were compared against high-throughput (HT) exposure predictions generated using probabilistic modeling and Bayesian approaches generated by the U.S. Environmental Protection Agency (EPA) ExpoCast program. This approach incorporated chemical-specific use and national production volume data with biomonitoring data to inform the exposure predictions. This HT exposure modeling approach provided predictions for all Phase II chemicals assessed in this study whereas estimates from regulatory sources were available for only 7% of chemicals. Of the 163 chemicals assessed in this study, 3 or 13 chemicals possessed AERs < 1 or < 100, respectively. Diverse bioactivities across a range of assays and concentrations were also noted across the wider chemical space surveyed. The availability of HT exposure estimation and bioactivity screening tools provides an opportunity to incorporate a risk-based strategy for use in testing prioritization. PMID:26251325

  5. Protein engineering by highly parallel screening of computationally designed variants

    PubMed Central

    Sun, Mark G. F.; Seo, Moon-Hyeong; Nim, Satra; Corbi-Verge, Carles; Kim, Philip M.

    2016-01-01

    Current combinatorial selection strategies for protein engineering have been successful at generating binders against a range of targets; however, the combinatorial nature of the libraries and their vast undersampling of sequence space inherently limit these methods due to the difficulty in finely controlling protein properties of the engineered region. Meanwhile, great advances in computational protein design that can address these issues have largely been underutilized. We describe an integrated approach that computationally designs thousands of individual protein binders for high-throughput synthesis and selection to engineer high-affinity binders. We show that a computationally designed library enriches for tight-binding variants by many orders of magnitude as compared to conventional randomization strategies. We thus demonstrate the feasibility of our approach in a proof-of-concept study and successfully obtain low-nanomolar binders using in vitro and in vivo selection systems. PMID:27453948

  6. Protein engineering by highly parallel screening of computationally designed variants.

    PubMed

    Sun, Mark G F; Seo, Moon-Hyeong; Nim, Satra; Corbi-Verge, Carles; Kim, Philip M

    2016-07-01

    Current combinatorial selection strategies for protein engineering have been successful at generating binders against a range of targets; however, the combinatorial nature of the libraries and their vast undersampling of sequence space inherently limit these methods due to the difficulty in finely controlling protein properties of the engineered region. Meanwhile, great advances in computational protein design that can address these issues have largely been underutilized. We describe an integrated approach that computationally designs thousands of individual protein binders for high-throughput synthesis and selection to engineer high-affinity binders. We show that a computationally designed library enriches for tight-binding variants by many orders of magnitude as compared to conventional randomization strategies. We thus demonstrate the feasibility of our approach in a proof-of-concept study and successfully obtain low-nanomolar binders using in vitro and in vivo selection systems. PMID:27453948

  7. Protein engineering by highly parallel screening of computationally designed variants.

    PubMed

    Sun, Mark G F; Seo, Moon-Hyeong; Nim, Satra; Corbi-Verge, Carles; Kim, Philip M

    2016-07-01

    Current combinatorial selection strategies for protein engineering have been successful at generating binders against a range of targets; however, the combinatorial nature of the libraries and their vast undersampling of sequence space inherently limit these methods due to the difficulty in finely controlling protein properties of the engineered region. Meanwhile, great advances in computational protein design that can address these issues have largely been underutilized. We describe an integrated approach that computationally designs thousands of individual protein binders for high-throughput synthesis and selection to engineer high-affinity binders. We show that a computationally designed library enriches for tight-binding variants by many orders of magnitude as compared to conventional randomization strategies. We thus demonstrate the feasibility of our approach in a proof-of-concept study and successfully obtain low-nanomolar binders using in vitro and in vivo selection systems.

  8. High-resolution mapping of protein sequence-function relationships.

    PubMed

    Fowler, Douglas M; Araya, Carlos L; Fleishman, Sarel J; Kellogg, Elizabeth H; Stephany, Jason J; Baker, David; Fields, Stanley

    2010-09-01

    We present a large-scale approach to investigate the functional consequences of sequence variation in a protein. The approach entails the display of hundreds of thousands of protein variants, moderate selection for activity and high-throughput DNA sequencing to quantify the performance of each variant. Using this strategy, we tracked the performance of >600,000 variants of a human WW domain after three and six rounds of selection by phage display for binding to its peptide ligand. Binding properties of these variants defined a high-resolution map of mutational preference across the WW domain; each position had unique features that could not be captured by a few representative mutations. Our approach could be applied to many in vitro or in vivo protein assays, providing a general means for understanding how protein function relates to sequence.

  9. Toxicant-induced leakage of germ cell-specific proteins from seminiferous tubules in the rat: relationship to blood-testis barrier integrity and prospects for biomonitoring.

    PubMed

    Elkin, Naomi D; Piner, Jacqui A; Sharpe, Richard M

    2010-10-01

    Evaluation of testicular toxicity during drug development is currently based on histopathological evaluation. A sensitive biomarker for testicular toxicology could provide an in-life and "early warning" measurement. Previous studies suggested that disruption of spermatogenesis induced leakage of germ cell proteins from seminiferous tubules (STs) into interstitial fluid (IF); such proteins have potential for use as biomarkers. To investigate this possibility further, adult male rats were treated with three testicular toxicants thought to have differing sites of action; cadmium chloride affects the blood-testis barrier (BTB), methoxyacetic acid (MAA) disrupts pachytene spermatocytes, and 1,3-dinitrobenzene (DNB) targets Sertoli cells. IF proteins were assessed by Coomassie-based dye-stained gels. Immunostaining was used to identify toxicant-induced damage (DAZL) and BTB integrity (ZO-1, occludin, N-cadherin, and β-catenin) and function (biotin). Cadmium chloride induced dose-dependent leakage of proteins from STs into IF coincident with loss of integrity and function of the BTB. Two of the "leaked" proteins were identified on Westerns as being germ cell specific, namely VASA and fatty acid-binding protein 9 (FABP9). In contrast, similar protein leakage was not evident after either MAA-induced or DNB-induced disruption of spermatogenesis and neither of these treatments affected BTB integrity or function. These results suggest that loss of BTB integrity is required for germ cell-specific proteins to leak from STs into IF, implying that use of such biomarkers has very limited potential for noninvasive monitoring of compound-induced disruption to spermatogenesis. PMID:20624998

  10. High-throughput identification of proteins with AMPylation using self-assembled human protein (NAPPA) microarrays.

    PubMed

    Yu, Xiaobo; LaBaer, Joshua

    2015-05-01

    AMPylation (adenylylation) has been recognized as an important post-translational modification that is used by pathogens to regulate host cellular proteins and their associated signaling pathways. AMPylation has potential functions in various cellular processes, and it is widely conserved across both prokaryotes and eukaryotes. However, despite the identification of many AMPylators, relatively few candidate substrates of AMPylation are known. This is changing with the recent development of a robust and reliable method for identifying new substrates using protein microarrays, which can markedly expand the list of potential substrates. Here we describe procedures for detecting AMPylated and auto-AMPylated proteins in a sensitive, high-throughput and nonradioactive manner. The approach uses high-density protein microarrays fabricated using nucleic acid programmable protein array (NAPPA) technology, which enables the highly successful display of fresh recombinant human proteins in situ. The modification of target proteins is determined via copper-catalyzed azide-alkyne cycloaddition (CuAAC). The assay can be accomplished within 11 h. PMID:25881200

  11. High-throughput identification of proteins with AMPylation using self-assembled human protein (NAPPA) microarrays.

    PubMed

    Yu, Xiaobo; LaBaer, Joshua

    2015-05-01

    AMPylation (adenylylation) has been recognized as an important post-translational modification that is used by pathogens to regulate host cellular proteins and their associated signaling pathways. AMPylation has potential functions in various cellular processes, and it is widely conserved across both prokaryotes and eukaryotes. However, despite the identification of many AMPylators, relatively few candidate substrates of AMPylation are known. This is changing with the recent development of a robust and reliable method for identifying new substrates using protein microarrays, which can markedly expand the list of potential substrates. Here we describe procedures for detecting AMPylated and auto-AMPylated proteins in a sensitive, high-throughput and nonradioactive manner. The approach uses high-density protein microarrays fabricated using nucleic acid programmable protein array (NAPPA) technology, which enables the highly successful display of fresh recombinant human proteins in situ. The modification of target proteins is determined via copper-catalyzed azide-alkyne cycloaddition (CuAAC). The assay can be accomplished within 11 h.

  12. Leptin is an endogenous protective protein against the toxicity exerted by tumor necrosis factor.

    PubMed

    Takahashi, N; Waelput, W; Guisez, Y

    1999-01-01

    Tumor necrosis factor (TNF) is a central mediator of a number of important pathologies such as the systemic inflammatory response syndrome. Administration of high TNF doses induces acute anorexia, metabolic derangement, inflammation, and eventually shock and death. The in vivo effects of TNF are largely mediated by a complex network of TNF-induced cytokines and hormones acting together or antagonistically. Since TNF also induces leptin, a hormone secreted by adipocytes that modulates food intake and metabolism, we questioned the role of leptin in TNF-induced pathology. To address this question, we tested mouse strains that were defective either in leptin gene (ob/ob) or in functional leptin receptor gene (db/db), and made use of a receptor antagonist of leptin. Ob/ob and db/db mice, as well as normal mice treated with antagonist, exhibited increased sensitivity to the lethal effect of TNF. Exogenous leptin afforded protection to TNF in ob/ob mice, but failed to enhance the protective effect of endogenous leptin in normal mice. We conclude that leptin is involved in the protective mechanisms that allow an organism to cope with the potentially autoaggressive effects of its immune system.

  13. Estimation of bisphenol A-Human toxicity by 3D cell culture arrays, high throughput alternatives to animal tests.

    PubMed

    Lee, Dong Woo; Oh, Woo-Yeon; Yi, Sang Hyun; Ku, Bosung; Lee, Moo-Yeal; Cho, Yoon Hee; Yang, Mihi

    2016-09-30

    Bisphenol A (BPA) has been widely used for manufacturing polycarbonate plastics and epoxy resins and has been extensively tested in animals to predict human toxicity. In order to reduce the use of animals for toxicity assessment and provide further accurate information on BPA toxicity in humans, we encapsulated Hep3B human hepatoma cells in alginate and cultured them in three dimensions (3D) on a micropillar chip coupled to a panel of metabolic enzymes on a microwell chip. As a result, we were able to assess the toxicity of BPA under various metabolic enzyme conditions using a high-throughput and micro assay; sample volumes were nearly 2,000 times less than that required for a 96-well plate. We applied a total of 28 different enzymes to each chip, including 10 cytochrome P450s (CYP450s), 10 UDP-glycosyltransferases (UGTs), 3 sulfotransferases (SULTs), alcohol dehydrogenase (ADH), and aldehyde dehydrogenase 2 (ALDH2). Phase I enzyme mixtures, phase II enzyme mixtures, and a combination of phase I and phase II enzymes were also applied to the chip. BPA toxicity was higher in samples containing CYP2E1 than controls, which contained no enzymes (IC50, 184±16μM and 270±25.8μM, respectively, p<0.01). However, BPA-induced toxicity was alleviated in the presence of ADH (IC50, 337±17.9μM), ALDH2 (335±13.9μM), and SULT1E1 (318±17.7μM) (p<0.05). CYP2E1-mediated cytotoxicity was confirmed by quantifying unmetabolized BPA using HPLC/FD. Therefore, we suggest the present micropillar/microwell chip platform as an effective alternative to animal testing for estimating BPA toxicity via human metabolic systems. PMID:27491884

  14. Can Gas Replace Protein Function? CO Abrogates the Oxidative Toxicity of Myoglobin

    PubMed Central

    Shaklai, Mati; Shaklai, Nurith

    2014-01-01

    Outside their cellular environments, hemoglobin (Hb) and myoglobin (Mb) are known to wreak oxidative damage. Using haptoglobin (Hp) and hemopexin (Hx) the body defends itself against cell-free Hb, yet mechanisms of protection against oxidative harm from Mb are unclear. Mb may be implicated in oxidative damage both within the myocyte and in circulation following rhabdomyolysis. Data from the literature correlate rhabdomyolysis with the induction of Heme Oxygenase-1 (HO-1), suggesting that either the enzyme or its reaction products are involved in oxidative protection. We hypothesized that carbon monoxide (CO), a product, might attenuate Mb damage, especially since CO is a specific ligand for heme iron. Low density lipoprotein (LDL) was chosen as a substrate in circulation and myosin (My) as a myocyte component. Using oxidation targets, LDL and My, the study compared the antioxidant potential of CO in Mb-mediated oxidation with the antioxidant potential of Hp in Hb-mediated oxidation. The main cause of LDL oxidation by Hb was found to be hemin which readily transfers from Hb to LDL. Hp prevented heme transfer by sequestering hemin within the Hp-Hb complex. Hemin barely transferred from Mb to LDL, and oxidation appeared to stem from heme iron redox in the intact Mb. My underwent oxidative crosslinking by Mb both in air and under N2. These reactions were fully arrested by CO. The data are interpreted to suit several circumstances, some physiological, such as high muscle activity, and some pathological, such as rhabdomyolysis, ischemia/reperfusion and skeletal muscle disuse atrophy. It appear that CO from HO-1 attenuates damage by temporarily binding to deoxy-Mb, until free oxygen exchanges with CO to restore the equilibrium. PMID:25111140

  15. Ag/ZnO nanomaterials as high performance sensors for flammable and toxic gases.

    PubMed

    Simon, Quentin; Barreca, Davide; Gasparotto, Alberto; Maccato, Chiara; Tondello, Eugenio; Sada, Cinzia; Comini, Elisabetta; Devi, Anjana; Fischer, Roland A

    2012-01-20

    Ag/ZnO nanocomposites supported on polycrystalline Al2O3 were synthesized by an unprecedented approach combining plasma enhanced chemical vapor deposition (PE-CVD) of ZnO matrices and the subsequent deposition of Ag nanoparticles (NPs) by radio frequency (RF) sputtering. The system structure, composition and morphology were investigated by glancing incidence x-ray diffraction (GIXRD), secondary ion mass spectrometry (SIMS), field emission scanning electron microscopy (FE-SEM) and energy dispersive x-ray spectroscopy (EDXS). A tailored dispersion and distribution of silver particles could be obtained under mild conditions by the sole variation of the sputtering time. Gas sensing properties toward flammable and toxic gases, both reducing (CH3CH2OH, CH3COCH3) and oxidizing (O3), were investigated in the temperature range 100-400 °C. Beside the high sensitivity, the developed sensors exhibited a response proportional to Ag content, thanks to catalytic and electronic effects promoted by silver NPs. In addition, discrimination between oxidizing and reducing analytes was enabled by a suitable choice of the adopted working temperature.

  16. Associations between Urinary Excretion of Cadmium and Proteins in a Nonsmoking Population: Renal Toxicity or Normal Physiology?

    PubMed Central

    Sallsten, Gerd; Lundh, Thomas; Barregard, Lars

    2012-01-01

    Background: Associations between cadmium (Cd) and kidney function have been reported even at low levels of exposure in the general population. Recently, the causality of these associations has been questioned. Objectives: We examined associations between urinary Cd (U-Cd; a biomarker of exposure) and urinary proteins that are used as biomarkers of kidney effects, based on repeated short-term sampling in healthy subjects. Methods: Twenty-four hour urine samples were collected on 2 separate days at six fixed times from 30 healthy nonsmoking men and women (median age 39 years). We analyzed the samples (N = 354) for Cd (i.e., U-Cd) and two proteins used as kidney function biomarkers: urinary albumin (U-Alb) and alpha-1-microglobulin (U-A1M). Concentrations were adjusted for creatinine concentration or for specific gravity, and excretion rates (mass per hour) were calculated. Possible associations were assessed within each individual participant, and mean correlations and regressions were evaluated. Results: We found clear positive mean associations within individuals between the excretion of U-Cd [mean, 0.11 µg/g creatinine (range, 0.01–0.52 µg/g creatinine)] and both U-Alb and U-A1M. The associations were stronger for excretion rates and concentrations adjusted for specific gravity than for concentrations adjusted for creatinine. We also found significant positive associations of urinary flow with excretion of U-Cd, U-Alb, and U-A1M. Conclusions: Associations between short-term changes in U-Cd and markers of kidney function within individual nonsmoking study participants are unlikely to reflect effects of Cd toxicity. A more likely explanation is that these associations result from normal variation in renal function, including changes in urinary flow, that influence the urinary excretion of both Cd and proteins in the same direction. These effects of normal variability may result in overestimation of the adverse effects of Cd on kidney function at low-level Cd

  17. ADAPTING THE MEDAKA EMBRYO ASSAY TO A HIGH-THROUGHPUT APPROACH FOR DEVELOPMENTAL TOXICITY TESTING.

    EPA Science Inventory

    Chemical exposure during embryonic development may cause persistent effects, yet developmental toxicity data exist for very few chemicals. Current testing procedures are time consuming and costly, underlining the need for rapid and low cost screening strategies. While in vitro ...

  18. Live cell cytoplasm staining and selective labeling of intracellular proteins by non-toxic cell-permeant thiophene fluorophores.

    PubMed

    Di Maria, F; Palamà, I E; Baroncini, M; Barbieri, A; Bongini, A; Bizzarri, R; Gigli, G; Barbarella, G

    2014-03-14

    A structurally correlated series of cell-permeant thiophene fluorophores, characterized by intense green or red fluorescence inside live mouse embryonic fibroblasts, was developed. The fluorophores displayed rapid internalization, excellent retention inside the cells, and high optical stability in the cytosolic environment and did not alter cell viability and reproducibility. Depending on the molecular structure, they experienced distinct fate inside the cells: from bright and lasting staining of the cytoplasm to selective tagging of a small set of globular proteins.

  19. Impact of Bone Marrow Radiation Dose on Acute Hematologic Toxicity in Cervical Cancer: Principal Component Analysis on High Dimensional Data

    SciTech Connect

    Yun Liang; Messer, Karen; Rose, Brent S.; Lewis, John H.; Jiang, Steve B.; Yashar, Catheryn M.; Mundt, Arno J.; Mell, Loren K.

    2010-11-01

    Purpose: To study the effects of increasing pelvic bone marrow (BM) radiation dose on acute hematologic toxicity in patients undergoing chemoradiotherapy, using a novel modeling approach to preserve the local spatial dose information. Methods and Materials: The study included 37 cervical cancer patients treated with concurrent weekly cisplatin and pelvic radiation therapy. The white blood cell count nadir during treatment was used as the indicator for acute hematologic toxicity. Pelvic BM radiation dose distributions were standardized across patients by registering the pelvic BM volumes to a common template, followed by dose remapping using deformable image registration, resulting in a dose array. Principal component (PC) analysis was applied to the dose array, and the significant eigenvectors were identified by linear regression on the PCs. The coefficients for PC regression and significant eigenvectors were represented in three dimensions to identify critical BM subregions where dose accumulation is associated with hematologic toxicity. Results: We identified five PCs associated with acute hematologic toxicity. PC analysis regression modeling explained a high proportion of the variation in acute hematologicity (adjusted R{sup 2}, 0.49). Three-dimensional rendering of a linear combination of the significant eigenvectors revealed patterns consistent with anatomical distributions of hematopoietically active BM. Conclusions: We have developed a novel approach that preserves spatial dose information to model effects of radiation dose on toxicity, which may be useful in optimizing radiation techniques to avoid critical subregions of normal tissues. Further validation of this approach in a large cohort is ongoing.

  20. Cysteine metabolism and metal toxicity.

    PubMed

    Quig, D

    1998-08-01

    Chronic, low level exposure to toxic metals is an increasing global problem. The symptoms associated with the slow accumulation of toxic metals are multiple and rather nondescript, and overt expression of toxic effects may not appear until later in life. The sulfhydryl-reactive metals (mercury, cadmium, lead, arsenic) are particularly insidious and can affect a vast array of biochemical and nutritional processes. The primary mechanisms by which the sulfhydryl-reactive metals elicit their toxic effects are summarized. The pro-oxidative effects of the metals are compounded by the fact that the metals also inhibit antioxidative enzymes and deplete intracellular glutathione. The metals also have the potential to disrupt the metabolism and biological activities of many proteins due to their high affinity for free sulfhydryl groups. Cysteine has a pivotal role in inducible, endogenous detoxication mechanisms in the body, and metal exposure taxes cysteine status. The protective effects of glutathione and the metallothioneins are discussed in detail. Basic research pertaining to the transport of toxic metals into the brain is summarized, and a case is made for the use of hydrolyzed whey protein to support metal detoxification and neurological function. Metal exposure also affects essential element status, which can further decrease antioxidation and detoxification processes. Early detection and treatment of metal burden is important for successful detoxification, and optimization of nutritional status is paramount to the prevention and treatment of metal toxicity.

  1. The RdgC protein of Escherichia coli binds DNA and counters a toxic effect of RecFOR in strains lacking the replication restart protein PriA.

    PubMed

    Moore, Timothy; McGlynn, Peter; Ngo, Hien-Ping; Sharples, Gary J; Lloyd, Robert G

    2003-02-01

    PriA protein provides a means to load the DnaB replicative helicase at DNA replication fork and D loop structures, and is therefore a key factor in the rescue of stalled or broken forks and subsequent replication restart. We show that the nucleoid-associated RdgC protein binds non-specifically to single-stranded (ss) DNA and double-stranded DNA. It is also essential for growth of a strain lacking PriA, indicating that it might affect replication fork progression or fork rescue. dnaC suppressors of priA overcome this inviability, especially when RecF, RecO or RecR is inactivated, indicating that RdgC avoids or counters a toxic effect of these proteins. Mutations modifying ssDNA-binding (SSB) protein also negate this toxic effect, suggesting that the toxicity reflects inappropriate loading of RecA on SSB-coated ssDNA, leading to excessive or untimely RecA activity. We suggest that binding of RdgC to DNA limits RecA loading, avoiding problems at replication forks that would otherwise require PriA to promote replication restart. Mutations in RNA polymerase also reduce the toxic effect of RecFOR, providing a further link between DNA replication, transcription and repair. PMID:12554673

  2. Identification of proteins that form specific complexes with the highly conserved protein Translin in Schizosaccharomyces pombe.

    PubMed

    Eliahoo, Elad; Litovco, Phyana; Ben Yosef, Ron; Bendalak, Keren; Ziv, Tamar; Manor, Haim

    2014-04-01

    Translin is a single-stranded DNA and RNA binding protein that has a high affinity for G-rich sequences. TRAX is a Translin paralog that associates with Translin. Both Translin and TRAX were highly conserved in eukaryotes. The nucleic acid binding form of Translin is a barrel-shaped homo-octamer. A Translin-TRAX hetero-octamer having a similar structure also binds nucleic acids. Previous reports suggested that Translin may be involved in chromosomal translocations, telomere metabolism and the control of mRNA transport and translation. More recent studies have indicated that Translin-TRAX hetero-octamers are involved in RNA silencing. To gain a further insight into the functions of Translin, we have undertaken to systematically search for proteins with which it forms specific complexes in living cells. Here we report the results of such a search conducted in the fission yeast Schizosaccharomyces pombe, a suitable model system. This search was carried out by affinity purification and immuno-precipitation techniques, combined with differential labeling of the intracellular proteins with the stable isotopes ¹⁵N and ¹⁴N. We identified for the first time two proteins containing an RNA Recognition Motif (RRM), which are specifically associated with the yeast Translin: (1) the pre-mRNA-splicing factor srp1 that belongs to the highly conserved SR family of proteins and (2) vip1, a protein conserved in fungi. Our data also support the presence of RNA in these intracellular complexes. Our experimental approach should be generally applicable to studies of weak intracellular protein-protein interactions and provides a clear distinction between false positive vs. truly interacting proteins.

  3. Characterization of seed storage proteins in high protein genotypes of cowpea [Vigna unguiculata (L.) Walp].

    PubMed

    Gupta, Prachi; Singh, Rohtas; Malhotra, S; Boora, K S; Singal, H R

    2010-01-01

    Twenty one genotypes and two check varieties viz. CS-88 and V-240 of cowpea [Vigna unguiculata (L.) Walp. ] were screened for total proteins. The total protein content ranged from 22.4 (HC-3) to 27.9 % (HC-98-64) in 21 genotypes whereas in check varieties it was 25.6 (V-240) and 26.0 % (CS-88). Seven genotypes viz. HC-6, HC-5, CP-21, LST-II-C-12, CP-16, COVU-702 and HC-98-64 having high protein content (26.7 to 27.9 %) were selected for further characterization of their seed storage proteins. Globulins were the major protein fraction ranging from 55.6 (LST-II-C-12) to 58.8 % (CP-16 and HC-6) of total protein. Glutelins was the second major fraction ranging from 14.4 to 15.6 % followed by albumins (8.2 to 11.9 %) and prolamins (2.3 to 5.0 %). Content of free amino acids also showed variations amongst genotypes with COVU-702 having maximum and LST-II-C-12 having minimum content. Essential amino acid analysis revealed that S-amino acids (cysteine and methionine) were the first limiting amino acids followed by tryptophan. From the results presented here it could be suggested that two genotypes viz. LST-II-C-12 and HC-5 be used in breeding programmes aimed at developing high protein moth bean varieties with good quality.

  4. Microelectrode array recordings of cardiac action potentials as a high throughput method to evaluate pesticide toxicity.

    PubMed

    Natarajan, A; Molnar, P; Sieverdes, K; Jamshidi, A; Hickman, J J

    2006-04-01

    The threat of environmental pollution, biological warfare agent dissemination and new diseases in recent decades has increased research into cell-based biosensors. The creation of this class of sensors could specifically aid the detection of toxic chemicals and their effects in the environment, such as pyrethroid pesticides. Pyrethroids are synthetic pesticides that have been used increasingly over the last decade to replace other pesticides like DDT. In this study we used a high-throughput method to detect pyrethroids by using multielectrode extracellular recordings from cardiac cells. The data from this cell-electrode hybrid system was compared to published results obtained with patch-clamp electrophysiology and also used as an alternative method to further understand pyrethroid effects. Our biosensor consisted of a confluent monolayer of cardiac myocytes cultured on microelectrode arrays (MEA) composed of 60 substrate-integrated electrodes. Spontaneous activity of these beating cells produced extracellular field potentials in the range of 100 microV to nearly 1200 microV with a beating frequency of 0.5-4 Hz. All of the tested pyrethroids; alpha-Cypermethrin, Tetramethrin and Tefluthrin, produced similar changes in the electrophysiological properties of the cardiac myocytes, namely reduced beating frequency and amplitude. The sensitivity of our toxin detection method was comparable to earlier patch-clamp studies, which indicates that, in specific applications, high-throughput extracellular methods can replace single-cell studies. Moreover, the similar effect of all three pyrethroids on the measured parameters suggests, that not only detection of the toxins but, their classification might also be possible with this method. Overall our results support the idea that whole cell biosensors might be viable alternatives when compared to current toxin detection methods. PMID:16198528

  5. Toxicity of seleno-l-methionine, seleno-dl-methionine, high selenium wheat, and selenized yeast to mallard ducklings

    USGS Publications Warehouse

    Heinz, G.H.; Hoffman, D.J.; LeCaptain, L.J.

    1996-01-01

    The toxicity of four chemical forms of selenium (seleno-L-methionine, seleno-DL-methionine, selenized yeast, and high selenium wheat) was compared in day-old mallard ducklings (Anas platyrhynchos). In the first experiment, in which the basal diet was 75% wheat, survival after 2 weeks was lower for ducklings fed 30 ?g/g selenium as seleno-L-methionine (36%) than for ducklings fed 30 ?g/g selenium as seleno-DL-methionine (100%) or 30 ?g/g selenium from high selenium yeast (88%). In a second experiment, in which the basal diet was a commercial duck feed, survival after 2 weeks was 100% in ducklings fed 30 ?g/g selenium as seleno-DL-methionine, seleno-L-methionine, or selenized yeast. The greater toxicity of the L form of selenomethionine was probably related to the palatability or nutritional nature of the wheat-based diet used in experiment 1, but the exact reason for the difference between the DL and L forms is unknown. Biologically incorporated selenium, derived from high selenium wheat was no more toxic than selenium derived from the two purified forms of selenomethionine, and the selenium in selenized yeast was not as toxic as that in the two forms of selenomethionine.

  6. Molecular Dynamics Simulations of Highly Charged Green Fluorescent Proteins

    SciTech Connect

    Lau, E Y; Phillips, J L; Colvin, M E

    2009-03-26

    A recent experimental study showed that green fluorescent protein (GFP) that has been mutated to have ultra-high positive or negative net charges, retain their native structure and fluorescent properties while gaining resistance to aggregation under denaturing conditions. These proteins also provide an ideal test case for studying the effects of surface charge on protein structure and dynamics. They have performed classical molecular dynamics (MD) simulations on the near-neutral wildtype GFP and mutants with net charges of -29 and +35. They analyzed the resulting trajectories to quantify differences in structure and dynamics between the three GFPs. This analyses shows that all three proteins are stable over the MD trajectory, with the near-neutral wild type GFP exhibiting somewhat more flexibility than the positive or negative GFP mutants, as measured by the order parameter and changes in phi-psi angles. There are more dramatic differences in the properties of the water and counter ions surrounding the proteins. The water diffusion constant near the protein surface is closer to the value for bulk water in the positively charged GFP than in the other two proteins. Additionally, the positively charged GFP shows a much greater clustering of the counter ions (CL-) near its surface than corresponding counter ions (Na+) near the negatively charged mutant.

  7. Protein electron transfer (mechanism and reproductive toxicity): iminium, hydrogen bonding, homoconjugation, amino acid side chains (redox and charged), and cell signaling.

    PubMed

    Kovacic, Peter

    2007-03-01

    This contribution presents novel biochemical perspectives of protein electron transfer (ET) with focus on the iminium nature of the peptide link, along with relationships to reproductive toxicity. The favorable influence of hydrogen bonding on protein ET has been widely documented. Hydrogen bonding of the zwitterionic peptide enhances iminium character. A wide array of such bonding agents is available in vivo, with many reports on the peptide link itself. ET proceeds along the backbone, due in part, to homoconjugation. Redox amino acids (AAs), mainly tyrosine (Tyr), tryptophan (Typ), histidine (His), cysteine (Cys), disulfide, and methionine (Met), are involved in the competing processes for radical formation: direct hydrogen atom abstraction versus electron and proton loss. It appears that the radical or radical cation generated during the redox process is capable of interacting with n-electrons of the backbone. Beneficial effects of cationic AAs impact the conduction process. A relationship apparently exists involving cell signaling, protein conduction, and radicals or electrons. In addition, the link between protein ET and reproductive toxicity is examined. A key element is the role of reactive oxygen species (ROS) generated by protein ET. There is extensive evidence for involvement of ROS in generation of birth defects. The radical species arise in protein mainly by ET transformations by enzymes, as illustrated in the case of alcoholism.

  8. High-Resolution Protein Structure Determination by Serial Femtosecond Crystallography

    PubMed Central

    Boutet, Sébastien; Lomb, Lukas; Williams, Garth J.; Barends, Thomas R. M.; Aquila, Andrew; Doak, R. Bruce; Weierstall, Uwe; DePonte, Daniel P.; Steinbrener, Jan; Shoeman, Robert L.; Messerschmidt, Marc; Barty, Anton; White, Thomas A.; Kassemeyer, Stephan; Kirian, Richard A.; Seibert, M. Marvin; Montanez, Paul A.; Kenney, Chris; Herbst, Ryan; Hart, Philip; Pines, Jack; Haller, Gunther; Gruner, Sol M.; Philipp, Hugh T.; Tate, Mark W.; Hromalik, Marianne; Koerner, Lucas J.; van Bakel, Niels; Morse, John; Ghonsalves, Wilfred; Arnlund, David; Bogan, Michael J.; Caleman, Carl; Fromme, Raimund; Hampton, Christina Y.; Hunter, Mark S.; Johansson, Linda C.; Katona, Gergely; Kupitz, Christopher; Liang, Mengning; Martin, Andrew V.; Nass, Karol; Redecke, Lars; Stellato, Francesco; Timneanu, Nicusor; Wang, Dingjie; Zatsepin, Nadia A.; Schafer, Donald; Defever, James; Neutze, Richard; Fromme, Petra; Spence, John C. H.; Chapman, Henry N.; Schlichting, Ilme

    2013-01-01

    Structure determination of proteins and other macromolecules has historically required the growth of high-quality crystals sufficiently large to diffract x-rays efficiently while withstanding radiation damage. We applied serial femtosecond crystallography (SFX) using an x-ray free-electron laser (XFEL) to obtain high-resolution structural information from microcrystals (less than 1 micrometer by 1 micrometer by 3 micrometers) of the well-characterized model protein lysozyme. The agreement with synchrotron data demonstrates the immediate relevance of SFX for analyzing the structure of the large group of difficult-to-crystallize molecules. PMID:22653729

  9. High-resolution protein structure determination by serial femtosecond crystallography.

    PubMed

    Boutet, Sébastien; Lomb, Lukas; Williams, Garth J; Barends, Thomas R M; Aquila, Andrew; Doak, R Bruce; Weierstall, Uwe; DePonte, Daniel P; Steinbrener, Jan; Shoeman, Robert L; Messerschmidt, Marc; Barty, Anton; White, Thomas A; Kassemeyer, Stephan; Kirian, Richard A; Seibert, M Marvin; Montanez, Paul A; Kenney, Chris; Herbst, Ryan; Hart, Philip; Pines, Jack; Haller, Gunther; Gruner, Sol M; Philipp, Hugh T; Tate, Mark W; Hromalik, Marianne; Koerner, Lucas J; van Bakel, Niels; Morse, John; Ghonsalves, Wilfred; Arnlund, David; Bogan, Michael J; Caleman, Carl; Fromme, Raimund; Hampton, Christina Y; Hunter, Mark S; Johansson, Linda C; Katona, Gergely; Kupitz, Christopher; Liang, Mengning; Martin, Andrew V; Nass, Karol; Redecke, Lars; Stellato, Francesco; Timneanu, Nicusor; Wang, Dingjie; Zatsepin, Nadia A; Schafer, Donald; Defever, James; Neutze, Richard; Fromme, Petra; Spence, John C H; Chapman, Henry N; Schlichting, Ilme

    2012-07-20

    Structure determination of proteins and other macromolecules has historically required the growth of high-quality crystals sufficiently large to diffract x-rays efficiently while withstanding radiation damage. We applied serial femtosecond crystallography (SFX) using an x-ray free-electron laser (XFEL) to obtain high-resolution structural information from microcrystals (less than 1 micrometer by 1 micrometer by 3 micrometers) of the well-characterized model protein lysozyme. The agreement with synchrotron data demonstrates the immediate relevance of SFX for analyzing the structure of the large group of difficult-to-crystallize molecules.

  10. High-resolution protein structure determination by serial femtosecond crystallography.

    PubMed

    Boutet, Sébastien; Lomb, Lukas; Williams, Garth J; Barends, Thomas R M; Aquila, Andrew; Doak, R Bruce; Weierstall, Uwe; DePonte, Daniel P; Steinbrener, Jan; Shoeman, Robert L; Messerschmidt, Marc; Barty, Anton; White, Thomas A; Kassemeyer, Stephan; Kirian, Richard A; Seibert, M Marvin; Montanez, Paul A; Kenney, Chris; Herbst, Ryan; Hart, Philip; Pines, Jack; Haller, Gunther; Gruner, Sol M; Philipp, Hugh T; Tate, Mark W; Hromalik, Marianne; Koerner, Lucas J; van Bakel, Niels; Morse, John; Ghonsalves, Wilfred; Arnlund, David; Bogan, Michael J; Caleman, Carl; Fromme, Raimund; Hampton, Christina Y; Hunter, Mark S; Johansson, Linda C; Katona, Gergely; Kupitz, Christopher; Liang, Mengning; Martin, Andrew V; Nass, Karol; Redecke, Lars; Stellato, Francesco; Timneanu, Nicusor; Wang, Dingjie; Zatsepin, Nadia A; Schafer, Donald; Defever, James; Neutze, Richard; Fromme, Petra; Spence, John C H; Chapman, Henry N; Schlichting, Ilme

    2012-07-20

    Structure determination of proteins and other macromolecules has historically required the growth of high-quality crystals sufficiently large to diffract x-rays efficiently while withstanding radiation damage. We applied serial femtosecond crystallography (SFX) using an x-ray free-electron laser (XFEL) to obtain high-resolution structural information from microcrystals (less than 1 micrometer by 1 micrometer by 3 micrometers) of the well-characterized model protein lysozyme. The agreement with synchrotron data demonstrates the immediate relevance of SFX for analyzing the structure of the large group of difficult-to-crystallize molecules. PMID:22653729

  11. Overexpression of alpha-synuclein at non-toxic levels increases dopaminergic cell death induced by copper exposure via modulation of protein degradation pathways.

    PubMed

    Anandhan, Annadurai; Rodriguez-Rocha, Humberto; Bohovych, Iryna; Griggs, Amy M; Zavala-Flores, Laura; Reyes-Reyes, Elsa M; Seravalli, Javier; Stanciu, Lia A; Lee, Jaekwon; Rochet, Jean-Christophe; Khalimonchuk, Oleh; Franco, Rodrigo

    2015-09-01

    Gene multiplications or point mutations in alpha (α)-synuclein are associated with familial and sporadic Parkinson's disease (PD). An increase in copper (Cu) levels has been reported in the cerebrospinal fluid and blood of PD patients, while occupational exposure to Cu has been suggested to augment the risk to develop PD. We aimed to elucidate the mechanisms by which α-synuclein and Cu regulate dopaminergic cell death. Short-term overexpression of wild type (WT) or mutant A53T α-synuclein had no toxic effect in human dopaminergic cells and primary midbrain cultures, but it exerted a synergistic effect on Cu-induced cell death. Cell death induced by Cu was potentiated by overexpression of the Cu transporter protein 1 (Ctr1) and depletion of intracellular glutathione (GSH) indicating that the toxic effects of Cu are linked to alterations in its intracellular homeostasis. Using the redox sensor roGFP, we demonstrated that Cu-induced oxidative stress was primarily localized in the cytosol and not in the mitochondria. However, α-synuclein overexpression had no effect on Cu-induced oxidative stress. WT or A53T α-synuclein overexpression exacerbated Cu toxicity in dopaminergic and yeast cells in the absence of α-synuclein aggregation. Cu increased autophagic flux and protein ubiquitination. Impairment of autophagy by overexpression of a dominant negative Atg5 form or inhibition of the ubiquitin/proteasome system (UPS) with MG132 enhanced Cu-induced cell death. However, only inhibition of the UPS stimulated the synergistic toxic effects of Cu and α-synuclein overexpression. Our results demonstrate that α-synuclein stimulates Cu toxicity in dopaminergic cells independent from its aggregation via modulation of protein degradation pathways.

  12. Overexpression of alpha-synuclein at non-toxic levels increases dopaminergic cell death induced by copper exposure via modulation of protein degradation pathways

    PubMed Central

    Anandhan, Annadurai; Rodriguez-Rocha, Humberto; Bohovych, Iryna; Griggs, Amy M.; Zavala-Flores, Laura; Reyes-Reyes, Elsa M.; Seravalli, Javier; Stanciu, Lia A.; Lee, Jaekwon; Rochet, Jean-Christophe; Khalimonchuk, Oleh; Franco, Rodrigo

    2014-01-01

    Gene multiplications or point mutations in alpha (α)-synuclein are associated with familial and sporadic Parkinson’s disease (PD). An increase in copper (Cu) levels has been reported in the cerebrospinal fluid and blood of PD patients, while occupational exposure to Cu has been suggested to augment the risk to develop PD. We aimed to elucidate the mechanisms by which α-synuclein and Cu regulate dopaminergic cell death. Short-term overexpression of WT or A53T α-synuclein had no toxic effect in human dopaminergic cells and primary midbrain cultures, but it exerted a synergistic effect on Cu-induced cell death. Cell death induced by Cu was potentiated by overexpression of the Cu transporter protein 1 (Ctr1) and depletion of intracellular glutathione (GSH) indicating that the toxic effects of Cu are linked to alterations in its intracellular homeostasis. Using the redox sensor roGFP, we demonstrated that Cu-induced oxidative stress was primarily localized in the cytosol and not in the mitochondria. However, α-synuclein overexpression had no effect on Cu-induced oxidative stress. WT or A53T α-synuclein overexpression exacerbated Cu toxicity in dopaminergic cells and yeast in the absence of α-synuclein aggregation. Cu increased autophagic flux and protein ubiquitination. Impairment of autophagy by overexpression of a dominant negative Atg5 form or inhibition of the ubiquitin/proteasome system (UPS) with MG132 enhanced Cu-induced cell death. However, only inhibition of the UPS stimulated the synergistic toxic effects of Cu and α-synuclein overexpression. Our results demonstrate that α-synuclein stimulates Cu toxicity in dopaminergic cells independent from its aggregation via modulation of protein degradation pathways. PMID:25497688

  13. High Throughput Screening Method to Explore Protein Interactions with Nanoparticles

    PubMed Central

    Nasir, Irem; Fatih, Warda; Svensson, Anja; Radu, Dennis; Linse, Sara; Cabaleiro Lago, Celia; Lundqvist, Martin

    2015-01-01

    The interactions of biological macromolecules with nanoparticles underlie a wide variety of current and future applications in the fields of biotechnology, medicine and bioremediation. The same interactions are also responsible for mediating potential biohazards of nanomaterials. Some applications require that proteins adsorb to the nanomaterial and that the protein resists or undergoes structural rearrangements. This article presents a screening method for detecting nanoparticle-protein partners and conformational changes on time scales ranging from milliseconds to days. Mobile fluorophores are used as reporters to study the interaction between proteins and nanoparticles in a high-throughput manner in multi-well format. Furthermore, the screening method may reveal changes in colloidal stability of nanomaterials depending on the physicochemical conditions. PMID:26313757

  14. High Throughput Screening Method to Explore Protein Interactions with Nanoparticles.

    PubMed

    Nasir, Irem; Fatih, Warda; Svensson, Anja; Radu, Dennis; Linse, Sara; Cabaleiro Lago, Celia; Lundqvist, Martin

    2015-01-01

    The interactions of biological macromolecules with nanoparticles underlie a wide variety of current and future applications in the fields of biotechnology, medicine and bioremediation. The same interactions are also responsible for mediating potential biohazards of nanomaterials. Some applications require that proteins adsorb to the nanomaterial and that the protein resists or undergoes structural rearrangements. This article presents a screening method for detecting nanoparticle-protein partners and conformational changes on time scales ranging from milliseconds to days. Mobile fluorophores are used as reporters to study the interaction between proteins and nanoparticles in a high-throughput manner in multi-well format. Furthermore, the screening method may reveal changes in colloidal stability of nanomaterials depending on the physicochemical conditions. PMID:26313757

  15. The highly efficient delivery of exogenous proteins into cells mediated by biodegradable chimaeric polymersomes.

    PubMed

    Liu, Guijing; Ma, Shoubao; Li, Shaoke; Cheng, Ru; Meng, Fenghua; Liu, Haiyan; Zhong, Zhiyuan

    2010-10-01

    Biodegradable chimaeric polymersomes based on asymmetric PEG-PCL-PDEA triblock copolymers were prepared and investigated for delivery of exogenous proteins into cells. PEG-PCL-PDEA copolymers with M(n)(PEG) = 5 kg/mol, M(n)(PCL) = 18.2 kg/mol, and short PDEA blocks ranging from 1.1, 2.7 to 4.1 kg/mol (denoted as copolymer 1, 2 and 3, respectively) were obtained by controlled reversible addition-fragmentation chain transfer (RAFT) polymerization. The direct hydration of copolymer thin films in MES buffer (pH 5.3) yielded uniform polymersomes with sizes of 130-175 nm. These polymersomes had close to neutral zeta potentials (-2 approximately +2.7 mV) at pH 7.4. The polymersomal structures were confirmed by confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), and catalytic activity experiment on 3,3',3''-phosphinidyne(trisbenzenesulfonic acid)-loaded polymersomes. MTT assays showed that these polymersomes were non-toxic up to a concentration of 0.5mg/mL. These chimaeric polymersomes, in particular polymersome 2, showed remarkably high protein loading efficiencies and loading contents for bovine serum albumin (BSA), cytochrome C (CC), lysozyme (Lys), ovalbumin (OVA) and immunoglobulin G (IgG). The encapsulation of proteins did not significantly alter the polymersome size distributions and zeta potentials. The protein release studies showed that both BSA and CC were released in a controlled manner. Importantly, the released CC fully maintained its activity. Notably, CLSM studies showed that FITC-CC loaded polymersomes efficiently delivered and released proteins into the cytoplasm of RAW 264.7 cells. Moreover, these chimaeric polymersomes were able to simultaneously load and transport proteins and doxorubicin into the cytoplasm as well as the cell nucleus. We are convinced that these biodegradable chimaeric polymersomes have great potentials in protein therapy.

  16. Linking high resolution mass spectrometry data with exposure and toxicity forecasts to advance high-throughput environmental monitoring.

    PubMed

    Rager, Julia E; Strynar, Mark J; Liang, Shuang; McMahen, Rebecca L; Richard, Ann M; Grulke, Christopher M; Wambaugh, John F; Isaacs, Kristin K; Judson, Richard; Williams, Antony J; Sobus, Jon R

    2016-03-01

    There is a growing need in the field of exposure science for monitoring methods that rapidly screen environmental media for suspect contaminants. Measurement and analysis platforms, based on high resolution mass spectrometry (HRMS), now exist to meet this need. Here we describe results of a study that links HRMS data with exposure predictions from the U.S. EPA's ExpoCast™ program and in vitro bioassay data from the U.S. interagency Tox21 consortium. Vacuum dust samples were collected from 56 households across the U.S. as part of the American Healthy Homes Survey (AHHS). Sample extracts were analyzed using liquid chromatography time-of-flight mass spectrometry (LC-TOF/MS) with electrospray ionization. On average, approximately 2000 molecular features were identified per sample (based on accurate mass) in negative ion mode, and 3000 in positive ion mode. Exact mass, isotope distribution, and isotope spacing were used to match molecular features with a unique listing of chemical formulas extracted from EPA's Distributed Structure-Searchable Toxicity (DSSTox) database. A total of 978 DSSTox formulas were consistent with the dust LC-TOF/molecular feature data (match score≥90); these formulas mapped to 3228 possible chemicals in the database. Correct assignment of a unique chemical to a given formula required additional validation steps. Each suspect chemical was prioritized for follow-up confirmation using abundance and detection frequency results, along with exposure and bioactivity estimates from ExpoCast and Tox21, respectively. Chemicals with elevated exposure and/or toxicity potential were further examined using a mixture of 100 chemical standards. A total of 33 chemicals were confirmed present in the dust samples by formula and retention time match; nearly half of these do not appear to have been associated with house dust in the published literature. Chemical matches found in at least 10 of the 56 dust samples include Piperine, N,N-Diethyl-m-toluamide (DEET

  17. Acclimation-induced changes in toxicity and induction of metallothionein-like proteins in the fathead minnow following sublethal exposure to cobalt, silver, and zinc

    SciTech Connect

    Hobson, J.F.

    1986-01-01

    Increases in tolerance and resistance to metal toxicity by aquatic organisms have been linked to elevated levels of low-molecular-weight metal-binding proteins (e.g., metallothioneins). Acclimation-induced changes in toxic response and the concentration of metallothionein-like proteins (MTP) were studied in laboratory populations of the fathead minnow, Pimephales promelas, following sublethal exposure to Co, Ag, and Zn. Following 7 and 14 days of sublethal exposure, tolerance and resistance, as measured by acute toxicity values, were altered in a dose dependent fashion. Acute toxicity values returned to control levels after 21 days of continuous exposure. Tolerance and resistance of Co- and Zn-acclimated animals were depressed after a 7-day post-acclimation period in control water. Tolerance and resistance of Ag-acclimated animals were temporarily enhanced after 7 days post-acclimation and returned to control levels after 14 days. Accumulation of Co, Ag, and Zn measured as wholebody residues appeared to be regulated in 4 of 6 exposure regimes with residues reaching stable levels after 7 to 14 days of exposure. MTP was induced by exposure to 1.8 mg Zn/L and 0.01 mg Ag/L, however, no sustained (i.e., post 21 days) tolerance or resistance were observed at these dose levels indicating that these two biological responses may not be directly related.

  18. Multidrug and toxic compound extrusion protein-1 (MATE1/SLC47A1) is a novel flavonoid transporter.

    PubMed

    Lee, Ji Hae; Lee, Jung Eun; Kim, Yeojin; Lee, Hojoung; Jun, Hee-Jin; Lee, Sung-Joon

    2014-10-01

    Dietary flavonoids have various biological functions. However, their cellular transport mechanisms are largely unknown. We have determined that the multidrug and toxic compound extrusion transporter-1 (MATE1) is a membrane transporter for flavonoids and has a high affinity for quercetin. HEK293T cells overexpressing MATE1 exhibited increased intracellular quercetin accumulation. This effect disappeared in the presence of a MATE1 inhibitor and after MATE1 gene knockdown. HepG2 cells expressed MATE1 significantly, with the uptake quercetin of which was dramatically reduced with MATE1 inhibition. On the basis of immunofluorescence analysis, MATE1 was highly expressed in peroxisomes and the endoplasmic reticulum (ER) as well as in plasma membranes in the liver and intestine, which suggests potential accumulation of quercetin in peroxisomes and the ER in these tissues. Fluorescent microscopic analysis confirmed selective accumulation of qurcetin in peroxisome. The effects of quercetin on cellular lipid reduction and glucose uptake were exaggerated with MATE1 overexpression. In conclusion, MATE1 is a membrane transporter for quercetin; its overexpression enhances the hypolipidemic activity of quercetin and cellular glucose transport. Considering the low bioavailability of quercetin, appropriate regulation of MATE1 expression may optimize cellular quercetin concentrations and promote health benefits.

  19. Ultra High Efficiency ESP for Fine Particulate and Air Toxics Control

    SciTech Connect

    Srinivasachar, Srivats; Pease, Benjamin R.; Porle, Kjell; Mauritzson, Christer; Haythornthwaite, Sheila

    1997-07-01

    Nearly ninety percent of U.S. coal-fired utility boilers are equipped with electrostatic precipitators (ESP). Cost effective retrofittable ESP technologies are the only means to accomplish Department of Energy's (DOE) goal of a major reduction in fine particulate and air toxic emissions from coal-fired power plants. Particles in the size range of 0.1 to 5 {micro}m typically escape ESPs. Metals, such as arsenic, cadmium, lead, molybdenum and antimony, concentrate on these particles. This is the main driver for improved fine particulate control. Vapor phase emissions of mercury, selenium and arsenic are also of major concern. Current dry ESPs, which operate at temperatures greater than 280 F, provide little control for vapor phase toxics. The need for inherent improvement to ESPs has to be considered keeping in perspective the current trend towards the use of low sulfur coals. Switching to low sulfur coals is the dominant approach for SO{sub 2} emission reduction in the utility industry. Low sulfur coals generate high resistivity ash, which can cause an undesirable phenomenon called ''back corona.'' Higher particulate emissions occur if there is back corona in the ESP. Results of the pilot-scale testing identified the ''low temperature ESP'' concept to have the biggest impact for the two low sulfur coals investigated. Lowering the flue gas temperature to 220 F provided the maximum impact in terms of decreased emissions. Intermediate operating temperatures (reduction from 340 to 270 F) also gave significant ESP performance improvement. A significant reduction in particulate emissions was also noted when the flue gas humidity was increased (temperature held constant) from the baseline condition for these moderately high resistivity ash coals. Independent control of flue gas humidity and temperature was an important and a notable element in this project. Mercury emissions were also measured as a function of flue gas temperature. Mercury emissions decreased as the flue

  20. High-Grade Acute Organ Toxicity as a Positive Prognostic Factor in Primary Radiochemotherapy for Anal Carcinoma

    SciTech Connect

    Wolff, Hendrik Andreas; Raus, Ismene; Jung, Klaus; Schueler, Phillip; Herrmann, Markus Karl; Hennies, Steffen; Vorwerk, Hilke; Hille, Andrea; Hess, Clemens Friedrich; Christiansen, Hans

    2011-04-01

    Purpose: To test for a possible correlation between high-grade acute organ toxicity during primary radiochemotherapy and treatment outcome for patients with anal carcinoma. Methods and Materials: From 1991 to 2009, 72 patients with anal carcinoma were treated at our department (10 patients had stage I, 28 patients had stage II, 11 patients had stage IIIA, and 13 patients had stage IIIB cancer [Union Internationale Contre le Cancer criteria]). All patients received normofractionated (1.8 Gy/day, five times/week) whole-pelvis irradiation including iliac and inguinal lymph nodes with a cumulative dose of 50.4 Gy. Concomitant chemotherapy regimen consisted of two cycles of 5-fluorouracil (1,000 mg/m{sup 2}total body surface area (TBSA)/day as continuous intravenous infusion on days 1-4 and 29-32) and mitomycin C (10 mg/m{sup 2}/TBSA, intravenously on days 1 and 29). Toxicity during treatment was monitored weekly, and any incidence of Common Toxicity Criteria (CTC) grade of {>=}3 for skin reaction, cystitis, proctitis, or enteritis was assessed as high-grade acute organ toxicity for later analysis. Results: We found significant correlation between high-grade acute organ toxicity and overall survival, locoregional control, and stoma-free survival, which was independent in multivariate analysis from other possible prognostic factors: patients with a CTC acute organ toxicity grade of {>=}3 had a 5-year overall survival rate of 97% compared to 30% in patients without (p < 0.01, multivariate analysis; 97% vs. 48%, p = 0.03 for locoregional control, and 95% vs. 59%, p = 0.05 for stoma-free survival). Conclusions: Our data indicate that normal tissue and tumor tissue may behave similarly with respect to treatment response, since high-grade acute organ toxicity during radiochemotherapy showed itself to be an independent prognostic marker in our patient population. This hypothesis should be further analyzed by using biomolecular and clinical levels in future clinical trials.

  1. An analysis of fosaprepitant-induced venous toxicity in patients receiving highly emetogenic chemotherapy

    PubMed Central

    Leal, Alexis D.; Grendahl, Darryl C.; Seisler, Drew K.; Sorgatz, Kristine M.; Anderson, Kari J.; Hilger, Crystal R.; Loprinzi, Charles L.

    2015-01-01

    Purpose Fosaprepitant is an antiemetic used for chemotherapy-induced nausea and vomiting. We recently reported increased infusion site adverse events (ISAE) in a cohort of breast cancer patients receiving chemotherapy with doxorubicin and cyclophosphamide (AC). In this current study, we evaluated the venous toxicity of fosaprepitant use with non-anthracycline platinum-based antineoplastic regimens. Methods A retrospective review was conducted of the first 81 patients initiated on fosaprepitant among patients receiving highly emetogenic chemotherapy, on or after January 1, 2011 at Mayo Clinic Rochester. None of these regimens included an anthracycline. Data collected included baseline demographics, chemotherapy regimen, type of intravenous access and type, and severity of ISAE. Data from these patients were compared to previously collected data from patients who had received AC. Statistical analysis using χ2 and univariate logistic regression was used to evaluate the association between treatment regimen, fosaprepitant, and risk of ISAE. Results Among these 81 patients, the incidence of ISAE was 7.4 % in the non-anthracycline platinum group. The most commonly reported ISAE were swelling (3 %), extravasation (3 %), and phlebitis (3 %). When stratified by regimen, fosaprepitant was associated with a statistically significant increased risk of ISAE in the anthracycline group (OR 8.1; 95 % CI 2.0–31.9) compared to the platinum group. Conclusions Fosaprepitant antiemetic therapy causes significant ISAE that are appreciably higher than previous reports. Patients receiving platinum-based chemotherapy appear to have less significant ISAE than do patients who receive anthracycline-based regimens. PMID:24964876

  2. Highly plastic genome of Microcystis aeruginosa PCC 7806, a ubiquitous toxic freshwater cyanobacterium

    PubMed Central

    Frangeul, Lionel; Quillardet, Philippe; Castets, Anne-Marie; Humbert, Jean-François; Matthijs, Hans CP; Cortez, Diego; Tolonen, Andrew; Zhang, Cheng-Cai; Gribaldo, Simonetta; Kehr, Jan-Christoph; Zilliges, Yvonne; Ziemert, Nadine; Becker, Sven; Talla, Emmanuel; Latifi, Amel; Billault, Alain; Lepelletier, Anthony; Dittmann, Elke; Bouchier, Christiane; Tandeau de Marsac, Nicole

    2008-01-01

    Background The colonial cyanobacterium Microcystis proliferates in a wide range of freshwater ecosystems and is exposed to changing environmental factors during its life cycle. Microcystis blooms are often toxic, potentially fatal to animals and humans, and may cause environmental problems. There has been little investigation of the genomics of these cyanobacteria. Results Deciphering the 5,172,804 bp sequence of Microcystis aeruginosa PCC 7806 has revealed the high plasticity of its genome: 11.7% DNA repeats containing more than 1,000 bases, 6.8% putative transposases and 21 putative restriction enzymes. Compared to the genomes of other cyanobacterial lineages, strain PCC 7806 contains a large number of atypical genes that may have been acquired by lateral transfers. Metabolic pathways, such as fermentation and a methionine salvage pathway, have been identified, as have genes for programmed cell death that may be related to the rapid disappearance of Microcystis blooms in nature. Analysis of the PCC 7806 genome also reveals striking novel biosynthetic features that might help to elucidate the ecological impact of secondary metabolites and lead to the discovery of novel metabolites for new biotechnological applications. M. aeruginosa and other large cyanobacterial genomes exhibit a rapid loss of synteny in contrast to other microbial genomes. Conclusion Microcystis aeruginosa PCC 7806 appears to have adopted an evolutionary strategy relying on unusual genome plasticity to adapt to eutrophic freshwater ecosystems, a property shared by another strain of M. aeruginosa (NIES-843). Comparisons of the genomes of PCC 7806 and other cyanobacterial strains indicate that a similar strategy may have also been used by the marine strain Crocosphaera watsonii WH8501 to adapt to other ecological niches, such as oligotrophic open oceans. PMID:18534010

  3. A micropatterned hepatocyte coculture model for assessment of liver toxicity using high-content imaging analysis.

    PubMed

    Trask, O Joseph; Moore, Amanda; LeCluyse, Edward L

    2014-01-01

    The current landscape of in vitro models used to identify drug- or chemical-induced hepatotoxicity relies heavily on cell culture models consisting of HepG2, induced pluripotent stem cell-derived, or primary hepatocytes. While these in vitro models offer powerful approaches for predicting toxicity, each system has challenges, including variable metabolic capacity, brief ex vivo life span in culture, and adoption with standard automated microscopy high-content screening (HCS) systems to measure reproducibility data at the single-cell level. In this report we introduce a novel primary hepatocyte coculture model, HepatoPac™, as an alternative to current model systems for evaluation of in vitro hepatotoxicity in 96-well microtiter plate format examined by HCS. The coculture model consists of primary hepatocytes that are micropatterned to form a discrete microarchitecture or "hepatocyte islands" that are surrounded by supporting fibroblasts resulting in long-term viability and metabolic function of primary hepatocytes. Using multiple HCS image capture and image analysis strategies, we established methods to interrogate various morphometric parameters, such as size, shape, and intensity, at the island or single-cell level. We applied these approaches to identify subpopulations of both fibroblasts and hepatocytes that exhibited alterations in nuclear parameters, cell permeability, mitochondria function, and apoptosis using known reference control compounds and an eight-point dose curve. Subpopulation analysis with additional bioprobe sets can provide a powerful means of addressing differential cell and tissue susceptibilities during compound profiling. Our data show that the HepatoPac is amendable for HCS imaging applications and provides a unique approach for studying hepatotoxicity over prolonged periods of time. PMID:24444127

  4. Toxic megacolon

    MedlinePlus

    ... disease - toxic megacolon; Crohn disease - toxic megacolon; Ulcerative colitis - toxic megacolon ... people with an inflamed colon due to: Ulcerative colitis , or Crohn disease that is not well controlled ...

  5. Whole-pelvic volumetric-modulated arc therapy for high-risk prostate cancer: treatment planning and acute toxicity.

    PubMed

    Ishii, Kentaro; Ogino, Ryo; Hosokawa, Yukinari; Fujioka, Chiaki; Okada, Wataru; Nakahara, Ryota; Kawamorita, Ryu; Tada, Takuhito; Hayashi, Yoshiki; Nakajima, Toshifumi

    2015-01-01

    The objectives of this study were to evaluate dosimetric quality and acute toxicity of volumetric-modulated arc therapy (VMAT) and daily image guidance in high-risk prostate cancer patients. A total of 100 consecutive high-risk prostate cancer patients treated with definitive VMAT with prophylactic whole-pelvic radiotherapy (WPRT) were enrolled. All patients were treated with a double-arc VMAT plan delivering 52 Gy to the prostate planning target volume (PTV), while simultaneously delivering 46.8 Gy to the pelvic nodal PTV in 26 fractions, followed by a single-arc VMAT plan delivering 26 Gy to the prostate PTV in 13 fractions. Image-guided RT was performed with daily cone-beam computed tomography. Dose-volume parameters for the PTV and the organs at risk (OARs), total number of monitor units (MUs) and treatment time were evaluated. Acute toxicity was assessed using the Common Terminology Criteria for Adverse Events, version 4.0. All dosimetric parameters met the present plan acceptance criteria. Mean MU and treatment time were 471 and 146 s for double-arc VMAT, respectively, and were 520 and 76 s for single-arc VMAT, respectively. No Grade 3 or higher acute toxicity was reported. Acute Grade 2 proctitis, diarrhea, and genitourinary toxicity occurred in 12 patients (12%), 6 patients (6%) and 13 patients (13%), respectively. The present study demonstrated that VMAT for WPRT in prostate cancer results in favorable PTV coverage and OAR sparing with short treatment time and an acceptable rate of acute toxicity. These findings support the use of VMAT for delivering WPRT to high-risk prostate cancer patients.

  6. Toxic metals and autophagy.

    PubMed

    Chatterjee, Sarmishtha; Sarkar, Shuvasree; Bhattacharya, Shelley

    2014-11-17

    The earth's resources are finite, and it can no longer be considered a source of inexhaustible bounty for the human population. However, this realization has not been able to contain the human desire for rapid industrialization. The collateral to overusing environmental resources is the high-level contamination of undesirable toxic metals, leading to bioaccumulation and cellular damage. Cytopathological features of biological systems represent a key variable in several diseases. A review of the literature revealed that autophagy (PCDII), a high-capacity process, may consist of selective elimination of vital organelles and/or proteins that intiate mechanisms of cytoprotection and homeostasis in different biological systems under normal physiological and stress conditions. However, the biological system does survive under various environmental stressors. Currently, there is no consensus that specifies a particular response as being a dependable biomarker of toxicology. Autophagy has been recorded as the initial response of a cell to a toxic metal in a concentration- and time-dependent manner. Various signaling pathways are triggered through cellular proteins and/or protein kinases that can lead to autophagy, apoptosis (or necroptosis), and necrosis. Although the role of autophagy in tumorigenesis is associated with promoting tumor cell survival and/or acting as a tumor suppressive mechanism, PCDII in metal-induced toxicity has not been extensively studied. The aim of this review is to analyze the comparative cytotoxicity of metals/metalloids and nanoparticles (As, Cd, Cr, Hg, Fe, and metal-NP) in cells enduring autophagy. It is noted that metals/metalloids and nanoparticles prefer ATG8/LC3 as a potent inducer of autophagy in several cell lines or animal cells. MAP kinases, death protein kinases, PI3K, AKT, mTOR, and AMP kinase have been found to be the major components of autophagy induction or inhibition in the context of cellular responses to metals/metalloids and

  7. Methanol extract of Ocimum gratissimum protects murine peritoneal macrophages from nicotine toxicity by decreasing free radical generation, lipid and protein damage and enhances antioxidant protection

    PubMed Central

    Mahapatra, Santanu Kar; Chakraborty, Subhankari Prasad; Das, Subhasis

    2009-01-01

    In the present study, methanol extract of Ocimum gratissimum Linn (ME-Og) was tested against nicotine-induced murine peritoneal macrophage in vitro. Phytochemical analysis of ME-Og shown high amount of flavonoid and phenolic compound present in it. The cytotoxic effect of ME-Og was studied in murine peritoneal macrophages at different concentrations (0.1 to 100 µg/ml) using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide (MTT) method. To establish the protective role of ME-Og against nicotine toxicity, peritoneal macrophages from mice were treated with nicotine (10 mM), nicotine + ME-Og (1 to 25 µg/ml) for 12 h in culture media. The significantly (p < 0.05) increased super oxide anion generation, reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, myeloperoxidase (MPO) activity, lipid peroxidation, protein carbonyls, oxidized glutathione levels were observed in nicotine-treated group as compared to control group; those were significantly (p < 0.05) reduced in ME-Og supplemented groups in concentration dependent manner. More over, significantly (p < 0.05) reduced antioxidant status due to nicotine exposure was effectively ameliorated by ME-Og supplementation in murine peritoneal macrophages. Among the different concentration of ME-Og, maximum protective effect was observed by 25 µg/ml, which does not produce significant cell cytotoxicity in murine peritoneal macrophages. These findings suggest the potential use and beneficial role of O. gratissimum as a modulator of nicotine-induced free radical generation, lipid-protein damage and antioxidant status in important immune cell, peritoneal macrophages. PMID:20716908

  8. Applications of nano-baskets in drug development: high solubility and low toxicity.

    PubMed

    Mokhtari, Bahram; Pourabdollah, Kobra

    2013-01-01

    The importance of calixarene nanobaskets in drug development and controlling their toxicity comes back to their ability to encapsulate the drug species and biological compounds. New complexes show improved physical characteristics, such as more solubility in water and enhanced chemical properties, including less toxicity in biological systems. Moreover, the covalent bonding, inclusion encapsulation, and induction activities of calixarenes lead to improving the activity of drug molecules. This review article deals with the importance of calixarene-based drugs and illustrates their potentials in drug development and toxicity control. The main aim and novelty of this study was to introduce the role and the action of calixarene scaffolds and their conjugated upper and lower rims as well as their binding mechanism in biomedical systems. The main aspects of this approach are classified in four categories and are discussed according to the results of recent researches.

  9. High durability and low toxicity antimicrobial coatings fabricated by quaternary ammonium silane copolymers.

    PubMed

    Li, Hairui; Bao, Hongqian; Bok, Ke Xin; Lee, Chi-Ying; Li, Bo; Zin, Melvin T; Kang, Lifeng

    2016-02-01

    Adhesion and subsequent growth of microorganisms on material surfaces are a major concern in many biomedical applications. Currently, various polymers are immobilized on material surfaces to prevent microbial colonization. However, there are several challenges with regard to the coating materials, including their inability to kill microorganisms, complexity of surface grafting, limited durability and toxicity towards humans. To address these challenges, we synthesize a novel quaternary ammonium silane (QAS) antimicrobial copolymer to confer the antimicrobial effect via a simple thermal-curing coating process. The QAS copolymers were less toxic to 3 human cell lines than a commercial antimicrobial QAS monomeric agent, namely, dimethyloctadecyl[3-(trimethoxysilyl) propyl]ammonium chloride (DTPAC). Moreover, the QAS coatings demonstrated superior antimicrobial efficacy and durability than those of the DTPAC coatings. In conclusion, the novel QAS copolymers are useful to prevent substrates from microbial infections, yet with low toxicity to humans and long durability. In addition, the synthetic process is potentially scalable for industrial applications.

  10. Effects of high external electric fields on protein conformation

    NASA Astrophysics Data System (ADS)

    Pompa, Pier Paolo; Bramanti, Alessandro; Maruccio, Giuseppe; del Mercato, Loretta Laureana; Chiuri, Rocco; Cingolani, Roberto; Rinaldi, Ross

    2005-06-01

    Resistance of biomolecules to high electric fields is a main concern for nanobioelectronics/nanobiosensing applications, and it is also a relevant issue from a fundamental perspective, to understand the dielectric properties and structural dynamics of proteins. In nanoscale devices, biomolecules may experience electric fields as high as 107 V/m in order to elicit charge transport/transfer. Understanding the effects of such fields on their structural integrity is thus crucial to assess the reliability of biomolecular devices. In this study, we show experimental evidence for the retention of native-like fold pattern by proteins embedded in high electric fields. We have tested the metalloprotein azurin, deposited onto SiO2 substrates in air with proper electrode configuration, by applying high static electric fields (up to 106-107 V/m). The effects on the conformational properties of protein molecules have been determined by means of intrinsic fluorescence measurements. Experimental results indicate that no significant field-induced conformational alteration occurs. This behavior is also discussed and supported by theoretical predictions of the intrinsic intra-protein electric fields. As the general features of such inner fields are not peculiar of azurin, the conclusions presented here should have general validity.

  11. Structural basis for the enhanced stability of highly fluorinated proteins

    PubMed Central

    Buer, Benjamin C.; Meagher, Jennifer L.; Stuckey, Jeanne A.; Marsh, E. Neil G.

    2012-01-01

    Noncanonical amino acids have proved extremely useful for modifying the properties of proteins. Among them, extensively fluorinated (fluorous) amino acids seem particularly effective in increasing protein stability; however, in the absence of structural data, the basis of this stabilizing effect remains poorly understood. To address this problem, we solved X-ray structures for three small proteins with hydrophobic cores that are packed with either fluorocarbon or hydrocarbon side chains and compared their stabilities. Although larger, the fluorinated residues are accommodated within the protein with minimal structural perturbation, because they closely match the shape of the hydrocarbon side chains that they replace. Thus, stability increases seem to be better explained by increases in buried hydrophobic surface area that accompany fluorination than by specific fluorous interactions between fluorinated side chains. This finding is illustrated by the design of a highly fluorinated protein that, by compensating for the larger volume and surface area of the fluorinated side chains, exhibits similar stability to its nonfluorinated counterpart. These structure-based observations should inform efforts to rationally modulate protein function using noncanonical amino acids. PMID:22411812

  12. An assessment of the behavioral toxicity of high-energy iron particles compared to other qualities of radiation

    SciTech Connect

    Rabin, B.M.; Hunt, W.A.; Joseph, J.A. )

    1989-07-01

    Conditioned taste aversion was used to evaluate the behavioral toxicity of exposure to high-energy iron particles ({sup 56}Fe, 600 MeV/amu) in comparison to that of gamma photons ({sup 60}Co), high-energy electrons, or fission neutrons. Exposure to high-energy iron particles (5-500 cGy) produced a dose-dependent taste aversion with a maximal effect achieved with a dose of 30 cGy. Gamma photons and electrons were the least effective stimuli for producing a conditioned taste aversion, with a maximal aversion obtained only after exposure to 500 cGy, while the effectiveness of fission neutrons was intermediate to that of photons and iron particles, and a maximal aversion was obtained with a dose of 100 cGy. In the second experiment, rats with lesions of the area postrema were exposed to iron particles (30 cGy), but failed to acquire a taste aversion. The results indicate that (1) high-energy iron particles are more toxic than other qualities of radiation and (2) similar mechanisms mediate the behavioral toxicity of gamma photons and high-energy iron particles.

  13. Assessment of the behavioral toxicity of high-energy iron particles compared to other qualities of radiation

    SciTech Connect

    Rabin, B.M.; Hunt, W.A.; Joseph, J.A.

    1989-01-01

    Conditioned taste aversion was used to evaluate the behavioral toxicity of exposure to high-energy iron particles (56Fe, 600 MeV/amu) in comparison to that of gamma photons (60 Co), high-energy electrons, or fission neutrons. Exposure to high-energy iron particles (5-500 cGy) produced a dose-dependent taste aversion with a maximal effect achieved with a dose of 30 cGy. Gamma photons and electrons were the least-effective stimuli for producing a conditioned taste aversion, with a maximal aversion obtained only after exposure to 500 cGy, while the effectiveness of fission neutrons was intermediate to that of photons and iron particles, and a maximal aversion was obtained with a dose of 100 CGy. In the second experiment, rats with lesions of the area postrema were exposed to iron particles (3- cGY), but failed to acquire a taste aversion. The results indicate that (1) high-energy iron particles are more toxic than other qualities of radiation and (2) similar mechanisms mediate the behavioral toxicity of gamma photons and high-energy iron particles.

  14. The effects of taurine on vigabatrin, high light intensity and mydriasis induced retinal toxicity in the pigmented rat.

    PubMed

    Rasmussen, Allan D; Truchot, Nathalie; Pickersgill, Nigel; Thale, Zia Irene; Rosolen, Serge G; Botteron, Catherine

    2015-01-01

    The overall purpose of this study was to establish a model that may be used for examining the effect of Vigabatrin-induced retinal toxicity in pigmented rats, and subsequently examine the possible effects of taurine on the retinal toxicity. In the first part of the study, pigmented Long Evans rats were subjected to combinations of induced mydriasis, low/high light intensities (40/2000 lx) and oral administration of near-MTD (Maximum Tolerated Dose) doses (200 mg/kg/day) of Vigabatrin for up to 6 weeks. The combination of mydriasis and high light intensity applied to Long Evans rats resulted in retinal damage that was increased by the administration of Vigabatrin. In the second part of the study Long Evans rats were subjected to combinations of induced mydriasis and high/low light intensity (40/2000 lx) while being orally administered low (30 mg/kg/day) or high (200 mg/kg/day) doses of Vigabatrin for up to 6 weeks. In addition, selected groups of animals were administered taurine via the drinking water (20 mg/ml), resulting in systemic taurine concentrations of approximately threefold the endogenous concentration. The combined results of the studies demonstrate that retinal damage can be induced in pigmented animals when combining mydriasis and high light intensity. Retinal damage was functionally evaluated by electroretinography (ERG), then confirmed by histopathology. While depending on mydriasis and high light intensity, administration of Vigabatrin increased the retinal toxicity and resulted in the formation of rosette-like structures in the retina in a dose-related manner. Administration of taurine did not alleviate the Vigabatrin-induced retinal toxicity, as demonstrated either functionally by ERG or morphologically, although systemic concentrations of 3-fold the endogenous levels were reached, and it was thus not possible to demonstrate a protective effect of taurine in these pigmented animals. PMID:25446799

  15. High-throughput protein crystallography and drug discovery.

    PubMed

    Tickle, Ian; Sharff, Andrew; Vinkovic, Mladen; Yon, Jeff; Jhoti, Harren

    2004-10-20

    Single crystal X-ray diffraction is the technique of choice for studying the interactions of small organic molecules with proteins by determining their three-dimensional structures; however the requirement for highly purified protein and lack of process automation have traditionally limited its use in this field. Despite these shortcomings, the use of crystal structures of therapeutically relevant drug targets in pharmaceutical research has increased significantly over the last decade. The application of structure-based drug design has resulted in several marketed drugs and is now an established discipline in most pharmaceutical companies. Furthermore, the recently published full genome sequences of Homo sapiens and a number of micro-organisms have provided a plethora of new potential drug targets that could be utilised in structure-based drug design programs. In order to take maximum advantage of this explosion of information, techniques have been developed to automate and speed up the various procedures required to obtain protein crystals of suitable quality, to collect and process the raw X-ray diffraction data into usable structural information, and to use three-dimensional protein structure as a basis for drug discovery and lead optimisation. This tutorial review covers the various technologies involved in the process pipeline for high-throughput protein crystallography as it is currently being applied to drug discovery. It is aimed at synthetic and computational chemists, as well as structural biologists, in both academia and industry, who are interested in structure-based drug design.

  16. Approaches to High-Performance Preparative Chromatography of Proteins

    NASA Astrophysics Data System (ADS)

    Sun, Yan; Liu, Fu-Feng; Shi, Qing-Hong

    Preparative liquid chromatography is widely used for the purification of chemical and biological substances. Different from high-performance liquid chromatography for the analysis of many different components at minimized sample loading, high-performance preparative chromatography is of much larger scale and should be of high resolution and high capacity at high operation speed and low to moderate pressure drop. There are various approaches to this end. For biochemical engineers, the traditional way is to model and optimize a purification process to make it exert its maximum capability. For high-performance separations, however, we need to improve chromatographic technology itself. We herein discuss four approaches in this review, mainly based on the recent studies in our group. The first is the development of high-performance matrices, because packing material is the central component of chromatography. Progress in the fabrication of superporous materials in both beaded and monolithic forms are reviewed. The second topic is the discovery and design of affinity ligands for proteins. In most chromatographic methods, proteins are separated based on their interactions with the ligands attached to the surface of porous media. A target-specific ligand can offer selective purification of desired proteins. Third, electrochromatography is discussed. An electric field applied to a chromatographic column can induce additional separation mechanisms besides chromatography, and result in electrokinetic transport of protein molecules and/or the fluid inside pores, thus leading to high-performance separations. Finally, expanded-bed adsorption is described for process integration to reduce separation steps and process time.

  17. High pressure-low temperature processing of food proteins.

    PubMed

    Dumay, Eliane; Picart, Laetitia; Regnault, Stéphanie; Thiebaud, Maryse

    2006-03-01

    High pressure-low temperature (HP-LT) processing is of interest in the food field in view of: (i) obtaining a "cold" pasteurisation effect, the level of microbial inactivation being higher after pressurisation at low or sub-zero than at ambient temperature; (ii) limiting the negative impact of atmospheric pressure freezing on food structures. The specific effects of freezing by fast pressure release on the formation of ice I crystals have been investigated on oil in water emulsions stabilized by proteins, and protein gels, showing the formation of a high number of small ice nuclei compared to the long needle-shaped crystals obtained by conventional freezing at 0.1 MPa. It was therefore of interest to study the effects of HP-LT processing on unfolding or dissociation/aggregation phenomena in food proteins, in view of minimizing or controlling structural changes and aggregation reactions, and/or of improving protein functional properties. In the present studies, the effects of HP-LT have been investigated on protein models such as (i) beta-lactoglobulin, i.e., a whey protein with a well known 3-D structure, and (ii) casein micelles, i.e., the main milk protein components, the supramolecular structure of which is not fully elucidated. The effects of HP-LT processing was studied up to 300 MPa at low or sub-zero temperatures and after pressure release, or up to 200 MPa by UV spectroscopy under pressure, allowing to follow reversible structural changes. Pressurisation of approximately 2% beta-lactoglobulin solutions up to 300 MPa at low/subzero temperatures minimizes aggregation reactions, as measured after pressure release. In parallel, such low temperature treatments enhanced the size reduction of casein micelles.

  18. Highly Effective, Low Toxicity, Low Environmental Impact Total Flooding Fire Suppressants

    NASA Technical Reports Server (NTRS)

    Glass, S. M.; Dhooge, P. M.; Nimitz, J. S.

    2001-01-01

    Halon 1301 (CF3Br) has been used for decades as the primary fire suppression agent for areas where powder agents cannot be used because of concerns for sensitive equipment. Halon 1301 is an excellent extinguishing agent, effective at about 3% in air and quite non-toxic. It has an effective exposure limit much greater than its extinguishing concentration, so it can be used in normally occupied areas. The ability of a chemical to destroy stratospheric ozone is its ozone-depletion potential (ODP). ODP is the amount of ozone destroyed per pound of a chemical, relative to the standard CFC-11 with an ODP = 1.0. Because halons have been implicated in stratospheric ozone depletion, their production was stopped at the end of 1995 under the provisions of the Montreal Protocol plus later amendments. In the US, the Clean Air Act Amendments of 1990, Presidential directives, and DoD Directive 6050.9 implemented this phaseout. These regulations and penalties have provided strong incentives for US businesses to decrease CFC use. The Omnibus Budget Reconciliation Act of 1989 mandates high Federal taxes on CFCs and halons, designed to price them out of the market. The taxes also capture for the government the windfall profits that would otherwise go to producers as scarcity drives up prices. Several replacements have been developed for Halon 1301. One is carbon dioxide, which has been used as a firefighting agent for many years. However, a high concentration of carbon dioxide is necessary to inert fuels. The effective concentration for inerting with carbon dioxide is approximately 29%, which is above the concentration lethal to humans. HFC-227ea is being used extensively to replace Halon 1301 systems in nominally occupied areas and some normally unoccupied areas. However, since the effective concentration of HFC-227ea is about three to four times that of Halon 1301 the extinguishing systems have to be larger and new extinguishing systems have to be installed. HFC-125 is also being

  19. Toxic myopathies.

    PubMed

    Pasnoor, Mamatha; Barohn, Richard J; Dimachkie, Mazen M

    2014-08-01

    Muscle tissue is highly sensitive to many substances. Early recognition of toxic myopathies is important, because they potentially are reversible on removal of the offending drug or toxin, with greater likelihood of complete resolution the sooner this is achieved. Clinical features range from mild muscle pain and cramps to severe weakness with rhabdomyolysis, renal failure, and even death. The pathogenic bases can be multifactorial. This article reviews some of the common toxic myopathies and their clinical presentation, histopathologic features, and possible underlying cellular mechanisms.

  20. Toxic Myopathies

    PubMed Central

    Pasnoor, Mamatha; Barohn, Richard J.; Dimachkie, Mazen M.

    2014-01-01

    Muscle tissue is highly sensitive to many substances. Early recognition of toxic myopathies is important, as they potentially are reversible on removal of the offending drug or toxin, with greater likelihood of complete resolution the sooner this is achieved. Clinical features range from mild muscle pain and cramps to severe weakness with rhabdomyolysis, renal failure, and even death. The pathogenic bases can be multifactorial. This article reviews some of the common toxic myopathies and their clinical presentation, histopathologic features and possible underlying cellular mechanisms. PMID:25037083

  1. Protein-directed modulation of high-LET hyperthermic radiosensitization

    SciTech Connect

    Chang, P.Y.

    1991-01-01

    A pair of Chinese Hamster Ovary cell lines, the wild-type CHO-SC1, and its temperature-sensitive mutant (CHO-tsH1) was used to examine the importance of protein synthesis in the development of thermotolerance. The classical biphasic thermotolerant survival response to hyperthermia was observed in the SC1 cells after continuous heating at 41.5C to 42.5C, while tsH1 showed no thermotolerance. In separate experiments, each cell line was triggered and challenged at 45C. The heat doses were separated with graded incubaton periods at 35C or 40C for thermotolerance development. SC1 cells expressed thermoresistance, with the synthesis of heat shock proteins, under both incubation conditions. tsH1 cells expressed thermotolerance similar to that seen in the SC1 cells when incubated at 35C, but the survival response with the non-permissive 40C incubation was much reduced in the absence of protein synthesis. The combined effects of heavy-ion radiation and hyperthermia were examined using the same cell system. A mild heat dose of 41.5C was used in conjunction with Neon particle radiation of various high LET values. The cell killing effects were highly dependent on the sequence of application of heat and Neon radiation. Heat applied immediately after Neon irradiation was more cytotoxic to SC1 cells than when heat was applied prior to the irradiation. The ability of cells to synthesize new proteins plays a key role in this sequence-dependent thermal radiosensitization. In the absence of protein synthesis in the tsH1 cells, the high-LET thermal enhancement for cell-killing was unchanged regardless of the sequence. In the presence of protein synthetic activity in the SC1 cells, the thermal enhancement of radiation-induced cell killing was LET-dependent.

  2. Melatonin prevents cytosolic calcium overload, mitochondrial damage and cell death due to toxically high doses of dexamethasone-induced oxidative stress in human neuroblastoma SH-SY5Y cells.

    PubMed

    Suwanjang, Wilasinee; Abramov, Andrey Y; Charngkaew, Komgrid; Govitrapong, Piyarat; Chetsawang, Banthit

    2016-07-01

    Stressor exposure activates the hypothalamic-pituitary-adrenal (HPA) axis and causes elevations in the levels of glucocorticoids (GC) from the adrenal glands. Increasing evidence has demonstrated that prolonged exposure to high GC levels can lead to oxidative stress, calcium deregulation, mitochondrial dysfunction and apoptosis in a number of cell types. However, melatonin, via its antioxidant activity, exhibits a neuroprotective effect against oxidative stress-induced cell death. Therefore, in the present study, we explored the protective effect of melatonin in GC-induced toxicity in human neuroblastoma SH-SY5Y cells. Cellular treatment with the toxically high doses of the synthetic GC receptor agonist, dexamethasone (DEX) elicited marked decreases in the levels of glutathione and increases in ROS production, lipid peroxidation and cell death. DEX toxicity also induced increases in the levels of cytosolic calcium and mitochondrial fusion proteins (Mfn1 and Opa1) but decreases in the levels of mitochondrial fission proteins (Fis1 and Drp1). Mitochondrial damage was observed in large proportions of the DEX-treated cells. Pretreatment of the cells with melatonin substantially prevented the DEX-induced toxicity. These results suggest that melatonin might exert protective effects against oxidative stress, cytosolic calcium overload and mitochondrial damage in DEX-induced neurotoxicity. PMID:27155536

  3. Influence of genetic polymorphisms in the folate pathway on toxicity after high-dose methotrexate treatment in pediatric osteosarcoma

    PubMed Central

    Park, Jeong A

    2016-01-01

    Background Methotrexate (MTX), one of the main drugs used to treat osteosarcoma, is a representative folic acid antagonist. Polymorphisms of various enzymes involved in the metabolism of MTX could contribute to differences in response to MTX in pediatric osteosarcoma patients. Methods Blood and tissue samples were obtained from 37 pediatric osteosarcoma patients who were treated with high-dose MTX therapy. The following 4 single nucleotide polymorphisms (SNPs) were analyzed: ATIC 347C>G, MTHFR 677C>T, MTHFR 1298A>C and SLC19A1 80G>A. Serial plasma MTX concentrations after high-dose MTX therapy and MTX-induced toxicities were evaluated. Correlations among polymorphisms, MTX concentrations and treatment-induced toxicities were assessed. Results Plasma MTX levels at 48 hours after high-dose MTX infusion were significantly associated with SLC19A1 80G>A (P=0.031). Higher plasma levels of MTX at 48 and 72 hours were significantly associated with MTX-induced mucositis (P=0.007 and P=0.046) and renal toxicity (P=0.002), respectively. SNP of SLC19A1 gene was associated with development of severe mucositis (P=0.026). Conclusion This study suggests that plasma levels of MTX are associated with GI and renal toxicities after high-dose MTX therapy, and genetic polymorphisms that affect the metabolism of MTX may influence drug concentrations and development of significant side effects in pediatric patients treated with high-dose MTX. PMID:27104192

  4. High-throughput functional genomics identifies genes that ameliorate toxicity due to oxidative stress in neuronal HT-22 cells: GFPT2 protects cells against peroxide.

    PubMed

    Zitzler, Jürgen; Link, Dieter; Schäfer, Rolf; Liebetrau, Wolfgang; Kazinski, Michael; Bonin-Debs, Angelika; Behl, Christian; Buckel, Peter; Brinkmann, Ulrich

    2004-08-01

    We describe a novel genetic screen that is performed by transfecting every individual clone of an expression clone collection into a separate population of cells in a high-throughput mode. We combined high-throughput functional genomics with experimental validation to discover human genes that ameliorate cytotoxic responses of neuronal HT-22 cells upon exposure to oxidative stress. A collection of 5,000 human cDNAs in mammalian expression vectors were individually transfected into HT-22 cells, which were then exposed to H(2)O(2). Five genes were found that are known to be involved in pathways of detoxification of peroxide (catalase, glutathione peroxidase-1, peroxiredoxin-1, peroxiredoxin-5, and nuclear factor erythroid-derived 2-like 2). The presence of those genes in our "hit list" validates our screening platform. In addition, a set of candidate genes was found that has not been previously described as involved in detoxification of peroxide. One of these genes, which was consistently found to reduce H(2)O(2) -induced toxicity in HT-22, was GFPT2. This gene is expressed at significant levels in the central nervous system (CNS) and encodes glutamine-fructose-6-phosphate transaminase (GFPT) 2, a rate-limiting enzyme in hexosamine biosynthesis. GFPT has recently also been shown to ameliorate the toxicity of methylmercury in Saccharomyces cerevisiae. Methylmercury causes neuronal cell death in part by protein modification as well as enhancing the production of reactive oxygen species (ROS). The protective effect of GFPT2 against H(2)O(2) toxicity in neuronal HT-22 cells may be similar to its protection against methylmercury in yeast. Thus, GFPT appears to be conserved among yeast and men as a critical target of methylmercury and ROS-induced cytotoxicity.

  5. A Preliminary Study on the Toxic Combustion Products Testing of Polymers Used in High-Pressure Oxygen Systems

    NASA Technical Reports Server (NTRS)

    Hshieh, Fu-Yu; Beeson, Harold D.

    2004-01-01

    One likely cause of polymer ignition in a high-pressure oxygen system is adiabatic-compression heating of polymers caused by pneumatic impact. Oxidative _ pyrolysis or combustion of polymers in a high-pressure oxygen system could generate toxic gases. This paper reports the preliminary results of toxic combustion product testing of selected polymers in a pneumatic-impact test system. Five polymers commonly used in high-pressure oxygen systems, Nylon 6/6, polychlorotrifluoroethylene (CTFE), polytetrafluoroethylene (PTFE), fluoroelastomer (Viton(TradeMark) A), and nitrile rubber (Buna N), were tested in a pneumatic-impact test system at 2500- or 3500-psia oxygen pressure. The polymers were ignited and burned, then combustion products were collected in a stainless-steel sample bottle and analyzed by GC/MS/IRD, GC/FID, and GC/Methanizer/FID. The results of adiabatic-compression tests show that combustion of hydrocarbon polymers, nitrogen-containing polymers, and halogenated polymers in high-pressure oxygen systems are relatively complete. Toxicity of the combustion product gas is presumably much lower than the combustion product gas generated from ambient-pressure oxygen (or air) environments. The NASA-Lewis equilibrium code was used to determine the composition of combustion product gas generated from a simulated, adiabatic-compression test of nine polymers. The results are presented and discussed.

  6. Toxicity of reduced nitrogen in eelgrass (Zostera marina) is highly dependent on shoot density and pH.

    PubMed

    van der Heide, T; Smolders, A J P; Rijkens, B G A; van Nes, E H; van Katwijk, M M; Roelofs, J G M

    2008-12-01

    In sheltered, eutrophicated estuaries, reduced nitrogen (NHx), and pH levels in the water layer can be greatly enhanced. In laboratory experiments, we studied the interactive effects of NHx, pH, and shoot density on the physiology and survival of eelgrass (Zostera marina). We tested long-term tolerance to NHx at pH 8 in a 5-week experiment. Short-term tolerance was tested for two shoot densities at both pH 8 and 9 in a 5-day experiment. At pH 8, eelgrass accumulated nitrogen as free amino acids when exposed to high loads of NHx, but showed no signs of necrosis. Low shoot density treatments became necrotic within days when exposed to NHx at pH 9. Increased NH3 intrusion and carbon limitation seemed to be the cause of this, as intracellular NHx could no longer be assimilated. Remarkably, experiments with high shoot densities at pH 9 showed hardly any necrosis, as the plants seemed to be able to alleviate the toxic effects of high NHx loads through joint NHx uptake. Our results suggest that NHx toxicity can be important in worldwide observed seagrass mass mortalities. We argue that the mitigating effect of high seagrass biomass on NHx toxicity is a positive feedback mechanism, potentially leading to alternative stable states in field conditions.

  7. Hydrogel Based 3-Dimensional (3D) System for Toxicity and High-Throughput (HTP) Analysis for Cultured Murine Ovarian Follicles.

    PubMed

    Zhou, Hong; Malik, Malika Amattullah; Arab, Aarthi; Hill, Matthew Thomas; Shikanov, Ariella

    2015-01-01

    Various toxicants, drugs and their metabolites carry potential ovarian toxicity. Ovarian follicles, the functional unit of the ovary, are susceptible to this type of damage at all stages of their development. However, despite of the large scale of potential negative impacts, assays that study ovarian toxicity are limited. Exposure of cultured ovarian follicles to toxicants of interest served as an important tool for evaluation of toxic effects for decades. Mouse follicles cultured on the bottom of a culture dish continue to serve an important approach for mechanistic studies. In this paper, we demonstrated the usefulness of a hydrogel based 3-dimensional (3D) mouse ovarian follicle culture as a tool to study ovarian toxicity in a different setup. The 3D in vitro culture, based on fibrin alginate interpenetrating network (FA-IPN), preserves the architecture of the ovarian follicle and physiological structure-function relationship. We applied the novel 3D high-throughput (HTP) in vitro ovarian follicle culture system to study the ovotoxic effects of an anti-cancer drug, Doxorobucin (DXR). The fibrin component in the system is degraded by plasmin and appears as a clear circle around the encapsulated follicle. The degradation area of the follicle is strongly correlated with follicle survival and growth. To analyze fibrin degradation in a high throughput manner, we created a custom MATLAB® code that converts brightfield micrographs of follicles encapsulated in FA-IPN to binary images, followed by image analysis. We did not observe any significant difference between manually processed images to the automated MATLAB® method, thereby confirming that the automated program is suitable to measure fibrin degradation to evaluate follicle health. The cultured follicles were treated with DXR at concentrations ranging from 0.005 nM to 200 nM, corresponding to the therapeutic plasma levels of DXR in patients. Follicles treated with DXR demonstrated decreased survival rate in

  8. Hydrogel Based 3-Dimensional (3D) System for Toxicity and High-Throughput (HTP) Analysis for Cultured Murine Ovarian Follicles

    PubMed Central

    Zhou, Hong; Malik, Malika Amattullah; Arab, Aarthi; Hill, Matthew Thomas; Shikanov, Ariella

    2015-01-01

    Various toxicants, drugs and their metabolites carry potential ovarian toxicity. Ovarian follicles, the functional unit of the ovary, are susceptible to this type of damage at all stages of their development. However, despite of the large scale of potential negative impacts, assays that study ovarian toxicity are limited. Exposure of cultured ovarian follicles to toxicants of interest served as an important tool for evaluation of toxic effects for decades. Mouse follicles cultured on the bottom of a culture dish continue to serve an important approach for mechanistic studies. In this paper, we demonstrated the usefulness of a hydrogel based 3-dimensional (3D) mouse ovarian follicle culture as a tool to study ovarian toxicity in a different setup. The 3D in vitro culture, based on fibrin alginate interpenetrating network (FA-IPN), preserves the architecture of the ovarian follicle and physiological structure-function relationship. We applied the novel 3D high-throughput (HTP) in vitro ovarian follicle culture system to study the ovotoxic effects of an anti-cancer drug, Doxorobucin (DXR). The fibrin component in the system is degraded by plasmin and appears as a clear circle around the encapsulated follicle. The degradation area of the follicle is strongly correlated with follicle survival and growth. To analyze fibrin degradation in a high throughput manner, we created a custom MATLAB® code that converts brightfield micrographs of follicles encapsulated in FA-IPN to binary images, followed by image analysis. We did not observe any significant difference between manually processed images to the automated MATLAB® method, thereby confirming that the automated program is suitable to measure fibrin degradation to evaluate follicle health. The cultured follicles were treated with DXR at concentrations ranging from 0.005 nM to 200 nM, corresponding to the therapeutic plasma levels of DXR in patients. Follicles treated with DXR demonstrated decreased survival rate in

  9. Evaluating the Toxicity Pathways Using High-Throughput Environmental Chemical Data

    EPA Science Inventory

    The application of HTS methods to the characterization of human phenotypic response to environmental chemicals is a largely unexplored area of pharmacogenomics. The U.S. Environmental Protection Agency (EPA), through its ToxCast program, is developing predictive toxicity approach...

  10. Cadmium toxicity in diazotrophic Anabaena spp. adjudged by hasty up-accumulation of transporter and signaling and severe down-accumulation of nitrogen metabolism proteins.

    PubMed

    Singh, Prashant Kumar; Shrivastava, Alok Kumar; Chatterjee, Antra; Pandey, Sarita; Rai, Snigdha; Singh, Shilpi; Rai, L C

    2015-09-01

    Present study demonstrates interspecies variation in proteome and survival strategy of three Anabaena species i.e., Anabaena L31, Anabaena sp. PCC 7120 and Anabaena doliolum subjected to respective LC50 doses of Cd at 0, 1, 3, 5 and 7day intervals. The proteome coverage with 452 differentially accumulated proteins unveiled species and time specific expression and interaction network of proteins involved in important cellular functions. Statistical analysis of protein abundance across Cd-treated proteomes clustered their co-expression pattern into four groups viz., (i) early (days 1 and 3) accumulated proteins, (ii) proteins up-accumulated for longer duration, (iii) late (days 5 and 7) accumulated proteins, and (iv) mostly down-accumulated proteins. Appreciable growth of Cd treated A L31 over other two species may be ascribed to proteins contained in the first and second groups (belonging to energy and carbohydrate metabolism (TK, G6-PI, PGD, FBA, PPA, ATP synthase)), sulfur metabolism (GR, GST, PGDH, PAPS reductase, GDC-P, and SAM synthetase), fatty acid metabolism (AspD, PspA, SQD-1), phosphorous metabolism (PhoD, PstB and SQD1), molecular chaperones (Gro-EL, FKBP-type peptidylprolyl isomerase), and antioxidative defense enzymes (SOD-A, catalase). Anabaena sp. PCC 7120 harboring proteins largely from the third group qualified as a late accumulator and A. doliolum housing majority of proteins from the fourth group emerged as the most sensitive species. Thus early up-accumulation of transporter and signaling category proteins and drastic reduction of nitrogen assimilation proteins could be taken as a vital indicator of cadmium toxicity in Anabaena spp. This article is part of a Special Issue entitled: Proteomics in India.

  11. High Pressure Cryocooling of Protein Crystals: The Enigma of Water

    NASA Astrophysics Data System (ADS)

    Gruner, Sol M.

    2010-03-01

    A novel high-pressure cryocooling technique for preparation biological samples for x-ray analysis is described. The method, high-pressure cryocooling, involves cooling samples to cryogenic temperatures (e.g., 100 K) in high-pressure Helium gas (up to 200 MPa). It bears both similarities and differences to high-pressure cooling methods that have been used to prepare samples for electron microscopy, and has been especially useful for cryocooling of macromolecular crystals for x-ray diffraction. Examples will be given where the method has been effective in providing high quality crystallographic data for difficult samples, such as cases where ligands needed to be stabilized in binding sites to be visualized, or where very high resolution data were required. The talk concludes with a discussion of data obtained by high-pressure cryocooling that pertains to two of the most important problems in modern science: the enigma of water and how water affects the activity of proteins.

  12. Nanosecond Responses of Proteins to Ultra-High Temperature Pulses

    PubMed Central

    Steel, Bradley C.; McKenzie, David R.; Bilek, Marcela M. M.; Nosworthy, Neil J.; dos Remedios, Cristobal G.

    2006-01-01

    Observations of fast unfolding events in proteins are typically restricted to <100°C. We use a novel apparatus to heat and cool enzymes within tens of nanoseconds to temperatures well in excess of the boiling point. The nanosecond temperature spikes are too fast to allow water to boil but can affect protein function. Spikes of 174°C for catalase and ∼290°C for horseradish peroxidase are required to produce irreversible loss of enzyme activity. Similar temperature spikes have no effect when restricted to 100°C or below. These results indicate that the “speed limit” for the thermal unfolding of large proteins is shorter than 10−8 s. The unfolding rate at high temperature is consistent with extrapolation of low temperature rates over 12 orders of magnitude using the Arrhenius relation. PMID:16844754

  13. Reproductive toxicity in rats with crystal nephropathy following high doses of oral melamine or cyanuric acid.

    PubMed

    Stine, Cynthia B; Reimschuessel, Renate; Keltner, Zachary; Nochetto, Cristina B; Black, Thomas; Olejnik, Nicholas; Scott, Michael; Bandele, Omari; Nemser, Sarah M; Tkachenko, Andriy; Evans, Eric R; Crosby, Tina C; Ceric, Olgica; Ferguson, Martine; Yakes, Betsy J; Sprando, Robert

    2014-06-01

    The industrial chemical melamine was used in 2007 and 2008 to raise the apparent protein content in pet feed and watered down milk, respectively. Because humans may be exposed to melamine via several different routes into the human diet as well as deliberate contamination, this study was designed to characterize the effect of high dose melamine or cyanuric acid oral exposure on the pregnant animal and developing fetus, including placental transfer. Clear rectangular crystals formed following a single triazine exposure which is a different morphology from the golden spherulites caused by combined exposure or the calculi formed when melamine combines with endogenous uric acid. Crystal nephropathy, regardless of cause, induces renal failure which in turn has reproductive sequelae. Specifically, melamine alone-treated dams had increased numbers of early and late fetal deaths compared to controls or cyanuric acid-treated dams. As melamine was found in the amniotic fluid, this study confirms transfer of melamine from mammalian mother to fetus and our study provides evidence that cyanuric acid also appears in the amniotic fluid if mothers are exposed to high doses.

  14. [Determination of serum proteins by high performance capillary zone electrophoresis].

    PubMed

    Zhang, N; Tang, Y; Hao, D M; Zheng, L; Qiu, G B

    1999-11-01

    The separation method of serum proteins was established with an untreared 50 microns i.d. x 47 cm (40 cm to detector) capillary and detection of absorbance at 200 nm. Analysis was performed by pressure injectction 17.23 kPa.s and by applying 23 kV in the constant voltage mode. Serum samples were diluted 40-folds with assay buffer (12.5 mmol/L sodium borate, 1 mmol/L calcium lactate, 0.7 mmol/L magnesium sulfate, 1 mmol/L EDTA were mixed). A normal control serum protein was separated into 6 fractions. In pregnant serum, the alpha 0 was an additionally unknown fraction. Comparison of capillary electrophoresis with conventional cellulose acetate electrophoresis for analysis of serum proteins from normal control, pregnant women multiple myeloma and tonic rachitis patients indicates that capillary clectrophoresis is a new technique for the analysis of serum proteins because of its high efficiency, on-line data processing and automation. Capillary electrophoresis is the reliable technique for clinical diagnosis of serum protein abnormalities.

  15. Comparison of Acute and Late Toxicities for Three Modern High-Dose Radiation Treatment Techniques for Localized Prostate Cancer

    SciTech Connect

    Mohammed, Nasiruddin; Kestin, Larry; Ghilezan, Mihai; Krauss, Daniel; Vicini, Frank; Brabbins, Donald; Gustafson, Gary; Ye Hong; Martinez, Alavaro

    2012-01-01

    Purpose: We compared acute and late genitourinary (GU) and gastrointestinal (GI) toxicities in prostate cancer patients treated with three different high-dose radiation techniques. Methods and Materials: A total of 1,903 patients with localized prostate cancer were treated with definitive RT at William Beaumont Hospital from 1992 to 2006: 22% with brachytherapy alone (BT), 55% with image-guided external beam (EB-IGRT), and 23% external beam with high-dose-rate brachytherapy boost (EBRT+HDR). Median dose with BT was 120 Gy for LDR and 38 Gy for HDR (9.5 Gy Multiplication-Sign 4). Median dose with EB-IGRT was 75.6 Gy (PTV) to prostate with or without seminal vesicles. For EBRT+HDR, the pelvis was treated to 46 Gy with an additional 19 Gy (9.5 Gy Multiplication-Sign 2) delivered via HDR. GI and GU toxicity was evaluated utilizing the NCI-CTC criteria (v.3.0). Median follow-up was 4.8 years. Results: The incidences of any acute {>=} Grade 2 GI or GU toxicities were 35%, 49%, and 55% for BT, EB-IGRT, and EBRT+HDR (p < 0.001). Any late GU toxicities {>=} Grade 2 were present in 22%, 21%, and 28% for BT, EB-IGRT, and EBRT+HDR (p = 0.01), respectively. Patients receiving EBRT+HDR had a higher incidence of urethral stricture and retention, whereas dysuria was most common in patients receiving BT. Any Grade {>=}2 late GI toxicities were 2%, 20%, and 9% for BT, EB-IGRT, and EBRT+HDR (p < 0.001). Differences were most pronounced for rectal bleeding, with 3-year rates of 0.9%, 20%, and 6% (p < 0.001) for BT, EB-IGRT, and EBRT+HDR respectively. Conclusions: Each of the three modern high-dose radiation techniques for localized prostate cancer offers a different toxicity profile. These data can help patients and physicians to make informed decisions regarding radiotherapy for prostate andenocarcinoma.

  16. High protein production of phytoplankton in the Amundsen Sea

    NASA Astrophysics Data System (ADS)

    Jung Song, Ho; Jung Kang, Jae; Kyung Kim, Bo; Joo, HuiTae; Jin Yang, Eun; Park, Jisoo; Hoon Lee, Sang; Heon Lee, Sang

    2016-01-01

    The Amundsen Sea polynya is one of the largest and most productive polynyas in the Southern Ocean and has recently experienced a rapid change in sea ice coverage. However, very little is known about current physiological status of phytoplankton and its quality as food for pelagic herbivores and consequently higher trophic levels in the Amundsen Sea. Using a 13C isotope tracer technique, macromolecular production measurements of phytoplankton at eleven stations were conducted at three light depths (100, 30, and 1%) onboard R/V ARAON in the Amundsen Sea, 2012. The concentrations of major inorganic nutrients were replete at all the productivity stations and no substantial difference in macromolecular production was found between polynya and non-polynya regions. Distinct vertical trends were not observed in low-molecular-weight metabolites (LMWM) and polysaccharide productions, but weak vertical patterns in lipid and protein productions were found during our cruise period. The vertical patterns of lipids slightly increased with depth whereas decreased for protein synthesis in this study, and these vertical trends were not consistent with the results reported previously in the Arctic Ocean. Overall, phytoplankton allocated more photosynthetic carbon into proteins (60.0%) than other macromolecules in the Amundsen Sea, which is markedly higher than those reported previously in the Antarctic Ocean, ranging from 7 to 23%. The high protein synthesis appears to be sustained by high concentrations of major nutrients, which might be a strong factor for general patterns of macromolecular productions of phytoplankton in polar oceans, even under potential iron limitation.

  17. Sterile Filtration of Highly Concentrated Protein Formulations: Impact of Protein Concentration, Formulation Composition, and Filter Material.

    PubMed

    Allmendinger, Andrea; Mueller, Robert; Huwyler, Joerg; Mahler, Hanns-Christian; Fischer, Stefan

    2015-10-01

    Differences in filtration behavior of concentrated protein formulations were observed during aseptic drug product manufacturing of biologics dependent on formulation composition. The present study investigates filtration forces of monoclonal antibody formulations in a small-scale set-up using polyvinylidene difluoride (PVDF) or polyethersulfone (PES) filters. Different factors like formulation composition and protein concentration related to differences in viscosity, as well as different filtration rates were evaluated. The present study showed that filtration behavior was influenced by the presence or absence of a surfactant in the formulation, which defines the interaction between filter membrane and surface active formulation components. This can lead to a change in filter resistance (PES filter) independent on the buffer system used. Filtration behavior was additionally defined by rheological non-Newtonian flow behavior. The data showed that high shear rates resulting from small pore sizes and filtration pressure up to 1.0 bar led to shear-thinning behavior for highly concentrated protein formulations. Differences in non-Newtonian behavior were attributed to ionic strength related to differences in repulsive and attractive interactions. The present study showed that the interplay of formulation composition, filter material, and filtration rate can explain differences in filtration behavior/filtration flux observed for highly concentrated protein formulations thus guiding filter selection.

  18. Sterile Filtration of Highly Concentrated Protein Formulations: Impact of Protein Concentration, Formulation Composition, and Filter Material.

    PubMed

    Allmendinger, Andrea; Mueller, Robert; Huwyler, Joerg; Mahler, Hanns-Christian; Fischer, Stefan

    2015-10-01

    Differences in filtration behavior of concentrated protein formulations were observed during aseptic drug product manufacturing of biologics dependent on formulation composition. The present study investigates filtration forces of monoclonal antibody formulations in a small-scale set-up using polyvinylidene difluoride (PVDF) or polyethersulfone (PES) filters. Different factors like formulation composition and protein concentration related to differences in viscosity, as well as different filtration rates were evaluated. The present study showed that filtration behavior was influenced by the presence or absence of a surfactant in the formulation, which defines the interaction between filter membrane and surface active formulation components. This can lead to a change in filter resistance (PES filter) independent on the buffer system used. Filtration behavior was additionally defined by rheological non-Newtonian flow behavior. The data showed that high shear rates resulting from small pore sizes and filtration pressure up to 1.0 bar led to shear-thinning behavior for highly concentrated protein formulations. Differences in non-Newtonian behavior were attributed to ionic strength related to differences in repulsive and attractive interactions. The present study showed that the interplay of formulation composition, filter material, and filtration rate can explain differences in filtration behavior/filtration flux observed for highly concentrated protein formulations thus guiding filter selection. PMID:26149748

  19. High dietary protein decreases fat deposition induced by high-fat and high-sucrose diet in rats.

    PubMed

    Chaumontet, Catherine; Even, Patrick C; Schwarz, Jessica; Simonin-Foucault, Angélique; Piedcoq, Julien; Fromentin, Gilles; Azzout-Marniche, Dalila; Tomé, Daniel

    2015-10-28

    High-protein diets are known to reduce adiposity in the context of high carbohydrate and Western diets. However, few studies have investigated the specific high-protein effect on lipogenesis induced by a high-sucrose (HS) diet or fat deposition induced by high-fat feeding. We aimed to determine the effects of high protein intake on the development of fat deposition and partitioning in response to high-fat and/or HS feeding. A total of thirty adult male Wistar rats were assigned to one of the six dietary regimens with low and high protein, sucrose and fat contents for 5 weeks. Body weight (BW) and food intake were measured weekly. Oral glucose tolerance tests and meal tolerance tests were performed after 4th and 5th weeks of the regimen, respectively. At the end of the study, the rats were killed 2 h after ingestion of a calibrated meal. Blood, tissues and organs were collected for analysis of circulating metabolites and hormones, body composition and mRNA expression in the liver and adipose tissues. No changes were observed in cumulative energy intake and BW gain after 5 weeks of dietary treatment. However, high-protein diets reduced by 20 % the adiposity gain induced by HS and high-sucrose high-fat (HS-HF) diets. Gene expression and transcriptomic analysis suggested that high protein intake reduced liver capacity for lipogenesis by reducing mRNA expressions of fatty acid synthase (fasn), acetyl-CoA carboxylase a and b (Acaca and Acacb) and sterol regulatory element binding transcription factor 1c (Srebf-1c). Moreover, ketogenesis, as indicated by plasma β-hydroxybutyrate levels, was higher in HS-HF-fed mice that were also fed high protein levels. Taken together, these results suggest that high-protein diets may reduce adiposity by inhibiting lipogenesis and stimulating ketogenesis in the liver.

  20. A sensitive and high throughput bacterial luminescence assay for assessing aquatic toxicity--the BLT-Screen.

    PubMed

    van de Merwe, Jason P; Leusch, Frederic D L

    2015-05-01

    Bioassays using naturally luminescent bacteria are commonly used to assess the toxicity of environmental contaminants, detected by a decrease in luminescence. Typically, this has involved the use of commercial test kits such as Microtox and ToxScreen. These commercial assays, however, have limitations for routine environmental monitoring, including the need for specialized equipment, a low throughput and high on-going costs. There is therefore a need to develop a bacteria bioassay that is sensitive, high-throughput and cost effective. This study presents the development and application of the BLT-Screen (Bacterial Luminescence Toxicity Screen), a 96-well plate bioassay using Photobacterium leiognathi. During development of the method, the concentration of the phosphate buffer in the experimental medium was adjusted to maximize the sensitivity of the assay, and protocols for analyzing both solid-phase extracts and raw water samples were established. A range of organic compounds and metals were analyzed in the assay, as well as extracts of various water samples, including drinking water, wastewater effluent and river water. The IC50 values of the organic compounds and metals tested in the BLT-Screen were comparable to previously published ToxScreen and Microtox data. In addition, the assay was sensitive enough to detect toxicity in all water types tested, and performed equally well for both solid-phase extracts and raw water samples. The BLT-Screen therefore presents a cost-effective, sensitive and high throughput method for testing the toxicity of environmental contaminants in a range of water types that has widespread applications for research, as well as for routine monitoring and operation of wastewater and drinking water plants.

  1. Automated High-Content Assay for Compounds Selectively Toxic to Trypanosoma cruzi in a Myoblastic Cell Line

    PubMed Central

    Alonso-Padilla, Julio; Cotillo, Ignacio; Presa, Jesús L.; Cantizani, Juan; Peña, Imanol; Bardera, Ana I.; Martín, Jose J.; Rodriguez, Ana

    2015-01-01

    Background Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, represents a very important public health problem in Latin America where it is endemic. Although mostly asymptomatic at its initial stage, after the disease becomes chronic, about a third of the infected patients progress to a potentially fatal outcome due to severe damage of heart and gut tissues. There is an urgent need for new drugs against Chagas disease since there are only two drugs available, benznidazole and nifurtimox, and both show toxic side effects and variable efficacy against the chronic stage of the disease. Methodology/Principal Findings Genetically engineered parasitic strains are used for high throughput screening (HTS) of large chemical collections in the search for new anti-parasitic compounds. These assays, although successful, are limited to reporter transgenic parasites and do not cover the wide T. cruzi genetic background. With the aim to contribute to the early drug discovery process against Chagas disease we have developed an automated image-based 384-well plate HTS assay for T. cruzi amastigote replication in a rat myoblast host cell line. An image analysis script was designed to inform on three outputs: total number of host cells, ratio of T. cruzi amastigotes per cell and percentage of infected cells, which respectively provides one host cell toxicity and two T. cruzi toxicity readouts. The assay was statistically robust (Z´ values >0.6) and was validated against a series of known anti-trypanosomatid drugs. Conclusions/Significance We have established a highly reproducible, high content HTS assay for screening of chemical compounds against T. cruzi infection of myoblasts that is amenable for use with any T. cruzi strain capable of in vitro infection. Our visual assay informs on both anti-parasitic and host cell toxicity readouts in a single experiment, allowing the direct identification of compounds selectively targeted to the parasite. PMID:25615687

  2. Using protein misfolding cyclic amplification generates a highly neurotoxic PrP dimer causing neurodegeneration.

    PubMed

    Yang, XiuJin; Yang, LiFeng; Zhou, XiangMei; Khan, Sher Hayat; Wang, HuiNuan; Yin, XiaoMin; Yuan, Zhen; Song, ZhiQi; Wu, WenYu; Zhao, DeMing

    2013-11-01

    Under the "protein-only" hypothesis, prion-based diseases are proposed to result from an infectious agent that is an abnormal isoform of the prion protein in the scrapie form, PrP(Sc). However, since PrP(Sc) is highly insoluble and easily aggregates in vivo, this view appears to be overly simplistic, implying that the presence of PrP(Sc) may indirectly cause neurodegeneration through its intermediate soluble form. We generated a neurotoxic PrP dimer with partial pathogenic characteristics of PrP(Sc) by protein misfolding cyclic amplification in the presence of 1-palmitoyl-2-oleoylphosphatidylglycerol consisting of recombinant hamster PrP (23-231). After intracerebral injection of the PrP dimer, wild-type hamsters developed signs of neurodegeneration. Clinical symptoms, necropsy findings, and histopathological changes were very similar to those of transmissible spongiform encephalopathies. Additional investigation showed that the toxicity is primarily related to cellular apoptosis. All results suggested that we generated a new neurotoxic form of PrP, PrP dimer, which can cause neurodegeneration. Thus, our study introduces a useful model for investigating PrP-linked neurodegenerative mechanisms.

  3. Detoxification of a highly toxic lead-loaded industrial solid waste by stabilization using apatites.

    PubMed

    Ioannidis, T A; Zouboulis, A I

    2003-02-28

    Apatites are known for their properties to immobilize lead contained in aqueous solutions or contaminated soils. In this study, apatites were examined as stabilization additives for lead-loaded industrial solid toxic wastes. The specific waste was the residue, obtained after thermal treatment of sludges (incineration), which was derived from tetraethyl lead fuel storage tanks. It was found to contain around 30 wt.% lead and 33 wt.% iron. Standard leaching tests (according to DIN 38414 S-4) were applied for the determination of leachability of metals from the ash and, thus, of chemical toxicity; the proposed leaching tests examined both initial and stabilized products in order to evaluate the effectiveness of the applied additives. The results obtained demonstrate the fact that lead concentrations in leachates, after the application of the proposed leaching tests using apatites as additives and with a ratio of 50% solid waste-50 wt.% apatite, could be reduced to the range of 1mg/l.

  4. A recombinant fusion protein containing a spider toxin specific for the insect voltage-gated sodium ion channel shows oral toxicity towards insects of different orders.

    PubMed

    Yang, Sheng; Pyati, Prashant; Fitches, Elaine; Gatehouse, John A

    2014-04-01

    Recombinant fusion protein technology allows specific insecticidal protein and peptide toxins to display activity in orally-delivered biopesticides. The spider venom peptide δ-amaurobitoxin-PI1a, which targets insect voltage-gated sodium channels, was fused to the "carrier" snowdrop lectin (GNA) to confer oral toxicity. The toxin itself (PI1a) and an amaurobitoxin/GNA fusion protein (PI1a/GNA) were produced using the yeast Pichia pastoris as expression host. Although both proteins caused mortality when injected into cabbage moth (Mamestra brassicae) larvae, the PI1a/GNA fusion was approximately 6 times as effective as recombinant PI1a on a molar basis. PI1a alone was not orally active against cabbage moth larvae, but a single 30 μg dose of the PI1a/GNA fusion protein caused 100% larval mortality within 6 days when fed to 3rd instar larvae, and caused significant reductions in survival, growth and feeding in 4th - 6th instar larvae. Transport of fusion protein from gut contents to the haemolymph of cabbage moth larvae, and binding to the nerve chord, was shown by Western blotting. The PI1a/GNA fusion protein also caused mortality when delivered orally to dipteran (Musca domestica; housefly) and hemipteran (Acyrthosiphon pisum; pea aphid) insects, making it a promising candidate for development as a biopesticide.

  5. A recombinant fusion protein containing a spider toxin specific for the insect voltage-gated sodium ion channel shows oral toxicity towards insects of different orders.

    PubMed

    Yang, Sheng; Pyati, Prashant; Fitches, Elaine; Gatehouse, John A

    2014-04-01

    Recombinant fusion protein technology allows specific insecticidal protein and peptide toxins to display activity in orally-delivered biopesticides. The spider venom peptide δ-amaurobitoxin-PI1a, which targets insect voltage-gated sodium channels, was fused to the "carrier" snowdrop lectin (GNA) to confer oral toxicity. The toxin itself (PI1a) and an amaurobitoxin/GNA fusion protein (PI1a/GNA) were produced using the yeast Pichia pastoris as expression host. Although both proteins caused mortality when injected into cabbage moth (Mamestra brassicae) larvae, the PI1a/GNA fusion was approximately 6 times as effective as recombinant PI1a on a molar basis. PI1a alone was not orally active against cabbage moth larvae, but a single 30 μg dose of the PI1a/GNA fusion protein caused 100% larval mortality within 6 days when fed to 3rd instar larvae, and caused significant reductions in survival, growth and feeding in 4th - 6th instar larvae. Transport of fusion protein from gut contents to the haemolymph of cabbage moth larvae, and binding to the nerve chord, was shown by Western blotting. The PI1a/GNA fusion protein also caused mortality when delivered orally to dipteran (Musca domestica; housefly) and hemipteran (Acyrthosiphon pisum; pea aphid) insects, making it a promising candidate for development as a biopesticide. PMID:24486516

  6. Phylogenetic analysis of microalgae based on highly abundant proteins using mass spectrometry.

    PubMed

    Lee, Hae-Won; Roh, Seong Woon; Cho, Kichul; Kim, Kil-Nam; Cha, In-Tae; Yim, Kyung June; Song, Hye Seon; Nam, Young-Do; Oda, Tatsuya; Chung, Young-Ho; Kim, Soo Jung; Choi, Jong-Soon; Kim, Daekyung

    2015-01-01

    The blooms of toxic phototrophic microorganisms, such as microalgae and cyanobacteria, which are typically found in freshwater and marine environments, are becoming more frequent and problematic in aquatic systems. Due to accumulation of toxic algae, harmful algal blooms (HABs) exert negative effects on aquatic systems. Therefore, rapid detection of harmful microalgae is important for monitoring the occurrence of HABs. Mass spectrometry-based methods have become sensitive, specific techniques for the identification and characterization of microorganisms. Matrix-assisted laser desorption/ionization (MALDI) with time-of-flight (TOF) mass spectrometry (MS) allows us to measure a unique molecular fingerprint of highly abundant proteins in a microorganism and has been used for the rapid, accurate identification of bacteria and fungi in clinical microbiology. Here, we tested the specificity of MALDI-TOF MS using microalgal strains (Heterocapsa, Alexandrium, Nannochloropsis, Chaetoceros, Chlorella, and Dunaliella spp.). Our research suggested that this method was comparable in terms of the rapid identification of microalgea to conventional methods based on genetic information and morphology. Thus, this efficient mass spectrometry-based technique may have applications in the rapid identification of harmful microorganisms from aquatic environmental samples.

  7. Phylogenetic analysis of microalgae based on highly abundant proteins using mass spectrometry.

    PubMed

    Lee, Hae-Won; Roh, Seong Woon; Cho, Kichul; Kim, Kil-Nam; Cha, In-Tae; Yim, Kyung June; Song, Hye Seon; Nam, Young-Do; Oda, Tatsuya; Chung, Young-Ho; Kim, Soo Jung; Choi, Jong-Soon; Kim, Daekyung

    2015-01-01

    The blooms of toxic phototrophic microorganisms, such as microalgae and cyanobacteria, which are typically found in freshwater and marine environments, are becoming more frequent and problematic in aquatic systems. Due to accumulation of toxic algae, harmful algal blooms (HABs) exert negative effects on aquatic systems. Therefore, rapid detection of harmful microalgae is important for monitoring the occurrence of HABs. Mass spectrometry-based methods have become sensitive, specific techniques for the identification and characterization of microorganisms. Matrix-assisted laser desorption/ionization (MALDI) with time-of-flight (TOF) mass spectrometry (MS) allows us to measure a unique molecular fingerprint of highly abundant proteins in a microorganism and has been used for the rapid, accurate identification of bacteria and fungi in clinical microbiology. Here, we tested the specificity of MALDI-TOF MS using microalgal strains (Heterocapsa, Alexandrium, Nannochloropsis, Chaetoceros, Chlorella, and Dunaliella spp.). Our research suggested that this method was comparable in terms of the rapid identification of microalgea to conventional methods based on genetic information and morphology. Thus, this efficient mass spectrometry-based technique may have applications in the rapid identification of harmful microorganisms from aquatic environmental samples. PMID:25476355

  8. Peritoneal Mesothelioma: An Unusual Cause of High-Protein Ascites

    PubMed Central

    2015-01-01

    We present a case illustrating the workup and diagnosis of peritoneal sarcomatous mesothelioma as an unusual etiology of intestinal obstruction and high-protein ascites in an otherwise healthy man. This rare disorder is diagnosed based on immunohistochemistry, which is necessary to differentiate it from other rare sarcomatous carcinomas. In many cases, localized disease can be treated to cure with surgery and intraperitoneal chemotherapy. Advanced disease is often treated for palliation of symptoms. PMID:26504886

  9. High-Sensitivity and Low-Toxicity Fucose Probe for Glycan Imaging and Biomarker Discovery.

    PubMed

    Kizuka, Yasuhiko; Funayama, Sho; Shogomori, Hidehiko; Nakano, Miyako; Nakajima, Kazuki; Oka, Ritsuko; Kitazume, Shinobu; Yamaguchi, Yoshiki; Sano, Masahiro; Korekane, Hiroaki; Hsu, Tsui-Ling; Lee, Hsiu-Yu; Wong, Chi-Huey; Taniguchi, Naoyuki

    2016-07-21

    Fucose, a terminal sugar in glycoconjugates, critically regulates various physiological and pathological phenomena, including cancer development and inflammation. However, there are currently no probes for efficient labeling and detection of this sugar. We chemically synthesized a novel series of alkynyl-fucose analogs as probe candidates and found that 7-alkynyl-fucose gave the highest labeling efficiency and low cytotoxicity. Among the fucose analogs, 7-alkynyl-fucose was the best substrate against all five fucosyltransferases examined. We confirmed its conversion to the corresponding guanosine diphosphate derivative in cells and found that cellular glycoproteins were labeled much more efficiently with 7-alkynyl-fucose than with an existing probe. 7-Alkynyl-fucose was detected in the N-glycan core by mass spectrometry, and 7-alkynyl-fucose-modified proteins mostly disappeared in core-fucose-deficient mouse embryonic fibroblasts, suggesting that this analog mainly labeled core fucose in these cells. These results indicate that 7-alkynyl-fucose is a highly sensitive and powerful tool for basic glycobiology research and clinical application for biomarker discovery. PMID:27447047

  10. High-Sensitivity and Low-Toxicity Fucose Probe for Glycan Imaging and Biomarker Discovery.

    PubMed

    Kizuka, Yasuhiko; Funayama, Sho; Shogomori, Hidehiko; Nakano, Miyako; Nakajima, Kazuki; Oka, Ritsuko; Kitazume, Shinobu; Yamaguchi, Yoshiki; Sano, Masahiro; Korekane, Hiroaki; Hsu, Tsui-Ling; Lee, Hsiu-Yu; Wong, Chi-Huey; Taniguchi, Naoyuki

    2016-07-21

    Fucose, a terminal sugar in glycoconjugates, critically regulates various physiological and pathological phenomena, including cancer development and inflammation. However, there are currently no probes for efficient labeling and detection of this sugar. We chemically synthesized a novel series of alkynyl-fucose analogs as probe candidates and found that 7-alkynyl-fucose gave the highest labeling efficiency and low cytotoxicity. Among the fucose analogs, 7-alkynyl-fucose was the best substrate against all five fucosyltransferases examined. We confirmed its conversion to the corresponding guanosine diphosphate derivative in cells and found that cellular glycoproteins were labeled much more efficiently with 7-alkynyl-fucose than with an existing probe. 7-Alkynyl-fucose was detected in the N-glycan core by mass spectrometry, and 7-alkynyl-fucose-modified proteins mostly disappeared in core-fucose-deficient mouse embryonic fibroblasts, suggesting that this analog mainly labeled core fucose in these cells. These results indicate that 7-alkynyl-fucose is a highly sensitive and powerful tool for basic glycobiology research and clinical application for biomarker discovery.

  11. Laser trapping and patterning of protein microcrystals: Toward highly integrated protein microarrays

    NASA Astrophysics Data System (ADS)

    Hosokawa, Yoichiroh; Matsumura, Satoshi; Masuhara, Hiroshi; Ikeda, Keiko; Shimo-oka, Ai; Mori, Hajime

    2004-09-01

    Some insect virus infections occlude into a crystalline matrix consisting of a protein named polyhedrin. The shape of the matrix is a cubic polyhedron of the size of a few micrometers. Recently it was shown that these polyhedra could immobilize various functional proteins within them. Therefore, the polyhedron is interesting as an element in a protein chip. In this work, individual polyhedra were arrayed and bonded under a microscope by focused laser beams, with the aim of fabricating a highly integrated protein chip. The polyhedron was trapped and transferred to a suitable position on a polymer substrate by optical trapping with a 1064nmNd3+:YAG (YAG, yttrium aluminum garnet) laser. To bond the polyhedron on the substrate, the polymer surface was mechanically and chemically modified by multiphoton absorption of a 120fs, 800nm femtosecond Ti: sapphire laser, which results in strong adhesion of the polyhedron to the substrate. The arraying and bonding of polyhedra were successful, to a precision of about 1μm, with this procedure. The biological activity of polyhedra after these laser irradiations was confirmed by the fluorescence of green fluorescent protein occluded in the polyhedrin matrix.

  12. [NADH:ubiquinone reductase and succinate dehydrogenase activity in the liver of rats with acetaminophen-induced toxic hepatitis on the background of alimentary protein deficiency].

    PubMed

    Kopylchuk, G P; Voloshchuk, O M

    2015-01-01

    The ratio between the redox forms of the nicotinamide coenzymes and key enzymatic activity of the I and II respiratory chain complexes in the liver cells mitochondria of rats with acetaminophen-induced hepatitis under the conditions of alimentary deprivation of protein was studied. It was estimated, that under the conditions of acute acetaminophen-induced hepatitis of rats kept on a low-protein diet during 4 weeks a significant decrease of the NADH:ubiquinone reductase and succinate dehydrogenase activity with simultaneous increase of the ratio between redox forms of the nicotinamide coenzymes (NAD+/NADH) is observed compared to the same indices in the liver cells of animals with experimental hepatitis kept on the ration balanced by all nutrients. Results of research may become basic ones for the biochemical rationale for the approaches directed to the correction and elimination of the consequences of energy exchange in the toxic hepatitis, induced on the background of protein deficiency. PMID:26036138

  13. Incorporating population variability and susceptible subpopulations into dosimetry for high-throughput toxicity testing.

    PubMed

    Wetmore, Barbara A; Allen, Brittany; Clewell, Harvey J; Parker, Timothy; Wambaugh, John F; Almond, Lisa M; Sochaski, Mark A; Thomas, Russell S

    2014-11-01

    Momentum is growing worldwide to use in vitro high-throughput screening (HTS) to evaluate human health effects of chemicals. However, the integration of dosimetry into HTS assays and incorporation of population variability will be essential before its application in a risk assessment context. Previously, we employed in vitro hepatic metabolic clearance and plasma protein binding data with in vitro in vivo extrapolation (IVIVE) modeling to estimate oral equivalent doses, or daily oral chemical doses required to achieve steady-state blood concentrations (Css) equivalent to media concentrations having a defined effect in an in vitro HTS assay. In this study, hepatic clearance rates of selected ToxCast chemicals were measured in vitro for 13 cytochrome P450 and five uridine 5'-diphospho-glucuronysyltransferase isozymes using recombinantly expressed enzymes. The isozyme-specific clearance rates were then incorporated into an IVIVE model that captures known differences in isozyme expression across several life stages and ethnic populations. Comparison of the median Css for a healthy population against the median or the upper 95th percentile for more sensitive populations revealed differences of 1.3- to 4.3-fold or 3.1- to 13.1-fold, respectively. Such values may be used to derive chemical-specific human toxicokinetic adjustment factors. The IVIVE model was also used to estimate subpopulation-specific oral equivalent doses that were directly compared with subpopulation-specific exposure estimates. This study successfully combines isozyme and physiologic differences to quantitate subpopulation pharmacokinetic variability. Incorporation of these values with dosimetry and in vitro bioactivities provides a viable approach that could be employed within a high-throughput risk assessment framework. PMID:25145659

  14. High-throughput insect cell protein expression applications.

    PubMed

    Buchs, Mirjam; Kim, Ernie; Pouliquen, Yann; Sachs, Michael; Geisse, Sabine; Mahnke, Marion; Hunt, Ian

    2009-01-01

    The Baculovirus Expression Vector System (BEVS) is one of the most efficient systems for production of recombinant proteins and consequently its application is wide-spread in industry as well as in academia. Since the early 1970s, when the first stable insect cell lines were established and the infectivity of bacu-lovirus in an in vitro culture system was demonstrated (1, 2), virtually thousands of reports have been published on the successful expression of proteins using this system as well as on method improvement. However, despite its popularity the system is labor intensive and time consuming. Moreover, adaptation of the system to multi-parallel (high-throughput) expression is much more difficult to achieve than with E. coli due to its far more complex nature. However, recent years have seen the development of strategies that have greatly enhanced the stream-lining and speed of baculovirus protein expression for increased throughput via use of automation and miniaturization. This chapter therefore tries to collate these developments in a series of protocols (which are modifications to standard procedure plus several new approaches) that will allow the user to expedite the speed and throughput of baculovirus-mediated protein expression and facilitate true multi-parallel, high-throughput protein expression profiling in insect cells. In addition we also provide a series of optimized protocols for small and large-scale transient insect cell expression that allow for both the rapid analysis of multiple constructs and the concomitant scale-up of those selected for on-going analysis. Since this approach is independent of viral propagation, the timelines for this approach are markedly shorter and offer a significant advantage over standard bacu-lovirus expression approach strategies in the context of HT applications.

  15. Application of Targeted Functional Assays to Assess a Putative Vascular Disruption Developmental Toxicity Pathway Informed By ToxCast High-Throughput Screening Data

    EPA Science Inventory

    Chemical perturbation of vascular development is a putative toxicity pathway which may result in developmental toxicity. EPA’s high-throughput screening (HTS) ToxCast program contains assays which measure cellular signals and biological processes critical for blood vessel develop...

  16. Combinatorial Synthesis of and high-throughput protein release from polymer film and nanoparticle libraries.

    PubMed

    Petersen, Latrisha K; Chavez-Santoscoy, Ana V; Narasimhan, Balaji

    2012-09-06

    antigenicity; in vitro cellular toxicity, cytokine production, surface marker expression, adhesion, proliferation and differentiation; and in vivo biodistribution and mucoadhesion(1-11). The combinatorial method developed herein enables high-throughput polymer synthesis and fabrication of protein-loaded nanoparticle and film libraries, which can, in turn, be screened in vitro and in vivo for optimization of biomaterial performance.

  17. Anti-Viral Antibody Profiling by High Density Protein Arrays

    PubMed Central

    Bian, Xiaofang; Wiktor, Peter; Kahn, Peter; Brunner, Al; Khela, Amritpal; Karthikeyan, Kailash; Barker, Kristi; Yu, Xiaobo; Magee, Mitch; Wasserfall, Clive H.; Gibson, David; Rooney, Madeleine E; Qiu, Ji; LaBaer, Joshua

    2015-01-01

    Viral infections elicit anti-viral antibodies and have been associated with various chronic diseases. Detection of these antibodies can facilitate diagnosis, treatment of infection and understanding of the mechanisms of virus associated diseases. In this work, we assayed anti-viral antibodies using a novel high density-nucleic acid programmable protein array (HD-NAPPA) platform. Individual viral proteins were expressed in situ directly from plasmids encoding proteins in an array of microscopic reaction chambers. Quality of protein display and serum response was assured by comparing intra- and inter- array correlation within or between printing batches with average correlation coefficients of 0.91 and 0.96, respectively. HD-NAPPA showed higher signal to background (S/B) ratio compared with standard NAPPA on planar glass slides and ELISA. Antibody responses to 761 antigens from 25 different viruses were profiled among patients with juvenile idiopathic arthritis (JIA) and type 1 diabetes (T1D). Common as well as unique antibody reactivity patterns were detected between patients and healthy controls. We believe HD-viral-NAPPA will enable the study of host-pathogen interactions at unprecedented dimensions and elucidate the role of pathogen infections in disease development. PMID:25758251

  18. Protein carbamylation renders high-density lipoprotein dysfunctional

    PubMed Central

    2012-01-01

    Aim Carbamylation of proteins through reactive cyanate has been demonstrated to predict an increased cardiovascular risk. Cyanate is formed in vivo by break-down of urea and at sites of inflammation by the phagocyte protein myeloperoxidase. Since myeloperoxidase (MPO) associates with high-density lipoprotein (HDL) in human atherosclerotic intima, we examined in the present study whether cyanate specifically targets HDL. Results Mass spectrometry analysis revealed that protein carbamylation is a major post-translational modification of HDL. The carbamyllysine content of lesion derived HDL was more than 20-fold higher in comparison to 3-chlorotyrosine levels, a specific oxidation product of MPO. Notable, the carbamyllysine content of lesion-derived HDL was 5 to 8-fold higher when compared to lesion derived low-density lipoprotein (LDL) or total lesion protein and increased with lesion severity. Importantly, the carbamyllysine content of HDL, but not of LDL, correlated with levels of 3-chlorotyrosine, suggesting MPO mediated carbamylation in the vessel wall. Remarkably, one carbamyllysine residue per HDL associated apolipoprotein A-I was sufficient to induce cholesterol accumulation and lipid droplet formation in macrophages through a pathway requiring the HDL receptor scavenger receptor class B, type I. Conclusion The present results raise the possibility that HDL carbamylation contributes to foam cell formation in atherosclerotic lesions. PMID:21235354

  19. How Toxic Is It?

    ERIC Educational Resources Information Center

    Crellin, John R.

    1989-01-01

    Discusses the relative danger from toxicity of some typical chemicals. Notes that some materials in solutions have low toxicity, but in dust form have high toxicity. Suggests that more chemical compounds should be treated as the dangerous compounds they are. Lists common compounds found in the lab. (MVL)

  20. Forced Protein Unfolding Leads to Highly Elastic and Tough Protein Hydrogels

    PubMed Central

    Fang, Jie; Mehlich, Alexander; Koga, Nobuyasu; Huang, Jiqing; Koga, Rie; Gao, Xiaoye; Hu, Chunguang; Jin, Chi; Rief, Matthias; Kast, Juergen; Baker, David; Li, Hongbin

    2014-01-01

    Protein-based hydrogels usually do not exhibit high stretchability or toughness, significantly limiting the scope of their potential biomedical applications. Here we report the engineering of a chemically crosslinked, highly elastic and tough protein hydrogel using a mechanically extremely labile, de novo designed protein that assumes the classical ferredoxin-like fold structure. Due to the low mechanical stability of the ferredoxin-like fold structure, swelling of hydrogels causes a significant fraction of the fold structure domains to unfold. Subsequent collapse and aggregation of unfolded ferredoxin-like fold structure domains leads to intertwining of physically and chemically crosslinked networks, entailing hydrogels with unusual physical and mechanical properties: a negative swelling ratio, high stretchability and toughness. These hydrogels can withstand an average strain of 450% before breaking and show massive energy dissipation. Upon relaxation, refolding of the ferredoxin-like fold structure domains enables the hydrogel to recover its massive hysteresis. This novel biomaterial may expand the scope of hydrogel applications in tissue engineering. PMID:24352111

  1. Identification of Reference Proteins for Western Blot Analyses in Mouse Model Systems of 2,3,7,8-Tetrachlorodibenzo-P-Dioxin (TCDD) Toxicity

    PubMed Central

    Prokopec, Stephenie D.; Watson, John D.; Pohjanvirta, Raimo; Boutros, Paul C.

    2014-01-01

    Western blotting is a well-established, inexpensive and accurate way of measuring protein content. Because of technical variation between wells, normalization is required for valid interpretation of results across multiple samples. Typically this involves the use of one or more endogenous controls to adjust the measured levels of experimental molecules. Although some endogenous controls are widely used, validation is required for each experimental system. This is critical when studying transcriptional-modulators, such as toxicants like 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).To address this issue, we examined hepatic tissue from 192 mice representing 47 unique combinations of strain, sex, Ahr-genotype, TCDD dose and treatment time. We examined 7 candidate reference proteins in each animal and assessed consistency of protein abundance through: 1) TCDD-induced fold-difference in protein content from basal levels, 2) inter- and intra- animal stability, and 3) the ability of each candidate to reduce instability of the other candidates. Univariate analyses identified HPRT as the most stable protein. Multivariate analysis indicated that stability generally increased with the number of proteins used, but gains from using >3 proteins were small. Lastly, by comparing these new data to our previous studies of mRNA controls on the same animals, we were able to show that the ideal mRNA and protein control-genes are distinct, and use of only 2–3 proteins provides strong stability, unlike in mRNA studies in the same cohort, where larger control-gene batteries were needed. PMID:25329058

  2. High Throughput Screening for Drugs that Modulate Intermediate Filament Proteins

    PubMed Central

    Sun, Jingyuan; Groppi, Vincent E.; Gui, Honglian; Chen, Lu; Xie, Qing; Liu, Li

    2016-01-01

    Intermediate filament (IF) proteins have unique and complex cell and tissue distribution. Importantly, IF gene mutations cause or predispose to more than 80 human tissue-specific diseases (IF-pathies), with the most severe disease phenotypes being due to mutations at conserved residues that result in a disrupted IF network. A critical need for the entire IF-pathy field is the identification of drugs that can ameliorate or cure these diseases, particularly since all current therapies target the IF-pathy complication, such as diabetes or cardiovascular disease, rather than the mutant IF protein or gene. We describe a high throughput approach to identify drugs that can normalize disrupted IF proteins. This approach utilizes transduction of lentivirus that expresses green-fluorescent-protein-tagged keratin 18 (K18) R90C in A549 cells. The readout is drug ‘hits’ that convert the dot-like keratin filament distribution, due to the R90C mutation, to a wildtype-like filamentous array. A similar strategy can be used to screen thousands of compounds and can be utilized for practically any IF protein with a filament-disrupting mutation, and could therefore potentially target many IF-pathies. ‘Hits’ of interest require validation in cell culture then using in vivo experimental models. Approaches to study the mechanism of mutant-IF normalization by potential drugs of interest are also described. The ultimate goal of this drug screening approach is to identify effective and safe compounds that can potentially be tested for clinical efficacy in patients. PMID:26795471

  3. Development of high protein and low calorie cookies.

    PubMed

    Kulthe, Amit Arjun; Pawar, Vithal Deorao; Kotecha, Pramod Mohanlal; Chavan, Uttam Dhyanu; Bansode, Venkatraman Vishwanath

    2014-01-01

    Cookies high in proteins and low in calories were prepared by substituting wheat maida with defatted soy flour (DSF) at 0, 10, 15, 20 and 25% levels and sugar with stevia leaves powder (SLP) at 0, 15, 20, 25 and 30% levels using traditional creamery method. Cookies were evaluated for physico-chemical and sensory quality parameters. The thickness and hardness of cookies increased; weight, diameter, spread ratio and spread factor decreased with increasing levels of DSF while there was increase in protein, crude fiber and ash content and decrease in fat and carbohydrate contents. The cookies with 20% substitution each of DSF and SLP scored maximum for all the sensory quality attributes. On storage of such cookies in LDPE, HDPE and PP for 90 days at ambient temperature, the sensory quality attributes were decreased, but the cookies were acceptable. The HDPE was better packaging material than LDPE and PP with regard to sensory quality of cookies during storage.

  4. High-resolution neutron spectroscopy on protein solution samples

    NASA Astrophysics Data System (ADS)

    Grimaldo, Marco; Roosen-Runge, Felix; Jalarvo, Niina; Zamponi, Michaela; Zanini, Fabio; Hennig, Marcus; Zhang, Fajun; Schreiber, Frank; Seydel, Tilo

    2015-01-01

    Proteins in solution move subject to a complex superposition of global translational and rotational diffusion as well as internal relaxations covering a wide range of time scales. With the advent of new high-flux neutron spectrometers in combination with enhanced analysis frameworks it has become possible to separate these different contributions. We discuss new approaches to the analysis by presenting example spectra and fits from data recorded on the backscattering spectrometers IN16, IN16B, and BASIS on the same protein solution sample. We illustrate the separation of the rotational and translational diffusion contribution, the accurate treatment of the solvent contribution, and the extraction of information on internal fluctuations. We also exemplify the progress made in passing from second- to third-generation backscattering spectrometers.

  5. A Parkinson's disease gene regulatory network identifies the signaling protein RGS2 as a modulator of LRRK2 activity and neuronal toxicity

    PubMed Central

    Dusonchet, Julien; Li, Hu; Guillily, Maria; Liu, Min; Stafa, Klodjan; Derada Troletti, Claudio; Boon, Joon Y.; Saha, Shamol; Glauser, Liliane; Mamais, Adamantios; Citro, Allison; Youmans, Katherine L.; Liu, LiQun; Schneider, Bernard L.; Aebischer, Patrick; Yue, Zhenyu; Bandopadhyay, Rina; Glicksman, Marcie A.; Moore, Darren J.; Collins, James J.; Wolozin, Benjamin

    2014-01-01

    Mutations in LRRK2 are one of the primary genetic causes of Parkinson's disease (PD). LRRK2 contains a kinase and a GTPase domain, and familial PD mutations affect both enzymatic activities. However, the signaling mechanisms regulating LRRK2 and the pathogenic effects of familial mutations remain unknown. Identifying the signaling proteins that regulate LRRK2 function and toxicity remains a critical goal for the development of effective therapeutic strategies. In this study, we apply systems biology tools to human PD brain and blood transcriptomes to reverse-engineer a LRRK2-centered gene regulatory network. This network identifies several putative master regulators of LRRK2 function. In particular, the signaling gene RGS2, which encodes for a GTPase-activating protein (GAP), is a key regulatory hub connecting the familial PD-associated genes DJ-1 and PINK1 with LRRK2 in the network. RGS2 expression levels are reduced in the striata of LRRK2 and sporadic PD patients. We identify RGS2 as a novel interacting partner of LRRK2 in vivo. RGS2 regulates both the GTPase and kinase activities of LRRK2. We show in mammalian neurons that RGS2 regulates LRRK2 function in the control of neuronal process length. RGS2 is also protective against neuronal toxicity of the most prevalent mutation in LRRK2, G2019S. We find that RGS2 regulates LRRK2 function and neuronal toxicity through its effects on kinase activity and independently of GTPase activity, which reveals a novel mode of action for GAP proteins. This work identifies RGS2 as a promising target for interfering with neurodegeneration due to LRRK2 mutations in PD patients. PMID:24794857

  6. DNA Repair Alterations in Children With Pediatric Malignancies: Novel Opportunities to Identify Patients at Risk for High-Grade Toxicities

    SciTech Connect

    Ruebe, Claudia E.

    2010-10-01

    Purpose: To evaluate, in a pilot study, the phosphorylated H2AX ({gamma}H2AX) foci approach for identifying patients with double-strand break (DSB) repair deficiencies, who may overreact to DNA-damaging cancer therapy. Methods and Materials: The DSB repair capacity of children with solid cancers was analyzed compared with that of age-matched control children and correlated with treatment-related normal-tissue responses (n = 47). Double-strand break repair was investigated by counting {gamma}H2AX foci in blood lymphocytes at defined time points after irradiation of blood samples. Results: Whereas all healthy control children exhibited proficient DSB repair, 3 children with tumors revealed clearly impaired DSB repair capacities, and 2 of these repair-deficient children developed life-threatening or even lethal normal-tissue toxicities. The underlying mutations affecting regulatory factors involved in DNA repair pathways were identified. Moreover, significant differences in mean DSB repair capacity were observed between children with tumors and control children, suggesting that childhood cancer is based on genetic alterations affecting DSB repair function. Conclusions: Double-strand break repair alteration in children may predispose to cancer formation and may affect children's susceptibility to normal-tissue toxicities. Phosphorylated H2AX analysis of blood samples allows one to detect DSB repair deficiencies and thus enables identification of children at risk for high-grade toxicities.

  7. High CJD infectivity remains after prion protein is destroyed.

    PubMed

    Miyazawa, Kohtaro; Emmerling, Kaitlin; Manuelidis, Laura

    2011-12-01

    The hypothesis that host prion protein (PrP) converts into an infectious prion form rests on the observation that infectivity progressively decreases in direct proportion to the decrease of PrP with proteinase K (PK) treatment. PrP that resists limited PK digestion (PrP-res, PrP(sc)) has been assumed to be the infectious form, with speculative types of misfolding encoding the many unique transmissible spongiform encephalopathy (TSE) agent strains. Recently, a PK sensitive form of PrP has been proposed as the prion. Thus we re-evaluated total PrP (sensitive and resistant) and used a cell-based assay for titration of infectious particles. A keratinase (NAP) known to effectively digest PrP was compared to PK. Total PrP in FU-CJD infected brain was reduced to ≤0.3% in a 2 h PK digest, yet there was no reduction in titer. Remaining non-PrP proteins were easily visualized with colloidal gold in this highly infectious homogenate. In contrast to PK, NAP digestion left 0.8% residual PrP after 2 h, yet decreased titer by >2.5 log; few residual protein bands remained. FU-CJD infected cells with 10× the infectivity of brain by both animal and cell culture assays were also evaluated. NAP again significantly reduced cell infectivity (>3.5 log). Extreme PK digestions were needed to reduce cell PrP to <0.2%, yet a very high titer of 8 logs survived. Our FU-CJD brain results are in good accord with the only other report on maximal PrP destruction and titer. It is likely that one or more residual non-PrP proteins may protect agent nucleic acids in infectious particles.

  8. Molecular Control of TiO2-NPs Toxicity Formation at Predicted Environmental Relevant Concentrations by Mn-SODs Proteins

    PubMed Central

    Wu, Qiuli; Li, Yiping; Tang, Meng; Ye, Boping; Wang, Dayong

    2012-01-01

    With growing concerns of the safety of nanotechnology, the in vivo toxicity of nanoparticles (NPs) at environmental relevant concentrations has drawn increasing attentions. We investigated the possible molecular mechanisms of titanium nanoparticles (Ti-NPs) in the induction of toxicity at predicted environmental relevant concentrations. In nematodes, small sizes (4 nm and 10 nm) of TiO2-NPs induced more severe toxicities than large sizes (60 nm and 90 nm) of TiO2-NPs on animals using lethality, growth, reproduction, locomotion behavior, intestinal autofluorescence, and reactive oxygen species (ROS) production as endpoints. Locomotion behaviors could be significantly decreased by exposure to 4-nm and 10-nm TiO2-NPs at concentration of 1 ng/L in nematodes. Among genes required for the control of oxidative stress, only the expression patterns of sod-2 and sod-3 genes encoding Mn-SODs in animals exposed to small sizes of TiO2-NPs were significantly different from those in animals exposed to large sizes of TiO2-NPs. sod-2 and sod-3 gene expressions were closely correlated with lethality, growth, reproduction, locomotion behavior, intestinal autofluorescence, and ROS production in TiO2-NPs-exposed animals. Ectopically expression of human and nematode Mn-SODs genes effectively prevented the induction of ROS production and the development of toxicity of TiO2-NPs. Therefore, the altered expression patterns of Mn-SODs may explain the toxicity formation for different sizes of TiO2-NPs at predicted environmental relevant concentrations. In addition, we demonstrated here a strategy to investigate the toxicological effects of exposure to NPs upon humans by generating transgenic strains in nematodes for specific human genes. PMID:22973466

  9. Select Small Core Structure Carbamates Exhibit High Contact Toxicity to “Carbamate-Resistant” Strain Malaria Mosquitoes, Anopheles gambiae (Akron)

    PubMed Central

    Wong, Dawn M.; Li, Jianyong; Chen, Qiao-Hong; Han, Qian; Mutunga, James M.; Wysinski, Ania; Anderson, Troy D.; Ding, Haizhen; Carpenetti, Tiffany L.; Verma, Astha; Islam, Rafique; Paulson, Sally L.; Lam, Polo C.-H.; Totrov, Maxim; Bloomquist, Jeffrey R.; Carlier, Paul R.

    2012-01-01

    Acetylcholinesterase (AChE) is a proven target for control of the malaria mosquito (Anopheles gambiae). Unfortunately, a single amino acid mutation (G119S) in An. gambiae AChE-1 (AgAChE) confers resistance to the AChE inhibitors currently approved by the World Health Organization for indoor residual spraying. In this report, we describe several carbamate inhibitors that potently inhibit G119S AgAChE and that are contact-toxic to carbamate-resistant An. gambiae. PCR-RFLP analysis was used to confirm that carbamate-susceptible G3 and carbamate-resistant Akron strains of An. gambiae carry wild-type (WT) and G119S AChE, respectively. G119S AgAChE was expressed and purified for the first time, and was shown to have only 3% of the turnover number (kcat) of the WT enzyme. Twelve carbamates were then assayed for inhibition of these enzymes. High resistance ratios (>2,500-fold) were observed for carbamates bearing a benzene ring core, consistent with the carbamate-resistant phenotype of the G119S enzyme. Interestingly, resistance ratios for two oxime methylcarbamates, and for five pyrazol-4-yl methylcarbamates were found to be much lower (4- to 65-fold). The toxicities of these carbamates to live G3 and Akron strain An. gambiae were determined. As expected from the enzyme resistance ratios, carbamates bearing a benzene ring core showed low toxicity to Akron strain An. gambiae (LC50>5,000 μg/mL). However, one oxime methylcarbamate (aldicarb) and five pyrazol-4-yl methylcarbamates (4a–e) showed good to excellent toxicity to the Akron strain (LC50 = 32–650 μg/mL). These results suggest that appropriately functionalized “small-core” carbamates could function as a resistance-breaking anticholinesterase insecticides against the malaria mosquito. PMID:23049714

  10. Highly chlorinated toxic contaminants in pesticide-treated wooden art objects.

    PubMed

    Covaci, Adrian; Kawaki, Peter; Indekeu, Charles; Schepens, Paul; Neels, Hugo

    2006-01-01

    Although the contamination of wooden art objects with pesticides is well known, to the authors' knowledge no attempt has yet been made to investigate the eventual presence of other toxic compounds that have been produced during the degradation of pesticides or that may be present in the technical formulations. Here, the authors report on the presence of polychlorinated biphenyls (PCBs) and polychlorinated terphenyls (PCTs) in scrapings from wooden antique art objects, namely printing blocks, sculptures, and masks. These antiques belong to 2 fine art museums in Belgium--Antwerp's Ethnographic Museum and the Plantin-Moretus Museum. It is documented that these art objects were treated with pesticides in the 1950s. In addition, 2-heptachlorodibenzo-p-dioxin (HpCDD) isomers and octachlorodibenzo-p-dioxin (OCDD) were also identified. The presence of these toxic compounds in these antiques requires a better understanding of safety for the persons (conservators, museum employees, restorers, and visitors) coming in contact with these objects. PMID:17967745

  11. Extracellular signal-related kinase 2/specificity protein 1/specificity protein 3/repressor element-1 silencing transcription factor pathway is involved in Aroclor 1254-induced toxicity in SH-SY5Y neuronal cells.

    PubMed

    Formisano, Luigi; Guida, Natascia; Laudati, Giusy; Boscia, Francesca; Esposito, Alba; Secondo, Agnese; Di Renzo, Gianfranco; Canzoniero, Lorella Maria Teresa

    2015-01-01

    Polychlorinated biphenyls (PCBs) cause a wide spectrum of toxic effects in the brain through undefined mechanisms. Exposure to the PCB mixture Aroclor-1254 (A1254) increases the repressor element-1 silencing transcription factor (REST) expression, leading to neuronal death. This study sought to understand the sequence of some molecular mechanisms to determine whether A1254 could increase REST expression and the cytoprotective effect of the phorbol ester tetradecanoylphorbol acetate (TPA) on A1254-induced toxicity in SH-SY5Y cells. As shown by Western blot analysis, A1254 (10 µg/ml) downregulates extracellular signal-related kinase 2 (ERK2) phosphorylation in a time-dependent manner, thereby triggering the binding of specificity protein 1 (Sp1) and Sp3 to the REST gene promoter as revealed by chromatin immunoprecipitation analysis. This chain of events results in an increase in REST mRNA and cell death, as assessed by quantitative real-time polymerase chain reaction and dimethylthiazolyl-2-5-diphenyltetrazolium-bromide assay, respectively. Accordingly, TPA prevented both the A1254-induced decrease in ERK2 phosphorylation and the A1254-induced increase in Sp1, Sp3, and REST protein expression. After 48 hr, TPA prevented A1254-induced cell death. ERK2 overexpression counteracted the A1254-induced increase in Sp1 and Sp3 protein expression and prevented A1254-induced Sp1 and Sp3 binding to the REST gene promoter, thus counteracting the increase in REST mRNA expression induced by the toxicant. In neuroblastoma SH-SY5Y cells, ERK2/Sp1/SP3/REST is a new pathway underlying the neurotoxic effect of PCB. The ERK2/Sp1/Sp3/REST pathway, which underlies A1254-induced neuronal death, might represent a new drug signaling cascade in PCB-induced neuronal toxicity. PMID:25093670

  12. Trichloroethene and cis-1,2-dichloroethene concentration-dependent toxicity model simulates anaerobic dechlorination at high concentrations. II: continuous flow and attached growth reactors.

    PubMed

    Sabalowsky, Andrew R; Semprini, Lewis

    2010-10-15

    A model that was used to describe toxicity from high concentrations of chlorinated aliphatic hydrocarbons (CAHs) on reductively dechlorinating cultures in batch reactors (Sabalowsky and Semprini (in press)) was extended here to simulate observations in continuous flow suspended and attached growth reactors. The reductively dechlorinating anaerobic Evanite subculture (EV-cDCE) was fed trichloroethene (TCE) and excess electron donor to accumulate cis-1,2-dichloroethene (cDCE) in a continuous flow stirred tank reactor (CFSTR); and an attached growth recirculating packed column (RPC). A concentration-dependent toxicity model used to simulate the results of batch reactors in part I (Sabalowsky and Semprini (in press) Biotechnol Bioeng) also simulated well the observations for the CFSTR and RPC growth modes. The toxicity model incorporates cDCE and TCE toxicity coefficients that directly increase the cell decay coefficient in proportion with cDCE and TCE concentrations. Simulated estimates of the cDCE and TCE toxicity coefficients indicate reductively dechlorinating cells are most sensitive to high concentrations of cDCE and TCE in batch-fed growth, followed by CFSTR, with attached growth being least sensitive. The greater toxicity of TCE than cDCE, and ratio of the modeled toxicity coefficients, agrees with previously proposed models relating toxicity to partitioning in the cell wall (K(M/B)), proportional to octanol-water partitioning (K(OW)) coefficients.

  13. CIP2A protein expression in high-grade, high-stage bladder cancer

    PubMed Central

    Huang, Lisa P; Savoly, Diana; Sidi, Abraham A; Adelson, Martin E; Mordechai, Eli; Trama, Jason P

    2012-01-01

    Bladder cancer is one of the most common cancers in the United States. Numerous markers have been evaluated for suitability of bladder cancer detection and surveillance. However, few of them are acceptable as a routine tool. Therefore, there exists a continuing need for an assay that detects the presence of bladder cancer in humans. It would be advantageous to develop an assay with a protein that is associated with the development of bladder cancer. We have identified the cancerous inhibitor of PP2A (CIP2A) protein as a novel bladder cancer biomarker. In this study, Western blot analysis was used to assess the expression level of CIP2A protein in bladder cancer cell lines and bladder cancer patient tissues (n = 43). Our studies indicated CIP2A protein was abundantly expressed in bladder cancer cell lines but not in nontumor epithelial cell lines. Furthermore, CIP2A was specifically expressed in transitional cell carcinoma (TCC) of the bladder tumor tissues but not in adjacent nontumor bladder tissue. Our data showed that CIP2A protein detection in high-grade TCC tissues had a sensitivity of 65%, which is 3.4-fold higher than that seen in low-grade TCC tissues (19%). The level of CIP2A protein expression increased with the stage of disease (12%, 27%, 67%, and 100% for pTa, pT1, pT2, and pT3 tumor, respectively). In conclusion, our studies suggest that CIP2A protein is specifically expressed in human bladder tumors. CIP2A is preferentially expressed in high-grade and high-stage TCC tumors, which are high-risk and invasive tumors. Our studies reported here support the role of CIP2A in bladder cancer progression and its usefulness for the surveillance of recurrence or progression of human bladder cancer. PMID:23342256

  14. Folding of a large protein at high structural resolution

    PubMed Central

    Walters, Benjamin T.; Mayne, Leland; Hinshaw, James R.; Sosnick, Tobin R.; Englander, S. Walter

    2013-01-01

    Kinetic folding of the large two-domain maltose binding protein (MBP; 370 residues) was studied at high structural resolution by an advanced hydrogen-exchange pulse-labeling mass-spectrometry method (HX MS). Dilution into folding conditions initiates a fast molecular collapse into a polyglobular conformation (<20 ms), determined by various methods including small angle X-ray scattering. The compaction produces a structurally heterogeneous state with widespread low-level HX protection and spectroscopic signals that match the equilibrium melting posttransition-state baseline. In a much slower step (7-s time constant), all of the MBP molecules, although initially heterogeneously structured, form the same distinct helix plus sheet folding intermediate with the same time constant. The intermediate is composed of segments that are distant in the MBP sequence but adjacent in the native protein where they close the longest residue-to-residue contact. Segments that are most HX protected in the early molecular collapse do not contribute to the initial intermediate, whereas the segments that do participate are among the less protected. The 7-s intermediate persists through the rest of the folding process. It contains the sites of three previously reported destabilizing mutations that greatly slow folding. These results indicate that the intermediate is an obligatory step on the MBP folding pathway. MBP then folds to the native state on a longer time scale (∼100 s), suggestively in more than one step, the first of which forms structure adjacent to the 7-s intermediate. These results add a large protein to the list of proteins known to fold through distinct native-like intermediates in distinct pathways. PMID:24191053

  15. Behaviour of five pharmaceuticals with high baseline toxicity in wastewater treatment

    NASA Astrophysics Data System (ADS)

    van Driezum, Inge; McArdell, Christa; Fenner, Kathrin; Helbling, Damian; van Breukelen, Boris

    2013-04-01

    Many pharmaceuticals enter the aquatic environment through sewer systems and are partially removed in wastewater treatment plants (WWTP) by sorption to sludge biomass or biodegradation. Biodegradation often does not lead to complete mineralization but to the formation of stable transformation products (TPs), which might still be harmful to the environment. Recently, a study was undertaken to assess the risk of the top 100 pharmaceuticals from wastewater of a hospital in Switzerland. The predicted toxicity was linked to the predicted environmental concentration in order to assess overall risk potential. In this study, biodegradation and sorption studies were carried out on the top five selected pharmaceuticals (amiodarone, atorvastatin, clotrimazole, meclozine and ritonavir). Potential TPs that are formed during activated sludge treatment were identified and concentrations of both the parent compounds and TPs were measured in the WWTP. With this data, the fate of these compounds was modeled in a WWTP system using a multi-reactor steady-state WWTP model. This study showed that sorption was the most important loss process for amiodarone and meclozine. They had an elimination of more than 99%. Sorption was also the main loss process for clotrimazole, but it was combined with some biodegradation. For ritonavir, both biodegradation and sorption played a role in the loss of this compound. The most important removal process for atorvastatin was biodegradation. Four TPs, formed through β-oxidation and monohydroxilation, were identified in both the activated sludge batch reactors and the WWTP effluent. In the WWTP effluent, only atorvastatin, clotrimazole and ritonavir were found. All identified TPs of atorvastatin were detected in the effluent. Risk quotients (RQ) of all five pharmaceuticals were estimated based on effluent concentration and baseline toxicity and ranged from zero to 2.14. Only ritonavir potentially poses an ecotoxicological risk for the aquatic environment.

  16. A Brief Study on Toxic Combustion Products of the Polymers Used in High-Pressure Oxygen Systems

    NASA Technical Reports Server (NTRS)

    Hshieh, Fu-Yu; Beeson, Harold D.

    2005-01-01

    One likely cause of polymer ignition in a high-pressure oxygen system is the adiabatic-compression heating of polymers caused by pneumatic impact. Oxidative pyrolysis or combustion of polymers in a high-pressure oxygen system could generate toxic gases. This paper investigates the feasibility of using the NASA pneumatic-impact system to conduct adiabatic-compression combustion tests and determines the toxic combustion products produced from the burning of five selected polymers. Five polymers commonly used in high-pressure oxygen systems, Zytel(Registered TradeMark) 42 (Nylon 6/6), Buna N (nitrile rubber), Witon(Registered TradeMark) A (copolymer of vinylidene fluoride and hexafluoropropylene), Neoflon(Registered TradeMark) (polychlorotrifluoroethylene), and Teflon(Registered TradeMark) (polytetrafluoroethylene), were tested in the NASA pneumatic-impact test system at 17.2-MPa oxygen pressure. The polymers were ignited and burned; combustion products were collected in a stainless-steel sample bottle and analyzed using various methods. The results show that the NASA pneumatic-impact system is an appropriate test system to conduct adiabatic-compression combustion tests and to collect combustion products for further chemical analysis. The composition of the combustion product gas generated from burning the five selected polymers are presented and discussed.

  17. Highly-sensitive electrocatalytic determination for toxic phenols based on coupled cMWCNT/cyclodextrin edge-functionalized graphene composite.

    PubMed

    Gao, Juanjuan; Liu, Maoxiang; Song, Haiou; Zhang, Shupeng; Qian, Yueyue; Li, Aimin

    2016-11-15

    Highly-sensitive electrocatalytic determination of toxic phenol compounds is of significance in environmental monitoring due to their low degradation and high toxicity to the environment and humans. In this paper, a rapid and sensitive electrochemical sensor based on coupled carboxyl-multi-walled carbon nanotube (cMWCNT) and cyclodextrin (CD) edge-functionalized graphene composite was successfully employed towards trace detection of three typical phenols (4-aminophenol, 4-AP; 4-chlorophenol, 4-CP; 4-nitrophenol, 4-NP). The morphology studies from scanning electron microscope and transmission electron microscope analysis revealed that cMWCNTs as conductive bridges were successfully incorporated into CD edge-functionalized graphene layers. Further, The electrocatalytic detection performance of the 3D simultaneously reduced and self-assembled sensing architecture (GN-CD-cMWCNT) with trace amounts of CDs was evaluated. The electrochemical studies demonstrated that GN-CD-cMWCNT displays excellent electrocatalytic activity, high sensitivity and stability. Under optimal conditions, the current responses of 4-AP, 4-CP and 4-NP are linear to concentrations over two different ranges, with low detection limit of 0.019, 0.017 and 0.027μM (S/N=3), respectively. And, GN-CD-cMWCNT shows an excellent anti-interference ability against electroactive species and metal ions. In addition, validation of the applicability of the presented sensor was also performed for the determination of three phenols in tap water sample with satisfactory results. PMID:27415597

  18. 20-Year Experience With Intraoperative High-Dose-Rate Brachytherapy for Pediatric Sarcoma: Outcomes, Toxicity, and Practice Recommendations

    SciTech Connect

    Folkert, Michael R.; Tong, William Y.; LaQuaglia, Michael P.; Wexler, Leonard H.; Chou, Alexander J.; Magnan, Heather; Zelefsky, Michael J.; Wolden, Suzanne L.

    2014-10-01

    Purpose: To assess outcomes and toxicity of high-dose-rate intraoperative radiation therapy (HDR-IORT) in the management of pediatric sarcoma. Methods and Materials: Seventy-five pediatric patients underwent HDR-IORT for sarcoma from May 1993 to November 2013. The median age was 9 years old (36 patients were ≤6 years old). HDR-IORT was part of initial therapy in 37 patients (49%) and for recurrent disease in 38 patients (51%). Forty-one patients (55%) received HDR-IORT and postoperative external beam RT (PORT), and 22 patients (29%) were previously treated with external beam radiation therapy to the IORT site. Local control (LC), overall survival (OS) and event-free survival (EFS) were estimated using Kaplan-Meier methods. Results: At a median follow-up of 7.8 years for surviving patients, 5-year projected rates of LC, EFS, and OS were 63% (95% confidence interval [CI] 50%-76%), 33% (95% CI 21%-45%), and 43% (95% CI 30%-55%), with a median survival of 3.1 years. The 5-year LC, EFS, and OS rates for patients with recurrent disease were 46% (95% CI, 28%-64%), 30% (95% CI, 13%-46%), and 36% (95% CI, 18%-54%). Acute toxicity ≥grade 3 occurred in 2 (2.5%) treatments; late toxicity ≥grade 3 occurred in 4 (5.3%) patients 0.3-9.9 years after HDR-IORT. The incidence of toxicity ≥grade 3 was not associated with HDR-IORT applicator size, HDR-IORT dose, prior RT or PORT, or prior or postoperative chemotherapy, but all toxicity ≥grade 3 occurred in patients ≤6 years treated with HDR-IORT doses ≥12 Gy. Conclusions: HDR-IORT is a well-tolerated component of multimodality therapy for pediatric sarcoma, allowing additional local treatment while reducing external beam exposure. Taking clinical considerations into account, doses between 8-12 Gy are appropriate for HDR-IORT in patients ≤6 years of age.

  19. Highly biocompatible multi-walled carbon nanotube-chitosan nanoparticle hybrids as protein carriers.

    PubMed

    Li, Cuiyun; Yang, Keqin; Zhang, Yingying; Tang, Hao; Yan, Feng; Tan, Liang; Xie, Qingji; Yao, Shouzhuo

    2011-08-01

    Carbon nanotube (CNT)-organic polymer hybrids have important potential applications in the immobilization of therapeutic biomolecules. Recently developed CNT-organic polymer composites require the use of organic solvents for their preparation and have limited polymer functionalization. To address these limitations, multi-walled CNT (MWCNT)-chitosan nanoparticle (CS NP) hybrids have been synthesized in situ by an ionotropic gelation process, which is extremely mild and involves the mixture of two aqueous solutions at room temperature. The MWCNT-CS NP hybrids were characterized by atomic force microscopy and thermogravimetric analysis. Under optimal conditions the CS NP can be tethered to the MWCNT surface in high density and with relatively uniform coverage. The MWCNT-CS NP hybrids show good dispersibility and stability in aqueous solutions. In order to evaluate the potential utilization of the hybrids as protein carries the cytotoxicity to HeLa cells and protein immobilization (of bovine serum albumin (BSA), used here as a model) capacity of the hybrids were investigated in detail. The results demonstrate that the MWCNT-CS NP hybrids are biocompatible at concentrations up to 100 μg mL(-1) for 24 h incubation. The MWCNT-CS NP hybrids improve the BSA immobilization efficiency 0.8 times and simultaneously decrease the cellular toxicity by about 50% compared with carboxylated MWCNT. PMID:21601019

  20. Yeast as a platform to explore polyglutamine toxicity and aggregation.

    PubMed

    Duennwald, Martin L

    2013-01-01

    Protein misfolding is associated with many neurodegenerative diseases, including neurodegenerative diseases caused by polyglutamine expansion proteins, such as Huntington's disease. The model organism baker's yeast (Saccharomyces cerevisiae) has provided important general insights into the basic cellular mechanisms underlying protein misfolding. Furthermore, experiments in yeast have identified cellular factors that modulate the toxicity and the aggregation associated with polyglutamine expansion proteins. Notably, many features discovered in yeast have been proven to be highly relevant in other model organisms and in human pathology. The experimental protocols depicted here serve to reliably determine polyglutamine toxicity and polyglutamine aggregation in yeast. PMID:23719914

  1. High Specificity of a Quantitative PCR Assay Targeting a Saxitoxin Gene for Monitoring Toxic Algae Associated with Paralytic Shellfish Toxins in the Yellow Sea

    PubMed Central

    Gao, Yan; Murray, Shauna A.; Chen, Jian-Hua; Kang, Zhen-Jun; Zhang, Qing-Chun; Kong, Fan-Zhou; Zhou, Ming-Jiang

    2015-01-01

    The identification of core genes involved in the biosynthesis of saxitoxin (STX) offers a great opportunity to detect toxic algae associated with paralytic shellfish toxins (PST). In the Yellow Sea (YS) in China, both toxic and nontoxic Alexandrium species are present, which makes it a difficult issue to specifically monitor PST-producing toxic algae. In this study, a quantitative PCR (qPCR) assay targeting sxtA4, a domain in the sxt gene cluster that encodes a unique enzyme involved in STX biosynthesis, was applied to analyze samples collected from the YS in spring of 2012. The abundance of two toxic species within the Alexandrium tamarense species complex, i.e., A. fundyense and A. pacificum, was also determined with TaqMan-based qPCR assays, and PSTs in net-concentrated phytoplankton samples were analyzed with high-performance liquid chromatography coupled with a fluorescence detector. It was found that the distribution of the sxtA4 gene in the YS was consistent with the toxic algae and PSTs, and the quantitation results of sxtA4 correlated well with the abundance of the two toxic species (r = 0.857). These results suggested that the two toxic species were major PST producers during the sampling season and that sxtA-based qPCR is a promising method to detect toxic algae associated with PSTs in the YS. The correlation between PST levels and sxtA-based qPCR results, however, was less significant (r = 0.552), implying that sxtA-based qPCR is not accurate enough to reflect the toxicity of PST-producing toxic algae. The combination of an sxtA-based qPCR assay and chemical means might be a promising method for monitoring toxic algal blooms. PMID:26231652

  2. High Specificity of a Quantitative PCR Assay Targeting a Saxitoxin Gene for Monitoring Toxic Algae Associated with Paralytic Shellfish Toxins in the Yellow Sea.

    PubMed

    Gao, Yan; Yu, Ren-Cheng; Murray, Shauna A; Chen, Jian-Hua; Kang, Zhen-Jun; Zhang, Qing-Chun; Kong, Fan-Zhou; Zhou, Ming-Jiang

    2015-10-01

    The identification of core genes involved in the biosynthesis of saxitoxin (STX) offers a great opportunity to detect toxic algae associated with paralytic shellfish toxins (PST). In the Yellow Sea (YS) in China, both toxic and nontoxic Alexandrium species are present, which makes it a difficult issue to specifically monitor PST-producing toxic algae. In this study, a quantitative PCR (qPCR) assay targeting sxtA4, a domain in the sxt gene cluster that encodes a unique enzyme involved in STX biosynthesis, was applied to analyze samples collected from the YS in spring of 2012. The abundance of two toxic species within the Alexandrium tamarense species complex, i.e., A. fundyense and A. pacificum, was also determined with TaqMan-based qPCR assays, and PSTs in net-concentrated phytoplankton samples were analyzed with high-performance liquid chromatography coupled with a fluorescence detector. It was found that the distribution of the sxtA4 gene in the YS was consistent with the toxic algae and PSTs, and the quantitation results of sxtA4 correlated well with the abundance of the two toxic species (r=0.857). These results suggested that the two toxic species were major PST producers during the sampling season and that sxtA-based qPCR is a promising method to detect toxic algae associated with PSTs in the YS. The correlation between PST levels and sxtA-based qPCR results, however, was less significant (r=0.552), implying that sxtA-based qPCR is not accurate enough to reflect the toxicity of PST-producing toxic algae. The combination of an sxtA-based qPCR assay and chemical means might be a promising method for monitoring toxic algal blooms.

  3. High Specificity of a Quantitative PCR Assay Targeting a Saxitoxin Gene for Monitoring Toxic Algae Associated with Paralytic Shellfish Toxins in the Yellow Sea.

    PubMed

    Gao, Yan; Yu, Ren-Cheng; Murray, Shauna A; Chen, Jian-Hua; Kang, Zhen-Jun; Zhang, Qing-Chun; Kong, Fan-Zhou; Zhou, Ming-Jiang

    2015-10-01

    The identification of core genes involved in the biosynthesis of saxitoxin (STX) offers a great opportunity to detect toxic algae associated with paralytic shellfish toxins (PST). In the Yellow Sea (YS) in China, both toxic and nontoxic Alexandrium species are present, which makes it a difficult issue to specifically monitor PST-producing toxic algae. In this study, a quantitative PCR (qPCR) assay targeting sxtA4, a domain in the sxt gene cluster that encodes a unique enzyme involved in STX biosynthesis, was applied to analyze samples collected from the YS in spring of 2012. The abundance of two toxic species within the Alexandrium tamarense species complex, i.e., A. fundyense and A. pacificum, was also determined with TaqMan-based qPCR assays, and PSTs in net-concentrated phytoplankton samples were analyzed with high-performance liquid chromatography coupled with a fluorescence detector. It was found that the distribution of the sxtA4 gene in the YS was consistent with the toxic algae and PSTs, and the quantitation results of sxtA4 correlated well with the abundance of the two toxic species (r=0.857). These results suggested that the two toxic species were major PST producers during the sampling season and that sxtA-based qPCR is a promising method to detect toxic algae associated with PSTs in the YS. The correlation between PST levels and sxtA-based qPCR results, however, was less significant (r=0.552), implying that sxtA-based qPCR is not accurate enough to reflect the toxicity of PST-producing toxic algae. The combination of an sxtA-based qPCR assay and chemical means might be a promising method for monitoring toxic algal blooms. PMID:26231652

  4. Comparison of the Folding Mechanism of Highly Homologous Proteins in the Lipid-binding Protein Family

    EPA Science Inventory

    The folding mechanism of two closely related proteins in the intracellular lipid binding protein family, human bile acid binding protein (hBABP) and rat bile acid binding protein (rBABP) were examined. These proteins are 77% identical (93% similar) in sequence Both of these singl...

  5. Effects of high-LET radiation on neural cells in culture: apoptosis induction, cell toxicity and gene expression

    NASA Astrophysics Data System (ADS)

    Vazquez, M.; Otto, S.; Estevez, L.; Rios, D.; Pena, L.; Anderson, C.

    Despite the fact that some in vivo studies suggest that chronic low-dose exposure to HZE particles might produce effects similar to aging and neurodegeneration, the basic mechanisms of HZE particle neurotoxicity remain to be elucidated. The goal of these experiments is to establish neural cellular models to evaluate the capacity of low- and high-LET radiation, to induce cell damage and apoptosis. In the present study we measured apoptosis, cell toxicity and gene expression induced by low fluences-doses of heavy ions, protons and photons using neuronal precursor cells (NT2, STRATAGENE) and post-mitotic neurons as models for adult neural cell system. Using heavy ions accelerated at AGS (BNL) and HIMAC (Chiba, Japan), and protons (Loma Linda) we study the neurotoxic effects of a variety of heavy particles (1 and 0.6 GeV/n Fe, 580 MeV/n Si, 290 MeV/n C, 550 MeV/n Ar; LET ranging from 13 to148 keV/μm), and 255 MeV/n protons. Apoptosis Induction: We measured the induction of apoptosis by flow cytometry using a FACSCalibur to detect the expression of Annexin V, as an early marker in the apoptotic pathway, in NT-2 cells. The ApoAlert Annexin V assay is based on the observation that soon after initiating apoptosis, most cell types translocate phosphatidylserine (PS) from the inner face of the plasma membrane to the cell surface. Once on the cell surface, PS can be easily detected by staining with a FITC conjugate of Annexin V, a protein that has a strong natural affinity for PS. Externalization of PS occurs earlier than the nuclear changes associated with apoptosis, so the ApoAlert Assay detects apoptotic cells significantly earlier than do DNA-based assays. Exposing NT-2 cells to Fe ions and protons induced a strong dose- and time-dependent induction of apoptosis with the peak of apoptosis appearing at 72 hours post-irradiation. It was determined that Fe ion exposure were more effective to induce apoptosis in comparison to protons and gamma rays, suggesting an high RBE

  6. Transgenic plants expressing the AaIT/GNA fusion protein show increased resistance and toxicity to both chewing and sucking pests.

    PubMed

    Liu, Shu-Min; Li, Jie; Zhu, Jin-Qi; Wang, Xiao-Wei; Wang, Cheng-Shu; Liu, Shu-Sheng; Chen, Xue-Xin; Li, Sheng

    2016-04-01

    The adoption of pest-resistant transgenic plants to reduce yield losses and decrease pesticide use has been successful. To achieve the goal of controlling both chewing and sucking pests in a given transgenic plant, we generated transgenic tobacco, Arabidopsis, and rice plants expressing the fusion protein, AaIT/GNA, in which an insecticidal scorpion venom neurotoxin (Androctonus australis toxin, AaIT) is fused to snowdrop lectin (Galanthus nivalis agglutinin, GNA). Compared with transgenic tobacco and Arabidopsis plants expressing AaIT or GNA, transgenic plants expressing AaIT/GNA exhibited increased resistance and toxicity to one chewing pest, the cotton bollworm, Helicoverpa armigera. Transgenic tobacco and rice plants expressing AaIT/GNA showed increased resistance and toxicity to two sucking pests, the whitefly, Bemisia tabaci, and the rice brown planthopper, Nilaparvata lugens, respectively. Moreover, in the field, transgenic rice plants expressing AaIT/GNA exhibited a significant improvement in grain yield when infested with N. lugens. This study shows that expressing the AaIT/GNA fusion protein in transgenic plants can be a useful approach for controlling pests, particularly sucking pests which are not susceptible to the toxin in Bt crops. PMID:25641865

  7. Determination of toxic cyclic heptapeptides by liquid chromatography with detection using ultra-violet, protein phosphatase assay and tandem mass spectrometry.

    PubMed

    Ortea, Paloma Muñiz; Allis, Orla; Healy, Brendan M; Lehane, Mary; Ní Shuilleabháin, Aine; Furey, Ambrose; James, Kevin J

    2004-06-01

    Microcystins, toxic cyclic heptapeptides and nodularin-R, a toxic cyclic pentapeptide, were determined using liquid chromatography (LC) with detection using photo-diode array ultra-violet (PDA-UV) and protein phosphatase (PP) assay. Positive fractions were analysed for toxins using collision-induced dissociation (CID) and tandem MS/MS experiments which were carried out simultaneously using electrospray ion-trap instrumentation. Reversed-phase liquid chromatography (LC) using an acetonitrile/water gradient was used for the LC-MS(2) determination of six microcystins standards and nodularin. The molecular related ion species, [M+H](+)([M+2H](2+) in the case of MC-RR), were used as the precursor ions for MS(2) experiments. Optimum calibration and reproducibility data were obtained for MC-LR using LC-MS(2); 0.1-5.0 microg/ml, r2 = 0.992 (n = 3); % RSD < or =7.3 at 0.25 microg MC-LR/ml (n = 3). The detection limit (S/N = 3) was better than 0.1 ng. Water samples for microcystin analysis were first screened using protein phosphatase (PP) assays and positives were concentrated using C-18 solid-phase extraction. The developed method was applied to examine a lake in Ireland contaminated by Microcystis sp. and MC-LR and MC-LA were identified.

  8. Metallothionein protection of cadmium toxicity

    SciTech Connect

    Klaassen, Curtis D. Liu, Jie; Diwan, Bhalchandra A.

    2009-08-01

    The discovery of the cadmium (Cd)-binding protein from horse kidney in 1957 marked the birth of research on this low-molecular weight, cysteine-rich protein called metallothionein (MT) in Cd toxicology. MT plays minimal roles in the gastrointestinal absorption of Cd, but MT plays important roles in Cd retention in tissues and dramatically decreases biliary excretion of Cd. Cd-bound to MT is responsible for Cd accumulation in tissues and the long biological half-life of Cd in the body. Induction of MT protects against acute Cd-induced lethality, as well as acute toxicity to the liver and lung. Intracellular MT also plays important roles in ameliorating Cd toxicity following prolonged exposures, particularly chronic Cd-induced nephrotoxicity, osteotoxicity, and toxicity to the lung, liver, and immune system. There is an association between human and rodent Cd exposure and prostate cancers, especially in the portions where MT is poorly expressed. MT expression in Cd-induced tumors varies depending on the type and the stage of tumor development. For instance, high levels of MT are detected in Cd-induced sarcomas at the injection site, whereas the sarcoma metastases are devoid of MT. The use of MT-transgenic and MT-null mice has greatly helped define the role of MT in Cd toxicology, with the MT-null mice being hypersensitive and MT-transgenic mice resistant to Cd toxicity. Thus, MT is critical for protecting human health from Cd toxicity. There are large individual variations in MT expression, which might in turn predispose some people to Cd toxicity.

  9. Protein denaturation of whey protein isolates (WPIs) induced by high intensity ultrasound during heat gelation.

    PubMed

    Frydenberg, Rikke P; Hammershøj, Marianne; Andersen, Ulf; Greve, Marie T; Wiking, Lars

    2016-02-01

    In this study, the impact of high intensity ultrasound (HIU) on proteins in whey protein isolates was examined. Effects on thermal behavior, secondary structure and nature of intra- and intermolecular bonds during heat-induced gelling were investigated. Ultrasonication (24 kHz, 300 W/cm(2), 2078 J/mL) significantly reduced denaturation enthalpies, whereas no change in secondary structure was detected by circular dichroism. The thiol-blocking agent N-ethylmaleimide was applied in order to inhibit formation of disulfide bonds during gel formation. Results showed that increased contents of α-lactalbumin (α-La) were associated with increased sensitivity to ultrasonication. The α-La:β-lactoglobulin (β-Lg) ratio greatly affected the nature of the interactions formed during gelation, where higher amounts of α-La lead to a gel more dependent on disulfide bonds. These results contribute to clarifying the mechanisms mediating the effects of HIU on whey proteins on the molecular level, thus moving further toward implementing HIU in the processing chain in the food industry.

  10. Cyanobacterial high-light-inducible proteins--Protectors of chlorophyll-protein synthesis and assembly.

    PubMed

    Komenda, Josef; Sobotka, Roman

    2016-03-01

    Cyanobacteria contain a family of genes encoding one-helix high-light-inducible proteins (Hlips) that are homologous to light harvesting chlorophyll a/b-binding proteins of plants and algae. Based on various experimental approaches used for their study, a spectrum of functions that includes regulation of chlorophyll biosynthesis, transient chlorophyll binding, quenching of singlet oxygen and non-photochemical quenching of absorbed energy is ascribed to Hlips. However, these functions had not been supported by conclusive experimental evidence until recently when it became clear that Hlips are able to quench absorbed light energy and assist during terminal step(s) of the chlorophyll biosynthesis and early stages of Photosystem II assembly. In this review we summarize and discuss the present knowledge about Hlips and provide a model of how individual members of the Hlip family operate during the biogenesis of chlorophyll-proteins, namely Photosystem II. This article is part of a Special Issue entitled Organization and dynamics of bioenergetic systems in bacteria, edited by Conrad Mullineaux.

  11. Consequences of inducing intrinsic disorder in a high-affinity protein-protein interaction.

    PubMed

    Papadakos, Grigorios; Sharma, Amit; Lancaster, Lorna E; Bowen, Rebecca; Kaminska, Renata; Leech, Andrew P; Walker, Daniel; Redfield, Christina; Kleanthous, Colin

    2015-04-29

    The kinetic and thermodynamic consequences of intrinsic disorder in protein-protein recognition are controversial. We address this by inducing one partner of the high-affinity colicin E3 rRNase domain-Im3 complex (K(d) ≈ 10(-12) M) to become an intrinsically disordered protein (IDP). Through a variety of biophysical measurements, we show that a single alanine mutation at Tyr507 within the hydrophobic core of the isolated colicin E3 rRNase domain causes the enzyme to become an IDP (E3 rRNase(IDP)). E3 rRNase(IDP) binds stoichiometrically to Im3 and forms a structure that is essentially identical to the wild-type complex. However, binding of E3 rRNase(IDP) to Im3 is 4 orders of magnitude weaker than that of the folded rRNase, with thermodynamic parameters reflecting the disorder-to-order transition on forming the complex. Critically, pre-steady-state kinetic analysis of the E3 rRNase(IDP)-Im3 complex demonstrates that the decrease in affinity is mostly accounted for by a drop in the electrostatically steered association rate. Our study shows that, notwithstanding the advantages intrinsic disorder brings to biological systems, this can come at severe kinetic and thermodynamic cost. PMID:25856265

  12. Expedient synthesis of highly potent antagonists of inhibitor of apoptosis proteins (IAPs) with unique selectivity for ML-IAP.

    PubMed

    Vamos, Mitchell; Welsh, Kate; Finlay, Darren; Lee, Pooi San; Mace, Peter D; Snipas, Scott J; Gonzalez, Monica L; Ganji, Santhi Reddy; Ardecky, Robert J; Riedl, Stefan J; Salvesen, Guy S; Vuori, Kristiina; Reed, John C; Cosford, Nicholas D P

    2013-04-19

    A series of novel, potent antagonists of the inhibitor of apoptosis proteins (IAPs) were synthesized in a highly convergent and rapid fashion (≤6 steps) using the Ugi four-component reaction as the key step, thus enabling rapid optimization of binding potency. These IAP antagonists compete with caspases 3, 7, and 9 for inhibition by X chromosome-linked IAP (XIAP) and bind strongly (nanomolar binding constants) to several crucial members of the IAP family of cancer pro-survival proteins to promote apoptosis, with a particularly unique selectivity for melanoma IAP (ML-IAP). Experiments in cell culture revealed powerful cancer cell growth inhibitory activity in multiple (breast, ovarian, and prostate) cell lines with single agent toxicity at low nanomolar levels against SKOV-3 human ovarian carcinoma cells. Administration of the compounds to human foreskin fibroblast cells revealed no general toxicity to normal cells. Furthermore, computational modeling was performed, revealing key contacts between the IAP proteins and antagonists, suggesting a structural basis for the observed potency.

  13. Anomalous Diffraction at Ultra-High Energy for Protein Crystallography

    SciTech Connect

    Jakoncic,J.; Di Michiel, M.; Zhong, Z.; Honkimaki, V.; Jouanneau, Y.; Stojanoff, V.

    2006-01-01

    Single-wavelength anomalous diffraction (SAD), multiwavelength anomalous diffraction (MAD) and single isomorphous replacement with anomalous scattering (SIRAS) phasing at ultra-high X-ray energy, 55 keV, are used successfully to determine a high-quality and high-resolution experimental electronic density map of hen egg-white lysozyme, a model protein. Several combinations, between single- and three-wavelength, with native data were exploited to demonstrate that standard phasing procedures with standard equipment and software can successfully be applied to three-dimensional crystal structure determination of a macromolecule, even at these very short wavelengths. For the first time, a high-quality three-dimensional molecular structure is reported from SAD phasing with ultra-high-energy X-rays. The quality of the crystallographic data and the experimental electron density maps meet current standards. The 2.7% anomalous signal from three Ho atoms, at the Ho K edge, was sufficient to obtain a remarkable electron density and build the first lanthanide structure for HEWL in its entirety.

  14. A differential protein solubility approach for the depletion of highly abundant proteins in plasma using ammonium sulfate.

    PubMed

    Bollineni, Ravi Chand; Guldvik, Ingrid J; Grönberg, Henrik; Wiklund, Fredrik; Mills, Ian G; Thiede, Bernd

    2015-12-21

    Depletion of highly abundant proteins is an approved step in blood plasma analysis by mass spectrometry (MS). In this study, we explored a precipitation and differential protein solubility approach as a fractionation strategy for abundant protein removal from plasma. Total proteins from plasma were precipitated with 90% saturated ammonium sulfate, followed by differential solubilization in 55% and 35% saturated ammonium sulfate solutions. Using a four hour liquid chromatography (LC) gradient and an LTQ-Orbitrap XL mass spectrometer, a total of 167 and 224 proteins were identified from the 55% and 35% ammonium sulfate fractions, whereas 235 proteins were found in the remaining protein fractions with at least two unique peptides. SDS-PAGE and exclusive total spectrum counts from LC-MS/MS analyses clearly showed that majority of the abundant plasma proteins were solubilized in 55% and 35% ammonium sulfate solutions, indicating that the remaining protein fraction is of potential interest for identification of less abundant plasma proteins. Serum albumin, serotransferrin, alpha-1-antitrypsin and transthyretin were the abundant proteins that were highly enriched in 55% ammonium sulfate fractions. Immunoglobulins, complement system proteins, and apolipoproteins were among other abundant plasma proteins that were enriched in 35% ammonium sulfate fractions. In the remaining protein fractions a total of 40 unique proteins were identified of which, 32 proteins were identified with at least 10 exclusive spectrum counts. According to PeptideAtlas, 9 of these 32 proteins were estimated to be present at low μg ml(-1) (0.12-1.9 μg ml(-1)) concentrations in the plasma, and 17 at low ng ml(-1) (0.1-55 ng ml(-1)) range.

  15. DNA damage in earthworms from highly contaminated soils: assessing resistance to arsenic toxicity by use of the Comet assay.

    PubMed

    Button, Mark; Jenkin, Gawen R T; Bowman, Karen J; Harrington, Chris F; Brewer, Tim S; Jones, George D D; Watts, Michael J

    2010-02-01

    Earthworms native to the former mine site of Devon Great Consols (DGC), UK reside in soils highly contaminated with arsenic (As). These earthworms are considered to have developed a resistance to As toxicity. The mechanisms underlying this resistance however, remain unclear. In the present study, non-resistant, commercially sourced Lumbricus terrestris were exposed to a typical DGC soil in laboratory mesocosms. The earthworms bio-accumulated As from the soil and incurred DNA-damage levels significantly above those observed in the control mesocosm (assessed using the Comet assay). A dose response was observed between DNA damage (% tail DNA) and As concentration in soil (control, 98, 183, 236, 324 and 436mgkg(-1)). As-resistant earthworms (Lumbricus rubellus, Dendrodrilus rubidus and L. terrestris) collected from contaminated soils at DGC (203 to 9025mgkg(-1) As) had also bio-accumulated high levels of As from their host soils, yet demonstrated low levels of DNA damage compared with earthworms from uncontaminated sites. The results demonstrate that the As-contaminated soils at DGC are genotoxic to non-native earthworms and much less so to earthworms native to DGC, thus providing further evidence of an acquired resistance to As toxicity in the native earthworms. PMID:20015476

  16. DNA damage in earthworms from highly contaminated soils: assessing resistance to arsenic toxicity by use of the Comet assay.

    PubMed

    Button, Mark; Jenkin, Gawen R T; Bowman, Karen J; Harrington, Chris F; Brewer, Tim S; Jones, George D D; Watts, Michael J

    2010-02-01

    Earthworms native to the former mine site of Devon Great Consols (DGC), UK reside in soils highly contaminated with arsenic (As). These earthworms are considered to have developed a resistance to As toxicity. The mechanisms underlying this resistance however, remain unclear. In the present study, non-resistant, commercially sourced Lumbricus terrestris were exposed to a typical DGC soil in laboratory mesocosms. The earthworms bio-accumulated As from the soil and incurred DNA-damage levels significantly above those observed in the control mesocosm (assessed using the Comet assay). A dose response was observed between DNA damage (% tail DNA) and As concentration in soil (control, 98, 183, 236, 324 and 436mgkg(-1)). As-resistant earthworms (Lumbricus rubellus, Dendrodrilus rubidus and L. terrestris) collected from contaminated soils at DGC (203 to 9025mgkg(-1) As) had also bio-accumulated high levels of As from their host soils, yet demonstrated low levels of DNA damage compared with earthworms from uncontaminated sites. The results demonstrate that the As-contaminated soils at DGC are genotoxic to non-native earthworms and much less so to earthworms native to DGC, thus providing further evidence of an acquired resistance to As toxicity in the native earthworms.

  17. Evaluation of heavy-metal ion toxicity in fish cells using a combined stress protein and cytotoxicity assay

    SciTech Connect

    Ryan, J.A.; Hightower, L.E. )

    1994-08-01

    All organisms, from bacteria and yeast to humans, respond to physical and chemical stressors by increasing the synthesis of a small group of cellular stress proteins.'' The authors have developed a simple in vitro system for quickly screening environmentally relevant stressors to detect stress-induced proteins that are good candidates for biomarkers. Polyacrylamide gel electrophoresis was used to detect stressor-induced, concentration-dependent changes in cellular stress protein levels in two fish cell culture systems, whereas simultaneous in vitro neutral red uptake cytotoxicity assays measured the stressors effect on cellular physiology. There was a direct concentration-dependent relationship between sublethal cytotoxic effects and the increases in stress protein levels. Increases of 50 to 200% were detected in stress proteins from desert topminnow, Poeciliopsis lucida, hepatoma-derived cell cultures exposed to cadmium or copper. Three proteins showed similar increases in winter flounder, Pleuronectes americanus, kidney cell cultures exposed to the same stressors. Increases in the evolutionarily conserved heat-shock protein hsp70 were detected in each experiment; its level increased with increasing stressor concentrations.

  18. High Resolution NMR Spectroscopy of Nanocrystalline Proteins at Ultra-High Magnetic Field

    PubMed Central

    Sperling, Lindsay J.; Nieuwkoop, Andrew J.; Lipton, Andrew S.; Berthold, Deborah A.; Rienstra, Chad M.

    2010-01-01

    Magic-angle spinning (MAS) solid-state NMR (SSNMR) spectroscopy of uniformly-13C,15N labeled protein samples provides insight into atomic-resolution chemistry and structure. Data collection efficiency has advanced remarkably in the last decade; however, the study of larger proteins is still challenged by relatively low resolution in comparison to solution NMR. In this study, we present a systematic analysis of SSNMR protein spectra acquired at 11.7, 17.6 and 21.1 Tesla (1H frequencies of 500, 750, and 900 MHz). For two protein systems—GB1, a 6 kDa nanocrystalline protein and DsbA, a 21 kDa nanocrystalline protein—line narrowing is demonstrated in all spectral regions with increasing field. Resolution enhancement is greatest in the aliphatic region, including methine, methylene and methyl sites. The resolution for GB1 increases markedly as a function of field, and for DsbA, resolution in the C-C region increases by 42%, according to the number of peaks that can be uniquely picked and integrated in the 900 MHz spectra when compared to the 500 MHz spectra. Additionally, chemical exchange is uniquely observed in the highest field spectra for at least two isoleucine Cδ1 sites in DsbA. These results further illustrate the benefits of high-field MAS SSNMR spectroscopy for protein structural studies. PMID:19953303

  19. Characterization of Diversity in Toxicity Mechanism Using In Vitro Cytotoxicity Assays in Quantitative High Throughput Screening

    PubMed Central

    Huang, Ruili; Southall, Noel; Cho, Ming-Hsuang; Xia, Menghang; Inglese, James; Austin, Christopher P.

    2009-01-01

    Assessing the potential health risks of environmental chemical compounds is an expensive undertaking which has motivated the development of new alternatives to traditional in vivo toxicological testing. One approach is to stage the evaluation, beginning with less expensive and higher throughput in vitro testing before progressing to more definitive trials. In vitro testing can be used to generate a hypothesis about a compound's mechanism of action, which can then be used to design an appropriate in vivo experiment. Here we begin to address the question of how to design such a battery of in vitro cell-based assays by combining data from two different types of assays, cell viability and caspase activation, with the aim of elucidating mechanism of action. Because caspase activation is a transient event during apoptosis, it is not possible to design a single end-point assay protocol that would identify all instances of compound-induced caspase activation. Nevertheless, useful information about compound mechanism of action can be obtained from these assays in combination with cell viability data. Unsupervised clustering in combination with Dunn's cluster validity index is a robust method for identifying mechanisms of action without requiring any a priori knowledge about mechanisms of toxicity. The performance of this clustering method is evaluated by comparing the clustering results against literature annotations of compound mechanisms. PMID:18281954

  20. A critical evaluation of in vitro cell culture models for high-throughput drug screening and toxicity.

    PubMed

    Astashkina, Anna; Mann, Brenda; Grainger, David W

    2012-04-01

    Drug candidate and toxicity screening processes currently rely on results from early-stage in vitro cell-based assays expected to faithfully represent essential aspects of in vivo pharmacology and toxicology. Several in vitro designs are optimized for high throughput to benefit screening efficiencies, allowing the entire libraries of potential pharmacologically relevant or possible toxin molecules to be screened for different types of cell signals relevant to tissue damage or to therapeutic goals. Creative approaches to multiplexed cell-based assay designs that select specific cell types, signaling pathways and reporters are routine. However, substantial percentages of new chemical and biological entities (NCEs/NBEs) that fail late-stage human drug testing, or receive regulatory "black box" warnings, or that are removed from the market for safety reasons after regulatory approvals all provide strong evidence that in vitro cell-based assays and subsequent preclinical in vivo studies do not yet provide sufficient pharmacological and toxicity data or reliable predictive capacity for understanding drug candidate performance in vivo. Without a reliable translational assay tool kit for pharmacology and toxicology, the drug development process is costly and inefficient in taking initial in vitro cell-based screens to in vivo testing and subsequent clinical approvals. Commonly employed methods of in vitro testing, including dissociated, organotypic, organ/explant, and 3-D cultures, are reviewed here with specific focus on retaining cell and molecular interactions and physiological parameters that determine cell phenotypes and their corresponding responses to bioactive agents. Distinct advantages and performance challenges for these models pertinent to cell-based assay and their predictive capabilities required for accurate correlations to in vivo mechanisms of drug toxicity are compared. PMID:22252140

  1. Toxicity challenges in environmental chemicals: Prediction of human plasma protein binding through quantitative structure-activity relationship (QSAR) models

    EPA Science Inventory

    The present study explores the merit of utilizing available pharmaceutical data to construct a quantitative structure-activity relationship (QSAR) for prediction of the fraction of a chemical unbound to plasma protein (Fub) in environmentally relevant compounds. Independent model...

  2. Associations of novel genetic variations in the folate-related and ARID5B genes with the pharmacokinetics and toxicity of high-dose methotrexate in paediatric acute lymphoblastic leukaemia.

    PubMed

    Csordas, Katalin; Lautner-Csorba, Orsolya; Semsei, Agnes F; Harnos, Andrea; Hegyi, Marta; Erdelyi, Daniel J; Eipel, Oliver T; Szalai, Csaba; Kovacs, Gabor T

    2014-08-01

    High-dose methotrexate (HD-MTX) plays an important role in the consolidation therapy of acute lymphoblastic leukaemia (ALL) in many treatment regimens worldwide. However, there is a large interpatient variability in the pharmacokinetics and toxicity of the drug. We investigated the influence of single nucleotide polymorphisms (SNPs) in genes of the folate metabolic pathway, transporter molecules and transcription proteins on the pharmacokinetics and toxicity of MTX and 7-hydroxy-methotrexate (7-OH-MTX). 63 SNPs of 14 genes were genotyped and a total of 463 HD-MTX courses (administered according to the ALL-BFM 95 and ALL IC-BFM 2002 protocols) were analysed. Haematological, hepatic and renal toxicities, estimated by routine laboratory parameters were evaluated. Random forest and regression trees were used for variable selection and model building. Linear mixed models were established to prove the significance of the selected variables. SNPs (rs4948502, rs4948496, rs4948487) of the ARID5B gene were associated with the serum levels of MTX (P < 0·02), serum levels and area under the curve of 7-OH-MTX (P < 0·02) and with hypoproteinaemia (P = 0·004). SLCO1B1 rs4149056 also showed a significant association with serum MTX levels (P < 0·001). Our findings confirm the association of novel genetic variations in folate-related and ARID5B genes with the serum MTX levels and acute toxicity.

  3. Highly purified mussel adhesive protein to secure biosafety for in vivo applications

    PubMed Central

    2014-01-01

    Background Unique adhesive and biocompatibility properties of mussel adhesive proteins (MAPs) are known for their great potential in many tissue engineering and biomedical applications. Previously, it was successfully demonstrated that redesigned hybrid type MAP, fp-151, mass-produced in Gram-negative bacterium Escherichia coli, could be utilized as a promising adhesive biomaterial. However, purification of recombinant fp-151 has been unsatisfactory due to its adhesive nature and polarity which make separation of contaminants (especially, lipopolysaccharide, a toxic Gram-negative cell membrane component) very difficult. Results In the present work, we devised a high resolution purification approach to secure safety standards of recombinant fp-151 for the successful use in in vivo applications. Undesirable impurities were remarkably eliminated as going through sequential steps including treatment with multivalent ion and chelating agent for cell membrane washing, mechanical cell disruption, non-ionic surfactant treatment for isolated inclusion body washing, acid extraction of washed inclusion body, and ion exchange chromatography purification of acid extracted sample. Through various analyses, such as high performance liquid chromatographic purity assay, limulus amoebocyte lysate endotoxin assay, and in vitro mouse macrophage cell tests on inflammation, viability, cytotoxicity, and apoptosis, we confirmed the biological safety of bacterial-derived purified recombinant fp-151. Conclusions Through this purification design, recombinant fp-151 achieved 99.90% protein purity and 99.91% endotoxin reduction that nearly no inflammation response was observed in in vitro experiments. Thus, the highly purified recombinant MAP would be successfully used as a safety-secured in vivo bioadhesive for tissue engineering and biomedical applications. PMID:24725543

  4. Evaluation of detoxification methods on toxic and antinutritional composition and nutritional quality of proteins in Jatropha curcas meal.

    PubMed

    Xiao, Jianhui; Zhang, Hui; Niu, Liya; Wang, Xingguo; Lu, Xia

    2011-04-27

    The Jatropha curcas meal was detoxified by different methods, and the effect of detoxification was evaluated in this study. The method that hydrolysis of enzymes (cellulase plus pectinase) followed by washing with ethanol (65%) had a significant (p < 0.05) effect on the toxin, antinutritional components, and nutritional quality of proteins. After this treatment, the phorbolesters (PEs) were decreased by 100%. The antinutritional components (phytates, tannins, saponins, protease inhibitor, and lectin activities) were decreased to tolerable levels, which were lower than those in soybean meal. The crude protein in detoxified meal was 74.68%, and the total content of amino acids was 66.87 g/100 g of dry matter. The in vitro protein digestibility (IVPD) increased from 82.14 to 92.37%. The pepsin-insoluble nitrogen was only 4.57% of the total nitrogen, and about 90% of the protein was true protein. The protein-digestibility-corrected amino acid score (PDCAAS) of the meal was 0.75. The results showed that this treatment was a promising way to detoxify J. curcas meal, and the nutritional quality of detoxified meal can be simultaneously enriched and improved. PMID:21410262

  5. High-strength silk protein scaffolds for bone repair

    PubMed Central

    Mandal, Biman B.; Grinberg, Ariela; Seok Gil, Eun; Panilaitis, Bruce; Kaplan, David L.

    2012-01-01

    Biomaterials for bone tissue regeneration represent a major focus of orthopedic research. However, only a handful of polymeric biomaterials are utilized today because of their failure to address critical issues like compressive strength for load-bearing bone grafts. In this study development of a high compressive strength (~13 MPa hydrated state) polymeric bone composite materials is reported, based on silk protein-protein interfacial bonding. Micron-sized silk fibers (10–600 µm) obtained utilizing alkali hydrolysis were used as reinforcement in a compact fiber composite with tunable compressive strength, surface roughness, and porosity based on the fiber length included. A combination of surface roughness, porosity, and scaffold stiffness favored human bone marrow-derived mesenchymal stem cell differentiation toward bone-like tissue in vitro based on biochemical and gene expression for bone markers. Further, minimal in vivo immunomodulatory responses suggested compatibility of the fabricated silk-fiber-reinforced composite matrices for bone engineering applications. PMID:22552231

  6. A microfluidic, high throughput protein crystal growth method for microgravity.

    PubMed

    Carruthers, Carl W; Gerdts, Cory; Johnson, Michael D; Webb, Paul

    2013-01-01

    The attenuation of sedimentation and convection in microgravity can sometimes decrease irregularities formed during macromolecular crystal growth. Current terrestrial protein crystal growth (PCG) capabilities are very different than those used during the Shuttle era and that are currently on the International Space Station (ISS). The focus of this experiment was to demonstrate the use of a commercial off-the-shelf, high throughput, PCG method in microgravity. Using Protein BioSolutions' microfluidic Plug Maker™/CrystalCard™ system, we tested the ability to grow crystals of the regulator of glucose metabolism and adipogenesis: peroxisome proliferator-activated receptor gamma (apo-hPPAR-γ LBD), as well as several PCG standards. Overall, we sent 25 CrystalCards™ to the ISS, containing ~10,000 individual microgravity PCG experiments in a 3U NanoRacks NanoLab (1U = 10(3) cm.). After 70 days on the ISS, our samples were returned with 16 of 25 (64%) microgravity cards having crystals, compared to 12 of 25 (48%) of the ground controls. Encouragingly, there were more apo-hPPAR-γ LBD crystals in the microgravity PCG cards than the 1g controls. These positive results hope to introduce the use of the PCG standard of low sample volume and large experimental density to the microgravity environment and provide new opportunities for macromolecular samples that may crystallize poorly in standard laboratories. PMID:24278480

  7. A Microfluidic, High Throughput Protein Crystal Growth Method for Microgravity

    PubMed Central

    Carruthers Jr, Carl W.; Gerdts, Cory; Johnson, Michael D.; Webb, Paul

    2013-01-01

    The attenuation of sedimentation and convection in microgravity can sometimes decrease irregularities formed during macromolecular crystal growth. Current terrestrial protein crystal growth (PCG) capabilities are very different than those used during the Shuttle era and that are currently on the International Space Station (ISS). The focus of this experiment was to demonstrate the use of a commercial off-the-shelf, high throughput, PCG method in microgravity. Using Protein BioSolutions’ microfluidic Plug Maker™/CrystalCard™ system, we tested the ability to grow crystals of the regulator of glucose metabolism and adipogenesis: peroxisome proliferator-activated receptor gamma (apo-hPPAR-γ LBD), as well as several PCG standards. Overall, we sent 25 CrystalCards™ to the ISS, containing ~10,000 individual microgravity PCG experiments in a 3U NanoRacks NanoLab (1U = 103 cm.). After 70 days on the ISS, our samples were returned with 16 of 25 (64%) microgravity cards having crystals, compared to 12 of 25 (48%) of the ground controls. Encouragingly, there were more apo-hPPAR-γ LBD crystals in the microgravity PCG cards than the 1g controls. These positive results hope to introduce the use of the PCG standard of low sample volume and large experimental density to the microgravity environment and provide new opportunities for macromolecular samples that may crystallize poorly in standard laboratories. PMID:24278480

  8. A microfluidic, high throughput protein crystal growth method for microgravity.

    PubMed

    Carruthers, Carl W; Gerdts, Cory; Johnson, Michael D; Webb, Paul

    2013-01-01

    The attenuation of sedimentation and convection in microgravity can sometimes decrease irregularities formed during macromolecular crystal growth. Current terrestrial protein crystal growth (PCG) capabilities are very different than those used during the Shuttle era and that are currently on the International Space Station (ISS). The focus of this experiment was to demonstrate the use of a commercial off-the-shelf, high throughput, PCG method in microgravity. Using Protein BioSolutions' microfluidic Plug Maker™/CrystalCard™ system, we tested the ability to grow crystals of the regulator of glucose metabolism and adipogenesis: peroxisome proliferator-activated receptor gamma (apo-hPPAR-γ LBD), as well as several PCG standards. Overall, we sent 25 CrystalCards™ to the ISS, containing ~10,000 individual microgravity PCG experiments in a 3U NanoRacks NanoLab (1U = 10(3) cm.). After 70 days on the ISS, our samples were returned with 16 of 25 (64%) microgravity cards having crystals, compared to 12 of 25 (48%) of the ground controls. Encouragingly, there were more apo-hPPAR-γ LBD crystals in the microgravity PCG cards than the 1g controls. These positive results hope to introduce the use of the PCG standard of low sample volume and large experimental density to the microgravity environment and provide new opportunities for macromolecular samples that may crystallize poorly in standard laboratories.

  9. Membrane Signaling Induced by High Doses of Ionizing Radiation in the Endothelial Compartment. Relevance in Radiation Toxicity

    PubMed Central

    Corre, Isabelle; Guillonneau, Maëva; Paris, François

    2013-01-01

    Tumor areas can now be very precisely delimited thanks to technical progress in imaging and ballistics. This has also led to the development of novel radiotherapy protocols, delivering higher doses of ionizing radiation directly to cancer cells. Despite this, radiation toxicity in healthy tissue remains a major issue, particularly with dose-escalation in these new protocols. Acute and late tissue damage following irradiation have both been linked to the endothelium irrigating normal tissues. The molecular mechanisms involved in the endothelial response to high doses of radiation are associated with signaling from the plasma membrane, mainly via the acid sphingomyelinase/ceramide pathway. This review describes this signaling pathway and discusses the relevance of targeting endothelial signaling to protect healthy tissues from the deleterious effects of high doses of radiation. PMID:24252908

  10. MOLECULAR CLONING, EXPRESSION PATTERN OF MULTIDRUG RESISTANCE ASSOCIATED PROTEIN 1 (MRP1, ABCC1) GENE, AND THE SYNERGISTIC EFFECTS OF VERAPAMIL ON TOXICITY OF TWO INSECTICIDES IN THE BIRD CHERRY-OAT APHID.

    PubMed

    Kang, Xin-Le; Zhang, Meng; Wang, Kang; Qiao, Xian-Feng; Chen, Mao-Hua

    2016-05-01

    The ATP-binding cassette (ABC) transporters are important transmembrane proteins encoded by a supergene family. The majority of ABC proteins are primary active transporters that bind and hydrolyze ATP to mediate the efflux of a diverse range of substrates across lipid membranes. In this study, we cloned and characterized a putative multidrug resistance associated protein 1 (MRP1) from Rhopalosiphum padi encoded by ABCC1. Structural analysis showed that this protein has structural features typical of the ABC transporter family. Phylogenetic analysis indicated that the amino acid sequence was highly similar that of the corresponding protein from Acyrthosiphon pisum. Real-time quantitative polymerase chain reaction (PCR) analysis showed that ABCC1 was expressed throughout all R. padi developmental stages, with the highest level of expression in the fourth larval instar. We also examined ABCC1 expression in four different tissue types and found that it was most highly expressed in the midgut. Exposing R. padi to imidacloprid and chlorpyrifos increased ABCC1 expression. Furthermore, ABCC1 expression was higher in the imidacloprid-resistant (IR) and chlorpyrifos-resistant (CR) strains than in an insecticide-susceptible strain (SS) of R. padi. Exposing R. padi to verapamil in combination with insecticides significantly increased the toxicity of the insecticides. The respective synergy factor of CR and IR R. padi strain was 1.33 and 1.26, which was lower than that (2.72 and 1.64, respectively) of the SS. Our results clarify the biological function of ABCC1 in R. padi, particularly its role in insecticide resistance, and suggest novel strategies for pest management that use ABC transporter inhibitors to increase the effectiveness of insecticides.

  11. Toxicities of busulfan/melphalan versus carboplatin/etoposide/melphalan for high-dose chemotherapy with stem cell rescue for high-risk neuroblastoma.

    PubMed

    Desai, A V; Heneghan, M B; Li, Y; Bunin, N J; Grupp, S A; Bagatell, R; Seif, A E

    2016-09-01

    The optimal autologous stem cell rescue (HDC-SCR) regimen for children with high-risk neuroblastoma (HR-NBL) is not defined. Carboplatin/etoposide/melphalan (CEM) is the current US standard; however, European data suggest busulfan/melphalan (Bu/Mel) may have less toxicity. Published data regarding toxicities associated with CEM and Bu/Mel are limited. We conducted a single-institution retrospective cohort study of children with HR-NBL who received CEM or Bu/Mel preparative regimens. Toxicity data were analyzed using χ(2) or Fisher's exact, Wilcoxon two-sample or log-rank tests. Sinusoidal obstruction syndrome (SOS) was observed in 7/44 CEM (15.9%) and 5/21 (24%) Bu/Mel patients (P=0.50). Median time to SOS was longer following Bu/Mel than CEM (20 versus 9 days, P=0.02). Pulmonary hypertension (PHTN) was observed in ~20% of children after Bu/Mel and none after CEM (P=0.01). CEM patients had more nephrotoxicity (P=0.001), packed red blood cell (P=0.02) and platelet transfusions (P=0.008), and days on maximum pain support (P=0.0007). Time to engraftment, length of stay, documented infection rates and HDC-SCR-related mortality were similar. Nephrotoxicity and resource utilization associated with cytopenias and mucositis were greater after CEM. Pulmonary toxicities were more severe after Bu/Mel, and increased vigilance for PHTN may be warranted, particularly in children with hypoxemia out of proportion to respiratory distress. PMID:27159174

  12. A high protein diet upregulated whole-body protein turnover during energy deficit

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The effects of higher protein diets and sustained energy deficit (ED) on whole-body protein turnover (WBPTO) are not well described. This study examined whether dietary protein level influences whole-body protein breakdown (Ra), non-oxidative leucine disposal (NOLD), and oxidation (Ox) during ED. ...

  13. How do membranes initiate Alzheimer's Disease? Formation of toxic amyloid fibrils by the amyloid β-protein on ganglioside clusters.

    PubMed

    Matsuzaki, Katsumi

    2014-08-19

    Alzheimer's disease (AD), a severe neurodegenerative disorder, causes more than half of dementia cases. According to the popular "Aβ hypothesis" to explain the mechanism of this disease, amyloid β-peptides (Aβ) of 39-43 amino acid residues aggregate and deposit onto neurons, igniting the neurotoxic cascade of the disease. Therefore, researchers studying AD would like to elucidate the mechanisms by which essentially water-soluble but hydrophobic Aβ aggregates under pathological conditions. Most researchers have investigated the aggregation of Aβ in aqueous solution, and they concluded that the final aggregation product, the amyloid fibrils, were less toxic than the component peptide oligomers. They consequently shifted their interests to more toxic "soluble oligomers", structures that form as intermediates or off-pathway products during the aggregation process. Some researchers have also investigated artificial oligomers prepared under nonphysiological conditions. In contrast to these "in solution" studies, we have focused on "membrane-mediated" amyloidogenesis. In an earlier study, other researchers identified a specific form of Aβ that was bound to monosialoganglioside GM1, a sugar lipid, in brains of patients who exhibited the early pathological changes associated with AD. This Account summarizes 15 years of our research on this topic. We have found that Aβ specifically binds to GM1 that occurs in clusters, but not when it is uniformly distributed. Clustering is facilitated by cholesterol. Upon binding, Aβ changes its conformation from a random coil to an α-helix-rich structure. A CH-π interaction between the aromatic side chains of Aβ and carbohydrate moieties appended to GM1 appears to be important for binding. In addition, as Aβ accumulates and reaches its first threshold concentration (Aβ/GM1 = ∼0.013), aggregated β-sheets of ∼15 molecules appear and coexist with the helical form. However, this β-structure is stable and does not form larger

  14. Toxicity of endophyte-infected ryegrass hay containing high ergovaline level in lactating ewes.

    PubMed

    Zbib, N; Repussard, C; Tardieu, D; Priymenko, N; Domange, C; Guerre, P

    2015-08-01

    The symbiotic association of var. (formerly named ) with perennial ryegrass () leads to the production of ergovaline (EV) and lolitrem B (LB) that are toxic for livestock. The objectives of this study were to determine the effects of feeding endophyte-infected ryegrass (SE+) hay on 16 lactating ewes (BW 80 ± 10 kg) in comparison with endophyte-free ryegrass (SE-) hay to investigate the putative mechanisms of action of EV and LB and to evaluate their persistence in milk and animal tissues. The mean EV and LB concentrations in SE+ hay were 851 and 884 μg/kg DM, respectively, whereas these alkaloids were below the limit of detection in SE- hay. No effect of SE+ was observed on animal health and skin temperature whereas prolactin decreased and significant differences between hays were observed from d 7 to 28 of the study ( < 0.03) but had no effect on milk production. Hematocrit and biochemical analyses of plasma revealed no significant difference between SE+ and SE-, whereas cortisol concentration differed significantly on d 28 ( = 0.001). Measurement of oxidative damage and antioxidant enzyme activities in plasma, liver, and kidneys revealed a slight increase in some enzyme activities involved in defense against oxidative damage in the SE+ fed ewes. Slight variations in the activities of hepatic and kidney flavin monooxygenase enzymes were observed, whereas in the kidney, glutathione -transferase activity decreased significantly ( = 0.002) in the SE+ fed ewes, whereas uridine diphosphate glucuronosyltransferase activity increased ( = 0.001). After 28 d of exposure of ewes to the SE+ hay, low EV and LB concentrations were measured in tissues. The highest concentration of EV was observed in the liver (0.68 μg/kg) whereas fat contained the highest concentration of LB (2.39 μg/kg). Both toxins were also identified at the trace level in milk. PMID:26440189

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

    PubMed Central

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

    2014-01-01

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

  16. Highly Luminescent Carbon Dots Synthesized by Microwave-Assisted Pyrolysis and Evaluation of Their Toxicity to Physa acuta.

    PubMed

    Sun, Xiaobo; Jin, Xiaozhe; Pan, Wei; Guo, Enmian; Liu, Weijian; Li, Denghui; Lu, Kunchao; Si, Shuxin; Zhang, Nianxing; Jia, Zhenzhen; Shi, Yanping; Li, Qianqian; Wang, Jinping

    2016-01-01

    As a newly emerging class of nanomaterials, carbon dots have increasingly attracted researchers' attention. However, their potentially adverse environmental effects are yet largely unknown. In this work, the highly luminescent carbon dots were synthesized by microwave-assisted pyrolysis of tris(hydroxymethyl)aminomethane (Tris) and citric acid. Then acute and chronic toxicities of carbon dots to Physa acuta (P. acuta), as well as their effect on reproduction, were evaluated using the as-synthesized dots as an example. The quantum yield of the as-synthesized carbon dots was up to 53.5% excited at 360 nm with the most fluorescent fraction of 82.6% after simple purification by gel column. The results showed that no acute but chronic toxicities to P. acuta exposed to different treatment concentrations of the as-synthesized carbon dots were observed with dose- dependence. In addition, the fecundity of P. acuta was promoted significantly by the carbon dots at the concentrations of 0.5 and 1.0 mg/mL, yet inhibited at the concentration of 3.0 mg/mL after 12-day exposure. Mainly distributing in the visceral mass might be responsible for the effects of the carbon dots on the survival and fecundity of P. acuta. And there was no further evidence to confirm that the carbon dots can cause malformation in developing embryos. PMID:27398502

  17. Assessing the mammalian toxicity of high-boiling petroleum substances under the rubric of the HPV program.

    PubMed

    Gray, Thomas M; Simpson, Barry J; Nicolich, Mark J; Murray, F Jay; Verstuyft, Allen W; Roth, Randy N; McKee, Richard H

    2013-11-01

    In 1998, the US EPA announced the HPV Challenge Program, a voluntary chemical data collection effort. The Petroleum HPV Testing Group (PHPVTG(1)) volunteered to provide data on approximately 110 high boiling petroleum substances (HBPS), i.e. substances with final boiling points ≥ approximately 650°F (343°C). These HBPS are substances of unknown and variable composition (UVCBs) that are composed of numerous individual constituents. Toxicity studies have shown that some HBPS can produce systemic (repeat-dose) and developmental effects, and some are mutagenic under in vitro conditions. The papers in this supplement show that these effects are related to the profiles of aromatic constituents in these substances. Further, it is shown that the effects on selected repeat-dose and developmental toxicity endpoints and mutagenic activity in bacterial assays can be predicted from compositional information using models based on the aromatic-ring class profile, "ARC profile" as defined by gas chromatographic separation of the DMSO-soluble fraction of the starting materials. This chromatographic method and the predictive models provide an efficient means of characterizing for screening purposes the potential for repeat-dose, developmental effects and bacterial mutagenicity of HBPS and can reduce the number of animal tests that would be required if these tests were conducted on all 110 HBPS.

  18. Highly Luminescent Carbon Dots Synthesized by Microwave-Assisted Pyrolysis and Evaluation of Their Toxicity to Physa acuta.

    PubMed

    Sun, Xiaobo; Jin, Xiaozhe; Pan, Wei; Guo, Enmian; Liu, Weijian; Li, Denghui; Lu, Kunchao; Si, Shuxin; Zhang, Nianxing; Jia, Zhenzhen; Shi, Yanping; Li, Qianqian; Wang, Jinping

    2016-01-01

    As a newly emerging class of nanomaterials, carbon dots have increasingly attracted researchers' attention. However, their potentially adverse environmental effects are yet largely unknown. In this work, the highly luminescent carbon dots were synthesized by microwave-assisted pyrolysis of tris(hydroxymethyl)aminomethane (Tris) and citric acid. Then acute and chronic toxicities of carbon dots to Physa acuta (P. acuta), as well as their effect on reproduction, were evaluated using the as-synthesized dots as an example. The quantum yield of the as-synthesized carbon dots was up to 53.5% excited at 360 nm with the most fluorescent fraction of 82.6% after simple purification by gel column. The results showed that no acute but chronic toxicities to P. acuta exposed to different treatment concentrations of the as-synthesized carbon dots were observed with dose- dependence. In addition, the fecundity of P. acuta was promoted significantly by the carbon dots at the concentrations of 0.5 and 1.0 mg/mL, yet inhibited at the concentration of 3.0 mg/mL after 12-day exposure. Mainly distributing in the visceral mass might be responsible for the effects of the carbon dots on the survival and fecundity of P. acuta. And there was no further evidence to confirm that the carbon dots can cause malformation in developing embryos.

  19. Assessing the mammalian toxicity of high-boiling petroleum substances under the rubric of the HPV program.

    PubMed

    Gray, Thomas M; Simpson, Barry J; Nicolich, Mark J; Murray, F Jay; Verstuyft, Allen W; Roth, Randy N; McKee, Richard H

    2013-11-01

    In 1998, the US EPA announced the HPV Challenge Program, a voluntary chemical data collection effort. The Petroleum HPV Testing Group (PHPVTG(1)) volunteered to provide data on approximately 110 high boiling petroleum substances (HBPS), i.e. substances with final boiling points ≥ approximately 650°F (343°C). These HBPS are substances of unknown and variable composition (UVCBs) that are composed of numerous individual constituents. Toxicity studies have shown that some HBPS can produce systemic (repeat-dose) and developmental effects, and some are mutagenic under in vitro conditions. The papers in this supplement show that these effects are related to the profiles of aromatic constituents in these substances. Further, it is shown that the effects on selected repeat-dose and developmental toxicity endpoints and mutagenic activity in bacterial assays can be predicted from compositional information using models based on the aromatic-ring class profile, "ARC profile" as defined by gas chromatographic separation of the DMSO-soluble fraction of the starting materials. This chromatographic method and the predictive models provide an efficient means of characterizing for screening purposes the potential for repeat-dose, developmental effects and bacterial mutagenicity of HBPS and can reduce the number of animal tests that would be required if these tests were conducted on all 110 HBPS. PMID:23247262

  20. Cell-surface Attachment of Bacterial Multienzyme Complexes Involves Highly Dynamic Protein-Protein Anchors*

    PubMed Central

    Cameron, Kate; Najmudin, Shabir; Alves, Victor D.; Bayer, Edward A.; Smith, Steven P.; Bule, Pedro; Waller, Helen; Ferreira, Luís M. A.; Gilbert, Harry J.; Fontes, Carlos M. G. A.

    2015-01-01

    Protein-protein interactions play a pivotal role in the assembly of the cellulosome, one of nature's most intricate nanomachines dedicated to the depolymerization of complex carbohydrates. The integration of cellulosomal components usually occurs through the binding of type I dockerin modules located at the C terminus of the enzymes to cohesin modules located in the primary scaffoldin subunit. Cellulosomes are typically recruited to the cell surface via type II cohesin-dockerin interactions established between primary and cell-surface anchoring scaffoldin subunits. In contrast with type II interactions, type I dockerins usually display a dual binding mode that may allow increased conformational flexibility during cellulosome assembly. Acetivibrio cellulolyticus produces a highly complex cellulosome comprising an unusual adaptor scaffoldin, ScaB, which mediates the interaction between the primary scaffoldin, ScaA, through type II cohesin-dockerin interactions and the anchoring scaffoldin, ScaC, via type I cohesin-dockerin interactions. Here, we report the crystal structure of the type I ScaB dockerin in complex with a type I ScaC cohesin in two distinct orientations. The data show that the ScaB dockerin displays structural symmetry, reflected by the presence of two essentially identical binding surfaces. The complex interface is more extensive than those observed in other type I complexes, which results in an ultra-high affinity interaction (Ka ∼1012 m). A subset of ScaB dockerin residues was also identified as modulating the specificity of type I cohesin-dockerin interactions in A. cellulolyticus. This report reveals that recruitment of cellulosomes onto the cell surface may involve dockerins presenting a dual binding mode to incorporate additional flexibility into the quaternary structure of highly populated multienzyme complexes. PMID:25855788

  1. Functional Aspects of the Solution Structure and Dynamics of PAF, a Highly Stable Antifungal Protein from Penicillium chrysogenum

    PubMed Central

    Batta, Gyula; Barna, Teréz; Gáspári, Zoltán; Sándor, Szabolcs; Kövér, Katalin E.; Binder, Ulrike; Sarg, Bettina; Kaiserer, Lydia; Chhillar, Anil Kumar; Eigentler, Andrea; Leiter, Éva; Hegedüs, Nikoletta; Pócsi, István; Lindner, Herbert; Marx, Florentine

    2015-01-01

    Summary PAF is a promising antimycotic without toxic effects on mammalian cells and therefore may be a drug candidate against the often lethal Aspergillus infections in human. The pathogenesis of PAF on sensitive fungi involves G-protein coupled signaling followed by apoptosis. Here, the solution structure of this small, cationic, antifungal protein from Penicillium chrysogenum is determined by solution NMR. We proved that PAF belongs to the SCOP fold class of its closest homologue AFP from Aspergillus giganteus. PAF comprises five β—strands forming two orthogonally packed β—sheets sharing a common interface. The ambiguity in the assignment of two disulfide bonds of three was investigated by NMR dynamics combined with restrained molecular dynamics calculations. The clue could not be resolved: two ensembles with different disulfide patterns and the one with no S-S bond exhibit essentially the same fold. 15N relaxation dispersion and interference experiments did not reveal disulfide bond rearrangements via slow exchange. The measured order parameters and the 3.0 ns correlation time is appropriate for a compact monomeric protein of this size. We demonstrated by site-directed mutagenesis that the highly conserved and positively charged lysine-rich domain region on the surface enhances the toxicity of PAF. However, the efficacy of the OB fold is reduced in PAF compared to AFP, due to less solvent exposed aromatic regions explaining the absence of chitobiose binding. The present work lends further support to the understanding of the documented substantial differences between the mode of action of two highly homologous antifungal proteins. PMID:19459942

  2. Cry4Ba and Cyt1Aa proteins from Bacillus thuringiensis israelensis: Interactions and toxicity mechanism against Aedes aegypti.

    PubMed

    Elleuch, Jihen; Jaoua, Samir; Darriet, Frédéric; Chandre, Fabrice; Tounsi, Slim; Zghal, Raida Zribi

    2015-09-15

    Individual crystal proteins from Bacillus thuringiensis israelensis exhibit variable levels of insecticidal activities against mosquito larvae. In all cases, they are much less active compared to the whole crystal proteins due to described complex synergistic interactions among them. In the present study we investigated the effects of Cyt1A98 (a Cyt1Aa type protein) on Cry4BLB (a Cry4Ba type toxin) insecticidal activity toward the dengue vector Aedes aegypti. The bioassay analyses demonstrated the ability of Cyt1A98 protein to enhance Cry4BLB toxin larvicidal activity even at a low proportion in the mixture (1%). In vitro interaction assays showed that Cyt1A98 provides supplementary binding sites for Cry4BLB in A. aegypti BBMVs. Moreover, it enhances the formation of Cry4BLB oligomeric structure. These results support that Cyt1A98 protein could act as a membrane-bound receptor fixing Cry4BLB δ-endotoxins and promoting its oligomerization.

  3. Haematopoietic stem cells require a highly regulated protein synthesis rate.

    PubMed

    Signer, Robert A J; Magee, Jeffrey A; Salic, Adrian; Morrison, Sean J

    2014-05-01

    Many aspects of cellular physiology remain unstudied in somatic stem cells, for example, there are almost no data on protein synthesis in any somatic stem cell. Here we set out to compare protein synthesis in haematopoietic stem cells (HSCs) and restricted haematopoietic progenitors. We found that the amount of protein synthesized per hour in HSCs in vivo was lower than in most other haematopoietic cells, even if we controlled for differences in cell cycle status or forced HSCs to undergo self-renewing divisions. Reduced ribosome function in Rpl24(Bst/+) mice further reduced protein synthesis in HSCs and impaired HSC function. Pten deletion increased protein synthesis in HSCs but also reduced HSC function. Rpl24(Bst/+) cell-autonomously rescued the effects of Pten deletion in HSCs; blocking the increase in protein synthesis, restoring HSC function, and delaying leukaemogenesis. Pten deficiency thus depletes HSCs and promotes leukaemia partly by increasing protein synthesis. Either increased or decreased protein synthesis impairs HSC function.

  4. Leaf-specific thionins of barley—a novel class of cell wall proteins toxic to plant-pathogenic fungi and possibly involved in the defence mechanism of plants

    PubMed Central

    Bohlmann, Holger; Clausen, Susanne; Behnke, Susanna; Giese, Henriette; Hiller, Claudia; Reimann-Philipp, Ulrich; Schrader, Gesine; Barkholt, Vibeke; Apel, Klaus

    1988-01-01

    A novel class of highly abundant polypeptides with antifungal activity has been detected in cell walls of barley leaves. Similar polypeptides known as thionins occur not only in monocotyledonous but also in various dictoyledonous plants. The leaf-specific thionins of barley are encoded by a complex multigene family, which consists of at least 50-100 members per haploid genome. All of these genes are confined to chromosome 6. The toxicity of these thionins for plant pathogenic fungi and the fact that their synthesis can also be triggered by pathogens strongly suggest that thionins are a naturally occurring, inducible plant protein possibly involved in the mechanism of plant defence against microbial infections. Images PMID:16453847

  5. A functional protein retention and release multilayer with high stability

    NASA Astrophysics Data System (ADS)

    Nie, Kun; An, Qi; Zhang, Yihe

    2016-04-01

    Effective and robust interfacial protein retention lies at the heart of the fabrication of protein-based functional interfaces, which is potentially applicable in catalysis, medical therapy, antifouling, and smart devices, but remains challenging due to the sensitive nature of proteins. This study reports a general protein retention strategy to spatial-temporally confine various types of proteins at interfacial regions. The proteins were preserved in mesoporous silica nanoparticles embedded in covalently woven multilayers. It is worth noting that the protein retention strategy effectively preserves the catalytic capabilities of the proteins, and the multilayer structure is robust enough to withstand the bubbling catalytic reactions and could be repeatedly used due to conservation of proteins. The spatiotemporal retention of proteins could be adjusted by varying the number of capping layers. Furthermore, we demonstrate that the protein-loaded interfacial layers could not only be used to construct catalytic-active interfaces, but also be integrated as the power-generating unit to propel a macroscopic floating device.Effective and robust interfacial protein retention lies at the heart of the fabrication of protein-based functional interfaces, which is potentially applicable in catalysis, medical therapy, antifouling, and smart devices, but remains challenging due to the sensitive nature of proteins. This study reports a general protein retention strategy to spatial-temporally confine various types of proteins at interfacial regions. The proteins were preserved in mesoporous silica nanoparticles embedded in covalently woven multilayers. It is worth noting that the protein retention strategy effectively preserves the catalytic capabilities of the proteins, and the multilayer structure is robust enough to withstand the bubbling catalytic reactions and could be repeatedly used due to conservation of proteins. The spatiotemporal retention of proteins could be adjusted by

  6. A high-affinity reversible protein stain for Western blots.

    PubMed

    Antharavally, Babu S; Carter, Brad; Bell, Peter A; Krishna Mallia, A

    2004-06-15

    We describe a reversible staining technique, using MemCode, a reversible protein stain by which proteins can be visualized on nitrocellulose and polyvinylidine fluoride (PVDF) membranes without being permanently fixed to the membrane itself. This allows subsequent immunoblot analysis of the proteins to be performed. The procedure is applicable only to protein blots on nitrocellulose and PVDF membranes. MemCode is a reversible protein stain composed of copper as a part of an organic complex that interacts noncovalently with proteins. MemCode shows rapid protein staining, taking 30s to 1 min for completion. The method is simple and utilizes convenient application conditions that are compatible with the matrix materials and the protein. The stain is more sensitive than any previously described dye-based universal protein staining system. The turquoise-blue-stained protein bands do not fade with time and are easy to photograph compared to those stained with Ponceau S. Absorbance in the blue region of the spectrum offers good properties for photo documentation and avoids interference from common biological chromophores. The stain on the protein is easily reversible in 2 min for nitrocellulose membrane and in 10 min for PVDF membrane with MemCode stain eraser. The stain is compatible with general Western blot detection systems, and membrane treatment with MemCode stain does not interfere with conventional chemiluminescent or chromogenic detection using horseradish peroxide and alkaline phosphatase substrates. The stain is also compatible with N-terminal sequence analysis of proteins.

  7. Inorganic–organic hybrids presenting high basic center content: SBA-15 incorporation, toxic metals sorption and energetic behavior

    SciTech Connect

    Oliveira, Fernando J.V.E.; Melo, Maurício A.; Airoldi, Claudio

    2013-03-15

    Highlights: ► Mesoporous SBA-15 silicas were organofunctionalized with new silylant agents. ► Thiocarbamate was used to enhance the silylating agent chains and basic centers. ► The synthesized pendant chains contain nitrogen and sulfur basic centers. ► The new hybrids sorb toxic cations from aqueous solutions with high efficiency. ► The thermodynamic data demonstrated favorable cation/basic center interactions. - Abstract: Mesoporous SBA-15 samples were organofunctionalized with mono, di- and tri-aminosilanes that previously reacted with thiocarbamide to enhance the organic chains and attach nitrogen and sulfur basic centers to the surface of the solids. These new organosilanes were synthesized through a non-solvent approach to reduce both cost and hazardous wastes. The high affinities for both hard and soft Lewis acids due to the combination of nitrogen and sulfur atoms attached to the same pendant chain enabled favorable sorption capacities for Cu{sup 2+}, Cd{sup 2+} and Pb{sup 2+} cations, with maximum capacities of 1.90, 3.48 and 5.30 mmol g{sup −1}, respectively, for the most efficient mesoporous silica. Microcalorimetric investigations allowed the calculation of the thermodynamic data at the solid/liquid interface. All Gibbs energy are negative as expected for spontaneous cation/basic center interactions and the positive entropic values from 49 ± 3 to 108 ± 5 J K{sup −1} mol{sup −1}, also reinforced this favorable interactive process in heterogeneous system. The designed organosilanes covalently bonded to the inorganic siliceous skeleton can be suggested as new materials for toxic metal removal from a wastewater with high efficiency.

  8. High-Pressure-High-Temperature Processing Reduces Maillard Reaction and Viscosity in Whey Protein-Sugar Solutions.

    PubMed

    Avila Ruiz, Geraldine; Xi, Bingyan; Minor, Marcel; Sala, Guido; van Boekel, Martinus; Fogliano, Vincenzo; Stieger, Markus

    2016-09-28

    The aim of the study was to determine the influence of pressure in high-pressure-high-temperature (HPHT) processing on Maillard reactions and protein aggregation of whey protein-sugar solutions. Solutions of whey protein isolate containing either glucose or trehalose at pH 6, 7, and 9 were treated by HPHT processing or conventional high-temperature (HT) treatments. Browning was reduced, and early and advanced Maillard reactions were retarded under HPHT processing at all pH values compared to HT treatment. HPHT induced a larger pH drop than HT treatments, especially at pH 9, which was not associated with Maillard reactions. After HPHT processing at pH 7, protein aggregation and viscosity of whey protein isolate-glucose/trehalose solutions remained unchanged. It was concluded that HPHT processing can potentially improve the quality of protein-sugar-containing foods, for which browning and high viscosities are undesired, such as high-protein beverages.

  9. Protein corona changes mediated by surface modification of amorphous silica nanoparticles suppress acute toxicity and activation of intrinsic coagulation cascade in mice

    NASA Astrophysics Data System (ADS)

    Yoshida, Tokuyuki; Yoshioka, Yasuo; Morishita, Yuki; Aoyama, Michihiko; Tochigi, Saeko; Hirai, Toshiro; Tanaka, Kota; Nagano, Kazuya; Kamada, Haruhiko; Tsunoda, Shin-ichi; Nabeshi, Hiromi; Yoshikawa, Tomoaki; Higashisaka, Kazuma; Tsutsumi, Yasuo

    2015-06-01

    Recently, nanomaterial-mediated biological effects have been shown to be governed by the interaction of nanomaterials with some kinds of proteins in biological fluids, and the physical characteristics of the nanomaterials determine the extent and type of their interactions with proteins. Here, we examined the relationships between the surface properties of amorphous silica nanoparticles with diameters of 70 nm (nSP70), their interactions with some proteins in biological fluids, and their toxicity in mice after intravenous administration. The surface modification of nSP70 with amino groups (nSP70-N) prevented acute lethality and abnormal activation of the coagulation cascade found in the nSP70-treated group of mice. Since our previous study showed that coagulation factor XII played a role in the nSP70-mediated abnormal activation of the coagulation cascade, we examined the interaction of nSP70 and nSP70-N with coagulation factor XII. Coagulation factor XII bonded to the surface of nSP70 to a greater extent than that observed for nSP70-N, and consequently more activation of coagulation factor XII was observed for nSP70 than for nSP70-N. Collectively, our results suggest that controlling the interaction of nSP70 with blood coagulation factor XII by modifying the surface properties would help to inhibit the nSP70-mediated abnormal activation of the blood coagulation cascade.

  10. Highly Accurate Prediction of Protein-Protein Interactions via Incorporating Evolutionary Information and Physicochemical Characteristics

    PubMed Central

    Li, Zheng-Wei; You, Zhu-Hong; Chen, Xing; Gui, Jie; Nie, Ru

    2016-01-01

    Protein-protein interactions (PPIs) occur at almost all levels of cell functions and play crucial roles in various cellular processes. Thus, identification of PPIs is critical for deciphering the molecular mechanisms and further providing insight into biological processes. Although a variety of high-throughput experimental techniques have been developed to identify PPIs, existing PPI pairs by experimental approaches only cover a small fraction of the whole PPI networks, and further, those approaches hold inherent disadvantages, such as being time-consuming, expensive, and having high false positive rate. Therefore, it is urgent and imperative to develop automatic in silico approaches to predict PPIs efficiently and accurately. In this article, we propose a novel mixture of physicochemical and evolutionary-based feature extraction method for predicting PPIs using our newly developed discriminative vector machine (DVM) classifier. The improvements of the proposed method mainly consist in introducing an effective feature extraction method that can capture discriminative features from the evolutionary-based information and physicochemical characteristics, and then a powerful and robust DVM classifier is employed. To the best of our knowledge, it is the first time that DVM model is applied to the field of bioinformatics. When applying the proposed method to the Yeast and Helicobacter pylori (H. pylori) datasets, we obtain excellent prediction accuracies of 94.35% and 90.61%, respectively. The computational results indicate that our method is effective and robust for predicting PPIs, and can be taken as a useful supplementary tool to the traditional experimental methods for future proteomics research. PMID:27571061

  11. Highly Accurate Prediction of Protein-Protein Interactions via Incorporating Evolutionary Information and Physicochemical Characteristics.

    PubMed

    Li, Zheng-Wei; You, Zhu-Hong; Chen, Xing; Gui, Jie; Nie, Ru

    2016-01-01

    Protein-protein interactions (PPIs) occur at almost all levels of cell functions and play crucial roles in various cellular processes. Thus, identification of PPIs is critical for deciphering the molecular mechanisms and further providing insight into biological processes. Although a variety of high-throughput experimental techniques have been developed to identify PPIs, existing PPI pairs by experimental approaches only cover a small fraction of the whole PPI networks, and further, those approaches hold inherent disadvantages, such as being time-consuming, expensive, and having high false positive rate. Therefore, it is urgent and imperative to develop automatic in silico approaches to predict PPIs efficiently and accurately. In this article, we propose a novel mixture of physicochemical and evolutionary-based feature extraction method for predicting PPIs using our newly developed discriminative vector machine (DVM) classifier. The improvements of the proposed method mainly consist in introducing an effective feature extraction method that can capture discriminative features from the evolutionary-based information and physicochemical characteristics, and then a powerful and robust DVM classifier is employed. To the best of our knowledge, it is the first time that DVM model is applied to the field of bioinformatics. When applying the proposed method to the Yeast and Helicobacter pylori (H. pylori) datasets, we obtain excellent prediction accuracies of 94.35% and 90.61%, respectively. The computational results indicate that our method is effective and robust for predicting PPIs, and can be taken as a useful supplementary tool to the traditional experimental methods for future proteomics research. PMID:27571061

  12. Highly Accurate Prediction of Protein-Protein Interactions via Incorporating Evolutionary Information and Physicochemical Characteristics.

    PubMed

    Li, Zheng-Wei; You, Zhu-Hong; Chen, Xing; Gui, Jie;