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Sample records for plant biotin-containing carboxylasesq

  1. Biotin decorated PLGA nanoparticles containing SN-38 designed for cancer therapy.

    Science.gov (United States)

    Mehdizadeh, Mozhdeh; Rouhani, Hasti; Sepehri, Nima; Varshochian, Reyhaneh; Ghahremani, Mohammad Hossein; Amini, Mohsen; Gharghabi, Mehdi; Ostad, Seyed Nasser; Atyabi, Fatemeh; Baharian, Azin; Dinarvand, Rassoul

    2017-05-01

    Active targeted chemotherapy is expected to provide more specific delivery of cytotoxic drugs to the tumor cells and hence reducing the side effects on healthy tissues. Due to the over expression of biotin receptors on cancerous cells as a result of further requirement for rapid proliferations, biotin can be a good candidate as a targeting agent. In this study, biotin decorated PLGA nanoparticles (NPs) containing SN-38 were prepared and in vitro studies were evaluated for their improved anti-cancer properties. In conclusion, biotin targeted PLGA NPs containing SN-38 showed preferential anticancer properties against tumor cells with biotin receptor over expression.

  2. Biotin Carboxyl Carrier Protein in Barley Chloroplast Membranes

    DEFF Research Database (Denmark)

    Kannangara, C. G.; Jense, C J

    1975-01-01

    Biotin localized in barley chloroplast lamellae is covalently bound to a single protein with an approximate molecular weight of 21000. It contains one mole of biotin per mole of protein and functions as a carboxyl carrier in the acetyl-CoA carboxylase reaction. The protein was obtained by solubil...... by solubilization of the lamellae in phenol/acetic acid/8 M urea. Feeding barley seedlings with [14C]-biotin revealed that the vitamin is not degraded into respiratory substrates by the plant, but is specifically incorporated into biotin carboxyl carrier protein....

  3. The Effects of Light and Temperature on Biotin Synthesis in Pea Sprouts.

    Science.gov (United States)

    Kamiyama, Shin; Ohnuki, Risa; Moriki, Aoi; Abe, Megumi; Ishiguro, Mariko; Sone, Hideyuki

    2016-01-01

    Biotin is an essential micronutrient, and is a cofactor for several carboxylases that are involved in the metabolism of glucose, fatty acids, and amino acids. Because plant cells can synthesize their own biotin, a wide variety of plant-based foods contains significant amounts of biotin; however, the influence of environmental conditions on the biotin content in plants remains largely unclear. In the present study, we investigated the effects of different cultivation conditions on the biotin content and biotin synthesis in pea sprouts (Pisum sativum). In the experiment, the pea sprouts were removed from their cotyledons and cultivated by hydroponics under five different lighting and temperature conditions (control [25ºC, 12-h light/12-h dark cycle], low light [25ºC, 4-h light/20-h dark cycle], dark [25ºC, 24 h dark], low temperature [12ºC, 12-h light/12-h dark cycle], and cold [6ºC, 12-h light/12-h dark cycle]) for 10 d. Compared to the biotin content of pea sprouts under the control conditions, the biotin contents of pea sprouts under the low-light, dark, and cold conditions had significantly decreased. The dark group showed the lowest biotin content among the groups. Expression of the biotin synthase gene (bio2) was also significantly decreased under the dark and cold conditions compared to the control condition, in a manner similar to that observed for the biotin content. No significant differences in the adenosine triphosphate content were observed among the groups. These results indicate that environmental conditions such as light and temperature modulate the biotin content of pea plant tissues by regulating the expression of biotin synthase.

  4. A versatile Escherichia coli strain for identification of biotin transporters and for biotin quantification

    Science.gov (United States)

    Finkenwirth, Friedrich; Kirsch, Franziska; Eitinger, Thomas

    2014-01-01

    Biotin is an essential cofactor of carboxylase enzymes in all kingdoms of life. The vitamin is produced by many prokaryotes, certain fungi, and plants. Animals depend on biotin uptake from their diet and in humans lack of the vitamin is associated with serious disorders. Many aspects of biotin metabolism, uptake, and intracellular transport remain to be elucidated. In order to characterize the activity of novel biotin transporters by a sensitive assay, an Escherichia coli strain lacking both biotin synthesis and its endogenous high-affinity biotin importer was constructed. This strain requires artificially high biotin concentrations for growth. When only trace levels of biotin are available, it is viable only if equipped with a heterologous high-affinity biotin transporter. This feature was used to ascribe transport activity to members of the BioY protein family in previous work. Here we show that this strain together with its parent is also useful as a diagnostic tool for wide-concentration-range bioassays. PMID:24256712

  5. Biotin

    OpenAIRE

    Zempleni, Janos; Wijeratne, Subhashinee S.K.; Hassan, Yousef I.

    2009-01-01

    Biotin is a water-soluble vitamin and serves as a coenzyme for five carboxylases in humans. Biotin is also covalently attached to distinct lysine residues in histones, affecting chromatin structure and mediating gene regulation. This review describes mammalian biotin metabolism, biotin analysis, markers of biotin status, and biological functions of biotin. Proteins such as holocarboxylase synthetase, biotinidase, and the biotin transporters SMVT and MCT1 play crucial roles in biotin homeostas...

  6. Laboratory evolution of a biotin-requiring Saccharomyces cerevisiae strain for full biotin prototrophy and identification of causal mutations

    NARCIS (Netherlands)

    Bracher, J.M.; de Hulster, A.F.; van den Broek, M.A.; Daran, J.G.; van Maris, A.J.A.; Pronk, J.T.

    2017-01-01

    Biotin prototrophy is a rare, incompletely understood, and industrially relevant characteristic of Saccharomyces cerevisiae strains. The genome of the haploid laboratory strain CEN.PK113-7D contains a full complement of biotin biosynthesis genes, but its growth in biotin-free synthetic medium is

  7. Increased expression of pyruvate carboxylase and biotin protein ligase increases lysine production in a biotin prototrophic Corynebacterium glutamicum strain

    DEFF Research Database (Denmark)

    Wang, Zhihao; Moslehi-Jenabian, Soloomeh; Solem, Christian

    2015-01-01

    , and achieved biotin prototrophy. We found that AHP-3, containing pBIO, was able to produce lysine in a medium lacking biotin and that the lysine yield on glucose was similar to what is obtained when using a medium containing biotin. However, there was a decrease in specific growth rate of 20% when the strain...... pimeloyl-Acyl Carrier Protein [ACP]) formation. Pyruvate carboxylase (pycA), a biotin-dependent enzyme needed for lysine biosynthesis and biotin ligase (birA), which is responsible for attaching biotin to pyruvate carboxylase, were overexpressed by replacing the native promoters with the strong superoxide...... dismutase (sod) promoter, to see whether growth could be restored. Neither pycA nor birA overexpression, whether alone or in combination, had an effect on specific growth rate, but they did have a positive effect on lysine yield, which increased by 55% in the strain overexpressing both enzymes....

  8. Expression of streptavidin gene in bacteria and plants

    International Nuclear Information System (INIS)

    Guan, Xueni; Wurtele, E.S.; Nikolau, B.J.

    1990-01-01

    Six biotin-containing proteins are present in plants, representing at least four different biotin enzymes. The physiological function of these biotin enzymes is not understood. Streptavidin, a protein from Streptomyces avidinii, binds tightly and specifically to biotin causing inactivation of biotin enzymes. One approach to elucidating the physiological function of biotin enzymes in plant metabolism is to create transgenic plants expressing the streptavidin gene. A plasmid containing a fused streptavidin-beta-galactosidase gene has been expressed in E. coli. We also have constructed various fusion genes that include an altered CaMV 35S promoter, signal peptides to target the streptavidin protein to specific organelles, and the streptavidin coding gene. We are examining the expression of these genes in cells of carrot

  9. Characterization of the biotin uptake system encoded by the biotin-inducible bioYMN operon of Corynebacterium glutamicum

    Science.gov (United States)

    2012-01-01

    Background The amino acid-producing Gram-positive Corynebacterium glutamicum is auxotrophic for biotin although biotin ring assembly starting from the precursor pimeloyl-CoA is still functional. It possesses AccBC, the α-subunit of the acyl-carboxylases involved in fatty acid and mycolic acid synthesis, and pyruvate carboxylase as the only biotin-containing proteins. Comparative genome analyses suggested that the putative transport system BioYMN encoded by cg2147, cg2148 and cg2149 might be involved in biotin uptake by C. glutamicum. Results By comparison of global gene expression patterns of cells grown with limiting or excess supply of biotin or with dethiobiotin as supplement replacing biotin revealed that expression of genes coding for enzymes of biotin ring assembly and for the putative uptake system was regulated according to biotin availability. RT-PCR and 5'-RACE experiments demonstrated that the genes bioY, bioM, and bioN are transcribed from one promoter as a single transcript. Biochemical analyses revealed that BioYMN catalyzes the effective uptake of biotin with a concentration of 60 nM biotin supporting a half-maximal transport rate. Maximal biotin uptake rates were at least five fold higher in biotin-limited cells as compared to cells grown with excess biotin. Overexpression of bioYMN led to an at least 50 fold higher biotin uptake rate as compared to the empty vector control. Overproduction of BioYMN alleviated biotin limitation and interfered with triggering L-glutamate production by biotin limitation. Conclusions The operon bioYMN from C. glutamicum was shown to be induced by biotin limitation. Transport assays with radio-labeled biotin revealed that BioYMN functions as a biotin uptake system. Overexpression of bioYMN affected L-glutamate production triggered by biotin limitation. PMID:22243621

  10. Characterization of the biotin uptake system encoded by the biotin-inducible bioYMN operon of Corynebacterium glutamicum.

    Science.gov (United States)

    Schneider, Jens; Peters-Wendisch, Petra; Stansen, K Corinna; Götker, Susanne; Maximow, Stanislav; Krämer, Reinhard; Wendisch, Volker F

    2012-01-13

    The amino acid-producing Gram-positive Corynebacterium glutamicum is auxotrophic for biotin although biotin ring assembly starting from the precursor pimeloyl-CoA is still functional. It possesses AccBC, the α-subunit of the acyl-carboxylases involved in fatty acid and mycolic acid synthesis, and pyruvate carboxylase as the only biotin-containing proteins. Comparative genome analyses suggested that the putative transport system BioYMN encoded by cg2147, cg2148 and cg2149 might be involved in biotin uptake by C. glutamicum. By comparison of global gene expression patterns of cells grown with limiting or excess supply of biotin or with dethiobiotin as supplement replacing biotin revealed that expression of genes coding for enzymes of biotin ring assembly and for the putative uptake system was regulated according to biotin availability. RT-PCR and 5'-RACE experiments demonstrated that the genes bioY, bioM, and bioN are transcribed from one promoter as a single transcript. Biochemical analyses revealed that BioYMN catalyzes the effective uptake of biotin with a concentration of 60 nM biotin supporting a half-maximal transport rate. Maximal biotin uptake rates were at least five fold higher in biotin-limited cells as compared to cells grown with excess biotin. Overexpression of bioYMN led to an at least 50 fold higher biotin uptake rate as compared to the empty vector control. Overproduction of BioYMN alleviated biotin limitation and interfered with triggering L-glutamate production by biotin limitation. The operon bioYMN from C. glutamicum was shown to be induced by biotin limitation. Transport assays with radio-labeled biotin revealed that BioYMN functions as a biotin uptake system. Overexpression of bioYMN affected L-glutamate production triggered by biotin limitation.

  11. Pharmacological Effects of Biotin in Animals.

    Science.gov (United States)

    Riveron-Negrete, Leticia; Fernandez-Mejia, Cristina

    2017-01-01

    In recent decades, it was found that vitamins affect biological functions in ways other than their long-known functions; niacin is the best example of a water-soluble vitamin known to possess multiple actions. Biotin, also known as vitamin B7 or vitamin H, is a water-soluble B-complex vitamin that serves as a covalently-bound coenzyme of carboxylases. It is now well documented that biotin has actions other than participating in classical enzyme catalysis reactions. Several lines of evidence have demonstrated that pharmacological concentrations of biotin affect glucose and lipid metabolism, hypertension, reproduction, development, and immunity. The effect of biotin on these functions is related to its actions at the transcriptional, translational, and post-translational levels. The bestsupported mechanism involved in the genetic effects of biotin is the soluble guanylate cyclase/protein kinase G (PKG) signaling cascade. Although there are commercially-available products containing pharmacological concentrations of biotin, the toxic effects of biotin have been poorly studied. This review summarizes the known actions and molecular mechanisms of pharmacological doses of biotin in animals and current information regarding biotin toxicity. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  12. Laboratory Evolution of a Biotin-Requiring Saccharomyces cerevisiae Strain for Full Biotin Prototrophy and Identification of Causal Mutations.

    Science.gov (United States)

    Bracher, Jasmine M; de Hulster, Erik; Koster, Charlotte C; van den Broek, Marcel; Daran, Jean-Marc G; van Maris, Antonius J A; Pronk, Jack T

    2017-08-15

    Biotin prototrophy is a rare, incompletely understood, and industrially relevant characteristic of Saccharomyces cerevisiae strains. The genome of the haploid laboratory strain CEN.PK113-7D contains a full complement of biotin biosynthesis genes, but its growth in biotin-free synthetic medium is extremely slow (specific growth rate [μ] ≈ 0.01 h -1 ). Four independent evolution experiments in repeated batch cultures and accelerostats yielded strains whose growth rates (μ ≤ 0.36 h -1 ) in biotin-free and biotin-supplemented media were similar. Whole-genome resequencing of these evolved strains revealed up to 40-fold amplification of BIO1 , which encodes pimeloyl-coenzyme A (CoA) synthetase. The additional copies of BIO1 were found on different chromosomes, and its amplification coincided with substantial chromosomal rearrangements. A key role of this gene amplification was confirmed by overexpression of BIO1 in strain CEN.PK113-7D, which enabled growth in biotin-free medium (μ = 0.15 h -1 ). Mutations in the membrane transporter genes TPO1 and/or PDR12 were found in several of the evolved strains. Deletion of TPO1 and PDR12 in a BIO1 -overexpressing strain increased its specific growth rate to 0.25 h -1 The effects of null mutations in these genes, which have not been previously associated with biotin metabolism, were nonadditive. This study demonstrates that S. cerevisiae strains that carry the basic genetic information for biotin synthesis can be evolved for full biotin prototrophy and identifies new targets for engineering biotin prototrophy into laboratory and industrial strains of this yeast. IMPORTANCE Although biotin (vitamin H) plays essential roles in all organisms, not all organisms can synthesize this vitamin. Many strains of baker's yeast, an important microorganism in industrial biotechnology, contain at least some of the genes required for biotin synthesis. However, most of these strains cannot synthesize biotin at all or do so at rates that are

  13. Laboratory Evolution of a Biotin-Requiring Saccharomyces cerevisiae Strain for Full Biotin Prototrophy and Identification of Causal Mutations

    Science.gov (United States)

    de Hulster, Erik; Koster, Charlotte C.; van den Broek, Marcel; van Maris, Antonius J. A.

    2017-01-01

    ABSTRACT Biotin prototrophy is a rare, incompletely understood, and industrially relevant characteristic of Saccharomyces cerevisiae strains. The genome of the haploid laboratory strain CEN.PK113-7D contains a full complement of biotin biosynthesis genes, but its growth in biotin-free synthetic medium is extremely slow (specific growth rate [μ] ≈ 0.01 h−1). Four independent evolution experiments in repeated batch cultures and accelerostats yielded strains whose growth rates (μ ≤ 0.36 h−1) in biotin-free and biotin-supplemented media were similar. Whole-genome resequencing of these evolved strains revealed up to 40-fold amplification of BIO1, which encodes pimeloyl-coenzyme A (CoA) synthetase. The additional copies of BIO1 were found on different chromosomes, and its amplification coincided with substantial chromosomal rearrangements. A key role of this gene amplification was confirmed by overexpression of BIO1 in strain CEN.PK113-7D, which enabled growth in biotin-free medium (μ = 0.15 h−1). Mutations in the membrane transporter genes TPO1 and/or PDR12 were found in several of the evolved strains. Deletion of TPO1 and PDR12 in a BIO1-overexpressing strain increased its specific growth rate to 0.25 h−1. The effects of null mutations in these genes, which have not been previously associated with biotin metabolism, were nonadditive. This study demonstrates that S. cerevisiae strains that carry the basic genetic information for biotin synthesis can be evolved for full biotin prototrophy and identifies new targets for engineering biotin prototrophy into laboratory and industrial strains of this yeast. IMPORTANCE Although biotin (vitamin H) plays essential roles in all organisms, not all organisms can synthesize this vitamin. Many strains of baker's yeast, an important microorganism in industrial biotechnology, contain at least some of the genes required for biotin synthesis. However, most of these strains cannot synthesize biotin at all or do so at rates

  14. Biotin dependency due to a defect in biotin transport

    OpenAIRE

    Mardach, Rebecca; Zempleni, Janos; Wolf, Barry; Cannon, Martin J.; Jennings, Michael L.; Cress, Sally; Boylan, Jane; Roth, Susan; Cederbaum, Stephen; Mock, Donald M.

    2002-01-01

    We describe a 3-year-old boy with biotin dependency not caused by biotinidase, holocarboxylase synthetase, or nutritional biotin deficiency. We sought to define the mechanism of his biotin dependency. The child became acutely encephalopathic at age 18 months. Urinary organic acids indicated deficiency of several biotin-dependent carboxylases. Symptoms improved rapidly following biotin supplementation. Serum biotinidase activity and Biotinidase gene sequence were normal. Activities of biotin-d...

  15. Use of a sensitive EnVision +-based detection system for Western blotting: avoidance of streptavidin binding to endogenous biotin and biotin-containing proteins in kidney and other tissues.

    Science.gov (United States)

    Banks, Rosamonde E; Craven, Rachel A; Harnden, Patricia A; Selby, Peter J

    2003-04-01

    Western blotting remains a central technique in confirming identities of proteins, their quantitation and analysis of various isoforms. The biotin-avidin/streptavidin system is often used as an amplification step to increase sensitivity but in some tissues such as kidney, "nonspecific" interactions may be a problem due to high levels of endogenous biotin-containing proteins. The EnVision system, developed for immunohistochemical applications, relies on binding of a polymeric conjugate consisting of up to 100 peroxidase molecules and 20 secondary antibody molecules linked directly to an activated dextran backbone, to the primary antibody. This study demonstrates that it is also a viable and sensitive alternative detection system in Western blotting applications.

  16. Intestinal absorption of biotin in the rat

    International Nuclear Information System (INIS)

    Bowman, B.B.; Selhub, J.; Rosenberg, I.H.

    1986-01-01

    We examined the absorption of biotin using the in vivo intestinal loop technique. Jejunal segments from male rats were filled with solutions containing [ 3 H]biotin and [ 14 C]inulin in Krebs-Ringer phosphate buffer, pH 6.5. Absorption was determined on the basis of luminal tritium disappearance after correction for inulin recovery. At biotin concentrations of 0.1 and 5.0 microM, luminal biotin disappearance was linear for at least 10 min. At biotin concentrations ranging from 2.3 nM to 75 microM, 10-28% of the administered dose was absorbed in 10 min. The concentration dependence of luminal biotin disappearance is consistent with the presence of both saturable and nonsaturable (linear) components of biotin uptake, with estimated Km = 9.6 microM and Jmax = 75.2 pmol/(2.5 cm loop X min). The rate constant for nonsaturable uptake is 3.1 pmol/(2.5 cm loop X min X microM). We conclude that at biotin concentrations less than 5 microM, biotin absorption proceeds largely by the saturable process, whereas at concentrations above 25 microM, nonsaturable uptake predominates. Additional studies demonstrated significantly less biotin uptake in the ileum than in the jejunum, a finding in agreement with previous in vitro studies

  17. Overexpression of biotin synthase and biotin ligase is required for efficient generation of sulfur-35 labeled biotin in E. coli.

    Science.gov (United States)

    Delli-Bovi, Teegan A; Spalding, Maroya D; Prigge, Sean T

    2010-10-11

    Biotin is an essential enzyme cofactor that acts as a CO2 carrier in carboxylation and decarboxylation reactions. The E. coli genome encodes a biosynthetic pathway that produces biotin from pimeloyl-CoA in four enzymatic steps. The final step, insertion of sulfur into desthiobiotin to form biotin, is catalyzed by the biotin synthase, BioB. A dedicated biotin ligase (BirA) catalyzes the covalent attachment of biotin to biotin-dependent enzymes. Isotopic labeling has been a valuable tool for probing the details of the biosynthetic process and assaying the activity of biotin-dependent enzymes, however there is currently no established method for 35S labeling of biotin. In this study, we produced [35S]-biotin from Na35SO4 and desthiobiotin with a specific activity of 30.7 Ci/mmol, two orders of magnitude higher than previously published methods. The biotinylation domain (PfBCCP-79) from the Plasmodium falciparum acetyl-CoA carboxylase (ACC) was expressed in E. coli as a biotinylation substrate. We found that overexpression of the E. coli biotin synthase, BioB, and biotin ligase, BirA, increased PfBCCP-79 biotinylation 160-fold over basal levels. Biotinylated PfBCCP-79 was purified by affinity chromatography, and free biotin was liberated using acid hydrolysis. We verified that we had produced radiolabeled biologically active [D]-biotin that specifically labels biotinylated proteins through reuptake in E. coli. The strategy described in our report provides a simple and effective method for the production of [35S]-biotin in E. coli based on affinity chromatography.

  18. Effect of biotin on activity and gene expression of biotin-dependent carboxylases in the liver of dairy cows.

    Science.gov (United States)

    Ferreira, G; Weiss, W P

    2007-03-01

    Biotin is a cofactor of the gluconeogenic enzymes pyruvate carboxylase (PC) and propionyl-coenzyme A carboxylase (PCC). We hypothesized that biotin supplementation increases the activity and gene expression of PC and PCC and the gene expression of phosphoenol-pyruvate carboxykinase (PEPCK) in the liver of lactating dairy cows. Eight multiparous Holstein cows (40 +/- 2 kg/d of milk yield and 162 +/- 35 d in milk) were randomly assigned to 1 of 2 diet sequences in a crossover design with two 22-d periods. Treatments consisted of a basal diet (60% concentrate) containing 0 or 0.96 mg/kg of supplemental biotin. On d 21 of each period, liver tissue was collected by percutaneous liver biopsy. Activities of PC and PCC were determined by measuring the fixation of [14C]O2 in liver homogenates. Abundance of mRNA for PCC, PC, and PEPCK was determined by quantitative reverse-transcription PCR. Biotin supplementation did not affect milk production or composition. Biotin supplementation increased the activity of PC but had no effect on PCC activity. Biotin supplementation did not affect the gene expression of PC, PCC, and PEPCK. The increased activity of PC without changes in mRNA abundance may have been caused by increased activation of the apoenzymes by holocarboxylase synthetase. In conclusion, biotin supplementation affected the activity of PC in the liver of lactating dairy cows, but whether biotin supplementation increases glucose production in the liver remains to be determined.

  19. Biosynthesis of biotin from dethiobiotin by the biotin auxotroph Lactobacillus plantarum.

    OpenAIRE

    Bowman, W C; DeMoll, E

    1993-01-01

    Lactobacillus plantarum requires biotin for growth. We show that in the presence of high levels of the biotin biosynthetic precursor, dethiobiotin, L. plantarum synthesizes biotin and grows in medium with dethiobiotin but without biotin. Lactobacillus casei also grew under similar conditions.

  20. Low serum biotin in Japanese children fed with hydrolysate formula.

    Science.gov (United States)

    Sato, Yasuhiro; Wakabayashi, Kenji; Ogawa, Eishin; Kodama, Hiroko; Mimaki, Masakazu

    2016-09-01

    Given that nutritional biotin deficiency in Japanese infants has been reported, a straightforward method for estimating biotin level is needed. The biotin content in infant formula, breast milk, and the sera of infants fed with various types of formula were measured using avidin-binding assay. A commercially available ELISA kit was used for the measurement of biotin in 54 types of formula, including hydrolysate formulas for milk allergy, as well as in breast milk and in the sera of 27 infants fed with these formulas. The biotin content reached the recommended value in only five formulas. All of the hydrolysate formulas and more than half of the special formulas contained biotin biotin was low in infants fed only with the hydrolysate formulas, and one of them had alopecia related to biotin deficiency. While many were asymptomatic, infants fed with formulas lacking biotin are at risk of developing symptomatic disease. The addition of biotin to breast milk substitutes was finally approved in the middle of 2014, however pediatricians in Japan should still be vigilant with regard to nutritional biotin deficiency in infants for the time being. © 2016 Japan Pediatric Society.

  1. In vivo studies of biotin absorption in distal rat intestine

    International Nuclear Information System (INIS)

    Bowman, B.B.; Rosenberg, I.H.

    1986-01-01

    The authors have extended their previous studies of biotin absorption in rat proximal jejunum (PJ) to examine biotin absorptive capacity of rat ileum (I) and proximal colon (PC) using in vivo intestinal loop technique. Intestinal loops (2.5 cm) were filled with 0.3 ml of solution containing ( 3 H)-biotin and ( 14 C)-inulin in phosphate buffer, pH 6.5. Biotin absorption was determined on the basis of luminal biotin disappearance after correction for inulin recovery and averaged (pmol/loop-10 min; X +/- SEM). In related experiments, 5-cm loops of PJ, distal I (DI), or PC were filled with 0.5 ml of solution of similar composition (1.0 μM biotin). The abdominal cavity was closed and the rats were allowed to recover from anesthesia, then sacrificed 3 hr after injection. Biotin absorption averaged 96.2% (PJ), 93.2% (DI), and 25.8% (PC) of the dose administered. These differences were reflected in the radioactive biotin content of plasma and intestinal loop, kidney, and liver. These data demonstrate significant biotin absorption in rat DI and PC, as required if the intestinal microflora are to be considered as a source of biotin for the host

  2. Microarray analysis of pancreatic gene expression during biotin repletion in biotin-deficient rats.

    Science.gov (United States)

    Dakshinamurti, Krishnamurti; Bagchi, Rushita A; Abrenica, Bernard; Czubryt, Michael P

    2015-12-01

    Biotin is a B vitamin involved in multiple metabolic pathways. In humans, biotin deficiency is relatively rare but can cause dermatitis, alopecia, and perosis. Low biotin levels occur in individuals with type-2 diabetes, and supplementation with biotin plus chromium may improve blood sugar control. The acute effect on pancreatic gene expression of biotin repletion following chronic deficiency is unclear, therefore we induced biotin deficiency in adult male rats by feeding them a 20% raw egg white diet for 6 weeks. Animals were then randomized into 2 groups: one group received a single biotin supplement and returned to normal chow lacking egg white, while the second group remained on the depletion diet. After 1 week, pancreata were removed from biotin-deficient (BD) and biotin-repleted (BR) animals and RNA was isolated for microarray analysis. Biotin depletion altered gene expression in a manner indicative of inflammation, fibrosis, and defective pancreatic function. Conversely, biotin repletion activated numerous repair and anti-inflammatory pathways, reduced fibrotic gene expression, and induced multiple genes involved in pancreatic endocrine and exocrine function. A subset of the results was confirmed by quantitative real-time PCR analysis, as well as by treatment of pancreatic AR42J cells with biotin. The results indicate that biotin repletion, even after lengthy deficiency, results in the rapid induction of repair processes in the pancreas.

  3. Nitric oxide signaling depends on biotin in Jurkat human lymphoma cells.

    Science.gov (United States)

    Rodriguez-Melendez, Rocio; Zempleni, Janos

    2009-03-01

    Biotin affects gene expression through a diverse array of cell signaling pathways. Previous studies provided evidence that cGMP-dependent signaling also depends on biotin, but the mechanistic sequence of cGMP regulation by biotin is unknown. Here we tested the hypothesis that the effects of biotin in cGMP-dependent cell signaling are mediated by nitric oxide (NO). Human lymphoid (Jurkat) cells were cultured in media containing deficient (0.025 nmol/L), physiological (0.25 nmol/L), and pharmacological (10 nmol/L) concentrations of biotin for 5 wk. Both levels of intracellular biotin and NO exhibited a dose-dependent relationship in regard to biotin concentrations in culture media. Effects of biotin on NO levels were disrupted by the NO synthase (NOS) inhibitor N-monomethyl-arginine. Biotin-dependent production of NO was linked with biotin-dependent expression of endothelial and neuronal NOS, but not inducible NOS. Previous studies revealed that NO is an activator of guanylate cyclase. Consistent with these previous observations, biotin-dependent generation of NO increased the abundance of cGMP in Jurkat cells. Finally, the biotin-dependent generation of cGMP increased protein kinase G activity. Collectively, the results of this study are consistent with the hypothesis that biotin-dependent cGMP signaling in human lymphoid cells is mediated by NO.

  4. Effect of endogenous biotin on the applications of streptavidin and biotin in mice

    International Nuclear Information System (INIS)

    Rusckowski, Mary; Fogarasi, Miklos; Fritz, Benjamin; Hnatowich, Donald J.

    1997-01-01

    The use of streptavidin-conjugated antibody to pretarget tumors in animals and patients, prior to administration of radiolabeled biotin, has provided encouraging results, in part because of the high affinity of biotin for streptavidin and the rapid whole-body clearance of biotin. However, binding of endogenous biotin to streptavidin may interfere with the clinical potential of this approach. This report evaluates the effect of endogenous biotin on an antibody-streptavidin conjugate in a mouse tumor model. Tumored nude mice were depleted of endogenous biotin by sequential intraperitoneal injections of streptavidin. The assay of serum biotin levels indicated less than 0.5 ng of biotin per mL of serum in treated mice versus 4 ng per mL in untreated animals. Flow cytometric analysis was used on single-cell suspensions of tumor from animals receiving streptavidin-conjugated IgG to detect the presence of the antibody on the cell membrane (with fluoroisothiocyanate-conjugated goat anti-mouse antibody), and to detect biotin binding sites on streptavidin (with biotin-phycoerythrin). Both treated and untreated mice demonstrated the presence of antibody on tumor cells through 48 h postadministration, but only in treated animals were biotin binding sites observed. These results in the mouse model suggest that the small concentration of streptavidin delivered to a tumor via a specific antibody may be saturated with endogenous biotin and therefore not able to be targeted subsequently with radiolabeled biotin

  5. Microbial biotin protein ligases aid in understanding holocarboxylase synthetase deficiency.

    Science.gov (United States)

    Pendini, Nicole R; Bailey, Lisa M; Booker, Grant W; Wilce, Matthew C; Wallace, John C; Polyak, Steven W

    2008-01-01

    The attachment of biotin onto the biotin-dependent enzymes is catalysed by biotin protein ligase (BPL), also known as holocarboxylase synthase HCS in mammals. Mammals contain five biotin-enzymes that participate in a number of important metabolic pathways such as fatty acid biogenesis, gluconeogenesis and amino acid catabolism. All mammalian biotin-enzymes are post-translationally biotinylated, and therefore activated, through the action of a single HCS. Substrate recognition by BPLs occurs through conserved structural cues that govern the specificity of biotinylation. Defects in biotin metabolism, including HCS, give rise to multiple carboxylase deficiency (MCD). Here we review the literature on this important enzyme. In particular, we focus on the new information that has been learned about BPL's from a number of recently published protein structures. Through molecular modelling studies insights into the structural basis of HCS deficiency in MCD are discussed.

  6. Effects of Biotin Deficiency on Biotinylated Proteins and Biotin-Related Genes in the Rat Brain.

    Science.gov (United States)

    Yuasa, Masahiro; Aoyama, Yuki; Shimada, Ryoko; Sawamura, Hiromi; Ebara, Shuhei; Negoro, Munetaka; Fukui, Toru; Watanabe, Toshiaki

    2016-01-01

    Biotin is a water-soluble vitamin that functions as a cofactor for biotin-dependent carboxylases. The biochemical and physiological roles of biotin in brain regions have not yet been investigated sufficiently in vivo. Thus, in order to clarify the function of biotin in the brain, we herein examined biotin contents, biotinylated protein expression (e.g. holocarboxylases), and biotin-related gene expression in the brain of biotin-deficient rats. Three-week-old male Wistar rats were divided into a control group, biotin-deficient group, and pair-fed group. Rats were fed experimental diets from 3 wk old for 8 wk, and the cortex, hippocampus, striatum, hypothalamus, and cerebellum were then collected. In the biotin-deficient group, the maintenance of total biotin and holocarboxylases, increases in the bound form of biotin and biotinidase activity, and the expression of an unknown biotinylated protein were observed in the cortex. In other regions, total and free biotin contents decreased, holocarboxylase expression was maintained, and bound biotin and biotinidase activity remained unchanged. Biotin-related gene (pyruvate carboxylase, sodium-dependent multivitamin transporter, holocarboxylase synthetase, and biotinidase) expression in the cortex and hippocampus also remained unchanged among the dietary groups. These results suggest that biotin may be related to cortex functions by binding protein, and the effects of a biotin deficiency and the importance of biotin differ among the different brain regions.

  7. Control of biotin biosynthesis in mycobacteria by a pyruvate carboxylase dependent metabolic signal.

    Science.gov (United States)

    Lazar, Nathaniel; Fay, Allison; Nandakumar, Madhumitha; Boyle, Kerry E; Xavier, Joao; Rhee, Kyu; Glickman, Michael S

    2017-12-01

    Biotin is an essential cofactor utilized by all domains of life, but only synthesized by bacteria, fungi and plants, making biotin biosynthesis a target for antimicrobial development. To understand biotin biosynthesis in mycobacteria, we executed a genetic screen in Mycobacterium smegmatis for biotin auxotrophs and identified pyruvate carboxylase (Pyc) as required for biotin biosynthesis. The biotin auxotrophy of the pyc::tn strain is due to failure to transcriptionally induce late stage biotin biosynthetic genes in low biotin conditions. Loss of bioQ, the repressor of biotin biosynthesis, in the pyc::tn strain reverted biotin auxotrophy, as did reconstituting the last step of the pathway through heterologous expression of BioB and provision of its substrate DTB. The role of Pyc in biotin regulation required its catalytic activities and could be supported by M. tuberculosis Pyc. Quantitation of the kinetics of depletion of biotinylated proteins after biotin withdrawal revealed that Pyc is the most rapidly depleted biotinylated protein and metabolomics revealed a broad metabolic shift in wild type cells upon biotin withdrawal which was blunted in cell lacking Pyc. Our data indicate that mycobacterial cells monitor biotin sufficiency through a metabolic signal generated by dysfunction of a biotinylated protein of central metabolism. © 2017 John Wiley & Sons Ltd.

  8. Biotin radioligand assay with an 125I-labeled biotin derivative, avidin, and avidin double-antibody reagents

    International Nuclear Information System (INIS)

    Livaniou, E.; Evangelatos, G.P.; Ithakissios, D.S.

    1987-01-01

    We describe a new radioligand assay for determining biotin in biological fluids by using a mixture of N-[beta-(4-OH-3-125I-phenyl)ethyl]- and N-[beta-(4-OH-3,5-di-125I-phenyl)ethyl]biotinamides as radiotracer, avidin as a binding protein, and an avidin double-antibody as a separation reagent. The radiotracer is synthesized by coupling (at pH 8.5, 20-22 degrees C, 90 min) N-hydroxysuccinimidobiotin to radioiodinated tyramine. The assay curve is linear and the assay itself is sensitive (less than 10 ng/L), reproducible (intra- and interassay CVs 4.1% and 7.0%, respectively), and allows the simultaneous handling of more than 100 samples in less than 4 h. Serum samples from apparently normal subjects contained 100-840 ng of biotin per liter (mean 340 ng/L). Pregnant women had low concentrations of biotin (100-300 ng/L) in their serum. Patients undergoing chronic hemodialysis treatment showed high concentrations (0.5-3.0 micrograms/L), which may be ascribable to the inability of avidin, which was used as the assay binding protein, to distinguish biotin from biotinyl derivatives with an intact ureido ring

  9. The Atypical Occurrence of Two Biotin Protein Ligases in Francisella novicida Is Due to Distinct Roles in Virulence and Biotin Metabolism

    Science.gov (United States)

    Feng, Youjun; Chin, Chui-Yoke; Chakravartty, Vandana; Gao, Rongsui; Crispell, Emily K.

    2015-01-01

    ABSTRACT The physiological function of biotin requires biotin protein ligase activity in order to attach the coenzyme to its cognate proteins, which are enzymes involved in central metabolism. The model intracellular pathogen Francisella novicida is unusual in that it encodes two putative biotin protein ligases rather than the usual single enzyme. F. novicida BirA has a ligase domain as well as an N-terminal DNA-binding regulatory domain, similar to the prototypical BirA protein in E. coli. However, the second ligase, which we name BplA, lacks the N-terminal DNA binding motif. It has been unclear why a bacterium would encode these two disparate biotin protein ligases, since F. novicida contains only a single biotinylated protein. In vivo complementation and enzyme assays demonstrated that BirA and BplA are both functional biotin protein ligases, but BplA is a much more efficient enzyme. BirA, but not BplA, regulated transcription of the biotin synthetic operon. Expression of bplA (but not birA) increased significantly during F. novicida infection of macrophages. BplA (but not BirA) was required for bacterial replication within macrophages as well as in mice. These data demonstrate that F. novicida has evolved two distinct enzymes with specific roles; BplA possesses the major ligase activity, whereas BirA acts to regulate and thereby likely prevent wasteful synthesis of biotin. During infection BplA seems primarily employed to maximize the efficiency of biotin utilization without limiting the expression of biotin biosynthetic genes, representing a novel adaptation strategy that may also be used by other intracellular pathogens. PMID:26060274

  10. Pharmacological effects of biotin.

    Science.gov (United States)

    Fernandez-Mejia, Cristina

    2005-07-01

    In the last few decades, more vitamin-mediated effects have been discovered at the level of gene expression. Increasing knowledge on the molecular mechanisms of these vitamins has opened new perspectives that form a connection between nutritional signals and the development of new therapeutic agents. Besides its role as a carboxylase prosthetic group, biotin regulates gene expression and has a wide repertoire of effects on systemic processes. The vitamin regulates genes that are critical in the regulation of intermediary metabolism: Biotin has stimulatory effects on genes whose action favors hypoglycemia (insulin, insulin receptor, pancreatic and hepatic glucokinase); on the contrary, biotin decreases the expression of hepatic phosphoenolpyruvate carboxykinase, a key gluconeogenic enzyme that stimulates glucose production by the liver. The findings that biotin regulates the expression of genes that are critical in the regulation of intermediary metabolism are in agreement with several observations that indicate that biotin supply is involved in glucose and lipid homeostasis. Biotin deficiency has been linked to impaired glucose tolerance and decreased utilization of glucose. On the other hand, the diabetic state appears to be ameliorated by pharmacological doses of biotin. Likewise, pharmacological doses of biotin appear to decrease plasma lipid concentrations and modify lipid metabolism. The effects of biotin on carbohydrate metabolism and the lack of toxic effects of the vitamin at pharmacological doses suggest that biotin could be used in the development of new therapeutics in the treatment of hyperglycemia and hyperlipidemia, an area that we are actively investigating.

  11. Biotin: From Nutrition to Therapeutics.

    Science.gov (United States)

    Mock, Donald M

    2017-08-01

    Although frank symptomatic biotin deficiency is rare, some evidence suggests that marginal biotin deficiency occurs spontaneously in a substantial proportion of women during normal human pregnancy and might confer an increased risk of birth defects. Herein I review 1 ) advances in assessing biotin status, including the relation between acylcarnitine excretion and biotin status; 2 ) recent studies of biotin status in pregnancy; 3 ) advances in understanding the role of biotin in gene expression and the potential roles of biotinylated proteins that are neither histones nor carboxylases; and 4 ) novel large-dose biotin supplementation as therapy for multiple sclerosis. The review concludes with a summary of recent studies that have reported potentially dangerous erroneous results in individuals consuming large amounts of biotin for measurements of various plasma hormones for common clinical assays that use streptavidin-biotin technology. © 2017 American Society for Nutrition.

  12. Dietary intake of high-dose biotin inhibits spermatogenesis in young rats.

    Science.gov (United States)

    Sawamura, Hiromi; Ikeda, Chieko; Shimada, Ryoko; Yoshii, Yui; Watanabe, Toshiaki

    2015-02-01

    To characterize a new function of the water-soluble vitamin, biotin, in reproduction and early growth in mammals, the effects of high dietary doses of biotin on early spermatogenesis were biochemically and histologically investigated in male rats. Weaned rats were fed a CE-2 (control) diet containing 0.00004% biotin, or a control diet supplemented with 0.01%, 0.1%, or 1.0% biotin. Pair-fed rats were fed a control diet that was equal in calories to the amount ingested by the 1.0% biotin group, because food intake was decreased in the 1.0% biotin group. Food intake and body weight gain were lower in the 1.0% biotin group than in the control group. The kidney, brain and testis weights were significantly lower in the 1.0% biotin group than in the pair-fed group after 6 weeks of feeding. The accumulation of biotin in the liver and testis increased in a dose-dependent manner. In the 1.0% biotin group, the number of mature sperm was markedly lower, that of sperm with morphologically abnormal heads, mainly consisting of round heads, had increased. In addition, the development of seminiferous tubules was inhibited, and few spermatogonia and no spermatocytes were histologically observed. These results demonstrated that the long-term intake of high-dose biotin inhibited spermatogenesis in young male rats. © 2014 Japanese Teratology Society.

  13. The influence of nitrogen and biotin interactions on the performance of Saccharomyces in alcoholic fermentations.

    Science.gov (United States)

    Bohlscheid, J C; Fellman, J K; Wang, X D; Ansen, D; Edwards, C G

    2007-02-01

    To study the impact of assimilable nitrogen, biotin and their interaction on growth, fermentation rate and volatile formation by Saccharomyces. Fermentations of synthetic grape juice media were conducted in a factorial design with yeast assimilable nitrogen (YAN) (60 or 250 mg l(-1)) and biotin (0, 1 or 10 microg l(-1)) as variables. All media contained 240 g l(-1) glucose + fructose (1 : 1) and were fermented using biotin-depleted Saccharomyces cerevisiae strains EC1118 or UCD 522. Both strains exhibited weak growth and sluggish fermentation rates without biotin. Increased nitrogen concentration resulted in higher maximum fermentation rates, while adjusting biotin from 1 to 10 microg l(-1) had no effect. Nitrogen x biotin interactions influenced fermentation time, production of higher alcohols and hydrogen sulfide (H(2)S). Maximum H(2)S production occurred in the medium containing 60 mg l(-1) YAN and 1 microg l(-1) biotin. Nitrogen x biotin interactions affect fermentation time and volatile production by Saccharomyces depending on strain. Biotin concentrations sufficient to complete fermentation may affect the organoleptic impact of wine. This study demonstrates the necessity to consider nutrient interactions when diagnosing problem fermentations.

  14. Serum biotin in Japanese children: Enzyme-linked immunosorbent assay measurement.

    Science.gov (United States)

    Wakabayashi, Kenji; Kodama, Hiroko; Ogawa, Eishin; Sato, Yasuhiro; Motoyama, Kahoko; Suzuki, Mitsuyoshi

    2016-09-01

    Biotin deficiency has been reported in Japanese infants fed special formulas for medical reasons, including those with milk allergy and congenital metabolic diseases, because these formulas contain little biotin. Serum biotin measurement is useful for diagnosing biotin deficiency. We applied a simple and rapid method to analyze serum biotin, and established normal ranges for children and adults. Serum biotin in 188 healthy Japanese children aged 0-4 years and in 25 healthy adults was analyzed using a Biotin ELISA Kit (immundiagnostik). The effects of various conditions on the measurement of serum biotin were also examined. Median biotin in children aged 0-4 years was 10.4 ng/dL (IQR, 7.9-13.4 ng/dL), and that in adults was 12.9 ng/dL (IQR, 10.8-15.8 ng/dL). Normal range was 4.7-22.0 ng/dL in children and 8.4-20.5 ng/dL in adults (calculated using two-sided 95%CI). Measurements obtained with this method were not affected by frozen storage, freeze-thaw, or hemolysis, indicating that serum biotin can be analyzed accurately under these conditions, with a possible application to plasma samples. Serum biotin was significantly lower in children than in adults, with the normal range being 4.7-22.0 ng/dL in children and 8.4-20.5 ng/dL in adults. This simple and accurate enzyme-linked immunosorbent assay method is useful for diagnosing biotin deficiency. © 2016 The Authors. Pediatrics International published by John Wiley & Sons Australia, Ltd on behalf of Japan Pediatric Society.

  15. Synthesis and detection of 3'-OH terminal biotin-labeled DNA probes

    International Nuclear Information System (INIS)

    Brakel, C.L.; Engelhardt, D.L.

    1985-01-01

    Nick translation has been used to prepare biotin-dUTP-containing DNA probes. These stable DNA probes have been identified, following hybridization to target DNA, by fluorescence using antibiotin antibodies or by enzyme reactions in which the enzyme has been linked to avidin or streptavidin. It is probable that this technology will be applicable to certain diagnostic determinations and that, with sufficient sensitivity, this technology might provide a system for obtaining rapid and specific diagnoses in situations presently requiring time-consuming growth assays. The sensitivity of this assay can be increased in two ways: (1) by increasing the amount of biotin contained in the DNA probes, and (2) by increasing the response to individual biotin molecules in the DNA probes. This report demonstrates that terminal deoxynucleotide transferase can be employed to increase the biotin content of DNA probes. We also introduce a new streptavidin-linked enzyme system that produces a greater response to biotinylated DNA probes than does streptavidin-linked horseradish peroxidase

  16. Biotin and biotinidase deficiency

    OpenAIRE

    Zempleni, Janos; Hassan, Yousef I; Wijeratne, Subhashinee SK

    2008-01-01

    Biotin is a water-soluble vitamin that serves as an essential coenzyme for five carboxylases in mammals. Biotin-dependent carboxylases catalyze the fixation of bicarbonate in organic acids and play crucial roles in the metabolism of fatty acids, amino acids and glucose. Carboxylase activities decrease substantially in response to biotin deficiency. Biotin is also covalently attached to histones; biotinylated histones are enriched in repeat regions in the human genome and appear to play a role...

  17. Sequential, solid-phase assay for biotin in physiologic fluids that correlates with expected biotin status

    International Nuclear Information System (INIS)

    Mock, D.M.; DuBois, D.B.

    1986-01-01

    Interest in accurate measurement of biotin concentrations in plasma and urine has been stimulated by recent advances in the understanding of biotin-responsive inborn errors of metabolism and by several reports describing acquired biotin deficiency during parenteral alimentation. This paper presents a biotin assay utilizing radiolabeled avidin in a sequential, solid-phase method; the assay has increased sensitivity compared to previous methods (greater than or equal to 10 fmol/tube), correlates with expected trends in biotin concentrations in blood and urine in a rat model of biotin deficiency, and can utilize commercially available radiolabeled avidin

  18. SUCROSE TRANSPORTER 5 supplies Arabidopsis embryos with biotin and affects triacylglycerol accumulation

    Science.gov (United States)

    Pommerrenig, Benjamin; Popko, Jennifer; Heilmann, Mareike; Schulmeister, Sylwia; Dietel, Katharina; Schmitt, Bianca; Stadler, Ruth; Feussner, Ivo; Sauer, Norbert

    2013-01-01

    The Arabidopsis SUC5 protein represents a classical sucrose/H+ symporter. Functional analyses previously revealed that SUC5 also transports biotin, an essential co-factor for fatty acid synthesis. However, evidence for a dual role in transport of the structurally unrelated compounds sucrose and biotin in plants was lacking. Here we show that SUC5 localizes to the plasma membrane, and that the SUC5 gene is expressed in developing embryos, confirming the role of the SUC5 protein as substrate carrier across apoplastic barriers in seeds. We show that transport of biotin but not of sucrose across these barriers is impaired in suc5 mutant embryos. In addition, we show that SUC5 is essential for the delivery of biotin into the embryo of biotin biosynthesis-defective mutants (bio1 and bio2). We compared embryo and seedling development as well as triacylglycerol accumulation and fatty acid composition in seeds of single mutants (suc5, bio1 or bio2), double mutants (suc5 bio1 and suc5 bio2) and wild-type plants. Although suc5 mutants were like the wild-type, bio1 and bio2 mutants showed developmental defects and reduced triacylglycerol contents. In suc5 bio1 and suc5 bio2 double mutants, developmental defects were severely increased and the triacylglycerol content was reduced to a greater extent in comparison to the single mutants. Supplementation with externally applied biotin helped to reduce symptoms in both single and double mutants, but the efficacy of supplementation was significantly lower in double than in single mutants, showing that transport of biotin into the embryo is lower in the absence of SUC5. PMID:23031218

  19. Selective accumulation of biotin in arterial chemoreceptors: requirement for carotid body exocytotic dopamine secretion.

    Science.gov (United States)

    Ortega-Sáenz, Patricia; Macías, David; Levitsky, Konstantin L; Rodríguez-Gómez, José A; González-Rodríguez, Patricia; Bonilla-Henao, Victoria; Arias-Mayenco, Ignacio; López-Barneo, José

    2016-12-15

    Biotin, a vitamin whose main role is as a coenzyme for carboxylases, accumulates at unusually large amounts within cells of the carotid body (CB). In biotin-deficient rats biotin rapidly disappears from the blood; however, it remains at relatively high levels in CB glomus cells. The CB contains high levels of mRNA for SLC5a6, a biotin transporter, and SLC19a3, a thiamine transporter regulated by biotin. Animals with biotin deficiency exhibit pronounced metabolic lactic acidosis. Remarkably, glomus cells from these animals have normal electrical and neurochemical properties. However, they show a marked decrease in the size of quantal dopaminergic secretory events. Inhibitors of the vesicular monoamine transporter 2 (VMAT2) mimic the effect of biotin deficiency. In biotin-deficient animals, VMAT2 protein expression decreases in parallel with biotin depletion in CB cells. These data suggest that dopamine transport and/or storage in small secretory granules in glomus cells depend on biotin. Biotin is a water-soluble vitamin required for the function of carboxylases as well as for the regulation of gene expression. Here, we report that biotin accumulates in unusually large amounts in cells of arterial chemoreceptors, carotid body (CB) and adrenal medulla (AM). We show in a biotin-deficient rat model that the vitamin rapidly disappears from the blood and other tissues (including the AM), while remaining at relatively high levels in the CB. We have also observed that, in comparison with other peripheral neural tissues, CB cells contain high levels of SLC5a6, a biotin transporter, and SLC19a3, a thiamine transporter regulated by biotin. Biotin-deficient rats show a syndrome characterized by marked weight loss, metabolic lactic acidosis, aciduria and accelerated breathing with normal responsiveness to hypoxia. Remarkably, CB cells from biotin-deficient animals have normal electrophysiological and neurochemical (ATP levels and catecholamine synthesis) properties; however

  20. Solitary BioY Proteins Mediate Biotin Transport into Recombinant Escherichia coli

    Science.gov (United States)

    Finkenwirth, Friedrich; Kirsch, Franziska

    2013-01-01

    Energy-coupling factor (ECF) transporters form a large group of vitamin uptake systems in prokaryotes. They are composed of highly diverse, substrate-specific, transmembrane proteins (S units), a ubiquitous transmembrane protein (T unit), and homo- or hetero-oligomeric ABC ATPases. Biotin transporters represent a special case of ECF-type systems. The majority of the biotin-specific S units (BioY) is known or predicted to interact with T units and ABC ATPases. About one-third of BioY proteins, however, are encoded in organisms lacking any recognizable T unit. This finding raises the question of whether these BioYs function as transporters in a solitary state, a feature ascribed to certain BioYs in the past. To address this question in living cells, an Escherichia coli K-12 derivative deficient in biotin synthesis and devoid of its endogenous high-affinity biotin transporter was constructed as a reference strain. This organism is particularly suited for this purpose because components of ECF transporters do not naturally occur in E. coli K-12. The double mutant was viable in media containing either high levels of biotin or a precursor of the downstream biosynthetic path. Importantly, it was nonviable on trace levels of biotin. Eight solitary bioY genes of proteobacterial origin were individually expressed in the reference strain. Each of the BioYs conferred biotin uptake activity on the recombinants, which was inferred from uptake assays with [3H]biotin and growth of the cells on trace levels of biotin. The results underscore that solitary BioY transports biotin across the cytoplasmic membrane. PMID:23836870

  1. Interaction Between the Biotin Carboxyl Carrier Domain and the Biotin Carboxylase Domain in Pyruvate Carboxylase from Rhizobium etli†

    Science.gov (United States)

    Lietzan, Adam D.; Menefee, Ann L.; Zeczycki, Tonya N.; Kumar, Sudhanshu; Attwood, Paul V.; Wallace, John C.; Cleland, W. Wallace; Maurice, Martin St.

    2011-01-01

    Pyruvate carboxylase (PC) catalyzes the ATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in mammalian tissues. To effect catalysis, the tethered biotin of PC must gain access to active sites in both the biotin carboxylase domain and the carboxyl transferase domain. Previous studies have demonstrated that a mutation of threonine 882 to alanine in PC from Rhizobium etli renders the carboxyl transferase domain inactive and favors the positioning of biotin in the biotin carboxylase domain. We report the 2.4 Å resolution X-ray crystal structure of the Rhizobium etli PC T882A mutant which reveals the first high-resolution description of the domain interaction between the biotin carboxyl carrier protein domain and the biotin carboxylase domain. The overall quaternary arrangement of Rhizobium etli PC remains highly asymmetrical and is independent of the presence of allosteric activator. While biotin is observed in the biotin carboxylase domain, its access to the active site is precluded by the interaction between Arg353 and Glu248, revealing a mechanism for regulating carboxybiotin access to the BC domain active site. The binding location for the biotin carboxyl carrier protein domain demonstrates that tethered biotin cannot bind in the biotin carboxylase domain active site in the same orientation as free biotin, helping to explain the difference in catalysis observed between tethered biotin and free biotin substrates in biotin carboxylase enzymes. Electron density located in the biotin carboxylase domain active site is assigned to phosphonoacetate, offering a probable location for the putative carboxyphosphate intermediate formed during biotin carboxylation. The insights gained from the T882A Rhizobium etli PC crystal structure provide a new series of catalytic snapshots in PC and offer a revised perspective on catalysis in the biotin-dependent enzyme family. PMID:21958016

  2. Profligate Biotin Synthesis in α-Proteobacteria – A Developing or Degenerating Regulatory System?

    Science.gov (United States)

    Feng, Youjun; Zhang, Huimin; Cronan, John E.

    2013-01-01

    Summary Biotin (vitamin H) is a key enzyme cofactor required in all three domains of life. Although this cofactor was discovered over 70 years ago and has long been recognized as an essential nutrient for animals, our knowledge of the strategies bacteria use to sense biotin demand is very limited. The paradigm mechanism is that of Escherichia coli in which BirA protein, the prototypical bi-functional biotin protein ligase, both covalently attaches biotin to the acceptor proteins of central metabolism and represses transcription of the biotin biosynthetic pathway in response to biotin demand. However, in other bacteria the biotin protein ligase lacks a DNA-binding domain which raises the question of how these bacteria regulate the synthesis of biotin, an energetically expensive molecule. A bioinformatic study by Rodionov and Gelfand (FEMS Microbiol Lett. (2006) 255:102–107) identified a protein termed BioR in α-proteobacteria and predicted that BioR would have the biotin operon regulatory role that in most other bacteria is fulfilled by the BirA DNA-binding domain. We have now tested this prediction in the plant pathogen Agrobacterium tumefaciens. As predicted the A. tumefaciens biotin protein ligase is a fully functional ligase that has no role in regulation of biotin synthesis whereas BioR represses transcription of the biotin synthesis genes. Moreover, as determined by electrophoretic mobility shift assays, BioR binds the predicted operator site, which is located downstream of the mapped transcription start site. qPCR measurements indicated that deletion of BioR resulted in a ca.15-fold increase of bio operon transcription in the presence of high biotin levels. Effective repression of a plasmid-borne bioB-lacZ reporter was seen only upon the overproduction of BioR. In contrast to E. coli and Bacillus subtilis where biotin synthesis is tightly controlled, A. tumefaciens synthesizes much more biotin than needed for modification of the biotin-requiring enzymes

  3. Streptavidin and its biotin complex at atomic resolution

    International Nuclear Information System (INIS)

    Le Trong, Isolde; Wang, Zhizhi; Hyre, David E.; Lybrand, Terry P.; Stayton, Patrick S.; Stenkamp, Ronald E.

    2011-01-01

    Analysis of atomic resolution crystal structures of wild-type streptavidin (1.03 Å) and its biotin complex (0.95 Å) indicate the range of conformational states taken on by this protein in the solid state. Most of the structural variation is found in the polypeptide loops between the strands in this β-sandwich protein. Atomic resolution crystallographic studies of streptavidin and its biotin complex have been carried out at 1.03 and 0.95 Å, respectively. The wild-type protein crystallized with a tetramer in the asymmetric unit, while the crystals of the biotin complex contained two subunits in the asymmetric unit. Comparison of the six subunits shows the various ways in which the protein accommodates ligand binding and different crystal-packing environments. Conformational variation is found in each of the polypeptide loops connecting the eight strands in the β-sandwich subunit, but the largest differences are found in the flexible binding loop (residues 45–52). In three of the unliganded subunits the loop is in an ‘open’ conformation, while in the two subunits binding biotin, as well as in one of the unliganded subunits, this loop ‘closes’ over the biotin–binding site. The ‘closed’ loop contributes to the protein’s high affinity for biotin. Analysis of the anisotropic displacement parameters included in the crystallographic models is consistent with the variation found in the loop structures and the view that the dynamic nature of the protein structure contributes to the ability of the protein to bind biotin so tightly

  4. Biotin in microbes, the genes involved in its biosynthesis, its biochemical role and perspectives for biotechnological production.

    Science.gov (United States)

    Streit, W R; Entcheva, P

    2003-03-01

    Biotin (vitamin H) is one of the most fascinating cofactors involved in central pathways in pro- and eukaryotic cell metabolism. Since its original discovery in 1901, research has led to the discovery of the complete biotin biosynthesis pathways in many different microbes and much work has been done on the highly intriguing and complex biochemistry of biotin biosynthesis. While humans and animals require several hundred micrograms of biotin per day, most microbes, plants and fungi appear to be able to synthesize the cofactor themselves. Biotin is added to many food, feed and cosmetic products, creating a world market of 10-30 t/year. However, the majority of the biotin sold is synthesized in a chemical process. Since the chemical synthesis is linked with a high environmental burden, much effort has been put into the development of biotin-overproducing microbes. A summary of biotin biosynthesis and its biological role is presented; and current strategies for the improvement of microbial biotin production using modern biotechnological techniques are discussed.

  5. The Role of Biotin in Bacterial Physiology and Virulence: a Novel Antibiotic Target for Mycobacterium tuberculosis.

    Science.gov (United States)

    Salaemae, Wanisa; Booker, Grant W; Polyak, Steven W

    2016-04-01

    Biotin is an essential cofactor for enzymes present in key metabolic pathways such as fatty acid biosynthesis, replenishment of the tricarboxylic acid cycle, and amino acid metabolism. Biotin is synthesized de novo in microorganisms, plants, and fungi, but this metabolic activity is absent in mammals, making biotin biosynthesis an attractive target for antibiotic discovery. In particular, biotin biosynthesis plays important metabolic roles as the sole source of biotin in all stages of the Mycobacterium tuberculosis life cycle due to the lack of a transporter for scavenging exogenous biotin. Biotin is intimately associated with lipid synthesis where the products form key components of the mycobacterial cell membrane that are critical for bacterial survival and pathogenesis. In this review we discuss the central role of biotin in bacterial physiology and highlight studies that demonstrate the importance of its biosynthesis for virulence. The structural biology of the known biotin synthetic enzymes is described alongside studies using structure-guided design, phenotypic screening, and fragment-based approaches to drug discovery as routes to new antituberculosis agents.

  6. High-dose biotin therapy leading to false biochemical endocrine profiles: validation of a simple method to overcome biotin interference.

    Science.gov (United States)

    Piketty, Marie-Liesse; Prie, Dominique; Sedel, Frederic; Bernard, Delphine; Hercend, Claude; Chanson, Philippe; Souberbielle, Jean-Claude

    2017-05-01

    High-dose biotin therapy is beneficial in progressive multiple sclerosis (MS) and is expected to be adopted by a large number of patients. Biotin therapy leads to analytical interference in many immunoassays that utilize streptavidin-biotin capture techniques, yielding skewed results that can mimic various endocrine disorders. We aimed at exploring this interference, to be able to remove biotin and avoid misleading results. We measured free triiodothyronine (fT3), free thyroxine (fT4), thyroid-stimulating hormone (TSH), parathyroid homrone (PTH), 25-hydroxyvitamin D (25OHD), follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin, C-peptide, cortisol (Roche Diagnostics assays), biotin and its main metabolites (liquid chromatography tandem mass spectrometry) in 23 plasmas from MS patients and healthy volunteers receiving high-dose biotin, and in 39 biotin-unsupplemented patients, before and after a simple procedure (designated N5) designed to remove biotin by means of streptavidin-coated microparticles. We also assayed fT4, TSH and PTH in the 23 high-biotin plasmas using assays not employing streptavidin-biotin binding. The biotin concentration ranged from 31.7 to 1160 µg/L in the 23 high-biotin plasmas samples. After the N5 protocol, the biotin concentration was below the detection limit in all but two samples (8.3 and 27.6 μg/L). Most hormones results were abnormal, but normalized after N5. All results with the alternative methods were normal except two slight PTH elevations. In the 39 biotin-unsupplemented patients, the N5 protocol did not affect the results for any of the hormones, apart from an 8.4% decrease in PTH. We confirm that most streptavidin-biotin hormone immunoassays are affected by high biotin concentrations, leading to a risk of misdiagnosis. Our simple neutralization method efficiently suppresses biotin interference.

  7. Time-resolved homo-FRET studies of biotin-streptavidin complexes.

    Science.gov (United States)

    Andreoni, Alessandra; Nardo, Luca; Rigler, Rudolf

    2016-09-01

    Förster resonance energy transfer is a mechanism of fluorescence quenching that is notably useful for characterizing properties of biomolecules and/or their interactions. Here we study water-solutions of Biotin-Streptavidin complexes, in which Biotin is labeled with a rigidly-bound fluorophore that can interact by Förster resonance energy transfer with the fluorophores labeling the other, up to three, Biotins of the same complex. The fluorophore, Atto550, is a Rhodamine analogue. We detect the time-resolved fluorescence decay of the fluorophores with an apparatus endowed with single-photon sensitivity and temporal resolution of ~30ps. The decay profiles we observe for samples containing constant Biotin-Atto550 conjugates and varying Streptavidin concentrations are multi-exponential. Each decay component can be associated with the rate of quenching exerted on each donor by each of the acceptors that label the other Biotin molecules, depending on the binding site they occupy. The main features that lead to this result are that (i) the transition dipole moments of the up-to-four Atto550 fluorophores that label the complexes are fixed as to both relative positions and mutual orientations; (ii) the fluorophores are identical and the role of donor in each Biotin-Streptavidin complex is randomly attributed to the one that has absorbed the excitation light (homo-FRET). Obviously the high-temporal resolution of the excitation-detection apparatus is necessary to discriminate among the fluorescence decay components. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Paracoccus denitrificans possesses two BioR homologs having a role in regulation of biotin metabolism.

    Science.gov (United States)

    Feng, Youjun; Kumar, Ritesh; Ravcheev, Dmitry A; Zhang, Huimin

    2015-08-01

    Recently, we determined that BioR, the GntR family of transcription factor, acts as a repressor for biotin metabolism exclusively distributed in certain species of α-proteobacteria, including the zoonotic agent Brucella melitensis and the plant pathogen Agrobacterium tumefaciens. However, the scenario is unusual in Paracoccus denitrificans, another closely related member of the same phylum α-proteobacteria featuring with denitrification. Not only does it encode two BioR homologs Pden_1431 and Pden_2922 (designated as BioR1 and BioR2, respectively), but also has six predictive BioR-recognizable sites (the two bioR homolog each has one site, whereas the two bio operons (bioBFDAGC and bioYB) each contains two tandem BioR boxes). It raised the possibility that unexpected complexity is present in BioR-mediated biotin regulation. Here we report that this is the case. The identity of the purified BioR proteins (BioR1 and BioR2) was confirmed with LC-QToF-MS. Phylogenetic analyses combined with GC percentage raised a possibility that the bioR2 gene might be acquired by horizontal gene transfer. Gel shift assays revealed that the predicted BioR-binding sites are functional for the two BioR homologs, in much similarity to the scenario seen with the BioR site of A. tumefaciens bioBFDAZ. Using the A. tumefaciens reporter system carrying a plasmid-borne LacZ fusion, we revealed that the two homologs of P. denitrificans BioR are functional repressors for biotin metabolism. As anticipated, not only does the addition of exogenous biotin stimulate efficiently the expression of bioYB operon encoding biotin transport/uptake system BioY, but also inhibits the transcription of the bioBFDAGC operon resembling the de novo biotin synthetic pathway. EMSA-based screening failed to demonstrate that the biotin-related metabolite is involved in BioR-DNA interplay, which is consistent with our former observation with Brucella BioR. Our finding defined a complex regulatory network for biotin

  9. Biotin protein ligase from Corynebacterium glutamicum: role for growth and L: -lysine production.

    Science.gov (United States)

    Peters-Wendisch, P; Stansen, K C; Götker, S; Wendisch, V F

    2012-03-01

    Corynebacterium glutamicum is a biotin auxotrophic Gram-positive bacterium that is used for large-scale production of amino acids, especially of L-glutamate and L-lysine. It is known that biotin limitation triggers L-glutamate production and that L-lysine production can be increased by enhancing the activity of pyruvate carboxylase, one of two biotin-dependent proteins of C. glutamicum. The gene cg0814 (accession number YP_225000) has been annotated to code for putative biotin protein ligase BirA, but the protein has not yet been characterized. A discontinuous enzyme assay of biotin protein ligase activity was established using a 105aa peptide corresponding to the carboxyterminus of the biotin carboxylase/biotin carboxyl carrier protein subunit AccBC of the acetyl CoA carboxylase from C. glutamicum as acceptor substrate. Biotinylation of this biotin acceptor peptide was revealed with crude extracts of a strain overexpressing the birA gene and was shown to be ATP dependent. Thus, birA from C. glutamicum codes for a functional biotin protein ligase (EC 6.3.4.15). The gene birA from C. glutamicum was overexpressed and the transcriptome was compared with the control strain revealing no significant gene expression changes of the bio-genes. However, biotin protein ligase overproduction increased the level of the biotin-containing protein pyruvate carboxylase and entailed a significant growth advantage in glucose minimal medium. Moreover, birA overexpression resulted in a twofold higher L-lysine yield on glucose as compared with the control strain.

  10. 21 CFR 182.8159 - Biotin.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Biotin. 182.8159 Section 182.8159 Food and Drugs... CONSUMPTION (CONTINUED) SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients § 182.8159 Biotin. (a) Product. Biotin. (b) Conditions of use. This substance is generally recognized as safe when used in accordance...

  11. BIOTIN INTERFERENCE WITH ROUTINE CLINICAL IMMUNOASSAYS: UNDERSTAND THE CAUSES AND MITIGATE THE RISKS.

    Science.gov (United States)

    Samarasinghe, Shanika; Meah, Farah; Singh, Vinita; Basit, Arshi; Emanuele, Nicholas; Emanuele, Mary Ann; Mazhari, Alaleh; Holmes, Earle W

    2017-08-01

    The objectives of this report are to review the mechanisms of biotin interference with streptavidin/biotin-based immunoassays, identify automated immunoassay systems vulnerable to biotin interference, describe how to estimate and minimize the risk of biotin interference in vulnerable assays, and review the literature pertaining to biotin interference in endocrine function tests. The data in the manufacturer's "Instructions for Use" for each of the methods utilized by seven immunoassay system were evaluated. We also conducted a systematic search of PubMed/MEDLINE for articles containing terms associated with biotin interference. Available original reports and case series were reviewed. Abstracts from recent scientific meetings were also identified and reviewed. The recent, marked, increase in the use of over-the-counter, high-dose biotin supplements has been accompanied by a steady increase in the number of reports of analytical interference by exogenous biotin in the immunoassays used to evaluate endocrine function. Since immunoassay methods of similar design are also used for the diagnosis and management of anemia, malignancies, autoimmune and infectious diseases, cardiac damage, etc., biotin-related analytical interference is a problem that touches every area of internal medicine. It is important for healthcare personnel to become more aware of immunoassay methods that are vulnerable to biotin interference and to consider biotin supplements as potential sources of falsely increased or decreased test results, especially in cases where a lab result does not correlate with the clinical scenario. FDA = U.S. Food & Drug Administration FT3 = free tri-iodothyronine FT4 = free thyroxine IFUs = instructions for use LH = luteinizing hormone PTH = parathyroid hormone SA/B = streptavidin/biotin TFT = thyroid function test TSH = thyroid-stimulating hormone.

  12. 21 CFR 582.5159 - Biotin.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Biotin. 582.5159 Section 582.5159 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS... § 582.5159 Biotin. (a) Product. Biotin. (b) Conditions of use. This substance is generally recognized as...

  13. Fake news? Biotin interference in thyroid immunoassays.

    Science.gov (United States)

    Koehler, Viktoria F; Mann, Ulrike; Nassour, Ayham; Alexander Mann, W

    2018-05-29

    We report on a 47 year old male patient with multiple sclerosis (MS) presenting in our outpatient neurology clinic in Frankfurt/Main for therapy evaluation. Before change of treatment laboratory investigations were performed. Thyroid function tests (TFTs) with a streptavidin/biotin based immunoassay revealed severe hyperthyroidism with positive thyroid autoantibodies suggestive for Graves' disease. Clinical presentation and thyroid sonography were unremarkable. Due to the discordance between clinical presentation and TFTs, we repeated medical history, in which the patient reported taking high-doses of biotin (300 mg/day) for MS. Recent studies with patients suffering from primary and secondary progressive MS, indicated promising effects of high-dose biotin on MS-related disability. In immunoassays relaying on streptavidin-biotin interaction, biotin intake can cause falsely high or low results. Two weeks after withdrawing biotin, biotin/streptavidin dependant assays showed no longer the biochemical picture of severe hyperthyroidism. Biotin intake should be paused for at least two to five days prior to the use of biotin/streptavidin dependant assays. Alternatively, non-biotin/streptavidin dependant assays (radioimmunoassay, gas chromatography-mass spectrometry/liquid chromatography-mass spectrometry) may be used. Copyright © 2017. Published by Elsevier B.V.

  14. Radioligand assay for biotin in liver tissues

    International Nuclear Information System (INIS)

    Rettenmaier, R.

    1979-01-01

    A radioligand assay for biotin in liver tissue is described. 3 H-biotin is used as tracer and avidin as binder. The biotin-loaded avidin is separated from free biotin on dextran-coated charcoal, which leaves the avidin-biotin complex in the supernatant liquid. Thus, the avidin-biotin complex can easily be utilized for determination of the radioactivity. Calibration with known additions of biotin in the range 0.25-8.0 ng per assay sample yields a linear logit-log plot. The biotin is extracted from liver tissues by enzymatic proteolysis with papain. This treatment is optimized to liberate the bound forms of the vitamin. Microbiological parallel assays with Lactobacillus plantarum were in good agreement with the radioligand assay giving a regression coefficient of 0.974(n=44). The coefficient of variation was found to be 4.2% in the range 500-1200 ng of biotin per g of liver tissue (n=46). The method is simple and reliable and allows the simultaneous analysis of a considerable number of samples. (Auth.)

  15. Biotin absorption by distal rat intestine

    International Nuclear Information System (INIS)

    Bowman, B.B.; Rosenberg, I.H.

    1987-01-01

    We used the in vivo intestinal loop approach, with short (10-min) and long (3-h) incubations, to examine biotin absorption in proximal jejunum, distal ileum, cecum and proximal colon. In short-term studies, luminal biotin disappearance from rat ileum was about half that observed in the jejunum, whereas absorption by proximal colon was about 12% of that in the jejunum. In 3-h closed-loop studies, the absorption of 1.0 microM biotin varied regionally. Biotin absorption was nearly complete in the small intestine after 3 h; however, only about 15% of the dose had been absorbed in the cecum and 27% in the proximal colon after 3 h. Independent of site of administration, the major fraction of absorbed biotin was recovered in the liver; measurable amounts of radioactive biotin were also present in kidney and plasma. The results support the potential nutritional significance for the rat of biotin synthesized by bacteria in the distal intestine, by demonstrating directly an absorptive capability of mammalian large bowel for this vitamin

  16. Improving the performance of solventogenic clostridia by reinforcing the biotin synthetic pathway.

    Science.gov (United States)

    Yang, Yunpeng; Lang, Nannan; Yang, Gaohua; Yang, Sheng; Jiang, Weihong; Gu, Yang

    2016-05-01

    An efficient production process is important for industrial microorganisms. The cellular efficiency of solventogenic clostridia, a group of anaerobes capable of producing a wealth of bulk chemicals and biofuels, must be improved for competitive commercialization. Here, using Clostridium acetobutylicum, a species of solventogenic clostridia, we revealed that the insufficient biosynthesis of biotin, a pivotal coenzyme for many important biological processes, is a major limiting bottleneck in this anaerobe's performance. To address this problem, we strengthened the biotin synthesis of C. acetobutylicum by overexpressing four relevant genes involved in biotin transport and biosynthesis. This strategy led to faster growth and improved the titer and productivity of acetone, butanol and ethanol (ABE solvents) of C. acetobutylicum in both biotin-containing and biotin-free media. Expressionally modulating these four genes by modifying the ribosome binding site further promoted cellular performance, achieving ABE solvent titer and productivity as high as 21.9g/L and 0.30g/L/h, respectively, in biotin-free medium; these values exceeded those of the wild-type strain by over 30%. More importantly, biotin synthesis reinforcement also conferred improved ability of C. acetobutylicum to use hexose and pentose sugars, further demonstrating the potential of this metabolic-engineering strategy in solventogenic clostridia. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  17. Design of Biotin-Functionalized Luminescent Quantum Dots

    Directory of Open Access Journals (Sweden)

    Kimihiro Susumu

    2007-01-01

    Full Text Available We report the design and synthesis of a tetraethylene glycol- (TEG- based bidentate ligand functionalized with dihydrolipoic acid (DHLA and biotin (DHLA—TEG—biotin to promote biocompatibility of luminescent quantum dots (QD's. This new ligand readily binds to CdSe—ZnS core-shell QDs via surface ligand exchange. QDs capped with a mixture of DHLA and DHLA—TEG—biotin or polyethylene glycol- (PEG- (molecular weight average ∼600 modified DHLA (DHLA—PEG600 and DHLA—TEG—biotin are easily dispersed in aqueous buffer solutions. In particular, homogeneous buffer solutions of QDs capped with a mixture of DHLA—PEG600 and DHLA—TEG—biotin that are stable over broad pH range have been prepared. QDs coated with mixtures of DHLA/DHLA—TEG—biotin and with DHLA—PEG600/DHLA—TEG—biotin were tested in surface binding assays and the results indicate that biotin groups on the QD surface interact specifically with NeutrAvidin-functionalized microtiter well plates.

  18. Effect of biotin and pantothenic acid on performance and concentrations of avidin-binding substances in blood and milk of lactating dairy cows.

    Science.gov (United States)

    Ferreira, Gonzalo; Brown, Alston N; Teets, Christy L

    2015-09-01

    We hypothesized that pantothenic acid reduces the absorption of biotin in lactating dairy cows. Therefore, the objective of this study was to evaluate the plausible interaction between biotin and pantothenic acid on production performance and concentration of avidin-binding substances (ABS), an indicator of biotin concentration, in blood and milk of lactating dairy cows. Eight primiparous and 16 multiparous Holstein cows were assigned to 1 of 4 diet sequences in a replicated 4×4 Latin square design with 18-d periods. Cows were housed in a freestall barn and fed once daily (0730 h) by means of a Calan gate system (American Calan Inc., Northwood, NH). Treatments consisted of a control diet that contained no B-vitamins, a biotin diet that contained 0.87 mg of biotin per kilogram of dry matter (DM), a pantothenic acid diet that contained 21 mg of pantothenic acid per kilogram of DM, and a biotin plus pantothenic acid diet that contained 0.87 mg of biotin and 21 mg of calcium pantothenic acid per kilogram of DM. Four different concentrates were prepared in a commercial feed mill. These concentrates were mixed with corn silage and grass hay and delivered ad libitum as a total mixed ration. Biotin supplementation did not affect DM intake, milk yield, or milk fat, protein, lactose, and milk-urea-nitrogen concentrations. Fat, protein, and lactose yields were not affected by treatments. The fat-to-protein ratio was Biotin supplementation did not increase the concentration of ABS in plasma. The supplementation of pantothenic acid did not affect the concentration of ABS in plasma when either supplemented alone or in combination with biotin. Biotin supplementation increased the concentration of ABS in milk relative to control. Contrary to our hypothesis, the supplementation of pantothenic acid did not decrease the concentration of ABS in milk relative to the control. When cows were supplemented with both biotin and pantothenic acid, the concentration of ABS in milk was similar

  19. The biotin repressor: modulation of allostery by corepressor analogs.

    Science.gov (United States)

    Brown, Patrick H; Cronan, John E; Grøtli, Morten; Beckett, Dorothy

    2004-04-02

    The Escherichia coli biotin repressor functions in biotin retention and regulation of biotin biosynthesis. Biotin retention is accomplished via the two-step biotinylation of the biotin-dependent enzyme, acetyl-CoA carboxylase. In the first step of this reaction the substrates biotin and ATP are utilized in synthesis of the activated biotin, biotinyl-5'-AMP, while in the second step this activated biotin is transferred to a unique lysine residue of the biotin carboxyl carrier protein subunit of the carboxylase. Regulation of biotin biosynthesis is accomplished through binding of the repressor to the transcription control region of the biotin biosynthetic operon. The adenylated or activated biotin functions as the corepressor in this DNA binding process. The activated biotin is a mixed anhydride and thus labile. In efforts to develop tools for structural and thermodynamic studies of the biotin regulatory interactions, two analogs of the adenylate, a sulfamoyl derivative and an ester derivative, have been synthesized and functionally characterized. Results of fluorescence measurements indicate that both analogs bind with high affinity to the repressor and that both are inactive in biotin transfer to the acceptor protein. Functional studies of their corepressor properties indicate that while the sulfamoyl is a weak allosteric activator, the ester closely mimics the physiological corepressor in activation of assembly of the transcription repression complex. Results of these studies also provide further insight into the allosteric mechanism of the biotin repressor.

  20. Effects of various spacers between biotin and the phospholipid headgroup on immobilization and sedimentation of biotinylated phospholipid-containing liposomes facilitated by avidin-biotin interactions.

    Science.gov (United States)

    Sakamoto, Yasuhisa; Kikuchi, Koji; Umeda, Kazuaki; Nakanishi, Hiroyuki

    2017-09-01

    Immobilization and sedimentation of liposomes (lipid vesicles) are used in liposome-protein binding assays, facilitated by avidin/streptavidin/NeutrAvidin and biotinylated phospholipid-containing liposomes. Here, we examined the effects of three spacers [six-carbon (X), polyethylene glycol (PEG) 180 (molecular weight 180) and PEG2000 (molecular weight 2,000)] between biotin and the phospholipid headgroup on the immobilization and sedimentation of small unilamellar liposomes/vesicles (SUVs). PEG180 and PEG2000 showed more efficient immobilization of biotinylated SUVs on NeutrAvidin-coated plates than X, but X and PEG180 showed more efficient sedimentation of biotinylated SUVs upon NeutrAvidin addition than PEG2000. Thus, the most appropriate spacers differed between immobilization and sedimentation. A spacer for biotinylated SUVs must be selected according to the particular liposome-protein binding assays examined. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  1. Biotin deficiency up-regulates TNF-alpha production in murine macrophages.

    Science.gov (United States)

    Kuroishi, Toshinobu; Endo, Yasuo; Muramoto, Koji; Sugawara, Shunji

    2008-04-01

    Biotin, a water-soluble vitamin of the B complex, functions as a cofactor of carboxylases that catalyze an indispensable cellular metabolism. Although significant decreases in serum biotin levels have been reported in patients with chronic inflammatory diseases, the biological roles of biotin in inflammatory responses are unclear. In this study, we investigated the effects of biotin deficiency on TNF-alpha production. Mice were fed a basal diet or a biotin-deficient diet for 8 weeks. Serum biotin levels were significantly lower in biotin-deficient mice than biotin-sufficient mice. After i.v. administration of LPS, serum TNF-alpha levels were significantly higher in biotin-deficient mice than biotin-sufficient mice. A murine macrophage-like cell line, J774.1, was cultured in a biotin-sufficient or -deficient medium for 4 weeks. Cell proliferation and biotinylation of intracellular proteins were decreased significantly in biotin-deficient cells compared with biotin-sufficient cells. Significantly higher production and mRNA expression of TNF-alpha were detected in biotin-deficient J774.1 cells than biotin-sufficient cells in response to LPS and even without LPS stimulation. Intracellular TNF-alpha expression was inhibited by actinomycin D, indicating that biotin deficiency up-regulates TNF-alpha production at the transcriptional level. However, the expression levels of TNF receptors, CD14, and TLR4/myeloid differentiation protein 2 complex were similar between biotin-sufficient and -deficient cells. No differences were detected in the activities of the NF-kappaB family or AP-1. The TNF-alpha induction by biotin deficiency was down-regulated by biotin supplementation in vitro and in vivo. These results indicate that biotin deficiency may up-regulate TNF-alpha production or that biotin excess down-regulates TNF-alpha production, suggesting that biotin status may influence inflammatory diseases.

  2. Regulation of immunological and inflammatory functions by biotin.

    Science.gov (United States)

    Kuroishi, Toshinobu

    2015-12-01

    Biotin is a water-soluble B-complex vitamin and is well-known as a co-factor for 5 indispensable carboxylases. Holocarboxylase synthetase (HLCS) catalyzes the biotinylation of carboxylases and other proteins, whereas biotinidase catalyzes the release of biotin from biotinylated peptides. Previous studies have reported that nutritional biotin deficiency and genetic defects in either HLCS or biotinidase induces cutaneous inflammation and immunological disorders. Since biotin-dependent carboxylases involve various cellular metabolic pathways including gluconeogenesis, fatty acid synthesis, and the metabolism of branched-chain amino acids and odd-chain fatty acids, metabolic abnormalities may play important roles in immunological and inflammatory disorders caused by biotin deficiency. Transcriptional factors, including NF-κB and Sp1/3, are also affected by the status of biotin, indicating that biotin regulates immunological and inflammatory functions independently of biotin-dependent carboxylases. An in-vivo analysis with a murine model revealed the therapeutic effects of biotin supplementation on metal allergies. The novel roles of biotinylated proteins and their related enzymes have recently been reported. Non-carboxylase biotinylated proteins induce chemokine production. HLCS is a nuclear protein involved in epigenetic and chromatin regulation. In this review, comprehensive knowledge on the regulation of immunological and inflammatory functions by biotin and its potential as a therapeutic agent is discussed.

  3. Fluorescence Enhancement of Fluorescent Unnatural Streptavidin by Binding of a Biotin Analogue with Spacer Tail and Its Application to Biotin Sensing

    Directory of Open Access Journals (Sweden)

    Xianwei Zhu

    2014-01-01

    Full Text Available We designed a novel molecular biosensing system for the detection of biotin, an important vitamin by the combination of fluorescent unnatural streptavidin with a commercialized biotin-(AC52-hydrazide. A fluorescent unnatural amino acid, BODIPY-FL-aminophenylalanine (BFLAF, was position-specifically incorporated into Trp120 of streptavidin by four-base codon method. Fluorescence of the Trp120BFLAF mutant streptavidin was enhanced by the addition of biotin-(AC52-hydrazide with the concentration dependent, whereas fluorescence enhancement was not observed at all by the addition of natural biotin. It was considered that the spacer tail of biotin-(AC52-hydrazide may disturb the fluorescence quenching of the Trp120BFLAF by Trp79 and Trp108 of the neighbor subunit. Therefore, biotin sensing was carried out by the competitive binding reaction of biotin-(AC52-hydrazide and natural biotin to the fluorescent mutant streptavidin. The fluorescence intensity decreased by increasing free biotin concentration. The result suggested that molecular biosensor for small ligand could be successfully designed by the pair of fluorescent mutant binding protein and ligand analogue.

  4. Consumption of a low-carbohydrate and high-fat diet (the ketogenic diet) exaggerates biotin deficiency in mice.

    Science.gov (United States)

    Yuasa, Masahiro; Matsui, Tomoyoshi; Ando, Saori; Ishii, Yoshie; Sawamura, Hiromi; Ebara, Shuhei; Watanabe, Toshiaki

    2013-10-01

    Biotin is a water-soluble vitamin that acts as a cofactor for several carboxylases. The ketogenic diet, a low-carbohydrate, high-fat diet, is used to treat drug-resistant epilepsy and promote weight loss. In Japan, the infant version of the ketogenic diet is known as the "ketone formula." However, as the special infant formulas used in Japan, including the ketone formula, do not contain sufficient amounts of biotin, biotin deficiency can develop in infants who consume the ketone formula. Therefore, the aim of this study was to evaluate the effects of the ketogenic diet on biotin status in mice. Male mice (N = 32) were divided into the following groups: control diet group, biotin-deficient (BD) diet group, ketogenic control diet group, and ketogenic biotin-deficient (KBD) diet group. Eight mice were used in each group. At 9 wk, the typical symptoms of biotin deficiency such as hair loss and dermatitis had only developed in the KBD diet group. The total protein expression level of biotin-dependent carboxylases and the total tissue biotin content were significantly decreased in the KBD and BD diet groups. However, these changes were more severe in the KBD diet group. These findings demonstrated that the ketogenic diet increases biotin bioavailability and consumption, and hence, promotes energy production by gluconeogenesis and branched-chain amino acid metabolism, which results in exaggerated biotin deficiency in biotin-deficient mice. Therefore, biotin supplementation is important for mice that consume the ketogenic diet. It is suggested that individuals that consume the ketogenic diet have an increased biotin requirement. Copyright © 2013 Elsevier Inc. All rights reserved.

  5. Recombinant Candida utilis for the production of biotin.

    Science.gov (United States)

    Hong, Yi-Ren; Chen, Ya-Lei; Farh, Lynn; Yang, Wen-Jen; Liao, Chen-Hua; Shiuan, David

    2006-06-01

    Biotin is an important nutritional supplement but is difficult to manufacture effectively. Here we present a trial of biotin production using the food yeast Candida utilis. In this system, we cloned the C. utilis biotin synthase (BIO2) gene, the gene of the rate-limiting enzyme for biotin biosynthesis, and assembled it under the control of a strong promoter. A series of plasmids were constructed to direct the integration of the BIO2 gene, either high-copy integration with 18S rDNA fragment or low-copy integration with URA3 or HIS3 fragment. The BIO2 gene can be successfully integrated into the C. utilis chromosome and can drive biotin production using these plasmids. The biotin yield in this system can reach 100-fold above the endogenous level in a small-scale culture. Although the biotin production is not stable if the selection pressure is removed, this system has the potential to produce biotin-rich feed or food additives directly without the requirement of further purification.

  6. Serum Biotin Levels in Women Complaining of Hair Loss.

    Science.gov (United States)

    Trüeb, Ralph M

    2016-01-01

    Biotin is a coenzyme for carboxylase enzymes that assist various metabolic reactions involved in fatty acid synthesis, branched-chain amino acid catabolism, and gluconeogenesis important for maintenance of healthy skin and hair. Due to its availability, affordability, and effective marketing for this purpose, biotin is a popular nutritional supplement for treatment of hair loss. However, there are little data on the frequency of biotin deficiency in patients complaining of hair loss and on the value of oral biotin for treatment of hair loss that is not due to an inborn error of biotin metabolism or deficiency. The aim of this study was to determine the frequency and significance of biotin deficiency in women complaining of hair loss. Biotin deficiency was found in 38% of women complaining of hair loss. Of those showing diffuse telogen effluvium in trichograms (24%), 35% had evidence of associated seborrheic-like dermatitis. About 11% of patients with biotin deficiency had a positive personal history for risk factors for biotin deficiency. The custom of treating women complaining of hair loss in an indiscriminate manner with oral biotin supplementation is to be rejected, unless biotin deficiency and its significance for the complaint of hair loss in an individual has been demonstrated on the basis of a careful patient history, clinical examination, determination of serum biotin levels, and exclusion of alternative factors responsible for hair loss.

  7. Marginal Biotin Deficiency Is Teratogenic in ICR Mice1,2

    OpenAIRE

    Mock, Donald M.; Mock, Nell I.; Stewart, Christopher W.; LaBorde, James B.; Hansen, Deborah K.

    2003-01-01

    The incidence of marginal biotin deficiency in normal human gestation is approximately one in three. In ICR mice, maternal biotin deficiency results in cleft palate, micrognathia, microglossia and limb hypoplasia. However, the relationships among the severity of maternal biotin deficiency, fetal biotin status and malformations have not been reported. This study utilized validated indices of biotin status to investigate the relationships among maternal biotin status, fetal biotin status and th...

  8. Development of Biotin-Prototrophic and -Hyperauxotrophic Corynebacterium glutamicum Strains

    Science.gov (United States)

    Miyamoto, Aya; Mutoh, Sumire; Kitano, Yuko; Tajima, Mei; Shirakura, Daisuke; Takasaki, Manami; Mitsuhashi, Satoshi; Takeno, Seiki

    2013-01-01

    To develop the infrastructure for biotin production through naturally biotin-auxotrophic Corynebacterium glutamicum, we attempted to engineer the organism into a biotin prototroph and a biotin hyperauxotroph. To confer biotin prototrophy on the organism, the cotranscribed bioBF genes of Escherichia coli were introduced into the C. glutamicum genome, which originally lacked the bioF gene. The resulting strain still required biotin for growth, but it could be replaced by exogenous pimelic acid, a source of the biotin precursor pimelate thioester linked to either coenzyme A (CoA) or acyl carrier protein (ACP). To bridge the gap between the pimelate thioester and its dedicated precursor acyl-CoA (or -ACP), the bioI gene of Bacillus subtilis, which encoded a P450 protein that cleaves a carbon-carbon bond of an acyl-ACP to generate pimeloyl-ACP, was further expressed in the engineered strain by using a plasmid system. This resulted in a biotin prototroph that is capable of the de novo synthesis of biotin. On the other hand, the bioY gene responsible for biotin uptake was disrupted in wild-type C. glutamicum. Whereas the wild-type strain required approximately 1 μg of biotin per liter for normal growth, the bioY disruptant (ΔbioY) required approximately 1 mg of biotin per liter, almost 3 orders of magnitude higher than the wild-type level. The ΔbioY strain showed a similar high requirement for the precursor dethiobiotin, a substrate for bioB-encoded biotin synthase. To eliminate the dependency on dethiobiotin, the bioB gene was further disrupted in both the wild-type strain and the ΔbioY strain. By selectively using the resulting two strains (ΔbioB and ΔbioBY) as indicator strains, we developed a practical biotin bioassay system that can quantify biotin in the seven-digit range, from approximately 0.1 μg to 1 g per liter. This bioassay proved that the engineered biotin prototroph of C. glutamicum produced biotin directly from glucose, albeit at a marginally

  9. Development of biotin-prototrophic and -hyperauxotrophic Corynebacterium glutamicum strains.

    Science.gov (United States)

    Ikeda, Masato; Miyamoto, Aya; Mutoh, Sumire; Kitano, Yuko; Tajima, Mei; Shirakura, Daisuke; Takasaki, Manami; Mitsuhashi, Satoshi; Takeno, Seiki

    2013-08-01

    To develop the infrastructure for biotin production through naturally biotin-auxotrophic Corynebacterium glutamicum, we attempted to engineer the organism into a biotin prototroph and a biotin hyperauxotroph. To confer biotin prototrophy on the organism, the cotranscribed bioBF genes of Escherichia coli were introduced into the C. glutamicum genome, which originally lacked the bioF gene. The resulting strain still required biotin for growth, but it could be replaced by exogenous pimelic acid, a source of the biotin precursor pimelate thioester linked to either coenzyme A (CoA) or acyl carrier protein (ACP). To bridge the gap between the pimelate thioester and its dedicated precursor acyl-CoA (or -ACP), the bioI gene of Bacillus subtilis, which encoded a P450 protein that cleaves a carbon-carbon bond of an acyl-ACP to generate pimeloyl-ACP, was further expressed in the engineered strain by using a plasmid system. This resulted in a biotin prototroph that is capable of the de novo synthesis of biotin. On the other hand, the bioY gene responsible for biotin uptake was disrupted in wild-type C. glutamicum. Whereas the wild-type strain required approximately 1 μg of biotin per liter for normal growth, the bioY disruptant (ΔbioY) required approximately 1 mg of biotin per liter, almost 3 orders of magnitude higher than the wild-type level. The ΔbioY strain showed a similar high requirement for the precursor dethiobiotin, a substrate for bioB-encoded biotin synthase. To eliminate the dependency on dethiobiotin, the bioB gene was further disrupted in both the wild-type strain and the ΔbioY strain. By selectively using the resulting two strains (ΔbioB and ΔbioBY) as indicator strains, we developed a practical biotin bioassay system that can quantify biotin in the seven-digit range, from approximately 0.1 μg to 1 g per liter. This bioassay proved that the engineered biotin prototroph of C. glutamicum produced biotin directly from glucose, albeit at a marginally

  10. Brucella BioR Regulator Defines a Complex Regulatory Mechanism for Bacterial Biotin Metabolism

    Science.gov (United States)

    Xu, Jie; Zhang, Huimin; Srinivas, Swaminath

    2013-01-01

    The enzyme cofactor biotin (vitamin H or B7) is an energetically expensive molecule whose de novo biosynthesis requires 20 ATP equivalents. It seems quite likely that diverse mechanisms have evolved to tightly regulate its biosynthesis. Unlike the model regulator BirA, a bifunctional biotin protein ligase with the capability of repressing the biotin biosynthetic pathway, BioR has been recently reported by us as an alternative machinery and a new type of GntR family transcriptional factor that can repress the expression of the bioBFDAZ operon in the plant pathogen Agrobacterium tumefaciens. However, quite unusually, a closely related human pathogen, Brucella melitensis, has four putative BioR-binding sites (both bioR and bioY possess one site in the promoter region, whereas the bioBFDAZ [bio] operon contains two tandem BioR boxes). This raised the question of whether BioR mediates the complex regulatory network of biotin metabolism. Here, we report that this is the case. The B. melitensis BioR ortholog was overexpressed and purified to homogeneity, and its solution structure was found to be dimeric. Functional complementation in a bioR isogenic mutant of A. tumefaciens elucidated that Brucella BioR is a functional repressor. Electrophoretic mobility shift assays demonstrated that the four predicted BioR sites of Brucella plus the BioR site of A. tumefaciens can all interact with the Brucella BioR protein. In a reporter strain that we developed on the basis of a double mutant of A. tumefaciens (the ΔbioR ΔbioBFDA mutant), the β-galactosidase (β-Gal) activity of three plasmid-borne transcriptional fusions (bioBbme-lacZ, bioYbme-lacZ, and bioRbme-lacZ) was dramatically decreased upon overexpression of Brucella bioR. Real-time quantitative PCR analyses showed that the expression of bioBFDA and bioY is significantly elevated upon removal of bioR from B. melitensis. Together, we conclude that Brucella BioR is not only a negative autoregulator but also a repressor of

  11. Identification and assessment of markers of biotin status in healthy adults

    Science.gov (United States)

    Eng, Wei Kay; Giraud, David; Schlegel, Vicki L.; Wang, Dong; Lee, Bo Hyun; Zempleni, Janos

    2016-01-01

    Human biotin requirements are unknown and the identification of reliable markers of biotin status is necessary to fill this knowledge gap. Here, we used an outpatient feeding protocol to create states of biotin deficiency, sufficiency and supplementation in sixteen healthy men and women. A total of twenty possible markers of biotin status were assessed, including the abundance of biotinylated carboxylases in lymphocytes, the expression of genes from biotin metabolism and the urinary excretion of biotin and organic acids. Only the abundance of biotinylated 3-methylcrotonyl-CoA carboxylase (holo-MCC) and propionyl-CoA carboxylase (holo-PCC) allowed for distinguishing biotin-deficient and biotin-sufficient individuals. The urinary excretion of biotin reliably identified biotin-supplemented subjects, but did not distinguish between biotin-depleted and biotin-sufficient individuals. The urinary excretion of 3-hydroxyisovaleric acid detected some biotin-deficient subjects, but produced a meaningful number of false-negative results and did not distinguish between biotin-sufficient and biotin-supplemented individuals. None of the other organic acids that were tested were useful markers of biotin status. Likewise, the abundance of mRNA coding for biotin transporters, holocarboxylase synthetase and biotin-dependent carboxylases in lymphocytes were not different among the treatment groups. Generally, datasets were characterised by variations that exceeded those seen in studies in cell cultures. We conclude that holo-MCC and holo-PCC are the most reliable, single markers of biotin status tested in the present study. PMID:23302490

  12. Role of the [2Fe-2S] cluster in recombinant Escherichia coli biotin synthase.

    Science.gov (United States)

    Jameson, Guy N L; Cosper, Michele Mader; Hernández, Heather L; Johnson, Michael K; Huynh, Boi Hanh

    2004-02-24

    Biotin synthase (BioB) converts dethiobiotin into biotin by inserting a sulfur atom between C6 and C9 of dethiobiotin in an S-adenosylmethionine (SAM)-dependent reaction. The as-purified recombinant BioB from Escherichia coli is a homodimeric molecule containing one [2Fe-2S](2+) cluster per monomer. It is inactive in vitro without the addition of exogenous Fe. Anaerobic reconstitution of the as-purified [2Fe-2S]-containing BioB with Fe(2+) and S(2)(-) produces a form of BioB that contains approximately one [2Fe-2S](2+) and one [4Fe-4S](2+) cluster per monomer ([2Fe-2S]/[4Fe-4S] BioB). In the absence of added Fe, the [2Fe-2S]/[4Fe-4S] BioB is active and can produce up to approximately 0.7 equiv of biotin per monomer. To better define the roles of the Fe-S clusters in the BioB reaction, Mössbauer and electron paramagnetic resonance (EPR) spectroscopy have been used to monitor the states of the Fe-S clusters during the conversion of dethiobiotin to biotin. The results show that the [4Fe-4S](2+) cluster is stable during the reaction and present in the SAM-bound form, supporting the current consensus that the functional role of the [4Fe-4S] cluster is to bind SAM and facilitate the reductive cleavage of SAM to generate the catalytically essential 5'-deoxyadenosyl radical. The results also demonstrate that approximately (2)/(3) of the [2Fe-2S] clusters are degraded by the end of the turnover experiment (24 h at 25 degrees C). A transient species with spectroscopic properties consistent with a [2Fe-2S](+) cluster is observed during turnover, suggesting that the degradation of the [2Fe-2S](2+) cluster is initiated by reduction of the cluster. This observed degradation of the [2Fe-2S] cluster during biotin formation is consistent with the proposed sacrificial S-donating function of the [2Fe-2S] cluster put forth by Jarrett and co-workers (Ugulava et al. (2001) Biochemistry 40, 8352-8358). Interestingly, degradation of the [2Fe-2S](2+) cluster was found not to parallel

  13. Autodisplay of an avidin with biotin-binding activity on the surface of Escherichia coli.

    Science.gov (United States)

    Pardavé-Alejandre, H D; Alvarado-Yaah, J E; Pompa-Mera, E N; Muñoz-Medina, J E; Sárquiz-Martínez, B; Santacruz-Tinoco, C E; Manning-Cela, R G; Ortíz-Navarrete, V; López-Macías, C; González-Bonilla, C R

    2018-03-01

    To display a recombinant avidin fused to the autotransporter ShdA to bind biotinylated molecules on the surface of Escherichia coli. Two chimeric protein constructs containing avidin fused to the autotransporter ShdA were expressed on the surface of Escherichia coli DH5α. One fusion protein contained 476 amino acids of the ShdA α and β domains, whereas the second consisted of a 314 amino acid from α and truncated β domains. Protein production was verified by SDS-PAGE using an antibody to the molecular FLAG-tag. The surface display of the avidin-shdA fusion protein was confirmed by confocal microscopy and flow cytometry analysis, and the biotin-binding activity was evaluated by fluorescence microscopy and flow cytometry using biotin-4-fluorescein and biotinylated-ovalbumin (OVA). Expression of a recombinant avidin with biotin-binding activity on the surface of E. coli was achieved using the autotransporter ShdA. This system is an alternative to bind biotinylated molecules to E. coli.

  14. Effects of excess biotin administration on the growth and urinary excretion of water-soluble vitamins in young rats.

    Science.gov (United States)

    Sawamura, Hiromi; Fukuwatari, Tsutomu; Shibata, Katsumi

    2007-12-01

    To determine the effects of excess biotin administration on growth and water-soluble vitamin metabolism, weaning rats were fed on a 20% casein diet containing 0.00002% biotin, or same diet with 0.04, 0.08, 0.10, 0.20, 0.50, 0.80 or 1.0% added biotin for 28 days. More than 0.08% biotin administration decreased the food intake and body weight gain compared with the levels in control rats. An accumulation of biotin in such tissues as the liver, brain and kidney increased in a dose-dependent manner, and the both bound and free biotin contents in the liver also increased in a dose-dependent manner. An excess administration of biotin did not affect the urinary excretion of other water-soluble vitamins, suggesting no effect on the metabolism of other water-soluble vitamins. The results of the food intake and body weight gain indicated that the lowest observed adverse effect level for young rats was 79.2 mg/kg body weight/day, while the no observed adverse effect level was 38.4 mg/kg/day. These results suggested immediately setting a tolerable upper intake level for biotin.

  15. On the intermediacy of carboxyphosphate in biotin-dependent carboxylations

    International Nuclear Information System (INIS)

    Ogita, Takeshi; Knowles, J.R.

    1988-01-01

    In the ATP-dependent carboxylation of biotin that is catalyzed by most biotin-dependent carboxylases, a fundamental mechanistic question is whether the ATP activates bicarbonate (via the formation of carboxyphosphate as an intermediate) or whether the ATP activates biotin (via the formation of O-phosphobiotin). The authors have resorted to three mechanistic tests using the biotin carboxylase subunit of acetyl-CoA carboxylase from Escherichia coli: positional isotope exchange, intermediate trapping, and 18 O tracer experiments on the ATPase activity. First, no catalysis of positional isotope exchange in adenosine 5'-([α,β- 18 O,β,β- 18 O 2 ]triphosphate) was observed when either biotin or bicarbonate was absent, nor was any exchange seen in the presence of both N-1-methylbiotin and bicarbonate. Second, the putative carboxyphosphate intermediate could not be trapped as its trimethyl ester, under conditions of incubation and analysis where the authentic triester was shown to be adequately stable. In the third test, however, they showed that the ATPase activity of biotin carboxylase that is seen in the absence of biotin, an activity that is known to parallel the normal carboxylase reaction when biotin is present, occurs with the transfer of an 18 O label directly from [ 18 O]bicarbonate into the product P i . This result suggests that the bicarbonate-dependent biotin-independent ATPase reaction catalyzed by biotin carboxylase goes via carboxyphosphate and that the carboxylation of biotin itself may proceed analogously

  16. Design and synthesis of biotin analogues reversibly binding with streptavidin.

    Science.gov (United States)

    Yamamoto, Tomohiro; Aoki, Kiyoshi; Sugiyama, Akira; Doi, Hirofumi; Kodama, Tatsuhiko; Shimizu, Yohei; Kanai, Motomu

    2015-04-01

    Two new biotin analogues, biotin carbonate 5 and biotin carbamate 6, have been synthesized. These molecules were designed to reversibly bind with streptavidin by replacing the hydrogen-bond donor NH group(s) of biotin's cyclic urea moiety with oxygen. Biotin carbonate 5 was synthesized from L-arabinose (7), which furnishes the desired stereochemistry at the 3,4-cis-dihydroxy groups, in 11% overall yield (over 10 steps). Synthesis of biotin carbamate 6 was accomplished from L-cysteine-derived chiral aldehyde 33 in 11% overall yield (over 7 steps). Surface plasmon resonance analysis of water-soluble biotin carbonate analogue 46 and biotin carbamate analogue 47 revealed that KD values of these compounds for binding to streptavidin were 6.7×10(-6)  M and 1.7×10(-10)  M, respectively. These values were remarkably greater than that of biotin (KD =10(-15)  M), and thus indicate the importance of the nitrogen atoms for the strong binding between biotin and streptavidin. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Crystallization and preliminary X-ray crystallographic studies of the biotin carboxyl carrier protein and biotin protein ligase complex from Pyrococcus horikoshii OT3

    International Nuclear Information System (INIS)

    Bagautdinov, Bagautdin; Matsuura, Yoshinori; Bagautdinova, Svetlana; Kunishima, Naoki

    2007-01-01

    A truncated form of biotin carboxyl carrier protein containing the C-terminal half fragment (BCCPΔN76) and the biotin protein ligase (BPL) with the mutation R48A (BPL*) or the double mutation R48A K111A (BPL**) were successfully cocrystallized in the presence of ATP and biotin. The BPL*–BCCPΔN76 and BPL**–BCCPΔN76 crystals belong to space group P2 1 and diffract X-rays to 2.7 and 2.0 Å resolution, respectively. Biotin protein ligase (BPL) catalyses the biotinylation of the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase. To elucidate the exact details of the protein–protein interactions in the biotinylation function, the C-terminal half fragment of BCCP (BCCPΔN76), the R48A mutant of BPL (BPL*) and the R48A K111A double mutant of BPL (BPL**), all of which are from Pyrococcus horikoshii OT3, have been expressed, purified and successfully cocrystallized. Cocrystals of the BPL*–BCCPΔN76 and BPL**–BCCPΔN76 complexes as well as crystals of BPL*, BPL** and BCCPΔN76 were obtained by the oil-microbatch method using PEG 20 000 as a precipitant at 295 K. Complete X-ray diffraction data sets for BPL*–BCCPΔN76 and BPL**–BCCPΔN76 crystals were collected at 100 K to 2.7 and 2.0 Å resolution, respectively, using synchrotron radiation. They belong to the monoclinic space group P2 1 , with similar unit-cell parameters a = 69.85, b = 63.12, c = 75.64 Å, β = 95.9°. Assuming two subunits of the complex per asymmetric unit gives a V M value of 2.45 Å 3 Da −1 and a solvent content of 50%

  18. Uptake of 153Sm-DTPA-bis-biotin and 99mTc-DTPA-bis-biotin in rat as-30D-hepatoma cells

    International Nuclear Information System (INIS)

    Correa-Gonzalez, Luis; Arteaga de Murphy, Consuelo; Ferro-Flores, Guillermina; Pedraza-Lopez, Martha; Murphy-Stack, Eduardo; Mino-Leon, Dolores; Perez-Villasenor, Graciela; Diaz-Torres, Yaneth; Munoz-Olvera, Rodrigo

    2003-01-01

    Labeled biotin has been used mainly for pretargeted therapy, an approach for increasing the amount of radioactivity delivered to a cancer cell. The aim of this investigation was to prepare 153 Sm-DTPA-bis-biotin and 99m Tc-DTPA-bis-biotin in order to study their in vitro and in vivo uptake in rat AS-30D hepatoma cells found in ascites and in implanted tumor. DTPA-bis-biotin (pH 8) was 153 Sm labeled with 153 SmCl 3 and 99m Tc-DTPA-bis-biotin was prepared via SnCl 2 reduction. Radiochemical purity was >98% in both cases. AS-30D hepatoma cells were obtained from ascites of a rat with hepatoma and were propagated in the peritoneum cavity of normal rats. In vitro ascites cell 153 Sm-DTPA-bis-biotin uptake was compared with 153 SmCl 3 cell uptake. The ratio cell 153 Sm-DTPA-bis-biotin/ 153 SmCl 3 was 39.6 and when avidin was added it increased to 50. The ratio 99m Tc-DTPA-bis-biotin/TcO 4 Na was 8.7. Concentration of 153 Sm-DTPA-bis-biotin in tumor 2, 3 and 24 h after administration, was 5, 15 and 3 times higher than in normal muscle (T/nT). Biodistribution in a 0.083-24 h time period showed that 153 Sm-DTPA-bis-biotin was taken up only by ascites tumor cells and hepatoma cells. Two and 3 h ratio ascites/liver (As/Lv) was 6.4 and 6.0. For 99m Tc-DTPA-bis-biotin 2 and 3 h T/nT was 15.7 and 4.7 and 2 h As/Lv was 1.4. In conclusion, both radiopharmaceuticals show high uptake in rat AS-30D hepatoma cells in ascites and in implanted tumor. Since lung, thyroid, kidney, liver or pancreas carcinomas are ascites producing cancers 153 Sm-DTPA-bis-biotin would be an adequate therapeutic radiopharmaceutical for these patients whose life quality would be enhanced with control of ascites, and a reduction of the primary tumor and its metastases

  19. Differential biotin labelling of the cell envelope proteins in lipopolysaccharidic diderm bacteria: Exploring the proteosurfaceome of Escherichia coli using sulfo-NHS-SS-biotin and sulfo-NHS-PEG4-bismannose-SS-biotin.

    Science.gov (United States)

    Monteiro, Ricardo; Chafsey, Ingrid; Leroy, Sabine; Chambon, Christophe; Hébraud, Michel; Livrelli, Valérie; Pizza, Mariagrazia; Pezzicoli, Alfredo; Desvaux, Mickaël

    2018-06-15

    Surface proteins are the major factor for the interaction between bacteria and its environment, playing an important role in infection, colonisation, virulence and adaptation. However, the study of surface proteins has proven difficult mainly due to their hydrophobicity and/or relatively low abundance compared with cytoplasmic proteins. To overcome these issues new proteomic strategies have been developed, such as cell-surface protein labelling using biotinylation reagents. Sulfo-NHS-SS-biotin is the most commonly used reagent to investigate the proteins expressed at the cell surface of various organisms but its use in lipopolysaccharidic diderm bacteria (archetypical Gram-negative bacteria) remains limited to a handful of species. While generally pass over in silence, some periplasmic proteins, but also some inner membrane lipoproteins, integral membrane proteins and cytoplasmic proteins (cytoproteins) are systematically identified following this approach. To limit cell lysis and diffusion of the sulfo-NHS-SS-biotin through the outer membrane, biotin labelling was tested over short incubation times and proved to be as efficient for 1 min at room temperature. To further limit labelling of protein located below the outer membrane, the use of high-molecular weight sulfo-NHS-PEG4-bismannose-SS-biotin appeared to recover differentially cell-envelope proteins compared to low-molecular weight sulfo-NHS-SS-biotin. Actually, the sulfo-NHS-SS-biotin recovers at a higher extent the proteins completely or partly exposed in the periplasm than sulfo-NHS-PEG4-bismannose-SS-biotin, namely periplasmic and integral membrane proteins as well as inner membrane and outer membrane lipoproteins. These results highlight that protein labelling using biotinylation reagents of different sizes provides a sophisticated and accurate way to differentially explore the cell envelope proteome of lipopolysaccharidic diderm bacteria. While generally pass over in silence, some periplasmic proteins

  20. Synthesis and evaluation of 99mTc/99Tc-MAG3-biotin conjugates for antibody pretargeting strategies

    International Nuclear Information System (INIS)

    Gog, Frank B. van; Visser, Gerard W.M.; Gowrising, Radjish W.A.; Snow, Gordon B.; Dongen, Guus A.M.S. van

    1998-01-01

    Four 99m Tc-MAG3-biotin conjugates were synthesized to determine their potential use in antibody pretargeting strategies for radioimmunoscintigraphy (RIS). To use these 99m Tc-MAG3-biotin conjugates as model compounds for 186 Re-MAG3-biotin conjugates for radioimmunotherapy (RIT), nanomolar amounts of 99 Tc were added as carrier to 99m Tc. The biotin derivatives used for the preparation of the conjugates - biocytin, biotin hydrazide, biotinyl-piperazine, and biotinyl-diaminosuccinic acid - differed at the site that is regarded to be susceptible to hydrolysis by biotinidase present in human plasma. All four conjugates were produced with high radiochemical purity, were stable in PBS, and demonstrated full binding capacity to streptavidin. The 99m Tc/ 99 Tc-MAG3-labeled biotinyl-piperazine and biotinyl-diaminosuccinic acid conjugates were stable in mouse as well as human plasma, whereas the corresponding biocytin and biotin hydrazide conjugates were rapidly degraded. The biodistribution in nude mice at 30 min after injection was similar for all conjugates, and a rapid blood clearance and high intestinal excretion were both observed. It is concluded that the metabolic routing of a conjugate containing biotin and MAG3 is dominated by these two moieties. For this reason, MAG3-biotin conjugates do not seem suited for pretargeted RIT, for which quantitative and fast renal excretion is a prerequisite to minimize radiation toxicity. However, in a pretargeted RIS approach the 99m Tc-MAG3-biotin conjugates might have potential

  1. Intercellular production of tamavidin 1, a biotin-binding protein from Tamogitake mushroom, confers resistance to the blast fungus Magnaporthe oryzae in transgenic rice.

    Science.gov (United States)

    Takakura, Yoshimitsu; Oka, Naomi; Suzuki, Junko; Tsukamoto, Hiroshi; Ishida, Yuji

    2012-05-01

    The blast fungus Magnaporthe oryzae, one of the most devastating rice pathogens in the world, shows biotin-dependent growth. We have developed a strategy for creating disease resistance to M. oryzae whereby intercellular production of tamavidin 1, a biotin-binding protein from Pleurotus cornucopiae occurs in transgenic rice plants. The gene that encodes tamavidin 1, fused to the sequence for a secretion signal peptide derived from rice chitinase gene, was connected to the Cauliflower mosaic virus 35S promoter, and the resultant construct was introduced into rice. The tamavidin 1 was accumulated at levels of 0.1-0.2% of total soluble leaf proteins in the transgenic rice and it was localized in the intercellular space of rice leaves. The tamavidin 1 purified from the transgenic rice was active, it bound to biotin and inhibited in vitro growth of M. oryzae by causing biotin deficiency. The transgenic rice plants showed a significant resistance to M. oryzae. This study shows the possibility of a new strategy to engineer disease resistance in higher plants by taking advantage of a pathogen's auxotrophy.

  2. Amplified voltammetric detection of glycoproteins using 4-mercaptophenylboronic acid/biotin-modified multifunctional gold nanoparticles as labels.

    Science.gov (United States)

    Liu, Lin; Xing, Yun; Zhang, Hui; Liu, Ruili; Liu, Huijing; Xia, Ning

    2014-01-01

    Ultrasensitive detection of protein biomarkers is essential for early diagnosis and therapy of many diseases. Glycoproteins, differing from other types of proteins, contain carbohydrate moieties in the oligosaccharide chains. Boronic acid can form boronate ester covalent bonds with diol-containing species. Herein, we present a sensitive and cost-effective electrochemical method for glycoprotein detection using 4-mercaptophenylboronic acid (MBA)/biotin-modified gold nanoparticles (AuNPs) (MBA-biotin-AuNPs) as labels. To demonstrate the feasibility and sensitivity of this method, recombinant human erythropoietin (rHuEPO) was tested as a model analyte. Specifically, rHuEPO was captured by the anti-rHuEPO aptamer-covered electrode and then derivatized with MBA-biotin-AuNPs through the boronic acid-carbohydrate interaction. The MBA-biotin-AuNPs facilitated the attachment of streptavidin-conjugated alkaline phosphatase for the production of electroactive p-aminophenol from p-aminophenyl phosphate substrate. A detection limit of 8 fmol L(-1) for rHuEPO detection was achieved. Other glycosylated and non-glycosylated proteins, such as horseradish peroxidase, prostate specific antigen, metallothionein, streptavidin, and thrombin showed no interference in the detection assay.

  3. The photostability of the commonly used biotin-4-fluorescein probe.

    Science.gov (United States)

    Haack, Richard A; Swift, Kerry M; Ruan, Qiaoqiao; Himmelsbach, Richard J; Tetin, Sergey Y

    2017-08-15

    Biotin-4-fluorescein (B4F) is a commonly used fluorescent probe for studying biotin-(strept)avidin interactions. During a characterization study of an anti-biotin antibody, using B4F as the probe, we noticed a discrepancy in the expected and experimentally determined number of biotin binding sites. Analytical testing showed that the biotin moiety in the probe undergoes a photosensitized oxidation to produce a mixture of biotin sulfoxides which has the potential to impact the quantitation of binding sites using this fluorescent probe. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Novel multi-biotin grafted poly(lactic acid) and its self-assembling nanoparticles capable of binding to streptavidin

    Science.gov (United States)

    Yan, Hao; Jiang, Weimin; Zhang, Yinxing; Liu, Ying; Wang, Bin; Yang, Li; Deng, Lihong; Singh, Gurinder K; Pan, Jun

    2012-01-01

    Targeted drug delivery requires novel biodegradable, specific binding systems with longer circulation time. The aim of this study was to prepare biotinylated poly(lactic acid) (PLA) nanoparticles (NPs) which can meet regular requirements as well conjugate more biotins in the polymer to provide better binding with streptavidin. A biotin-graft-PLA was synthesized based on previously published biodegradable poly(ethylene glycol) (PEG)-graft-PLA, with one polymer molecule containing three PEG molecules. Newly synthesized biotin-graft-PLA had three biotins per polymer molecule, higher than the previous biotinylated PLA (≤1 biotin per polymer molecule). A PEG with a much lower molecular weight (MW ~1900) than the previous biotinylated PLA (PEG MW ≥ 3800), and thus more biocompatible, was used which supplied good nonspecific protein-resistant property compatible to PEG-graft-PLA, suggesting its possible longer stay in the bloodstream. Biotin-graft-PLA specifically bound to streptavidin and self-assembled into NPs, during which naproxen, a model small molecule (MW 230 Da) and hydrophobic drug, was encapsulated (encapsulation efficiency 51.88%). The naproxen-loaded NPs with particle size and zeta potential of 175 nm and −27.35 mV realized controlled release within 170 hours, comparable to previous studies. The biotin-graft-PLA NPs adhered approximately two-fold more on streptavidin film and on biotin film via a streptavidin arm both in static and dynamic conditions compared with PEG-graft-PLA NPs, the proven nonspecific protein-resistant NPs. The specific binding of biotin-graft-PLA NPs with streptavidin and with biotin using streptavidin arm, as well as its entrapment and controlled release for naproxen, suggest potential applications in targeted drug delivery. PMID:22334778

  5. Radioimmunocytochemistry with [3H]biotin

    International Nuclear Information System (INIS)

    Hunt, S.P.; Mantyh, P.W.

    1984-01-01

    The authors exploit the high affinity of biotin for avidin in the design of radioimmunocytochemical methods using [ 3 H]biotin. [ 3 H]Biotin and avidin D form a radioactive complex which can be linked onto a primary antibody by means of a biotinylated anti-rabbit IgG or biotinylated protein A link. With both approaches it was possible to localize a number of antigens such as somatostatin, substance P, avian pancreatic polypeptide, tyrosine hydroxylase, and enkephalin-like immunoreactivity in various regions of the rat and human brain. By using tritium-sensitive film, large regions of the brain could be studied and analyzed semiquantitatively using computerized microdensitometry. The technique was also taken to the electron microscope level, and in the case of substance P immunoreactivity within the rat substantia nigra silver grains were found to be highly localized over axons and axon terminals. It was also possible to demonstrate co-existence or lack of co-existence of a number of different antigens within neurones. The first primary antibody was localized with biotinylated protein A followed by avidin-peroxidase, while the second primary antibody was linked to the [ 3 H]biotin again with biotinylated protein A. As an example of the potential of these methods for semiquantification, the distribution of substance P within postmortem human spinal cord was examined 24 months after amputation. A 49% loss of peptide was found in the corresponding dorsal horn. In summary these methods using [ 3 H]biotin have proved successful in quantification, electron microscopy and double labelling studies. (Auth.)

  6. Neuroprotective potential of high-dose biotin.

    Science.gov (United States)

    McCarty, Mark F; DiNicolantonio, James J

    2017-11-01

    A recent controlled trial has established that high-dose biotin supplementation - 100 mg, three times daily - has a stabilizing effect on progression of multiple sclerosis (MS). Although this effect has been attributed to an optimization of biotin's essential cofactor role in the brain, a case can be made that direct stimulation of soluble guanylate cyclase (sGC) by pharmacological concentrations of biotin plays a key role in this regard. The utility of high-dose biotin in MS might reflect an anti-inflammatory effect of cGMP on the cerebral microvasculature, as well on oligodendrocyte differentiation and on Schwann cell production of neurotrophic factors thought to have potential for managing MS. But biotin's ability to boost cGMP synthesis in the brain may have broader neuroprotective potential. In many types of neurons and neural cells, cGMP exerts neurotrophic-mimetic effects - entailing activation of the PI3K-Akt and Ras-ERK pathways - that promote neuron survival and plasticity. Hippocampal long term potentiation requires nitric oxide synthesis, which in turn promotes an activating phosphorylation of CREB via a pathway involving cGMP and protein kinase G (PKG). In Alzheimer's disease (AD), amyloid beta suppresses this mechanism by inhibiting sGC activity; agents which exert a countervailing effect by boosting cGMP levels tend to restore effective long-term potentiation in rodent models of AD. Moreover, NO/cGMP suppresses amyloid beta production within the brain by inhibiting expression of amyloid precursor protein and BACE1. In conjunction with cGMP's ability to oppose neuron apoptosis, these effects suggest that high-dose biotin might have potential for the prevention and management of AD. cGMP also promotes neurogenesis, and may lessen stroke risk by impeding atherogenesis and hypertrophic remodeling in the cerebral vasculature. The neuroprotective potential of high-dose biotin likely could be boosted by concurrent administration of brain

  7. The role of CH/π interactions in the high affinity binding of streptavidin and biotin.

    Science.gov (United States)

    Ozawa, Motoyasu; Ozawa, Tomonaga; Nishio, Motohiro; Ueda, Kazuyoshi

    2017-08-01

    The streptavidin-biotin complex has an extraordinarily high affinity (Ka: 10 15 mol -1 ) and contains one of the strongest non-covalent interactions known. This strong interaction is widely used in biological tools, including for affinity tags, detection, and immobilization of proteins. Although hydrogen bond networks and hydrophobic interactions have been proposed to explain this high affinity, the reasons for it remain poorly understood. Inspired by the deceased affinity of biotin observed for point mutations of streptavidin at tryptophan residues, we hypothesized that a CH/π interaction may also contribute to the strong interaction between streptavidin and biotin. CH/π interactions were explored and analyzed at the biotin-binding site and at the interface of the subunits by the fragment molecular orbital method (FMO) and extended applications: PIEDA and FMO4. The results show that CH/π interactions are involved in the high affinity for biotin at the binding site of streptavidin. We further suggest that the involvement of CH/π interactions at the subunit interfaces and an extended CH/π network play more critical roles in determining the high affinity, rather than involvement at the binding site. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Selection of variants with high levels of biotin from cultured green Lavandula vera cells irradiated with gamma rays

    International Nuclear Information System (INIS)

    Watanabe, K.; Yamada, Y.

    1982-01-01

    Cultured green Lavandula vera cells were irradiated with various dosages of gamma rays which increased the variation in the amount of free biotin produced by the cell clones. Variant sublines containing much more free biotin than the original line were obtained by repeated selection. The effectiveness of gamma rays for the induction of the variant sublines is described

  9. Biotin deficiency enhances the inflammatory response of human dendritic cells.

    Science.gov (United States)

    Agrawal, Sudhanshu; Agrawal, Anshu; Said, Hamid M

    2016-09-01

    The water-soluble biotin (vitamin B7) is indispensable for normal human health. The vitamin acts as a cofactor for five carboxylases that are critical for fatty acid, glucose, and amino acid metabolism. Biotin deficiency is associated with various diseases, and mice deficient in this vitamin display enhanced inflammation. Previous studies have shown that biotin affects the functions of adaptive immune T and NK cells, but its effect(s) on innate immune cells is not known. Because of that and because vitamins such as vitamins A and D have a profound effect on dendritic cell (DC) function, we investigated the effect of biotin levels on the functions of human monocyte-derived DCs. Culture of DCs in a biotin-deficient medium (BDM) and subsequent activation with LPS resulted in enhanced secretion of the proinflammatory cytokines TNF-α, IL-12p40, IL-23, and IL-1β compared with LPS-activated DCs cultured in biotin-sufficient (control) and biotin-oversupplemented media. Furthermore, LPS-activated DCs cultured in BDM displayed a significantly higher induction of IFN-γ and IL-17 indicating Th1/Th17 bias in T cells compared with cells maintained in biotin control or biotin-oversupplemented media. Investigations into the mechanisms suggested that impaired activation of AMP kinase in DCs cultured in BDM may be responsible for the observed increase in inflammatory responses. In summary, these results demonstrate for the first time that biotin deficiency enhances the inflammatory responses of DCs. This may therefore be one of the mechanism(s) that mediates the observed inflammation that occurs in biotin deficiency.

  10. Plasma Levels of Biotin Metabolites Are Elevated in Hemodialysis Patients with Cramps.

    Science.gov (United States)

    Fujiwara, Masako; Ando, Itiro; Yagi, Shigeaki; Nishizawa, Manabu; Oguma, Shiro; Satoh, Keisuke; Sato, Hiroshi; Imai, Yutaka

    2016-08-01

    Patients with renal failure undergoing hemodialysis (HD) are susceptible to muscle cramps during and after HD. Muscle cramps are defined as the sudden onset of a prolonged involuntary muscle contraction accompanied by severe pain. Through HD, water-soluble vitamins are drawn out with water. Since biotin, a water-soluble vitamin, plays an essential role as one of the coenzymes in producing energy, we have hypothesized that deficiency of biotin may be responsible for HD-associated cramps. We previously reported that biotin administration ameliorated the muscle cramps, despite the elevated plasma biotin levels before HD and biotin administration, as judged by an enzyme-linked immunosorbent assay (ELISA). However, the ELISA measures not only biotin but also total avidin-binding substances (TABS) including biotin metabolites. In the present study, we determined biotin in HD patients as well as healthy controls, using a newly developed method with ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The plasma samples were collected from 28 HD patients (16 patients with cramps and 12 patients without cramps) before HD and biotin administration and from 11 controls. The results showed that the accumulation of biotin and TABS in plasma of HD patients compared to controls. Importantly, the levels of biotin metabolites, i.e. TABS subtracted by biotin, increased significantly in patients with cramps over those without cramps. Moreover, the levels of biotin metabolites were significantly higher in patients with a poor response to administered biotin, compared to those with a good response. We propose that accumulated biotin metabolites impair biotin's functions as a coenzyme.

  11. Conjugate of biotin with silicon(IV) phthalocyanine for tumor-targeting photodynamic therapy.

    Science.gov (United States)

    Li, Ke; Qiu, Ling; Liu, Qingzhu; Lv, Gaochao; Zhao, Xueyu; Wang, Shanshan; Lin, Jianguo

    2017-09-01

    In order to improve the efficacy of photodynamic therapy (PDT), biotin was axially conjugated with silicon(IV) phthalocyanine (SiPc) skeleton to develop a new tumor-targeting photosensitizer SiPc-biotin. The target compound SiPc-biotin showed much higher binding affinity toward BR-positive (biotin receptor overexpressed) HeLa human cervical carcinoma cells than its precursor SiPc-pip. However, when the biotin receptors of HeLa cells were blocked by free biotin, >50% uptake of SiPc-biotin was suppressed, demonstrating that SiPc-biotin could selectively accumulate in BR-positive cancer cells via the BR-mediated internalization. The confocal fluorescence images further confirmed the target binding ability of SiPc-biotin. As a consequence of specificity of SiPc-biotin toward BR-positive HeLa cells, the photodynamic effect was also largely dependent on the BR expression level of HeLa cells. The photodynamic activities of SiPc-biotin against HeLa cells were dramatically reduced when the biotin receptors were blocked by the free biotin (IC 50 : 0.18μM vs. 0.46μM). It is concluded that SiPc-biotin can selectively damage BR-positive cancer cells under irradiation. Furthermore, the dark toxicity of SiPc-biotin toward human normal liver cell lines LO2 was much lower than that of its precursor SiPc-pip. The targeting photodynamic activity and low dark toxicity suggest that SiPc-biotin is a promising photosensitizer for tumor-targeting photodynamic therapy. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Engineering of biotin-prototrophy in Pichia pastoris for robust production processes.

    Science.gov (United States)

    Gasser, Brigitte; Dragosits, Martin; Mattanovich, Diethard

    2010-11-01

    Biotin plays an essential role as cofactor for biotin-dependent carboxylases involved in essential metabolic pathways. The cultivation of Pichia pastoris, a methylotrophic yeast that is successfully used as host for the production of recombinant proteins, requires addition of high dosage of biotin. As biotin is the only non-salt media component used during P. pastoris fermentation (apart from the carbon source), nonconformities during protein production processes are usually attributed to poor quality of the added biotin. In order to avoid dismissed production runs due to biotin quality issues, we engineered the biotin-requiring yeast P. pastoris to become a biotin-prototrophic yeast. Integration of four genes involved in the biotin biosynthesis from brewing yeast into the P. pastoris genome rendered P. pastoris biotin-prototrophic. The engineered strain has successfully been used as production host for both intracellular and secreted heterologous proteins in fed-batch processes, employing mineral media without vitamins. Another field of application for these truly prototrophic hosts is the production of biochemicals and small metabolites, where defined mineral media leads to easier purification procedures. Copyright © 2010 Elsevier Inc. All rights reserved.

  13. Development of a tetrameric streptavidin mutein with reversible biotin binding capability: engineering a mobile loop as an exit door for biotin.

    Directory of Open Access Journals (Sweden)

    Valerie J O'Sullivan

    Full Text Available A novel form of tetrameric streptavidin has been engineered to have reversible biotin binding capability. In wild-type streptavidin, loop(3-4 functions as a lid for the entry and exit of biotin. When biotin is bound, interactions between biotin and key residues in loop(3-4 keep this lid in the closed state. In the engineered mutein, a second biotin exit door is created by changing the amino acid sequence of loop(7-8. This door is mobile even in the presence of the bound biotin and can facilitate the release of biotin from the mutein. Since loop(7-8 is involved in subunit interactions, alteration of this loop in the engineered mutein results in an 11° rotation between the two dimers in reference to wild-type streptavidin. The tetrameric state of the engineered mutein is stabilized by a H127C mutation, which leads to the formation of inter-subunit disulfide bonds. The biotin binding kinetic parameters (k(off of 4.28×10(-4 s(-1 and K(d of 1.9×10(-8 M make this engineered mutein a superb affinity agent for the purification of biotinylated biomolecules. Affinity matrices can be regenerated using gentle procedures, and regenerated matrices can be reused at least ten times without any observable reduction in binding capacity. With the combination of both the engineered mutein and wild-type streptavidin, biotinylated biomolecules can easily be affinity purified to high purity and immobilized to desirable platforms without any leakage concerns. Other potential biotechnological applications, such as development of an automated high-throughput protein purification system, are feasible.

  14. Development of a Tetrameric Streptavidin Mutein with Reversible Biotin Binding Capability: Engineering a Mobile Loop as an Exit Door for Biotin

    Science.gov (United States)

    O'Sullivan, Valerie J.; Barrette-Ng, Isabelle; Hommema, Eric; Hermanson, Greg T.; Schofield, Mark; Wu, Sau-Ching; Honetschlaeger, Claudia; Ng, Kenneth K.-S.; Wong, Sui-Lam

    2012-01-01

    A novel form of tetrameric streptavidin has been engineered to have reversible biotin binding capability. In wild-type streptavidin, loop3–4 functions as a lid for the entry and exit of biotin. When biotin is bound, interactions between biotin and key residues in loop3–4 keep this lid in the closed state. In the engineered mutein, a second biotin exit door is created by changing the amino acid sequence of loop7–8. This door is mobile even in the presence of the bound biotin and can facilitate the release of biotin from the mutein. Since loop7–8 is involved in subunit interactions, alteration of this loop in the engineered mutein results in an 11° rotation between the two dimers in reference to wild-type streptavidin. The tetrameric state of the engineered mutein is stabilized by a H127C mutation, which leads to the formation of inter-subunit disulfide bonds. The biotin binding kinetic parameters (koff of 4.28×10−4 s−1 and Kd of 1.9×10−8 M) make this engineered mutein a superb affinity agent for the purification of biotinylated biomolecules. Affinity matrices can be regenerated using gentle procedures, and regenerated matrices can be reused at least ten times without any observable reduction in binding capacity. With the combination of both the engineered mutein and wild-type streptavidin, biotinylated biomolecules can easily be affinity purified to high purity and immobilized to desirable platforms without any leakage concerns. Other potential biotechnological applications, such as development of an automated high-throughput protein purification system, are feasible. PMID:22536357

  15. Pharmacokinetics and biodistribution of radiolabeled avidin, streptavidin and biotin

    International Nuclear Information System (INIS)

    Rosebrough, S.F.

    1993-01-01

    The extraordinarily high affinity of avidin and streptavidin for biotin may be exploited in a two-step approach for delivering radiolabeled biotin derivatives suitable for imaging and therapy to target-bound streptavidin or avidin conjugated monoclonal antibodies (MAbs). The in vivo pharmacokinetics and biodistribution of radiolabeled avidin, streptavidin (SA) and DTPA-biocytinamide (DTPA-biotin) were studied in the rabbit and dog. SA circulated in the blood similar to other 60 kDa proteins, avidin cleared immediately and DTPA-biotin exhibited plasma clearance by glomerular filtration. (author)

  16. The Binding of Biotin to Sepharose-Avidin Column: Demonstration of the Affinity Chromatography Technique

    Science.gov (United States)

    Landman, A. D.; Landman, N. N.

    1976-01-01

    Describes a biochemistry experiment that illustrates the methodology of affinity chromatography by attaching avidin, a glycoprotein in egg white, to a Sepharose matrix in order to bind biotin-containing proteins. (MLH)

  17. Changes in biotin levels during production of natto, Japanese fermented soybean

    Directory of Open Access Journals (Sweden)

    Makoto Muratsugu

    2017-09-01

    Full Text Available The change of biotin level during production of natto (Japanese fermented soybean was investigated in this study.  The total biotin level was measured by an agar plate bioassay using Lactobacillus plantarum ATCC 8014.  The total biotin level decreased during water soaking, but increased after the fermentation of soybeans using Bacillus subtilis var. natto (B. natto and reached a maximum level.  The increase of total biotin was not affected by Asp, Arg, and Ile which promoted the growth of L. plantarum in high concentrations.  The peak level of biotin in the fermented soybeans was significantly higher than that of dry soybeans.  The fermented soybeans at the biotin peak level were adequate for food.  In addition, we detected 9 and 4 biotinylated polypeptides in the soybeans and B. natto used in this study, respectively.  We speculated that the increase of biotin level may depend on the increase of the 4 biotinylated polypeptides and free biotin in B. natto.

  18. A conserved regulatory mechanism in bifunctional biotin protein ligases.

    Science.gov (United States)

    Wang, Jingheng; Beckett, Dorothy

    2017-08-01

    Class II bifunctional biotin protein ligases (BirA), which catalyze post-translational biotinylation and repress transcription initiation, are broadly distributed in eubacteria and archaea. However, it is unclear if these proteins all share the same molecular mechanism of transcription regulation. In Escherichia coli the corepressor biotinoyl-5'-AMP (bio-5'-AMP), which is also the intermediate in biotin transfer, promotes operator binding and resulting transcription repression by enhancing BirA dimerization. Like E. coli BirA (EcBirA), Staphylococcus aureus, and Bacillus subtilis BirA (Sa and BsBirA) repress transcription in vivo in a biotin-dependent manner. In this work, sedimentation equilibrium measurements were performed to investigate the molecular basis of this biotin-responsive transcription regulation. The results reveal that, as observed for EcBirA, Sa, and BsBirA dimerization reactions are significantly enhanced by bio-5'-AMP binding. Thus, the molecular mechanism of the Biotin Regulatory System is conserved in the biotin repressors from these three organisms. © 2017 The Protein Society.

  19. Biotin-deficient diet induces chromosome misalignment and spindle defects in mouse oocytes.

    Science.gov (United States)

    Tsuji, Ai; Nakamura, Toshinobu; Shibata, Katsumi

    2015-01-01

    Increased abnormal oocytes due to meiotic chromosome misalignment and spindle defects lead to elevated rates of infertility, miscarriage, and trisomic conceptions. Here, we investigated the effect of biotin deficiency on oocyte quality. Three-week-old female ICR mice were fed a biotin-deficient or control diet (0, 0.004 g biotin/kg diet) for 21 days. On day 22, these mouse oocytes were analyzed by immunofluorescence. Due to biotin, undernutrition increased the frequency of abnormal oocytes (the biotin deficient vs. control: 40 vs. 16%). Next, the remaining mice in the biotin-deficient group were fed a control or biotin-deficient diet from day 22 to 42. Although biotin nutritional status in the recovery group was restored, the frequency of abnormal oocytes in the recovery group was still higher than that in the control group (48 vs. 18%). Our results indicate that steady, sufficient biotin intake is required for the production of high-quality oocytes in mice.

  20. Avidin/PSS membrane microcapsules with biotin-binding activity.

    Science.gov (United States)

    Endo, Yoshihiro; Sato, Katsuhiko; Sugimoto, Kentaro; Anzai, Jun-ichi

    2011-08-15

    Polyelectrolyte microcapsules with avidin-poly(styrene sulfonate) (PSS) membrane were prepared by a layer-by-layer deposition technique. The uptake and release of biotin-labeled fluorescein (b-FITC) as well as immobilization of biotin-labeled glucose oxidase (b-GOx) to the microcapsule were studied. The polyelectrolyte microcapsules were prepared by coating the surface of calcium carbonate (CaCO(3)) microparticles with an avidin/PSS multilayer membrane, followed by dissolution of CaCO(3) core in an ethylenediaminetetraacetic acid solution. Inner and outer poly(allylamine)/PSS films were required to isolate the microcapsules, whereas microcapsules could not be formed without the support. The uptake of b-FITC into the microcapsule was highly enhanced through a strong binding of b-FITC to avidin as compared with the uptake of biotin-free FITC. Release of b-FITC from the microcapsule was accelerated upon addition of biotin due to a competitive binding of the added biotin to the binding site of avidin. Similarly, the surface of microcapsule was modified with b-GOx with retaining its catalytic activity. Copyright © 2011 Elsevier Inc. All rights reserved.

  1. Treatment of biotin-responsive basal ganglia disease: Open comparative study between the combination of biotin plus thiamine versus thiamine alone.

    Science.gov (United States)

    Tabarki, Brahim; Alfadhel, Majid; AlShahwan, Saad; Hundallah, Khaled; AlShafi, Shatha; AlHashem, Amel

    2015-09-01

    To compare the combination of biotin plus thiamine to thiamine alone in treating patients with biotin-responsive basal ganglia disease in an open-label prospective, comparative study. twenty patients with genetically proven biotin-responsive basal ganglia disease were enrolled, and received for at least 30 months a combination of biotin plus thiamine or thiamine alone. The outcome measures included duration of the crisis, number of recurrence/admissions, the last neurological examination, the severity of dystonia using the Burke-Fahn-Marsden Dystonia Rating Scale (BFMDRS), and the brain MRI findings during the crisis and after 30 months of follow-up. Ten children with a mean age of 6 years(1/2) were recruited in the biotin plus thiamine group (group 1) and ten children (6 females and 4 males) with a mean age of 6 years and 2 months were recruited in the thiamine group (group 2). After 2 years of follow-up treatment, 6 of 20 children achieved complete remission, 10 had minimal sequelae in the form of mild dystonia and dysarthria (improvement of the BFMDRS, mean: 80%), and 4 had severe neurologic sequelae. All these 4 patients had delayed diagnosis and management. Regarding outcome measures, both groups have a similar outcome regarding the number of recurrences, the neurologic sequelae (mean BFMDS score between the groups, p = 0.84), and the brain MRI findings. The only difference was the duration of the acute crisis: group 1 had faster recovery (2 days), versus 3 days in group 2 (p = 0.005). Our study suggests that over 30 months of treatment, the combination of biotin plus thiamine is not superior to thiamine alone in the treatment of biotin-responsive basal ganglia disease. Copyright © 2015 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.

  2. Biotin-independent strains of Escherichia coli for enhanced streptavidin production.

    Science.gov (United States)

    Jeschek, Markus; Bahls, Maximilian O; Schneider, Veronika; Marlière, Philippe; Ward, Thomas R; Panke, Sven

    2017-03-01

    Biotin is an archetypal vitamin used as cofactor for carboxylation reactions found in all forms of life. However, biotin biosynthesis is an elaborate multi-enzymatic process and metabolically costly. Moreover, many industrially relevant organisms are incapable of biotin synthesis resulting in the requirement to supplement defined media. Here we describe the creation of biotin-independent strains of Escherichia coli and Corynebacterium glutamicum through installation of an optimized malonyl-CoA bypass, which re-routes natural fatty acid synthesis, rendering the previously essential vitamin completely obsolete. We utilize biotin-independent E. coli for the production of the high-value protein streptavidin which was hitherto restricted because of toxic effects due to biotin depletion. The engineered strain revealed significantly improved streptavidin production resulting in the highest titers and productivities reported for this protein to date. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  3. The control of fruiting body formation in the ascomycete Sordaria macrospora Auersw. by arginine and biotin: a two-factor analysis.

    Science.gov (United States)

    Molowitz, R; Bahn, M; Hock, B

    1976-01-01

    Fruiting body formation of Sordaria macrospora Auersw. is controlled by L-arginine and biotin when the fungus is grown on a synthetic nutrient medium containing optimal concentrations of fructose, KNO3, KH2PO4, MgSO4, and ZnSO4. Arginine and biotin operate in very low concentrations which exclude unspecific nutrient effects. In spite of the complicated interactions of arginine and biotin which are shown qualitatively (Figs. 3 and 4a) and quantitatively (Figs. 2 and 4b), the following conclusions are reached: 1. In the absence of biotin, the development of Sordaria macrospora is blocked at the stage of small protoperithecia. The external addition of biotin (optimal concentration: 3-12 μg/l) allows the formation of fertile fruiting bodies. This effect cannot be imitated by arginine. The biotin effect is discussed in connection with stimulated RNA synthesis.-2. The developmental velocity is influenced by the external addition of arginine. Without arginine but at permissible biotin concentrations, the total life cycle takes about 10 days, in the presence of arginine (1 mM), however, about 6 days.-3. The hyphal density, as well as the total number of fruiting bodies being produced, is controlled in a similar manner by biotin and arginine. The induction of fruiting body formation obviously takes place after the transgression of a critical hyphal density.

  4. Biotin-specific synthetic receptors prepared using molecular imprinting

    Energy Technology Data Exchange (ETDEWEB)

    Piletska, Elena; Piletsky, Sergey; Karim, Kal; Terpetschnig, Ewald; Turner, Anthony

    2004-02-16

    The composition of new molecularly imprinted polymers (MIPs) specific for biotin was optimised using molecular modelling software. Three functional monomers: methacrylic acid (MAA), 2-(trifluoromethyl)acrylic acid (TFAA) and 2-acrylamido-2-methylpropanesulfonic acid (AMPSA), which demonstrated the highest binding scores with biotin, were tested on their ability to generate specific binding sites. The imprinted polymers were photografted to the surface of polystyrene microspheres in water. The affinity of the synthetic 'receptor' sites was evaluated in binding experiments using horseradish peroxidase-labelled biotin. Good correlation was found between the modelling results and the performance of the materials in the template re-binding study. The dissociation constants for all MIPs were 1.4-16.8 nM, which is sufficient for most analytical applications where biotin is used as a label.

  5. Biotin-specific synthetic receptors prepared using molecular imprinting

    International Nuclear Information System (INIS)

    Piletska, Elena; Piletsky, Sergey; Karim, Kal; Terpetschnig, Ewald; Turner, Anthony

    2004-01-01

    The composition of new molecularly imprinted polymers (MIPs) specific for biotin was optimised using molecular modelling software. Three functional monomers: methacrylic acid (MAA), 2-(trifluoromethyl)acrylic acid (TFAA) and 2-acrylamido-2-methylpropanesulfonic acid (AMPSA), which demonstrated the highest binding scores with biotin, were tested on their ability to generate specific binding sites. The imprinted polymers were photografted to the surface of polystyrene microspheres in water. The affinity of the synthetic 'receptor' sites was evaluated in binding experiments using horseradish peroxidase-labelled biotin. Good correlation was found between the modelling results and the performance of the materials in the template re-binding study. The dissociation constants for all MIPs were 1.4-16.8 nM, which is sufficient for most analytical applications where biotin is used as a label

  6. 18F-PEG-biotin: Precursor (boroaryl-PEG-biotin) synthesis, 18F-labelling and an in-vitro assessment of its binding with NeutravidinTM-trastuzumab pre-treated cells

    International Nuclear Information System (INIS)

    Smith, Tim A.D.; Simpson, Michael; Cheyne, Richard; Trembleau, Laurent

    2011-01-01

    In terms of nuclear decay 18 F is the most ideal PET nuclide but its short t 1/2 precludes its use for directly labelling whole antibodies due to their long blood residence times. Pre-targeted imaging using affinity systems such as Neutravidin TM -biotin facilitates the application of short-lived nuclides by their attachment to biotin for imaging cell surface proteins targeted with Neutravidin TM -conjugated antibodies. Methods: Boroaryl functionalised biotin was prepared with a PEG linker and radiolabelled by incubation with 18 F in acidified aqueous solution. Cells expressing high (SKBr3), medium (MDA-MB-453) and low (MDA-MB-468) levels of HER-2 were pre-incubated with Neutravidin TM -conjugated trastuzumab, washed, and then incubated with 18 F-PEG-biotin. Results: The 18 F-fluorination of boroaryl-PEG-biotin was much more efficient than reported for other versions of boroaryl-biotin. The novel 18 F-PEG-biotin was demonstrated to bind to HER-2-expressing cells in-vitro pre-incubated with Neutravidin TM -conjugated trastuzumab. Conclusion: Biotin can be functionalised with boroaryl and readily 18 F-radiolabelled in aqueous solution and will bind to cells pre-incubated with Neutravidin TM -antibody conjugates. - Highlights: → Boroaryl-biotin precursor is prepared. → Rapid 18 F-fluorination is demonstrated. → HER-2 expressing breast cancer cells pre-treated with trastuzumab-Neutravidin TM . → 18 F-PEG-biotin binding to pre-treated cells corresponds with HER-2 expression.

  7. Biotin conjugated organic molecules and proteins for cancer therapy: A review.

    Science.gov (United States)

    Maiti, Santanu; Paira, Priyankar

    2018-02-10

    The main transporter for biotin is sodium dependent multivitamin transporter (SMVT), which is overexpressed in various aggressive cancer cell lines such as ovarian (OV 2008, ID8), leukemia (L1210FR), mastocytoma (P815), colon (Colo-26), breast (4T1, JC, MMT06056), renal (RENCA, RD0995), and lung (M109) cancer cell lines. Furthermore, its overexpression was found higher to that of folate receptor. Therefore, biotin demand in the rapidly growing tumors is higher than normal tissues. Several biotin conjugated organic molecules has been reported here for selective delivery of the drug in cancer cell. Biotin conjugated molecules are showing higher fold of cytotoxicity in biotin positive cancer cell lines than the normal cell. Nanoparticles and polymer surface modified drugs and biotin mediated cancer theranostic strategy was highlighted in this review. The cytotoxicity and selectivity of the drug in cancer cells has enhanced after biotin conjugation. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  8. Transcarboxylase (TC): demonstration by site-directed mutagenesis that methionines at the biotin site are essential for catalysis

    International Nuclear Information System (INIS)

    Wood, H.G.; Shenoy, B.C.; Kumar, G.K.; Paranjape, S.; Murtif, V.; Samols, D.

    1987-01-01

    All biotin enzymes that have thus far been sequenced contain a conserved region ALA MET BCT MET. Two possible roles of the conserved region are (i) for recognition of the specific lysine of the enzyme that is to be biotinated posttranslationally by the synthetase or (ii) for activation of the biotin to function as a carboxyl carrier. The BCT of TC is at residue 89 of the 1.3S subunit. By site-directed mutagenesis, single amino acid substitutions have been made giving LEU 88, THR 88 and LEU 90 and these mutant subunits have been expressed in E. coli and isolated. Catalysis by TC involves Partial Reactions: (1) - 00 14 CCH 2 COCOO - + 1.3S biotin pyruvate + 1.3S biotin-COO - catalyzed by the 5S subunit (2) 14 CH 3 CH( 14 COO - )COSCoA + 1.3S biotin CH 3 CH 2 COSCoA + 1.3S biotin- 14 COO - , catalyzed by the 12S subunit. The mutant subunits LEU 88 and THR 88 are inactive in Reaction 1. In Reaction 2, they are 8% as active as the 1.3S wild type. At 10 times the concentration of the wild type, they are 40% as active. The LEU 90 subunit is about 40% as active as wild type in both Reactions 1 and 2. Thus, the two METS are functionally not equivalent. What their catalytic roles are remains to be determined. Shenoy et al. have shown these modifications do not effect the synthetase reaction

  9. Preparation of 177Lu-DTPA-BIS-BIOTIN and biodistribution evaluation in normal mice

    International Nuclear Information System (INIS)

    Deng Xinrong; Luo Zhifu; Du Jin

    2010-01-01

    The labeling method for 177 Lu-DTPA-BIS-BIOTIN was established, and the biodistribution of 177 Lu-DTPA-BIS-BIOTIN in normal mice was carried out as well. Under the optimal experimental condition (DTPA-BIS-BIOTIN 25 μg, pH=4.5 reacting at 80 degree C for 20 min), the labeling yield of 177 Lu-DTPA-BIS-BIOTIN is more than 99.0%. 177 Lu-DTPA-BIS-BIOTIN shows pretty good in vitro stability. The biodistribution of 177 Lu-DTPA-BIS-BIOTIN in normal mice shows a rapid blood clearance. The uptake of 177 Lu-DTPA-BIS-BIOTIN is mainly accumulated in liver, spleen and kidney. 177 Lu-DTPA-BIS-BIOTIN is excreted by kidney. The results provide the basis for further study on 177 Lu-DTPA-BIS-BIOTIN used in pretargeted radioimage and radiotherapy of cancer. (authors)

  10. Protein labelling with avidin-biotin systems

    International Nuclear Information System (INIS)

    Hernandez B, B.E.

    1998-01-01

    The stability of connection in avidin-biotin system is very important due to the quadruple connections with avidin established with the same number of biotin molecules, which can amplify damage on cancer cells and increase specific activity of radio immuno conjugate in white cell. If between the first and second step (Ac Mo-biotin + avidin) enough time is left so that the monoclonal antibody accumulates in a therapeutic concentration required for the tumor or cancerous cells, then upon application of the third step (biotin-DTPA- 153 Sm) it is hoped that in the first 30 minutes after application, only radioactivity remains with tumor. However, so that the amount radioactivity is enough to destroy a tumor, it would be necessary to use 153 Sm with an activity of approximately 370 GBq (10 Ci)/ (mg). Since 99m Tc has similar chemistry to that of the 188 Re, it is possible to propose their conjugates with biotin-avidin-Ac Mo- 188 Re as a powerful option for therapeutic applications, this is, recommending the use of biotinylated labelled monoclonal antibody and the further injection of avidin to decrease of desirable effects on several other organs and bone marrow and high specific and selective action on tumor. On the other hand, we postulate the hypothesis in the sense that 188 Re complexes tend to be more stable than those of 99m Tc, probably due to their metabolism, in which radioactivity of 188 Re, not captured by tumor, is cleared easily from blood stream which results in a decrease of total and liver total dose in patient. (Author)

  11. Engineering biotin prototrophic Corynebacterium glutamicum strains for amino acid, diamine and carotenoid production.

    Science.gov (United States)

    Peters-Wendisch, P; Götker, S; Heider, S A E; Komati Reddy, G; Nguyen, A Q; Stansen, K C; Wendisch, V F

    2014-12-20

    The Gram-positive Corynebacterium glutamicum is auxotrophic for biotin. Besides the biotin uptake system BioYMN and the transcriptional regulator BioQ, this bacterium possesses functional enzymes for the last three reactions of biotin synthesis starting from pimeloyl-CoA. Heterologous expression of bioF from the Gram-negative Escherichia coli enabled biotin synthesis from pimelic acid added to the medium, but expression of bioF together with bioC and bioH from E. coli did not entail biotin prototrophy. Heterologous expression of bioWAFDBI from Bacillus subtilis encoding another biotin synthesis pathway in C. glutamicum allowed for growth in biotin-depleted media. Stable growth of the recombinant was observed without biotin addition for eight transfers to biotin-depleted medium while the empty vector control stopped growth after the first transfer. Expression of bioWAFDBI from B. subtilis in C. glutamicum strains overproducing the amino acids l-lysine and l-arginine, the diamine putrescine, and the carotenoid lycopene, respectively, enabled formation of these products under biotin-depleted conditions. Thus, biotin-prototrophic growth and production by recombinant C. glutamicum were achieved. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Reactivity comparison of biological material after radiolabeling with avidin-biotin system

    International Nuclear Information System (INIS)

    Fan Wo; Qian Jianhua; Zhu Benxing

    2003-01-01

    To find a method for determining the immunoreactivity of monoclonal antibodies after radiolabeling avidin is unlabeled and labeled with Rodamine, 131 I and 188 Re, respectively. The affinities and half-desorbed amounts of biotin and four kinds of avidin are determined by the biotin columns plus non-labeled avidin (cold avidin). The affinities of biotin and avidin unlabeled and labeled with Rodamine, 188 Re and 131 I are decreased in turn. Their half-desorbed amounts from biotin are 21.9, 19.5, 25.7 and 47.9 μg of cold avidin. Two kinds of radiolabeled avidin have lower affinity with biotin than that of avidin unlabeled and labeled with Rodamine. There is a possibility to evaluate the reactivity of biological materials with different labeling methods by avidin-biotin system

  13. A radiochemical assay for biotin in biological materials

    International Nuclear Information System (INIS)

    Hood, R.L.

    1975-01-01

    A radiochemical assay for biotin is described. The assay was sensitive to one nanogram and simple enough for routine biotin analyses. The assay yielded results which were comparable to those obtained from a microbiological assay using Lactobacillus plantarum. (author)

  14. Dietary Biotin Supplementation Modifies Hepatic Morphology without Changes in Liver Toxicity Markers

    Directory of Open Access Journals (Sweden)

    Leticia Riverón-Negrete

    2016-01-01

    Full Text Available Pharmacological concentrations of biotin have pleiotropic effects. Several reports have documented that biotin supplementation decreases hyperglycemia. We have shown that a biotin-supplemented diet increased insulin secretion and the mRNA abundance of proteins regulating insulin transcription and secretion. We also found enlarged pancreatic islets and modified islet morphology. Other studies have shown that pharmacological concentrations of biotin modify tissue structure. Although biotin administration is considered safe, little attention has been given to its effect on tissue structure. In this study, we investigated the effect of biotin supplementation on hepatic morphology and liver toxicity markers. Male BALB/cAnN Hsd mice were fed a control or a biotin-supplemented diet for 8 weeks. Versus the control mice, biotin-supplemented mice had an altered portal triad with dilated sinusoids, increased vascularity, and bile conducts. Furthermore, we observed an increased proportion of nucleomegaly and binucleated hepatocytes. In spite of the liver morphological changes, no differences were observed in the serum liver damage indicators, oxidative stress markers, or antioxidant enzymes. Our data demonstrate for the first time that biotin supplementation affects liver morphology in normal mice, and that these modifications are not paralleled with damage markers.

  15. Dietary Biotin Supplementation Modifies Hepatic Morphology without Changes in Liver Toxicity Markers.

    Science.gov (United States)

    Riverón-Negrete, Leticia; Sicilia-Argumedo, Gloria; Álvarez-Delgado, Carolina; Coballase-Urrutia, Elvia; Alcántar-Fernández, Jonathan; Fernandez-Mejia, Cristina

    2016-01-01

    Pharmacological concentrations of biotin have pleiotropic effects. Several reports have documented that biotin supplementation decreases hyperglycemia. We have shown that a biotin-supplemented diet increased insulin secretion and the mRNA abundance of proteins regulating insulin transcription and secretion. We also found enlarged pancreatic islets and modified islet morphology. Other studies have shown that pharmacological concentrations of biotin modify tissue structure. Although biotin administration is considered safe, little attention has been given to its effect on tissue structure. In this study, we investigated the effect of biotin supplementation on hepatic morphology and liver toxicity markers. Male BALB/cAnN Hsd mice were fed a control or a biotin-supplemented diet for 8 weeks. Versus the control mice, biotin-supplemented mice had an altered portal triad with dilated sinusoids, increased vascularity, and bile conducts. Furthermore, we observed an increased proportion of nucleomegaly and binucleated hepatocytes. In spite of the liver morphological changes, no differences were observed in the serum liver damage indicators, oxidative stress markers, or antioxidant enzymes. Our data demonstrate for the first time that biotin supplementation affects liver morphology in normal mice, and that these modifications are not paralleled with damage markers.

  16. Labeling of biotin with Dy-166/Ho-166 as a stable in vivo generator system

    International Nuclear Information System (INIS)

    Ferro-Flores, G.; Monroy-Guzman, F.; Jimenez-Varela, R.; Tendilla, J.I.; Arteaga-Murphy, C.; Pedraza-Lopez, M.

    2002-01-01

    Aim: Biotin is a vitamin found in low concentration in blood and tissue. In radioimmunodiagnosis and radioimmunotherapy practice, the pre-targeting avidin-biotin strategy has shown that target-to-nontarget radioactivity ratios can be significantly improved. In addition, the biotin content of cancerous tumors is higher than that of normal tissue and it has been found in the cellular nucleus due to a specific transfer of biotin to histones by human serum biotinidase. Because of its nuclear properties, the 166 Dy/ 166 Ho radionuclide pair can be considered as an in vivo generator system. The aim of this work was to synthesize 166 Dy/ 166 Ho-DTPA-bisBiotin to evaluate its potential as a new radiopharmaceutical for targeted radiotherapy. Material and Methods: Dysprosium-166/Holmium-166 chloride was obtained by neutron irradiation of 50 mg of 99% enriched 164 Dy 2 O 3 in a TRIGA Mark III reactor at a flux in the central thimble of 3.10 13 n. cm -2 s -1 for 20 h. Following irradiation, the target was allowed to decay for 2 days, 100 μL of 12 N chloride acid were added and the mixture stirred for 3 min. To this suspension 1.0 mL of injectable water were added and heated for 2 min at 90 0 C. The biotin used in this investigation was diethylenetriaminepentaacetic-α,ω-bis(biocytinamide)(DTPA-bisBiotin, Sigma). Sterile and apyrogenic V-vial was prepared to contain 16.0 mg (1.52 x 10 -4 mmol) of the DTPA-bisBiotin in 4.0 mL of 0.05 M bicarbonate buffer (pH 8.5), then 50 μL of the radiochloride solution were added to the preparation. TLC aluminum cellulose sheets were used as the stationary phase and a ternary mixture of methanol: water: ammonium hydroxide (20:40:2) as the mobile phase. 166 Dy/ 166 Ho-DTPA-bisBiotin traveled with the solvent front R f 0.9-1.0 and the Dy +3 /Ho +3 species remained at the origin (R f 0.0). HPLC reverse phase was also used to evaluate radiochemical purity. The biological integrity of labeled biotin was achieved evaluating its avidity for avidin

  17. Biotin-dependent functions in adiposity: a study of monozygotic twin pairs.

    Science.gov (United States)

    Järvinen, E; Ismail, K; Muniandy, M; Bogl, L H; Heinonen, S; Tummers, M; Miettinen, S; Kaprio, J; Rissanen, A; Ollikainen, M; Pietiläinen, K H

    2016-05-01

    Biotin acts as a coenzyme for carboxylases regulating lipid and amino-acid metabolism. We investigated alterations of the biotin-dependent functions in obesity and the downstream effects of biotin restriction in adipocytes in vitro. Twenty-four monozygotic twin pairs discordant for body mass index (BMI). Mean within-pair difference (heavy-lean co-twin, Δ) of BMI was 6.0 kg m(-2) (range 3.1-15.2 kg m(-)(2)). Adipose tissue (AT) DNA methylation, gene expression of AT and adipocytes, and leukocytes (real-time quantitative PCR), serum biotin, C-reactive protein (CRP) and triglycerides were measured in the twins. Human adipocytes were cultured in low and control biotin concentrations and analyzed for lipid droplet content, mitochondrial morphology and mitochondrial respiration. The gene expression levels of carboxylases, PCCB and MCCC1, were upregulated in the heavier co-twins' leukocytes. ΔPCCB (r=0.91, P=0.0046) and ΔMCCC1 (r=0.79, P=0.036) correlated with ΔCRP within-pairs. Serum biotin levels were lower in the heavier (274 ng l(-1)) than in the lean co-twins (390 ng l(-1), P=0.034). ΔBiotin correlated negatively with Δtriglycerides (r=-0.56, P=0.045) within-pairs. In AT, HLCS and ACACB were hypermethylated and biotin cycle genes HLCS and BTD were downregulated (PBiotin-dependent carboxylases were downregulated (ACACA, ACACB, PCCB, MCCC2 and PC; Pbiotin had decreased lipid accumulation, altered mitochondrial morphology and deficient mitochondrial respiration. Biotin-dependent functions are modified by adiposity independent of genetic effects, and correlate with inflammation and hypertriglyceridemia. Biotin restriction decreases lipid accumulation and respiration, and alters mitochondrial morphology in adipocytes.

  18. Effects of biotin supplementation on peripartum performance and metabolites of Holstein cows.

    Science.gov (United States)

    Rosendo, O; Staples, C R; McDowell, L R; McMahon, R; Badinga, L; Martin, F G; Shearer, J F; Seymour, W M; Wilkinson, N S

    2004-08-01

    Fifty-two multiparous Holstein cows were randomly assigned to receive 0 or 20 mg of biotin/d starting at an average of 16 d prepartum and then switched to 0 or 30 mg of biotin/d from calving through 70 d postpartum to determine whether supplemental biotin would affect cow performance, hepatic lipidosis, and plasma metabolites. Mean concentration of biotin in plasma sampled weekly was greater in cows fed biotin (4.3 vs. 9.4 nmol/L). Postpartum dry matter intake as a percentage of body weight (3.9% vs. 4.0%), milk production (35.8 vs. 34.8 kg/d), and milk fat concentrations (3.59% vs. 3.69%) were similar between treatment groups. Milk from biotin-supplemented cows tended to have a greater concentration of protein (2.73% vs. 2.83%). Concentrations of plasma nonesterified fatty acids were lower at wk 2 (652 vs. 413 microEq/mL) and 4 (381 vs. 196 microEq/mL) postpartum in cows fed supplemental biotin. However, mean plasma concentrations of beta-hydroxybutyric acid were not affected by biotin supplementation. Mean concentration of plasma glucose was greater for lactating cows fed supplemental biotin (63.4 vs. 66.6 mg/dL). Biopsies of liver were taken at 2, 16, and 30 d postpartum. The triacylglycerol concentration in liver (wet basis) tended to decrease at a faster rate after d 2 postpartum with biotin supplementation compared with control cows. The potential mechanisms that link improved glucose status and decreased lipid mobilization in cows supplemented with biotin warrant further investigation.

  19. File list: Oth.Brs.50.Biotin.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Brs.50.Biotin.AllCell hg19 TFs and others Biotin Breast SRX673718,SRX673712,SRX...RX673713 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Brs.50.Biotin.AllCell.bed ...

  20. File list: Oth.Brs.05.Biotin.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Brs.05.Biotin.AllCell hg19 TFs and others Biotin Breast SRX673718,SRX673712,SRX...RX673714 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Brs.05.Biotin.AllCell.bed ...

  1. File list: Oth.Brs.10.Biotin.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Brs.10.Biotin.AllCell hg19 TFs and others Biotin Breast SRX673718,SRX673715,SRX...RX673713 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Brs.10.Biotin.AllCell.bed ...

  2. Distinct roles of two anaplerotic pathways in glutamate production induced by biotin limitation in Corynebacterium glutamicum.

    Science.gov (United States)

    Sato, Hiroki; Orishimo, Keita; Shirai, Tomokazu; Hirasawa, Takashi; Nagahisa, Keisuke; Shimizu, Hiroshi; Wachi, Masaaki

    2008-07-01

    Corynebacterium glutamicum is a biotin auxotrophic bacterium in which glutamate production is induced under biotin-limited conditions. During glutamate production, anaplerotic reactions catalyzed by phosphoenolpyruvate carboxylase (PEPC) and a biotin-containing enzyme pyruvate carboxylase (PC) are believed to play an important role in supplying oxaloacetate in the tricarboxylic acid cycle. To understand the distinct roles of PEPC and PC on glutamate production by C. glutamicum, we observed glutamate production induced under biotin-limited conditions in the disruptants of the genes encoding PEPC (ppc) and PC (pyc), respectively. The pyc disruptant retained the ability to produce high amounts of glutamate, and lactate was simultaneously produced probably due to the increased intracellular pyruvate levels. On the other hand, the ppc knockout mutant could not produce glutamate. Additionally, glutamate production in the pyc disruptant was enhanced by overexpression of ppc rather than disruption of the lactate dehydrogenase gene (ldh), which is involved in lactate production. Metabolic flux analysis based on the 13C-labeling experiment and measurement of 13C-enrichment in glutamate using nuclear magnetic resonance spectroscopy revealed that the flux for anaplerotic reactions in the pyc disruptant was lower than that in the wild type, concomitantly increasing the flux for lactate formation. Moreover, overexpression of ppc increased this flux in both the pyc disruptant and the wild type. Our results suggest that the PEPC-catalyzed anaplerotic reaction is necessary for glutamate production induced under biotin-limited conditions, because PC is not active during glutamate production, and overexpression of ppc effectively enhances glutamate production under biotin-limited conditions.

  3. Rich biotin content in lignocellulose biomass plays the key role in determining cellulosic glutamic acid accumulation by Corynebacterium glutamicum.

    Science.gov (United States)

    Wen, Jingbai; Xiao, Yanqiu; Liu, Ting; Gao, Qiuqiang; Bao, Jie

    2018-01-01

    Lignocellulose is one of the most promising alternative feedstocks for glutamic acid production as commodity building block chemical, but the efforts by the dominant industrial fermentation strain Corynebacterium glutamicum failed for accumulating glutamic acid using lignocellulose feedstock. We identified the existence of surprisingly high biotin concentration in corn stover hydrolysate as the determining factor for the failure of glutamic acid accumulation by Corynebacterium glutamicum . Under excessive biotin content, induction by penicillin resulted in 41.7 ± 0.1 g/L of glutamic acid with the yield of 0.50 g glutamic acid/g glucose. Our further investigation revealed that corn stover contained 353 ± 16 μg of biotin per kg dry solids, approximately one order of magnitude greater than the biotin in corn grain. Most of the biotin remained stable during the biorefining chain and the rich biotin content in corn stover hydrolysate almost completely blocked the glutamic acid accumulation. This rich biotin existence was found to be a common phenomenon in the wide range of lignocellulose biomass and this may be the key reason why the previous studies failed in cellulosic glutamic acid fermentation from lignocellulose biomass. The extended recording of the complete members of all eight vitamin B compounds in lignocellulose biomass further reveals that the major vitamin B members were also under the high concentration levels even after harsh pretreatment. The high content of biotin in wide range of lignocellulose biomass feedstocks and the corresponding hydrolysates was discovered and it was found to be the key factor in determining the cellulosic glutamic acid accumulation. The highly reserved biotin and the high content of their other vitamin B compounds in biorefining process might act as the potential nutrients to biorefining fermentations. This study creates a new insight that lignocellulose biorefining not only generates inhibitors, but also keeps nutrients

  4. Anion binding by biotin[6]uril in water

    DEFF Research Database (Denmark)

    Lisbjerg, Micke; Nielsen, Bjarne Enrico; Milhøj, Birgitte Olai

    2015-01-01

    In this contribution we show that the newly discovered 6 + 6 biotin-formaldehyde macrocycle Biotin[6]uril binds a variety of anionic guest molecules in water. We discuss how and why the anions are bound based on data obtained using NMR spectroscopy, mass spectrometry, isothermal titration...

  5. File list: Oth.Neu.20.Biotin.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Neu.20.Biotin.AllCell mm9 TFs and others Biotin Neural SRX1057041,SRX1057049,SR...X1057045,SRX1057047,SRX1057043,SRX1057051 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Neu.20.Biotin.AllCell.bed ...

  6. File list: Oth.Neu.50.Biotin.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Neu.50.Biotin.AllCell mm9 TFs and others Biotin Neural SRX1057041,SRX1057049,SR...X1057045,SRX1057047,SRX1057043,SRX1057051 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Neu.50.Biotin.AllCell.bed ...

  7. File list: Oth.Neu.05.Biotin.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Neu.05.Biotin.AllCell mm9 TFs and others Biotin Neural SRX1057045,SRX1057047,SR...X1057049,SRX1057041,SRX1057051,SRX1057043 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Neu.05.Biotin.AllCell.bed ...

  8. Tetracycline is back. Three-step tetracycline-biotin tumour targeting

    International Nuclear Information System (INIS)

    Salehi, N.; Lichtenstein, M.

    1998-01-01

    Full text: In the 1960s, investigators attempted to use radiolabelled tetracycline for the detection of tumours. This was limited by bone and gastrointestinal uptake. The monoclonal antibody Avidin Biotin technology has been used for 10 years to target tumours. We have improved a novel mechanism using three step targeting, to demonstrate tumour cells in (C57B1/6X balb-c) F1 mice with subcutaneously implanted E-3 thymoma. The three steps were (1) i.p. injection of Biotin Tetracycline conjugate (t:1) ratio, (2) 96 h later Avidin was injected, and (3) 24 h after (2) 99m Tc-CDTPA-Biotin was injected. Avidin has four high affinity (Km 10-15) Biotin binding sites, hence step (2) couples the Avidin to Tetracycline-Biotin in the tumour. The Avidin then provides a high affinity target for the otherwise rapidly urinary excreted 99m Tc-CDTPA-Biotin. Mice were sacrificed 16-24h after (3) by cervical dislocation. Biodistribution of radioactivity tumour to blood, liver, bone and stomach were: T:BL= 7.2, T:LI= 3.35, TBO= 9.65, T:ST= 0.93. The percentage of injected dose/g was T = 4.49%, BL = 0.62%. E-3 Thymoma is a rapid growing tumour. At day 1 (step 1) the tumour size was 0.45 cm, six days later (step 3) each dimension was doubled. Hence, percentage of injected dose per gram is artefactually reduced eight-fold. With a slowly growing tumour using the same method the results may be better. The conclusions reached are that Tetracycline-Biotin 3-stage method of tumour targeting is worthy of further development

  9. Carrier-mediated system for transport of biotin in rat intestine in vitro

    International Nuclear Information System (INIS)

    Said, H.M.; Redha, R.

    1987-01-01

    Transport of biotin was examined in rat intestine using the everted sac technique. Transport of 0.1 μM biotin was linear with time for at least 30 min of incubation and occurred at a rate 3.7 pmol g initial tissue wet wt -1 min -1 . Transport of biotin was higher in the jejunum than the ileum and was minimum in the colon (85 +/- 6, 36 +/- 6, and 2.8 +/- 0.6 pmol x g initial tissue wet wt -1 x 25 min -1 , respectively). In the jejunum, transport of biotin was saturable at low concentrations but linear at higher concentrations. The transport of low concentrations of biotin was 1) inhibited by structural analogues (desthiobiotin, biotin methyl ester, diaminobiotin, and biocytin), 2) Na + dependent, 3) energy dependent, 4) temperature dependent, and 5) proceeded against a concentration gradient in the serosal compartment. No metabolic alteration occurs to the biotin molecule during transport. This study demonstrates that biotin transport in rat intestine occurs by a carrier-mediated process at low concentrations and by simple diffusion at high concentrations. Furthermore, the carrier-mediated process is Na + , energy, and temperature dependent

  10. File list: Oth.ALL.50.Biotin.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.ALL.50.Biotin.AllCell mm9 TFs and others Biotin All cell types SRX477548,SRX273...57049,SRX1057045,SRX1057047,SRX019779,SRX1057043,SRX1057051 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.ALL.50.Biotin.AllCell.bed ...

  11. File list: Oth.ALL.20.Biotin.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.ALL.20.Biotin.AllCell mm9 TFs and others Biotin All cell types SRX477548,SRX312...7041,SRX1057049,SRX1057045,SRX1057047,SRX1057043,SRX1057051 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.ALL.20.Biotin.AllCell.bed ...

  12. File list: Oth.ALL.10.Biotin.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.ALL.10.Biotin.AllCell mm9 TFs and others Biotin All cell types SRX218273,SRX477...7041,SRX1057049,SRX1057045,SRX1057047,SRX1057043,SRX1057051 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.ALL.10.Biotin.AllCell.bed ...

  13. File list: Oth.ALL.05.Biotin.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.ALL.05.Biotin.AllCell mm9 TFs and others Biotin All cell types SRX218273,SRX148...57047,SRX148805,SRX1057049,SRX1057041,SRX1057051,SRX1057043 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.ALL.05.Biotin.AllCell.bed ...

  14. Discordant Analytical Results Caused by Biotin Interference on Diagnostic Immunoassays in a Pediatric Hospital.

    Science.gov (United States)

    Ali, Mahesheema; Rajapakshe, Deepthi; Cao, Liyun; Devaraj, Sridevi

    2017-09-01

    Recent studies have reported that biotin interferes with certain immunoassays. In this study, we evaluated the analytical interference of biotin on immunoassays that use streptavidin-biotin in our pediatric hospital. We tested the effect of different concentrations of biotin (1.5-200 ng/ml) on TSH, Prolactin, Ferritin, CK-MB, β-hCG, Troponin I, LH, FSH, Cortisol, Anti-HAV antibody (IgG and IgM), assays on Ortho Clinical Diagnostic Vitros 5600 Analyzer. Biotin (up to 200 ng/mL) did not significantly affect Troponin I and HAV assays. Biotin (up to 12.5 ng/ml) resulted in biotin >6.25 ng/mL significantly affected TSH (>20% bias) assay. Prolactin was significantly affected even at low levels (Biotin 1.5 ng/mL). Thus, we recommend educating physicians about biotin interference in common immunoassays and adding an electronic disclaimer. © 2017 by the Association of Clinical Scientists, Inc.

  15. Connecting DNA origami structures using the biotin- streptavidin ...

    African Journals Online (AJOL)

    Aghomotsegin

    carbon nanotubes on DNA origami. In order to reconfigure DNA origami pliers, Kuzuya (Kuzuya et al.,. 2011) and colleagues used the strong binding biotin- streptavidin interaction. All these researchers made use of the biotin- streptavidin interaction to functionalize the DNA strand or. DNA origami structures. In this work, we ...

  16. Biological behavior of 188Re-biotin chelate for multistep therapy with the avidin-biotin system

    International Nuclear Information System (INIS)

    Choi, T. H.; Ahn, S. H.; Choi, C. W.; Woo, K. S.; Jung, W. S.; Lim, S. J.; Lim, S. M.

    1999-01-01

    The purpose of this study was to test the three-step targeting of tumors in mice using biotinylated antibody, streptavidin and radiolabeled biotin for radioimmunotherapy (RAIT). Three-step pretargetting can potentially decreases harmful radiation to normal tissues in radioimmunotherapy. 188 Re from 188 W- 188 Re generator, is recently introduced in therapeutic nuclear medicine and made it possible to use whenever needed. We studied biotin-chelates MGB for use in the avidin/biotin pretargetting system. Chelates that hold radiometals with high stability under physiological conditions are essential to avoid excessive radiation damage to non-target cells. We synthesized MAG 2 GABA-Biocytin (MGB), labeled with 188 Re and evaluated biological behavior of 188 Re-MGB. biotinyl MAG 2 GABA bind the therapeutic radiometal 188 Re with excellent in vitro stability and have the required physiological properties for pretargetted therapy. In normal mice, 188 Re-MGB was excreted via hepatobiliary pathway, %ID/g of GI tract was 52.1 at 120min. In Raji cells tumor bearing nude mice, liver and colon were higher than those of normal mouse. Tumor uptake at 120min was 0.05%ID/g. 188 Re-MGB may have a role in pretargetted radioimmunotherapy

  17. File list: Oth.ALL.05.Biotin.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.ALL.05.Biotin.AllCell hg19 TFs and others Biotin All cell types SRX731138,SRX31...X673717,SRX673711,SRX673719,SRX673720,SRX673713,SRX673714,SRX1091033 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.ALL.05.Biotin.AllCell.bed ...

  18. File list: Oth.ALL.10.Biotin.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.ALL.10.Biotin.AllCell hg19 TFs and others Biotin All cell types SRX731138,SRX31...X673714,SRX673717,SRX673719,SRX673720,SRX673711,SRX673713,SRX1091033 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.ALL.10.Biotin.AllCell.bed ...

  19. File list: Oth.ALL.50.Biotin.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.ALL.50.Biotin.AllCell hg19 TFs and others Biotin All cell types SRX731138,SRX31...X673719,SRX673717,SRX673711,SRX673714,SRX1091033,SRX673713,SRX315187 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.ALL.50.Biotin.AllCell.bed ...

  20. File list: Oth.PSC.05.Biotin.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.PSC.05.Biotin.AllCell mm9 TFs and others Biotin Pluripotent stem cell SRX218273...67,SRX115147,SRX312228,SRX984569,SRX984573,SRX984572,SRX984568,SRX218274,SRX172568 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.PSC.05.Biotin.AllCell.bed ...

  1. File list: Oth.PSC.20.Biotin.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.PSC.20.Biotin.AllCell mm9 TFs and others Biotin Pluripotent stem cell SRX477548...44,SRX115145,SRX984568,SRX172568,SRX218274,SRX327702,SRX312228,SRX213794,SRX327701 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.PSC.20.Biotin.AllCell.bed ...

  2. File list: Oth.ALL.20.Biotin.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.ALL.20.Biotin.AllCell hg19 TFs and others Biotin All cell types SRX731138,SRX31...X673711,SRX673716,SRX673717,SRX673719,SRX673713,SRX673714,SRX1091033 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.ALL.20.Biotin.AllCell.bed ...

  3. File list: Oth.PSC.50.Biotin.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.PSC.50.Biotin.AllCell mm9 TFs and others Biotin Pluripotent stem cell SRX477548...68,SRX172568,SRX218274,SRX327702,SRX213792,SRX213794,SRX172567,SRX312228,SRX327701 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.PSC.50.Biotin.AllCell.bed ...

  4. File list: Oth.PSC.10.Biotin.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.PSC.10.Biotin.AllCell mm9 TFs and others Biotin Pluripotent stem cell SRX218273...69,SRX984573,SRX115147,SRX327702,SRX984572,SRX984568,SRX115145,SRX172568,SRX218274 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.PSC.10.Biotin.AllCell.bed ...

  5. Biotin/Folate-decorated Human Serum Albumin Nanoparticles of Docetaxel: Comparison of Chemically Conjugated Nanostructures and Physically Loaded Nanoparticles for Targeting of Breast Cancer.

    Science.gov (United States)

    Nateghian, Navid; Goodarzi, Navid; Amini, Mohsen; Atyabi, Fatemeh; Khorramizadeh, Mohammad Reza; Dinarvand, Rassoul

    2016-01-01

    Docetaxel (DTX) is a widely used chemotherapeutic agent with very low water solubility. Conjugation of DTX to human serum albumin (HSA) is an effective way to increase its water solubility. Attachment of folic acid (FA) or biotin as targeting moieties to DTX-HSA conjugates may lead to active targeting and specific uptake by cancer cells with overexpressed FA or biotin receptors. In this study, FA or biotin molecules were attached to DTX-HSA conjugates by two different methods. In one method, FA or biotin molecules were attached to remaining NH2 residues of HSA in DTX-HSA conjugate by covalent bonds. In the second method, HSA-FA or HSA-biotin conjugates were synthesized separately and then combined by DTX-HSA conjugate in proper ratio to prepare nanoparticles containing DTX-HSA plus HSA-FA or HSA-biotin. Cell viability of different nanoparticle was evaluated on MDA-MB-231 (folate receptor positive), A549 (folate receptor negative), and 4T1 (biotin receptor positive) and showed superior cytotoxicity compared with free docetaxel (Taxotere). In vivo studies of DTX-HSA-FA and DTX-HSA-biotin conjugates in BULB/c mice, tumorized by 4T1 cell line, showed the conjugates prepared in this study were more powerful in the reduction in tumor size and increasing the survival rate when compared to free docetaxel. © 2015 John Wiley & Sons A/S.

  6. Effects of dietary biotin supplementation on glucagon production, secretion, and action.

    Science.gov (United States)

    Lazo-de-la-Vega-Monroy, Maria-Luisa; Larrieta, Elena; Tixi-Verdugo, Wilma; Ramírez-Mondragón, Rafael; Hernández-Araiza, Ileana; German, Michael S; Fernandez-Mejia, Cristina

    Despite increasing evidence that pharmacologic concentrations of biotin modify glucose metabolism, to our knowledge there have not been any studies addressing the effects of biotin supplementation on glucagon production and secretion, considering glucagon is one of the major hormones in maintaining glucose homeostasis. The aim of this study was to investigate the effects of dietary biotin supplementation on glucagon expression, secretion, and action. Male BALB/cAnN Hsd mice were fed a control or a biotin-supplemented diet (1.76 or 97.7 mg biotin/kg diet) for 8 wk postweaning. Glucagon gene mRNA expression was measured by the real-time polymerase chain reaction. Glucagon secretion was assessed in isolated islets and by glucagon concentration in plasma. Glucagon action was evaluated by glucagon tolerance tests, phosphoenolpyruvate carboxykinase (Pck1) mRNA expression, and glycogen degradation. Compared with the control group, glucagon mRNA and secretion were increased from the islets of the biotin-supplemented group. Fasting plasma glucagon levels were higher, but no differences between the groups were observed in nonfasting glucagon levels. Despite the elevated fasting glucagon levels, no differences were found in fasting blood glucose concentrations, fasting/fasting-refeeding glucagon tolerance tests, glycogen content and degradation, or mRNA expression of the hepatic gluconeogenic rate-limiting enzyme, Pck1. These results demonstrated that dietary biotin supplementation increased glucagon expression and secretion without affecting fasting blood glucose concentrations or glucagon tolerance and provided new insights into the effect of biotin supplementation on glucagon production and action. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Effects of defaunation on fermentation characteristics and biotin balance in an artificial rumen-simulation system (RUSITEC) receiving diets with different amounts and types of cereal.

    Science.gov (United States)

    Abel, H; Schröder, B; Lebzien, P; Flachowsky, G

    2006-01-01

    Biotin is required by rumen microbes for efficient fermentation. To evaluate the role of protozoa in ruminal biotin metabolism, five diets composed of grass hay or of grass hay/cereal grain mixtures were supplied to faunated or defaunated RUSITEC fermenters. In the mixed diets, hay was replaced to 33:67 or 67:33 w/w on an air-dried basis by either wheat or maize grain in order to simulate different cellulolytic and amylolytic fermentation conditions. Defaunation increased SCFA production, whereas NH4 concentration and the release of CH4 were reduced. Biotin input declined when cereal grain was used to replace the hay. With the exception of the high-wheat treatment, defaunated fermenters yielded higher biotin outputs than faunated fermenters. The biotin balance, calculated as the difference between the total biotin output (biotin in the solid residue contained in the nylon bags after fermentation plus the biotin in the effluent) and the biotin input with the feed, was negative for all the dietary treatments apart from fermenters supplied with the high-maize diet. It was less negative or, in the case of the high-maize diets, more positive for defaunated compared with faunated fermenters. It was concluded that, under normal faunated conditions, protozoa directly utilise or indirectly affect the bacterial synthesis and/or utilisation of biotin. With diets of a high fermentation potential, as realised with the high-wheat diet, protozoa prevent the development of a bacterial population that would utilise high or synthesise low amounts of biotin.

  8. {sup 18}F-PEG-biotin: Precursor (boroaryl-PEG-biotin) synthesis, {sup 18}F-labelling and an in-vitro assessment of its binding with Neutravidin{sup TM}-trastuzumab pre-treated cells

    Energy Technology Data Exchange (ETDEWEB)

    Smith, Tim A.D., E-mail: t.smith@abdn.ac.uk [Biomedical Physics Building, John Mallard PET Unit, Aberdeen Biomedical Imaging Centre, School of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD (United Kingdom); Simpson, Michael; Cheyne, Richard [Biomedical Physics Building, John Mallard PET Unit, Aberdeen Biomedical Imaging Centre, School of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD (United Kingdom); School of Natural and Computing Sciences, University of Aberdeen, Aberdeen AB24 3UE (United Kingdom); Trembleau, Laurent [School of Natural and Computing Sciences, University of Aberdeen, Aberdeen AB24 3UE (United Kingdom)

    2011-10-15

    In terms of nuclear decay {sup 18}F is the most ideal PET nuclide but its short t{sub 1/2} precludes its use for directly labelling whole antibodies due to their long blood residence times. Pre-targeted imaging using affinity systems such as Neutravidin{sup TM}-biotin facilitates the application of short-lived nuclides by their attachment to biotin for imaging cell surface proteins targeted with Neutravidin{sup TM}-conjugated antibodies. Methods: Boroaryl functionalised biotin was prepared with a PEG linker and radiolabelled by incubation with {sup 18}F in acidified aqueous solution. Cells expressing high (SKBr3), medium (MDA-MB-453) and low (MDA-MB-468) levels of HER-2 were pre-incubated with Neutravidin{sup TM}-conjugated trastuzumab, washed, and then incubated with {sup 18}F-PEG-biotin. Results: The {sup 18}F-fluorination of boroaryl-PEG-biotin was much more efficient than reported for other versions of boroaryl-biotin. The novel {sup 18}F-PEG-biotin was demonstrated to bind to HER-2-expressing cells in-vitro pre-incubated with Neutravidin{sup TM}-conjugated trastuzumab. Conclusion: Biotin can be functionalised with boroaryl and readily {sup 18}F-radiolabelled in aqueous solution and will bind to cells pre-incubated with Neutravidin{sup TM}-antibody conjugates. - Highlights: > Boroaryl-biotin precursor is prepared. > Rapid {sup 18}F-fluorination is demonstrated. > HER-2 expressing breast cancer cells pre-treated with trastuzumab-Neutravidin{sup TM}. > {sup 18}F-PEG-biotin binding to pre-treated cells corresponds with HER-2 expression.

  9. Structural Analysis of Substrate, Reaction Intermediate, and Product Binding in Haemophilus influenzae Biotin Carboxylase

    Science.gov (United States)

    Broussard, Tyler C.; Pakhomova, Svetlana; Neau, David B.; Bonnot, Ross; Waldrop, Grover L.

    2015-01-01

    Acetyl-CoA carboxylase catalyzes the first and regulated step in fatty acid synthesis. In most Gram-negative and Gram-positive bacteria, the enzyme is composed of three proteins: biotin carboxylase, a biotin carboxyl carrier protein (BCCP), and carboxyltransferase. The reaction mechanism involves two half-reactions with biotin carboxylase catalyzing the ATP-dependent carboxylation of biotin-BCCP in the first reaction. In the second reaction, carboxyltransferase catalyzes the transfer of the carboxyl group from biotin-BCCP to acetyl-CoA to form malonyl-CoA. In this report, high-resolution crystal structures of biotin carboxylase from Haemophilus influenzae were determined with bicarbonate, the ATP analogue AMPPCP; the carboxyphosphate intermediate analogues, phosphonoacetamide and phosphonoformate; the products ADP and phosphate; and the carboxybiotin analogue N1′-methoxycarbonyl biotin methyl ester. The structures have a common theme in that bicarbonate, phosphate, and the methyl ester of the carboxyl group of N1′-methoxycarbonyl biotin methyl ester all bound in the same pocket in the active site of biotin carboxylase and as such utilize the same set of amino acids for binding. This finding suggests a catalytic mechanism for biotin carboxylase in which the binding pocket that binds tetrahedral phosphate also accommodates and stabilizes a tetrahedral dianionic transition state resulting from direct transfer of CO2 from the carboxyphosphate intermediate to biotin. PMID:26020841

  10. A SNARE-Like Protein and Biotin Are Implicated in Soybean Cyst Nematode Virulence.

    Directory of Open Access Journals (Sweden)

    Sadia Bekal

    Full Text Available Phytoparasitic nematodes that are able to infect and reproduce on plants that are considered resistant are referred to as virulent. The mechanism(s that virulent nematodes employ to evade or suppress host plant defenses are not well understood. Here we report the use of a genetic strategy (allelic imbalance analysis to associate single nucleotide polymorphisms (SNPs with nematode virulence genes in Heterodera glycines, the soybean cyst nematode (SCN. To accomplish this analysis, a custom SCN SNP array was developed and used to genotype SCN F3-derived populations grown on resistant and susceptible soybean plants. Three SNPs reproducibly showed allele imbalances between nematodes grown on resistant and susceptible plants. Two candidate SCN virulence genes that were tightly linked to the SNPs were identified. One SCN gene encoded biotin synthase (HgBioB, and the other encoded a bacterial-like protein containing a putative SNARE domain (HgSLP-1. The two genes mapped to two different linkage groups. HgBioB contained sequence polymorphisms between avirulent and virulent nematodes. However, the gene encoding HgSLP-1 had reduced copy number in virulent nematode populations and appears to produce multiple forms of the protein via intron retention and alternative splicing. We show that HgSLP-1 is an esophageal-gland protein that is secreted by the nematode during plant parasitism. Furthermore, in bacterial co-expression experiments, HgSLP-1 co-purified with the SCN resistance protein Rhg1 α-SNAP, suggesting that these two proteins physically interact. Collectively our data suggest that multiple SCN genes are involved in SCN virulence, and that HgSLP-1 may function as an avirulence protein and when absent it helps SCN evade host defenses.

  11. Pregnancy and lactation alter biomarkers of biotin metabolism in women consuming a controlled diet.

    Science.gov (United States)

    Perry, Cydne A; West, Allyson A; Gayle, Antoinette; Lucas, Lauren K; Yan, Jian; Jiang, Xinyin; Malysheva, Olga; Caudill, Marie A

    2014-12-01

    Biotin functions as a cofactor for several carboxylase enzymes with key roles in metabolism. At present, the dietary requirement for biotin is unknown and intake recommendations are provided as Adequate Intakes (AIs). The biotin AI for adults and pregnant women is 30 μg/d, whereas 35 μg/d is recommended for lactating women. However, pregnant and lactating women may require more biotin to meet the demands of these reproductive states. The current study sought to quantify the impact of reproductive state on biotin status response to a known dietary intake of biotin. To achieve this aim, we measured a panel of biotin biomarkers among pregnant (gestational week 27 at study entry; n = 26), lactating (postnatal week 5 at study entry; n = 28), and control (n = 21) women who participated in a 10- to 12-wk feeding study providing 57 μg of dietary biotin/d as part of a mixed diet. Over the course of the study, pregnant women excreted 69% more (vs. control; P biotin-dependent methylcrotonyl-coenzyme A carboxylase is impaired. Interestingly, urinary excretion of 3-hydroxyisovaleryl-carnitine (3-HIA-carnitine), a downstream metabolite of 3-HIA, was 27% lower (P = 0.05) among pregnant (vs. control) women, a finding that may arise from carnitine inadequacy during gestation. No differences (P > 0.05) were detected in plasma biotin, urinary biotin, or urinary bisnorbiotin between pregnant and control women. Lactating women excreted 76% more (vs. control; P = 0.001) of the biotin catabolite bisnorbiotin, indicating that lactation accelerates biotin turnover and loss. Notably, with respect to control women, lactating women excreted 23% less (P = 0.04) urinary 3-HIA and 26% less (P = 0.05) urinary 3-HIA-carnitine, suggesting that lactation reduces leucine catabolism and that these metabolites may not be useful indicators of biotin status during lactation. Overall, these data demonstrate significant alterations in markers of biotin metabolism during pregnancy and lactation and

  12. Pantothenic acid and biotin

    Science.gov (United States)

    Pantothenic acid and biotin are types of B vitamins. They are water-soluble, which means that the body can't store them. If the body can't use all of the vitamin, the extra vitamins leave the body through the ...

  13. Labeling of biotin with 166Dy/166Ho as a stable in vivo generator system

    International Nuclear Information System (INIS)

    Ferro-Flores, G.; Monroy-Guzman, F.; Tendilla, J.I.; Garcia-Salinas, L.; Villarreal-Barajas, J.E.; Arteaga-Murphy, C.

    2002-01-01

    Biotin (cis-tetrahydro-2-oxothieno[3,4-d]imidazoline-4-valeric acid) is a 244 Da vitamin found in low concentration in blood and tissue (vitamin H). The aim of this work was to synthesize 166 Dy/ 166 Ho-DTPA-bisBiotin to evaluate its potential as a new radiopharmaceutical for targeted radiotherapy. Dysprosium-166/ holmium-166 chloride was obtained by neutron irradiation of 20 mg of enriched Dy 2 O 3 ( 164 Dy, 99 %, from Oak Ridge NL) in a Triga Mark III reactor at a flux in the central thimble of 3.10 13 n. cm -2 s -1 for 20 h. Following irradiation, the target was allowed to decay for 2 days, then 100 μL of 12 N chloride acid were added and stirred for 1 min. To this solution was added 500 μL of injectable water and the whole was also stirred for 2 min. The average radioactive concentration was 332 MBq/ml. The biotin used in this investigation was covalently conjugated to diethylenetriamine pentaacetic acid (DTPA) through the use of the cyclic anhydride and lysine conjugate to biotin (biocytin) to produce DTPA-α,ω-bis(biocytin amide)(DTPA-bisBiotin). Sterile and apyrogenic V-vial was prepared to contain 2.0 mg (1.9 x 10 -3 mmol) of the DTPA-bisbiotin compound in 1.0 ml of 0.05 M bicarbonate buffer (pH 8.0) and then 20 μL of 166 Dy2Cl3 solution were added to the preparation. Thin Layer Chromatography aluminum cellulose sheets were utilised as the stationary phase and a ternary mixture of methanol:water:ammonium hydroxide (20:40:2) as the mobile phase. 166 Dy/ 166 Ho-DTPA-bisBiotin travelled with the solvent front R f 0.9-1.0 and the Dy +3 /Ho +3 species remained at the origin (R f = 0). The biological integrity of labelled biotin was achieved evaluating its avidity for avidin in an agarose column. Stability studies against dilution were carried out by diluting the radiocomplex solution with saline and with human serum at 310 K. After 10 min and 24 h the radiochemical purity of each 166 Dy/ 166 Ho complex solution was determined by TLC. The complex 166 Dy/ 166

  14. Development of an internet based system for modeling biotin metabolism using Bayesian networks.

    Science.gov (United States)

    Zhou, Jinglei; Wang, Dong; Schlegel, Vicki; Zempleni, Janos

    2011-11-01

    Biotin is an essential water-soluble vitamin crucial for maintaining normal body functions. The importance of biotin for human health has been under-appreciated but there is plenty of opportunity for future research with great importance for human health. Currently, carrying out predictions of biotin metabolism involves tedious manual manipulations. In this paper, we report the development of BiotinNet, an internet based program that uses Bayesian networks to integrate published data on various aspects of biotin metabolism. Users can provide a combination of values on the levels of biotin related metabolites to obtain the predictions on other metabolites that are not specified. As an inherent feature of Bayesian networks, the uncertainty of the prediction is also quantified and reported to the user. This program enables convenient in silico experiments regarding biotin metabolism, which can help researchers design future experiments while new data can be continuously incorporated. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  15. Oligonucleotide-stabilized fluorescent silver nanoclusters for the specific and sensitive detection of biotin.

    Science.gov (United States)

    Xiong, Xiaoli; Tang, Yan; Zhao, Jingjin; Zhao, Shulin

    2016-02-21

    A novel biotin fluorescent probe based on oligonucleotide-stabilized silver nanoclusters (DNA-AgNCs) was synthesized by employing a biotinylated cytosine-rich sequence as a synthesized template. The fluorescence properties of the DNA-AgNCs are related to the modified position of the DNA. When biotin is linked to the middle thymine base of the DNA sequence, the DNA-AgNCs emit the strongest fluorescence. Moreover, the stability of the DNA-AgNCs was affected by avidin through biotin-avidin binding, quenching the fluorescence of the DNA-AgNCs. In contrast, if free biotin is further introduced into this system, the quenching is apparently weakened by competition, leading to the restoration of fluorescence. This phenomenon can be utilized for the detection of biotin. Under the optimal conditions, the fluorescence recovery is linearly proportional to the concentration of biotin in the range of 10 nM-1.0 μM with a detection limit of 6.0 nM. This DNA-AgNCs probe with excellent fluorescent properties is sensitive and selective for the detection of biotin and has been applied for the determination of biotin in wheat flour.

  16. Radiolabeled biotin amides from triazenyl precursors: synthesis, binding, and in-vivo properties

    International Nuclear Information System (INIS)

    Kortylewicz, Z.P.; Baranowska-Kortylewicz, J.; Adelstein, S.J.; Carmel, A.D.; Kassis, A.I.

    1994-01-01

    The synthesis of N-(4-[ 127/125/123 I]iodobenzyl)biotin amides 4a - 4c performed by the direct decomposition of N-[4-(3',3'-dimethyltriazenyl)benzyl]biotin amide with sodium iodide in the presence of CF 3 COOH is described. Iodinated in this way biotin formed a stable complex with avidin (K d = 2.84 ± 0.45 x 10 -15 M, n = 3.9 ± 0.6) which dissociated in the presence of excess native biotin with a rate constant of 0.034 ± 0.006 hr -1 . Blood clearance studies and the lack of thyroid uptake indicated that this compound was not deiodinated in vivo and behaved in circulation much like native biotin. This aryltriazene precursor method is suitable for labeling with short-lived radiohalides. It can be used to produce no-carrier-added derivatives of biotin for use in biologic studies and assays involving avidin or streptavidin. (author)

  17. Salmonella infection inhibits intestinal biotin transport: cellular and molecular mechanisms.

    Science.gov (United States)

    Ghosal, Abhisek; Jellbauer, Stefan; Kapadia, Rubina; Raffatellu, Manuela; Said, Hamid M

    2015-07-15

    Infection with the nontyphoidal Salmonella is a common cause of food-borne disease that leads to acute gastroenteritis/diarrhea. Severe/prolonged cases of Salmonella infection could also impact host nutritional status, but little is known about its effect on intestinal absorption of vitamins, including biotin. We examined the effect of Salmonella enterica serovar Typhimurium (S. typhimurium) infection on intestinal biotin uptake using in vivo (streptomycin-pretreated mice) and in vitro [mouse (YAMC) and human (NCM460) colonic epithelial cells, and human intestinal epithelial Caco-2 cells] models. The results showed that infecting mice with wild-type S. typhimurium, but not with its nonpathogenic isogenic invA spiB mutant, leads to a significant inhibition in jejunal/colonic biotin uptake and in level of expression of the biotin transporter, sodium-dependent multivitamin transporter. In contrast, infecting YAMC, NCM460, and Caco-2 cells with S. typhimurium did not affect biotin uptake. These findings suggest that the effect of S. typhimurium infection is indirect and is likely mediated by proinflammatory cytokines, the levels of which were markedly induced in the intestine of S. typhimurium-infected mice. Consistent with this hypothesis, exposure of NCM460 cells to the proinflammatory cytokines TNF-α and IFN-γ led to a significant inhibition of biotin uptake, sodium-dependent multivitamin transporter expression, and activity of the SLC5A6 promoter. The latter effects appear to be mediated, at least in part, via the NF-κB signaling pathway. These results demonstrate that S. typhimurium infection inhibits intestinal biotin uptake, and that the inhibition is mediated via the action of proinflammatory cytokines.

  18. Preparation of Conjugates of Cytotoxic Lupane Triterpenes with Biotin.

    Science.gov (United States)

    Soural, Miroslav; Hodon, Jiri; Dickinson, Niall J; Sidova, Veronika; Gurska, Sona; Dzubak, Petr; Hajduch, Marian; Sarek, Jan; Urban, Milan

    2015-12-16

    To better understand the mechanism of action of antitumor triterpenes, we are developing methods to identify their molecular targets. A promising method is based on combination of quantitative proteomics with SILAC and uses active compounds anchored to magnetic beads via biotin-streptavidin interaction. We developed a simple and fast solid-phase synthetic technique to connect terpenes to biotin through a linker. Betulinic acid was biotinylated from three different conjugation sites for use as a standard validation tool since many molecular targets of this triterpene are already known. Then, a set of four other cytotoxic triterpenoids was biotinylated. Biotinylated terpenes were similarly cytotoxic to their nonbiotinylated parents, which suggests that the target identification should not be influenced by linker or biotin. The developed solid-phase synthetic approach is the first attempt to use solid-phase synthesis to connect active triterpenes to biotin and is applicable as a general procedure for routine conjugation of triterpenes with other molecules of choice.

  19. A Biotin Biosynthesis Gene Restricted to Helicobacter

    Science.gov (United States)

    Bi, Hongkai; Zhu, Lei; Jia, Jia; Cronan, John E.

    2016-01-01

    In most bacteria the last step in synthesis of the pimelate moiety of biotin is cleavage of the ester bond of pimeloyl-acyl carrier protein (ACP) methyl ester. The paradigm cleavage enzyme is Escherichia coli BioH which together with the BioC methyltransferase allows synthesis of the pimelate moiety by a modified fatty acid biosynthetic pathway. Analyses of the extant bacterial genomes showed that bioH is absent from many bioC-containing bacteria and is replaced by other genes. Helicobacter pylori lacks a gene encoding a homologue of the known pimeloyl-ACP methyl ester cleavage enzymes suggesting that it encodes a novel enzyme that cleaves this intermediate. We isolated the H. pylori gene encoding this enzyme, bioV, by complementation of an E. coli bioH deletion strain. Purified BioV cleaved the physiological substrate, pimeloyl-ACP methyl ester to pimeloyl-ACP by use of a catalytic triad, each member of which was essential for activity. The role of BioV in biotin biosynthesis was demonstrated using a reconstituted in vitro desthiobiotin synthesis system. BioV homologues seem the sole pimeloyl-ACP methyl ester esterase present in the Helicobacter species and their occurrence only in H. pylori and close relatives provide a target for development of drugs to specifically treat Helicobacter infections. PMID:26868423

  20. The electrochemical reduction of biotin (vitamin B7) and conversion into its ester

    International Nuclear Information System (INIS)

    Lauw, Sherman J.L.; Ganguly, Rakesh; Webster, Richard D.

    2013-01-01

    Highlights: •Biotin can be reduced electrochemically, by one-electron, at a platinum electrode. •The reduction likely follows a direct discharge mechanism of the carboxyl group. •Electrochemically generated biotin carboxylate was reacted with iodomethane (91%). •ATR–FTIR characterization of biotin, its carboxylate anion, and its methyl ester. -- Abstract: An electrochemical study on biotin (vitamin B7), performed in aprotic solvents and at a platinum electrode, revealed that at approximately E f 0 =−1.6to−1.8 vs. (Fc/Fc + )/V (E f 0 =formal reduction potential and Fc=ferrocene), biotin is reduced by one-electron to form its carboxylate anion and dihydrogen via a direct discharge of the carboxylic acid at the platinum surface. The electrochemical reduction process appeared to be chemically reversible on the time-frame of cyclic voltammetry (CV) (t ≤ s), but not over the extended period of controlled potential electrolysis (CPE) (t ≥ min) where the conversion of biotin into its carboxylate anion was found to be chemically irreversible. A strategy to functionalize biotin's carboxyl group was established by performing a bulk reductive electrolysis, and then reacting the electrochemically generated carboxylate anion with iodomethane to afford biotin methyl ester in excellent yield (91%). Attenuated total reflectance–Fourier transform infrared (ATR–FTIR) spectroscopy was successful in identifying several distinct and characteristic carbonyl absorbance peaks associated with the analogous forms of biotin available before electrolysis, after electrolysis, and after methylation

  1. Factitious Graves' Disease Due to Biotin Immunoassay Interference-A Case and Review of the Literature.

    Science.gov (United States)

    Elston, Marianne S; Sehgal, Shekhar; Du Toit, Stephen; Yarndley, Tania; Conaglen, John V

    2016-09-01

    Biotin (vitamin B7) is an essential co-factor for four carboxylases involved in fatty acid metabolism, leucine degradation, and gluconeogenesis. The recommended daily intake (RDI) of biotin is approximately 30 μg per day. Low-moderate dose biotin is a common component of multivitamin preparations, and high-dose biotin (10 000 times RDI) has been reported to improve clinical outcomes and quality of life in patients with progressive multiple sclerosis. Biotin is also a component of immunoassays, and supplementation may cause interference in both thyroid and non-thyroid immunoassays. To assess whether biotin ingestion caused abnormal thyroid function tests (TFTs) in a patient through assay interference. We report a patient with biotin-associated abnormal TFTs and a systematic review of the literature. A tertiary endocrine service in Hamilton, New Zealand. The patient had markedly abnormal TFTs that did not match the clinical context. After biotin cessation, TFTs normalized far more rapidly than possible given the half-life of T4, consistent with assay interference by biotin. Multiple other analytes also tested abnormal in the presence of biotin. Biotin ingested in moderate to high doses can cause immunoassay interference. Depending on the assay format, biotin interference can result in either falsely high or low values. Interference is not limited to thyroid tests and has the potential to affect a wide range of analytes. It is important for clinicians to be aware of this interaction to prevent misdiagnosis and inappropriate treatment.

  2. Pregnancy and Lactation Alter Biomarkers of Biotin Metabolism in Women Consuming a Controlled Diet123

    Science.gov (United States)

    Perry, Cydne A; West, Allyson A; Gayle, Antoinette; Lucas, Lauren K; Yan, Jian; Jiang, Xinyin; Malysheva, Olga; Caudill, Marie A

    2014-01-01

    Background: Biotin functions as a cofactor for several carboxylase enzymes with key roles in metabolism. At present, the dietary requirement for biotin is unknown and intake recommendations are provided as Adequate Intakes (AIs). The biotin AI for adults and pregnant women is 30 μg/d, whereas 35 μg/d is recommended for lactating women. However, pregnant and lactating women may require more biotin to meet the demands of these reproductive states. Objective: The current study sought to quantify the impact of reproductive state on biotin status response to a known dietary intake of biotin. Methods: To achieve this aim, we measured a panel of biotin biomarkers among pregnant (gestational week 27 at study entry; n = 26), lactating (postnatal week 5 at study entry; n = 28), and control (n = 21) women who participated in a 10- to 12-wk feeding study providing 57 μg of dietary biotin/d as part of a mixed diet. Results: Over the course of the study, pregnant women excreted 69% more (vs. control; P biotin-dependent methylcrotonyl–coenzyme A carboxylase is impaired. Interestingly, urinary excretion of 3-hydroxyisovaleryl-carnitine (3-HIA-carnitine), a downstream metabolite of 3-HIA, was 27% lower (P = 0.05) among pregnant (vs. control) women, a finding that may arise from carnitine inadequacy during gestation. No differences (P > 0.05) were detected in plasma biotin, urinary biotin, or urinary bisnorbiotin between pregnant and control women. Lactating women excreted 76% more (vs. control; P = 0.001) of the biotin catabolite bisnorbiotin, indicating that lactation accelerates biotin turnover and loss. Notably, with respect to control women, lactating women excreted 23% less (P = 0.04) urinary 3-HIA and 26% less (P = 0.05) urinary 3-HIA-carnitine, suggesting that lactation reduces leucine catabolism and that these metabolites may not be useful indicators of biotin status during lactation. Conclusions: Overall, these data demonstrate significant alterations in markers of

  3. Pre-targeted tumor imaging with avidin-McAb and 99Tcm-DTPA-Biotin

    International Nuclear Information System (INIS)

    Zhang Jinming; Tian Jiahe; Wang Yuqi; Liu Xi; Sun Xin

    2002-01-01

    Objective: Biotin-avidin is used as pre-targeting system (BAS) in radioimmunoimaging in order to decrease radiation background and dose associated with the use of directly labelled McAb. The authors tried to use 99 Tc m to substitute 111 In to label DTPA-biotin to evaluate the value of the 99 Tc m -DTPA-biotin in BAS. Methods: DTPA-biotin solution was mixed with SnCl 2 and then fresh eluted 99 Tc m . The solution incubated for 10 min at room temperature. Mice bearing lung tumor (LA-795) with and without metastases in lung underwent 3-step pre-targeting test. Briefly, biotin-C50 was injected first, then avidin and 99 Tc m -DTPA-biotin was respectively given 1 day, 2 days later. Directly labelled C50 with 99 Tc m was used as control agent. Results: The labelling yield of 99 Tc m -DTPA-biotin was over 90%. The amount of SnCl 2 was the key feature in labelling. The tumor could be seen at 2 h after injection of 99 Tc m -DTPA-biotin with γ camera in 3- step groups. The tracer uptake in tumor was (1.35 +- 0.45)% ID/g at 2 h after injection, Tumor/Blood (T/B) was 5.86, T/Muscle (T/M) was 8.43. In control group which received 99 Tc m -DTPA-biotin only, the T/B was 0.85, T/M 1.1. For the directly labelled C50, the T/B was 1.65, T/M was 2.0 at 8 h after injection. Conclusion: Avidin-biotin pre-targeting system can be labelled with 99 Tc m , and the BAS can image the tumor as early as 2 h after injection

  4. Effects of biotin supplementation on performance and claw lesions on a commercial dairy farm.

    Science.gov (United States)

    Bergsten, C; Greenough, P R; Gay, J M; Seymour, W M; Gay, C C

    2003-12-01

    A controlled 14-mo field trial was conducted to evaluate the effect of biotin supplementation on hoof lesions, milk production, and reproductive performance in a commercial dairy herd. One hundred seventy cows were studied and supplemented with either 0 or 20 mg/d of biotin by computer feeder. All were housed in the same free-stall facility with the same environment, base diet, and management. The feet of 99 cows were trimmed three times at 6-mo intervals, and hoof health was evaluated. Milk production and fertility data were captured monthly by the Dairy Herd Improvement Association. At the final hoof trimming, sole hemorrhages were significantly higher in control (50%) vs. biotin-supplemented animals (24%). The incidents of cows affected with double soles, hoof wall grooves, and heel horn erosion did not differ between control and biotin-supplemented animals. Biotin supplementation of trimmed cows resulted in 878 kg more milk than control cows when compared with previous lactation yield (n = 46 biotin supplemented, n = 48 control cows). At the end of the study, for both trimmed and untrimmed animals, biotin supplemented cows (n = 81) produced 481 kg more milk and 25 kg more fat than the controls (n = 81). There was no interaction between biotin supplementation and hoof trimming on milk production. There were variations in the response of fertility to biotin between age groups. First lactation heifers fed supplemental biotin had significantly fewer days from calving to conception and required fewer inseminations per pregnancy than controls of the same parity.

  5. A biotin-triggered genetic switch in mammalian cells and mice.

    Science.gov (United States)

    Weber, Wilfried; Lienhart, Cédric; Baba, Marie Daoud-El; Fussenegger, Martin

    2009-03-01

    Adjustable and reversible transgene expression systems enabling precise control of metabolic pathways and tunable production of specific target proteins have been essential for conditional reprogramming of mammalian cells to achieve progress in basic and applied bioengineering disciplines. Most of the currently available transgene control modalities have been designed to be responsive to clinically licensed pharmacologically active drugs which were expected to prevail in future clinical trials yet raised concerns about side effects when administered long term at subclinical doses. We have chosen vitamin H, also known as biotin, to control target gene transcription in mammalian cells in a potentially side effect-free manner. BirA, the Escherichia coli repressor of the biotin biosynthesis operon, was fused to the Herpes simplex transactivation domain to generate a biotin-dependent transactivator(BIT), which, in the presence of biotin, binds and activates chimeric target promoters (P(BIT)) harboring BirA-specific operator sites 5' of a minimal promoter. Biotin-inducible transgene expression was functional in a variety of rodent, monkey and human cell lines, showed excellent adjustability and reversibility in transgenic Chinese hamster ovary cell lines, provided precise product gene control in standard bioreactor cultures and enabled dose-dependent vitamin H control of a human glycoprotein in mice. The combination of a side effect-free inducer, precise and reversible transcription tunability and broad functionality in different cell types as well as in entire animals represents a unique asset for the use of biotin-inducible transgene control in future gene therapy, tissue engineering and biopharmaceutical manufacturing scenarios.

  6. Identification of total reversible cysteine oxidation in an atherosclerosis model using a modified biotin switch assay.

    Science.gov (United States)

    Li, Ru; Huang, Jiqing; Kast, Juergen

    2015-05-01

    Oxidative stress due to the imbalance of reactive oxygen species (ROS) and the resulting reversible cysteine oxidation (CysOX) are involved in the early proatherogenic aspect of atherosclerosis. Given that the corresponding redox signaling pathways are still unclear, a modified biotin switch assay was developed to quantify the reversible CysOX in an atherosclerosis model established by using a monocytic cell line treated with platelet releasate. The accumulation of ROS was observed in the model system and validated in human primary monocytes. Through the application of the modified biotin switch assay, we obtained the first reversible CysOX proteome for this model. A total of 75 peptides, corresponding to 53 proteins, were quantified with oxidative modification. The bioinformatics analysis of these CysOX-containing proteins highlighted biological processes including glycolysis, cytoskeleton arrangement, and redox regulation. Moreover, the reversible oxidation of three glycolysis enzymes was observed using this method, and the regulation influence was verified by an enzyme activity assay. NADPH oxidase (NOX) inhibition treatment, in conjunction with the modified biotin switch method, was used to evaluate the global CysOX status. In conclusion, this versatile modified biotin switch assay provides an approach for the quantification of all reversible CysOX and for the study of redox signaling in atherosclerosis as well as in diseases in other biological systems.

  7. Triazole biotin: a tight-binding biotinidase-resistant conjugate.

    Science.gov (United States)

    Germeroth, Anne I; Hanna, Jill R; Karim, Rehana; Kundel, Franziska; Lowther, Jonathan; Neate, Peter G N; Blackburn, Elizabeth A; Wear, Martin A; Campopiano, Dominic J; Hulme, Alison N

    2013-11-28

    The natural amide bond found in all biotinylated proteins has been replaced with a triazole through CuAAC reaction of an alkynyl biotin derivative. The resultant triazole-linked adducts are shown to be highly resistant to the ubiquitous hydrolytic enzyme biotinidase and to bind avidin with dissociation constants in the low pM range. Application of this strategy to the production of a series of biotinidase-resistant biotin-Gd-DOTA contrast agents is demonstrated.

  8. Efficient synthesis of a fluorine-18 labeled biotin derivative

    International Nuclear Information System (INIS)

    Claesener, Michael; Breyholz, Hans-Jörg; Hermann, Sven; Faust, Andreas; Wagner, Stefan; Schober, Otmar; Schäfers, Michael; Kopka, Klaus

    2012-01-01

    Introduction: The natural occurring vitamin biotin, also known as vitamin H or vitamin B 7 , plays a major role in various metabolic reactions. Caused by its high binding affinity to the protein avidin with a dissociation constant of about 10 -15 M the biotin-avidin system was extensively examined for multiple applications. We have synthesized a fluorine-18 labeled biotin derivative [ 18 F]4 for a potential application in positron emission tomography (PET). Methods: Mesylate precursor 3 was obtained by an efficient two-step reaction via a copper catalyzed azide-alkyne cycloaddition (CuAAC) from easily accessible starting materials. [ 18 F]4 was successfully synthesized by a nucleophilic radiofluorination of precursor 3. A biodistribution study by means of small-animal PET imaging in wt-mice was performed and serum stability was examined. Results: Compound [ 18 F]4 was obtained from precursor compound 3 with an average specific activity of 16 GBq/μmol within 45 min and a radiochemical yield of 45 ± 5% (decay corrected). [ 18 F]4 demonstrated only negligible decomposition in human serum. A qualitative binding study revealed the high affinity of the synthesized biotin derivative to avidin. Blocking experiments with native biotin showed that binding was site-specific. Biodistribution studies showed that [ 18 F]4 was cleared quickly and efficiently from the body by hepatobiliary and renal elimination. Conclusion: An efficient synthesis for [ 18 F]4 was established. In vivo characteristics were determined and demonstrated the pharmacokinetic behaviour of [ 18 F]4.

  9. Holocarboxylase Synthetase: A Moonlighting Transcriptional Coregulator of Gene Expression and a Cytosolic Regulator of Biotin Utilization.

    Science.gov (United States)

    León-Del-Río, Alfonso; Valadez-Graham, Viviana; Gravel, Roy A

    2017-08-21

    The vitamin biotin is an essential nutrient for the metabolism and survival of all organisms owing to its function as a cofactor of enzymes collectively known as biotin-dependent carboxylases. These enzymes use covalently attached biotin as a vector to transfer a carboxyl group between donor and acceptor molecules during carboxylation reactions. In human cells, biotin-dependent carboxylases catalyze key reactions in gluconeogenesis, fatty acid synthesis, and amino acid catabolism. Biotin is attached to apocarboxylases by a biotin ligase: holocarboxylase synthetase (HCS) in mammalian cells and BirA in microbes. Despite their evolutionary distance, these proteins share structural and sequence similarities, underscoring their importance across all life forms. However, beyond its role in metabolism, HCS participates in the regulation of biotin utilization and acts as a nuclear transcriptional coregulator of gene expression. In this review, we discuss the function of HCS and biotin in metabolism and human disease, a putative role for the enzyme in histone biotinylation, and its participation as a nuclear factor in chromatin dynamics. We suggest that HCS be classified as a moonlighting protein, with two biotin-dependent cytosolic metabolic roles and a distinct biotin-independent nuclear coregulatory function.

  10. Misdiagnosis of Graves' Disease with Apparent Severe Hyperthyroidism in a Patient Taking Biotin Megadoses.

    Science.gov (United States)

    Barbesino, Giuseppe

    2016-06-01

    Accurate immunoassays measuring minute quantities of hormones are the cornerstone of the practice of endocrinology. Despite tremendous advances in this field, novel pitfalls in these tests emerge from time to time. Oral biotin can interfere with immunoassays of several hormones. The purpose of this report is to relate an extreme case of such interference. A patient with progressive multiple sclerosis was found to have extremely elevated free thyroxine, triiodothyronine, and suppressed thyrotropin (TSH) levels. His TSH receptor binding inhibiting antibody level was also elevated. This constellation of laboratory findings suggested a diagnosis of severe Graves' disease. All of the assays yielding abnormal results employed the biotin-streptavidin affinity in their design. The patient had no symptoms of hyperthyroidism, and detailed review of his medications revealed intake of megadoses of biotin. Temporary discontinuation of biotin treatment resulted in complete resolution of the biochemical abnormalities. Non-physiologic biotin supplementation may interfere with several immunoassays, including thyroid hormones, TSH, thyroglobulin, and TSH receptor binding inhibiting antibody, leading to erroneous diagnoses. Questioning for biotin intake should be part of the evaluation for patients undergoing endocrine tests. Interruption of biotin supplementation for at least two days prior to biotin-sensitive tests should be sufficient to avoid major misdiagnoses.

  11. In HepG2 cells, coexisting carnitine deficiency masks important indicators of marginal biotin deficiency.

    Science.gov (United States)

    Bogusiewicz, Anna; Boysen, Gunnar; Mock, Donald M

    2015-01-01

    A large number of birth defects are related to nutrient deficiencies; concern that biotin deficiency is teratogenic in humans is reasonable. Surprisingly, studies indicate that increased urinary 3-hydroxyisovalerylcarnitine (3HIAc), a previously validated marker of biotin deficiency, is not a valid biomarker in pregnancy. In this study we hypothesized that coexisting carnitine deficiency can prevent the increase in 3HIAc due to biotin deficiency. We used a 2-factor nutrient depletion design to induce isolated and combined biotin and carnitine deficiency in HepG2 cells and then repleted cells with carnitine. To elucidate the metabolic pathogenesis, we quantitated intracellular and extracellular free carnitine, acylcarnitines, and acylcarnitine ratios using liquid chromatography-tandem mass spectrometry. Relative to biotin-sufficient, carnitine-sufficient cells, intracellular acetylcarnitine increased by 90%, propionylcarnitine more than doubled, and 3HIAc increased by >10-fold in biotin-deficient, carnitine-sufficient (BDCS) cells, consistent with a defensive mechanism in which biotin-deficient cells transesterify the acyl-coenzyme A (acyl-CoA) substrates of the biotin-dependent carboxylases to the related acylcarnitines. Likewise, in BDCS cells, the ratio of acetylcarnitine to malonylcarnitine and the ratio of propionylcarnitine to methylmalonylcarnitine both more than tripled, and the ratio of 3HIAc to 3-methylglutarylcarnitine (MGc) increased by >10-fold. In biotin-deficient, carnitine-deficient (BDCD) cells, the 3 substrate-derived acylcarnitines changed little, but the substrate:product ratios were masked to a lesser extent. Moreover, carnitine repletion unmasked biotin deficiency in BDCD cells as shown by increases in acetylcarnitine, propionylcarnitine, and 3HIAc (each increased by >50-fold). Likewise, ratios of acetylcarnitine:malonylcarnitine, propionylcarnitine:methylmalonylcarnitine, and 3HIAc:MGc all increased by >8-fold. Our findings provide strong

  12. Medium composition influence on Biotin and Riboflavin production by newly isolated Candida sp.

    Science.gov (United States)

    Suzuki, Gaby Tiemi; Macedo, Juliana Alves; Macedo, Gabriela Alves

    2011-07-01

    Complex B vitamins as Biotin and Riboflavin are required by living organisms, not only for growth but also for metabolite production, and the feed market classifies them as growth promoters. Since Brazil will soon be one of the world's biggest animal protein producers, feed production is a large consumer of vitamins and micronutrients. The industry requires 10 mg riboflavin/0.2 mg biotin per kilogram of feed; a ratio of 40 ~ 50:1. Although few studies have been conducted specifically on riboflavin production using factorial design and surface response method as an optimization strategy, it is a common practice in biotechnology with many research reports available. However, there are no reports on the use of statistical design for biotin production. This study set out to evaluate medium composition influence on biotin and riboflavin production using a statistical design. There are no studies relating biotin and riboflavin production by Candida sp LEB 130. In this preliminary study to improve the simultaneous production of biotin and riboflavin, the maximum riboflavin/biotin ratio of 8.3 μg/mL was achieved with medium component concentrations of: sucrose 30 g/L, KH2PO4 2 g/L, MgSO4 1 g/L and ZnSO4 0.5mL/L.

  13. The Staphylococcus aureus group II biotin protein ligase BirA is an effective regulator of biotin operon transcription and requires the DNA binding domain for full enzymatic activity.

    Science.gov (United States)

    Henke, Sarah K; Cronan, John E

    2016-11-01

    Group II biotin protein ligases (BPLs) are characterized by the presence of an N-terminal DNA binding domain that functions in transcriptional regulation of the genes of biotin biosynthesis and transport. The Staphylococcus aureus Group II BPL which is called BirA has been reported to bind an imperfect inverted repeat located upstream of the biotin synthesis operon. DNA binding by other Group II BPLs requires dimerization of the protein which is triggered by synthesis of biotinoyl-AMP (biotinoyl-adenylate), the intermediate in the ligation of biotin to its cognate target proteins. However, the S. aureus BirA was reported to dimerize and bind DNA in the absence of biotin or biotinoyl-AMP (Soares da Costa et al. (2014) Mol Microbiol 91: 110-120). These in vitro results argued that the protein would be unable to respond to the levels of biotin or acceptor proteins and thus would lack the regulatory properties of the other characterized BirA proteins. We tested the regulatory function of the protein using an in vivo model system and examined its DNA binding properties in vitro using electrophoretic mobility shift and fluorescence anisotropy analyses. We report that the S. aureus BirA is an effective regulator of biotin operon transcription and that the prior data can be attributed to artifacts of mobility shift analyses. We also report that deletion of the DNA binding domain of the S. aureus BirA results in loss of virtually all of its ligation activity. © 2016 John Wiley & Sons Ltd.

  14. High dietary levels of biotin and zinc to improve health of foot pads in broilers exposed experimentally to litter with critical moisture content.

    Science.gov (United States)

    Abd El-Wahab, A; Radko, D; Kamphues, J

    2013-07-01

    Foot pad dermatitis (FPD) is a widespread problem in poultry production and constitutes a welfare issue. The objective of this study was to test potentially prophylactic effects of higher biotin and Zn levels in the diet of broilers exposed to critical litter moisture content (35% water) on the development of FPD. Two trials were performed in each 4 groups of 1-wk-old male broilers (Ross 708) during 33 d. The pens of all groups (25 birds in each) were littered with wood shavings of critical moisture content. Two groups were fed high levels of Zn as zinc-oxide (150 mg/kg of diet), with normal levels of biotin (300 µg/kg of diet) or high biotin (2,000 µg/kg of diet). The other 2 groups were fed Zn as zinc-methionine (150 mg/kg of diet), with normal levels of biotin (300 µg/kg of diet) or high biotin (2,000 µg/kg of diet). External assessment of foot pads and measurements the moisture contents of excreta and litter were performed weekly. The signs of foot pad lesions were recorded on a 7-point scale (0 = normal skin; 7 = more than half of the foot pad is necrotic). High biotin supplementation resulted in a reduction of 30 and 18% of cases of foot pad lesions in trials 1 and 2, respectively. The combination of Zn-methionine and high biotin supplementation led to a decreased severity of FPD in a range of about 50 and 30% in trials 1 and 2, respectively. In broilers fed the diet containing zinc-oxide and normal biotin levels about 28 and 24% of the birds had the scores of 6 and 7 (= high foot pad alterations), whereas in birds fed Zn-methionine and high biotin no high alterations (score = 7) in the foot pad (0%) occurred in either trial. The presented results suggest that it is advisable to combine the maximum levels of Zn (especially of Zn-methionine) and high levels of biotin when clinically relevant alterations in the foot pad occur.

  15. The biodistribution and kinetics of the 153Sm labelled avidin, streptavidin and biotin

    International Nuclear Information System (INIS)

    Li Guiping; Zhu Chengmo; Jiang Xufeng; Feng Guowei; Zhang Shengguo

    1999-01-01

    Due to the high affinity of biotin to Av or SA. The authors labelled a biotin derivative (DTPA-biotin) with 153 Sm and then bound this 153 Sm labelled DTPA-biotin to Av or SA. The in vivo kinetics and biodistribution of 153 Sm labelled Av, SA and DTPA-biotin were studied in the rat and mice. The results demonstrated that 153 Sm-Av cleared from the blood rapidly with high liver and renal uptake; 153 Sm-SA cleared from blood slowly with high retention in liver, spleen and kidney, whereas 153 Sm metabolize more fast, and excreted mainly through the kidney. Thereby, the biodistribution difference of SA and Av mentioned above provided an experimental basis for the selection of different components of A-V system in pre-targeting radio-immuno imaging and radioimmunotherapy

  16. Association of Biotin Ingestion With Performance of Hormone and Nonhormone Assays in Healthy Adults.

    Science.gov (United States)

    Li, Danni; Radulescu, Angela; Shrestha, Rupendra T; Root, Matthew; Karger, Amy B; Killeen, Anthony A; Hodges, James S; Fan, Shu-Ling; Ferguson, Angela; Garg, Uttam; Sokoll, Lori J; Burmeister, Lynn A

    2017-09-26

    Biotinylated antibodies and analogues, with their strong binding to streptavidin, are used in many clinical laboratory tests. Excess biotin in blood due to supplemental biotin ingestion may affect biotin-streptavidin binding, leading to potential clinical misinterpretation. However, the degree of interference remains undefined in healthy adults. To assess performance of specific biotinylated immunoassays after 7 days of ingesting 10 mg/d of biotin, a dose common in over-the-counter supplements for healthy adults. Nonrandomized crossover trial involving 6 healthy adults who were treated at an academic medical center research laboratory. Administration of 10 mg/d of biotin supplementation for 7 days. Analyte concentrations were compared with baseline (day 0) measures on the seventh day of biotin treatment and 7 days after treatment had stopped (day 14). The 11 analytes included 9 hormones (ie, thyroid-stimulating hormone, total thyroxine, total triiodothyronine, free thyroxine, free triiodothyronine, parathyroid hormone, prolactin, N-terminal pro-brain natriuretic peptide, 25-hydroxyvitamin D) and 2 nonhormones (prostate-specific antigen and ferritin). A total of 37 immunoassays for the 11 analytes were evaluated on 4 diagnostic systems, including 23 assays that incorporated biotin and streptavidin components and 14 assays that did not include biotin and streptavidin components and served as negative controls. Among the 2 women and 4 men (mean age, 38 years [range, 31-45 years]) who took 10 mg/d of biotin for 7 days, biotin ingestion-associated interference was found in 9 of the 23 (39%) biotinylated assays compared with none of the 14 nonbiotinylated assays (P = .007). Results from 5 of 8 biotinylated (63%) competitive immunoassays tested falsely high and results from 4 out of 15 (27%) biotinylated sandwich immunoassays tested falsely low. In this preliminary study of 6 healthy adult participants and 11 hormone and nonhormone analytes measured by 37 immunoassays

  17. False biochemical diagnosis of hyperthyroidism in streptavidin-biotin-based immunoassays: the problem of biotin intake and related interferences.

    Science.gov (United States)

    Piketty, Marie-Liesse; Polak, Michel; Flechtner, Isabelle; Gonzales-Briceño, Laura; Souberbielle, Jean-Claude

    2017-05-01

    Immunoassays are now commonly used for hormone measurement, in high throughput analytical platforms. Immunoassays are generally robust to interference. However, endogenous analytical error may occur in some patients; this may be encountered in biotin supplementation or in the presence of anti-streptavidin antibody, in immunoassays involving streptavidin-biotin interaction. In these cases, the interference may induce both false positive and false negative results, and simulate a seemingly coherent hormonal profile. It is to be feared that this type of errors will be more frequently observed. This review underlines the importance of keeping close interactions between biologists and clinicians to be able to correlate the hormonal assay results with the clinical picture.

  18. Efficient synthesis of a fluorine-18 labeled biotin derivative.

    Science.gov (United States)

    Claesener, Michael; Breyholz, Hans-Jörg; Hermann, Sven; Faust, Andreas; Wagner, Stefan; Schober, Otmar; Schäfers, Michael; Kopka, Klaus

    2012-11-01

    The natural occurring vitamin biotin, also known as vitamin H or vitamin B(7), plays a major role in various metabolic reactions. Caused by its high binding affinity to the protein avidin with a dissociation constant of about 10(-15)M the biotin-avidin system was extensively examined for multiple applications. We have synthesized a fluorine-18 labeled biotin derivative [(18)F]4 for a potential application in positron emission tomography (PET). Mesylate precursor 3 was obtained by an efficient two-step reaction via a copper catalyzed azide-alkyne cycloaddition (CuAAC) from easily accessible starting materials. [(18)F]4 was successfully synthesized by a nucleophilic radiofluorination of precursor 3. A biodistribution study by means of small-animal PET imaging in wt-mice was performed and serum stability was examined. Compound [(18)F]4 was obtained from precursor compound 3 with an average specific activity of 16GBq/μmol within 45min and a radiochemical yield of 45±5% (decay corrected). [(18)F]4 demonstrated only negligible decomposition in human serum. A qualitative binding study revealed the high affinity of the synthesized biotin derivative to avidin. Blocking experiments with native biotin showed that binding was site-specific. Biodistribution studies showed that [(18)F]4 was cleared quickly and efficiently from the body by hepatobiliary and renal elimination. An efficient synthesis for [(18)F]4 was established. In vivo characteristics were determined and demonstrated the pharmacokinetic behaviour of [(18)F]4. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. Semi-synthetic biotin imprinting onto avidin crosslinked gold-silver nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    At Latin-Small-Letter-Dotless-I l Latin-Small-Letter-Dotless-I r Oezcan, Ayca, E-mail: aatilir@anadolu.edu.tr; Ersoez, Arzu; Huer, Deniz; Y Latin-Small-Letter-Dotless-I lmaz, Filiz [Anadolu University, Department of Chemistry (Turkey); Gueltekin, Aytac [Karamanoglu Mehmetbey University, Department of Engineering of Energy Systems (Turkey); Denizli, Adil [Hacettepe University, Department of Chemistry (Turkey); Say, R Latin-Small-Letter-Dotless-I dvan [Anadolu University, Department of Chemistry (Turkey)

    2012-06-15

    This study is a different and new application of molecular imprinted polymers (MIPs) based on sensor technologies. In this study, semi-synthetic biotin imprinted polymeric shell has been decorated onto the surface of avidin crosslinked Au/Ag nanoclusters using bis (2-2 Prime -bipyridyl) MATyr-MATrp-ruthenium(II) (MATyr-Ru-MATrp) as photosensitive monomer. The synthesized nanoclusters have been used the recognition of biotin by flourometric method. Synthesis of the photosensitive monomers has been realized by AmiNoAcid (monomer) Decorated and Light Underpinning Conjugation Approach (ANADOLUCA) method. This method provides a strategy for the preparation of photosensitive ruthenium based aminoacid monomers and oligomers, aminoacid monomer-protein crosslinking using photosensitation and conjugation approach on micro and nano-structures by ruthenium-chelate based monomers. The affinity constant (K{sub a}) of biotin imprinted Au/Ag nanoclusters has been determined using the Scatchard method and found to be 3.89 Multiplication-Sign 10{sup 5} M{sup -1}. The obtained calibration graph is linear for the range of 0.051 and 2.50 {mu}M of biotin. The detection limit of biotin has been found to be 15 nM. Also, the reusability of these nanoclusters has been investigated and it has been observed that the same clusters could be used 10 times during a long period without any binding capacity decreasing.

  20. Semi-synthetic biotin imprinting onto avidin crosslinked gold–silver nanoparticles

    International Nuclear Information System (INIS)

    Atılır Özcan, Ayça; Ersöz, Arzu; Hür, Deniz; Yılmaz, Filiz; Gültekin, Aytaç; Denizli, Adil; Say, Rıdvan

    2012-01-01

    This study is a different and new application of molecular imprinted polymers (MIPs) based on sensor technologies. In this study, semi-synthetic biotin imprinted polymeric shell has been decorated onto the surface of avidin crosslinked Au/Ag nanoclusters using bis (2-2′-bipyridyl) MATyr-MATrp-ruthenium(II) (MATyr-Ru-MATrp) as photosensitive monomer. The synthesized nanoclusters have been used the recognition of biotin by flourometric method. Synthesis of the photosensitive monomers has been realized by AmiNoAcid (monomer) Decorated and Light Underpinning Conjugation Approach (ANADOLUCA) method. This method provides a strategy for the preparation of photosensitive ruthenium based aminoacid monomers and oligomers, aminoacid monomer-protein crosslinking using photosensitation and conjugation approach on micro and nano-structures by ruthenium-chelate based monomers. The affinity constant (K a ) of biotin imprinted Au/Ag nanoclusters has been determined using the Scatchard method and found to be 3.89 × 10 5 M −1 . The obtained calibration graph is linear for the range of 0.051 and 2.50 μM of biotin. The detection limit of biotin has been found to be 15 nM. Also, the reusability of these nanoclusters has been investigated and it has been observed that the same clusters could be used 10 times during a long period without any binding capacity decreasing.

  1. Deciphering a unique biotin scavenging pathway with redundant genes in the probiotic bacterium Lactococcus lactis.

    Science.gov (United States)

    Zhang, Huimin; Wang, Qingjing; Fisher, Derek J; Cai, Mingzhu; Chakravartty, Vandana; Ye, Huiyan; Li, Ping; Solbiati, Jose O; Feng, Youjun

    2016-05-10

    Biotin protein ligase (BPL) is widespread in the three domains of the life. The paradigm BPL is the Escherichia coli BirA protein, which also functions as a repressor for the biotin biosynthesis pathway. Here we report that Lactococcus lactis possesses two different orthologues of birA (birA1_LL and birA2_LL). Unlike the scenario in E. coli, L. lactis appears to be auxotrophic for biotin in that it lacks a full biotin biosynthesis pathway. In contrast, it retains two biotin transporter-encoding genes (bioY1_LL and bioY2_LL), suggesting the use of a scavenging strategy to obtain biotin from the environment. The in vivo function of the two L. lactis birA genes was judged by their abilities to complement the conditional lethal E. coli birA mutant. Thin-layer chromatography and mass spectroscopy assays demonstrated that these two recombinant BirA proteins catalyze the biotinylation reaction of the acceptor biotin carboxyl carrier protein (BCCP), through the expected biotinoyl-AMP intermediate. Gel shift assays were used to characterize bioY1_LL and BirA1_LL. We also determined the ability to uptake (3)H-biotin by L. lactis. Taken together, our results deciphered a unique biotin scavenging pathway with redundant genes present in the probiotic bacterium L. lactis.

  2. Chloride ion-dependent surface-enhanced Raman scattering study of biotin on the silver surface

    International Nuclear Information System (INIS)

    Liu Fangfang; Gu Huaimin; Yuan Xiaojuan; Dong Xiao; Lin Yue

    2011-01-01

    In the present paper, the surface enhanced Raman scattering (SERS) technique was employed to study the SERS spectra of biotin molecules formed on the silver surface. The adsorption geometries of biotin molecules on the silver surface were analyzed based on the SERS data. It can be found that most vibration modes show a Raman shift in silver sol after the addition of sodium chloride solution. In addition, The Raman signals of biotin become weaker and weaker with the increase of the concentration of sodium chloride. This may be due to that the interaction between chloride ions and silver particles is stronger than the interaction between biotin molecules and silver particles. When the concentration of sodium chloride in silver colloid is higher than 0.05mol/L, superfluous chloride ions may form an absorption layer so that biotin can not be adsorbed on silver surface directly. The changes in intensity and profile shape in the SERS spectra suggest different adsorption behavior and surface-coverage of biotin on silver surface. The SERS spectra of biotin suggest that the contribution of the charge transfer mechanism to SERS may be dominant.

  3. Intestinal Dysbiosis and Biotin Deprivation Induce Alopecia through Overgrowth of Lactobacillus murinus in Mice.

    Science.gov (United States)

    Hayashi, Atsushi; Mikami, Yohei; Miyamoto, Kentaro; Kamada, Nobuhiko; Sato, Toshiro; Mizuno, Shinta; Naganuma, Makoto; Teratani, Toshiaki; Aoki, Ryo; Fukuda, Shinji; Suda, Wataru; Hattori, Masahira; Amagai, Masayuki; Ohyama, Manabu; Kanai, Takanori

    2017-08-15

    Metabolism by the gut microbiota affects host physiology beyond the gastrointestinal tract. Here, we find that antibiotic-induced dysbiosis, in particular, overgrowth of Lactobacillus murinus (L. murinus), impaired gut metabolic function and led to the development of alopecia. While deprivation of dietary biotin per se did not affect skin physiology, its simultaneous treatment with vancomycin resulted in hair loss in specific pathogen-free (SPF) mice. Vancomycin treatment induced the accumulation of L. murinus in the gut, which consumes residual biotin and depletes available biotin in the gut. Consistently, L. murinus induced alopecia when monocolonized in germ-free mice fed a biotin-deficient diet. Supplementation of biotin can reverse established alopecia symptoms in the SPF condition, indicating that L. murinus plays a central role in the induction of hair loss via a biotin-dependent manner. Collectively, our results indicate that luminal metabolic alterations associated with gut dysbiosis and dietary modifications can compromise skin physiology. Copyright © 2017. Published by Elsevier Inc.

  4. Altered Regulation of Escherichia coli Biotin Biosynthesis in BirA Superrepressor Mutant Strains

    Science.gov (United States)

    Chakravartty, Vandana

    2012-01-01

    Transcription of the Escherichia coli biotin (bio) operon is directly regulated by the biotin protein ligase BirA, the enzyme that covalently attaches biotin to its cognate acceptor proteins. Binding of BirA to the bio operator requires dimerization of the protein, which is triggered by BirA-catalyzed synthesis of biotinoyl-adenylate (biotinoyl-5′-AMP), the obligatory intermediate of the ligation reaction. Although several aspects of this regulatory system are well understood, no BirA superrepressor mutant strains had been isolated. Such superrepressor BirA proteins would repress the biotin operon transcription in vivo at biotin concentrations well below those needed for repression by wild-type BirA. We isolated mutant strains having this phenotype by a combined selection-screening approach and resolved multiple mutations to give several birA superrepressor alleles, each having a single mutation, all of which showed repression dominant over that of the wild-type allele. All of these mutant strains repressed bio operon transcription in vivo at biotin concentrations that gave derepression of the wild-type strain and retained sufficient ligation activity for growth when overexpressed. All of the strains except that encoding G154D BirA showed derepression of bio operon transcription upon overproduction of a biotin-accepting protein. In BirA, G154D was a lethal mutation in single copy, and the purified protein was unable to transfer biotin from enzyme-bound biotinoyl-adenylate either to the natural acceptor protein or to a biotin-accepting peptide sequence. Consistent with the transcriptional repression data, each of the purified mutant proteins showed increased affinity for the biotin operator DNA in electrophoretic mobility shift assays. Surprisingly, although most of the mutations were located in the catalytic domain, all of those tested, except G154D BirA, had normal ligase activity. Most of the mutations that gave superrepressor phenotypes altered residues

  5. Plants cultivation in controlled containments

    International Nuclear Information System (INIS)

    2000-01-01

    The plants cultivation in controlled containments permits to the - Departement d'Ecophysiologie Vegetale et de Microbiologie (DVEM) - of the CEA to lead several topics of research. The works of DVEM which are based on the molecular labelling, technique adapted to plants, contribute to understand the plant - soil relationships and the plant growth process. In addition, the staff of DVEM study the impact of pollutant heavy metals, existing in the soil, on plants and the plant stress induced by oxygen, light, ionizing radiations,... and defence mechanisms of plants (F. M.)

  6. Biotin-Streptavidin Competition Mediates Sensitive Detection of Biomolecules in Enzyme Linked Immunosorbent Assay.

    Science.gov (United States)

    Lakshmipriya, Thangavel; Gopinath, Subash C B; Tang, Thean-Hock

    2016-01-01

    Enzyme Linked Immunosorbent Assay (ELISA) is the gold standard assay for detecting and identifying biomolecules using antibodies as the probe. Improving ELISA is crucial for detecting disease-causing agents and facilitating diagnosis at the early stages of disease. Biotinylated antibody and streptavidin-conjugated horse radish peroxide (streptavidin-HRP) often are used with ELISA to enhance the detection of various kinds of targets. In the present study, we used a competition-based strategy in which we pre-mixed free biotin with streptavidin-HRP to generate high-performance system, as free biotin occupies some of the biotin binding sites on streptavidin, thereby providing more chances for streptavidin-HRP to bind with biotinylated antibody. ESAT-6, which is a protein secreted early during tuberculosis infection, was used as the model target. We found that 8 fM of free biotin mixed with streptavidin-HRP anchored the higher detection level of ESAT-6 by four-fold compared with detection without free biotin (only streptavidin-HRP), and the limit of detection of the new method was 250 pM. These results suggest that biotin-streptavidin competition can be used to improve the diagnosis of analytes in other types of sensors.

  7. Biotin Switch Assays for Quantitation of Reversible Cysteine Oxidation.

    Science.gov (United States)

    Li, R; Kast, J

    2017-01-01

    Thiol groups in protein cysteine residues can be subjected to different oxidative modifications by reactive oxygen/nitrogen species. Reversible cysteine oxidation, including S-nitrosylation, S-sulfenylation, S-glutathionylation, and disulfide formation, modulate multiple biological functions, such as enzyme catalysis, antioxidant, and other signaling pathways. However, the biological relevance of reversible cysteine oxidation is typically underestimated, in part due to the low abundance and high reactivity of some of these modifications, and the lack of methods to enrich and quantify them. To facilitate future research efforts, this chapter describes detailed procedures to target the different modifications using mass spectrometry-based biotin switch assays. By switching the modification of interest to a biotin moiety, these assays leverage the high affinity between biotin and avidin to enrich the modification. The use of stable isotope labeling and a range of selective reducing agents facilitate the quantitation of individual as well as total reversible cysteine oxidation. The biotin switch assay has been widely applied to the quantitative analysis of S-nitrosylation in different disease models and is now also emerging as a valuable research tool for other oxidative cysteine modifications, highlighting its relevance as a versatile, robust strategy for carrying out in-depth studies in redox proteomics. © 2017 Elsevier Inc. All rights reserved.

  8. In HepG2 Cells, Coexisting Carnitine Deficiency Masks Important Indicators of Marginal Biotin Deficiency123

    Science.gov (United States)

    Bogusiewicz, Anna; Boysen, Gunnar; Mock, Donald M

    2015-01-01

    Background: A large number of birth defects are related to nutrient deficiencies; concern that biotin deficiency is teratogenic in humans is reasonable. Surprisingly, studies indicate that increased urinary 3-hydroxyisovalerylcarnitine (3HIAc), a previously validated marker of biotin deficiency, is not a valid biomarker in pregnancy. Objective: In this study we hypothesized that coexisting carnitine deficiency can prevent the increase in 3HIAc due to biotin deficiency. Methods: We used a 2-factor nutrient depletion design to induce isolated and combined biotin and carnitine deficiency in HepG2 cells and then repleted cells with carnitine. To elucidate the metabolic pathogenesis, we quantitated intracellular and extracellular free carnitine, acylcarnitines, and acylcarnitine ratios using liquid chromatography–tandem mass spectrometry. Results: Relative to biotin-sufficient, carnitine-sufficient cells, intracellular acetylcarnitine increased by 90%, propionylcarnitine more than doubled, and 3HIAc increased by >10-fold in biotin-deficient, carnitine-sufficient (BDCS) cells, consistent with a defensive mechanism in which biotin-deficient cells transesterify the acyl-coenzyme A (acyl-CoA) substrates of the biotin-dependent carboxylases to the related acylcarnitines. Likewise, in BDCS cells, the ratio of acetylcarnitine to malonylcarnitine and the ratio of propionylcarnitine to methylmalonylcarnitine both more than tripled, and the ratio of 3HIAc to 3-methylglutarylcarnitine (MGc) increased by >10-fold. In biotin-deficient, carnitine-deficient (BDCD) cells, the 3 substrate-derived acylcarnitines changed little, but the substrate:product ratios were masked to a lesser extent. Moreover, carnitine repletion unmasked biotin deficiency in BDCD cells as shown by increases in acetylcarnitine, propionylcarnitine, and 3HIAc (each increased by >50-fold). Likewise, ratios of acetylcarnitine:malonylcarnitine, propionylcarnitine:methylmalonylcarnitine, and 3HIAc:MGc all increased

  9. Use of biotin targeted methotrexate–human serum albumin conjugated nanoparticles to enhance methotrexate antitumor efficacy

    Science.gov (United States)

    Taheri, Azade; Dinarvand, Rassoul; Nouri, Faranak Salman; Khorramizadeh, Mohammad Reza; Borougeni, Atefeh Taheri; Mansoori, Pooria; Atyabi, Fatemeh

    2011-01-01

    Biotin molecules could be used as suitable targeting moieties in targeted drug delivery systems against tumors. To develop a biotin targeted drug delivery system, we employed human serum albumin (HSA) as a carrier. Methotrexate (MTX) molecules were conjugated to HSA. MTX-HSA nanoparticles (MTX-HSA NPs) were prepared from these conjugates by cross-linking the HSA molecules. Biotin molecules were then conjugated on the surface of MTX-HSA NPs. The anticancer efficacy of biotin targeted MTX-HSA NPs was evaluated in mice bearing 4T1 breast carcinoma. A single dose of biotin targeted MTX-HSA NPs showed stronger in vivo antitumor activity than non-targeted MTX-HSA NPs and free MTX. By 7 days after treatment, average tumor volume in the biotin targeted MTX-HSA NPs-treated group decreased to 17.6% of the initial tumor volume when the number of attached biotin molecules on MTX-HSA-NPs was the highest. Average tumor volume in non-targeted MTX-HSA NPs-treated mice grew rapidly and reached 250.7% of the initial tumor volume. Biotin targeted MTX-HSA NPs increased the survival of tumor-bearing mice to 47.5 ± 0.71 days and increased their life span up to 216.7%. Mice treated with biotin targeted MTX-HSA NPs showed slight body weight loss (8%) 21 days after treatment, whereas non-targeted MTX-HSA NPs treatment at the same dose caused a body weight loss of 27.05% ± 3.1%. PMID:21931482

  10. Psychological Assessment of Patients With Biotin-Thiamine-Responsive Basal Ganglia Disease.

    Science.gov (United States)

    Alfadhel, Majid; Al-Bluwi, Amal

    2017-01-01

    Biotin-thiamine-responsive basal ganglia disease is a devastating autosomal recessive inherited neurological disorder. We conducted a retrospective chart review of all patients with biotin-thiamine-responsive basal ganglia disease who underwent a formal psychological assessment. Six females and 3 males were included. Five patients (56%) had an average IQ, two patients (22%) had mild delay, and two (22%) had severe delay. A normal outcome was directly related to the time of diagnosis and initiation of treatment. Early diagnosis and immediate commencement of treatment were associated with a favorable outcome and vice versa. The most affected domain was visual motor integration, while understanding and mathematical problem-solving were the least affected. In summary, this is the first study discussing the psychological assessment of patients with biotin-thiamine-responsive basal ganglia disease. The results of this study alert clinicians to consider prompt initiation of biotin and thiamine in any patient presenting with neuroregression and a basal ganglia lesion on a brain magnetic resonance imaging.

  11. Characterization of the scope and magnitude of biotin interference in susceptible Roche Elecsys competitive and sandwich immunoassays.

    Science.gov (United States)

    Trambas, Christina; Lu, Zhong; Yen, Tina; Sikaris, Ken

    2018-03-01

    Background Biotin interference is a significant problem to which at-risk laboratories must now be attuned. We sought to systematically characterize the nature of this interference in Roche immunoassays. Methods Known concentrations of biotin were titrated into serum samples and the effects on competitive and sandwich immunoassays were analysed. The maximum and minimum concentrations examined reflect those likely to be achieved in individuals on 5 to 10 mg supplements at the lower end, and 100 to 300 mg biotin at the high end. Results A high variability in biotin tolerance was observed. Some assays, such as troponin T, TSH and antithyroid antibodies, were extremely sensitive to the lower concentrations of biotin (15.6 and 31.3 ng/mL), whereas the majority of assays were relatively resistant. At concentrations ≥500 ng/mL, all assays showed significant interference from biotin but, again, the magnitude of the interference was variable. The more sensitive assays showed profound analytical bias at biotin concentrations that occur with high-dose therapy. Conclusion Our data demonstrate high variability in biotin tolerance across Roche immunoassays. The shape of the dose-response curves provides more detailed information than the single manufacturer-quoted figure for biotin tolerance. Accordingly, these data may be used by laboratories for more accurate risk assessment in predicting the effects of biotin. Our data may also be extrapolated to guide timing of blood tests in patients on high-dose biotin therapy: it demonstrates the number of half-lives required to withhold biotin in order to decrease its concentration to below a given assay tolerance.

  12. Biotin deprivation impairs mitochondrial structure and function and has implications for inherited metabolic disorders.

    Science.gov (United States)

    Ochoa-Ruiz, Estefanía; Díaz-Ruiz, Rodrigo; Hernández-Vázquez, Alaín de J; Ibarra-González, Isabel; Ortiz-Plata, Alma; Rembao, Daniel; Ortega-Cuéllar, Daniel; Viollet, Benoit; Uribe-Carvajal, Salvador; Corella, José Ahmed; Velázquez-Arellano, Antonio

    2015-11-01

    Certain inborn errors of metabolism result from deficiencies in biotin containing enzymes. These disorders are mimicked by dietary absence or insufficiency of biotin, ATP deficit being a major effect,whose responsible mechanisms have not been thoroughly studied. Here we show that in rats and cultured cells it is the result of reduced TCA cycle flow, partly due to deficient anaplerotic biotin-dependent pyruvate carboxylase. This is accompanied by diminished flow through the electron transport chain, augmented by deficient cytochrome c oxidase (complex IV) activity with decreased cytochromes and reduced oxidative phosphorylation. There was also severe mitochondrial damage accompanied by decrease of mitochondria, associated with toxic levels of propionyl CoA as shown by carnitine supplementation studies, which explains the apparently paradoxical mitochondrial diminution in the face of the energy sensor AMPK activation, known to induce mitochondria biogenesis. This idea was supported by experiments on AMPK knockout mouse embryonic fibroblasts (MEFs). The multifactorial ATP deficit also provides a plausible basis for the cardiomyopathy in patients with propionic acidemia, and other diseases.Additionally, systemic inflammation concomitant to the toxic state might explain our findings of enhanced IL-6, STAT3 and HIF-1α, associated with an increase of mitophagic BNIP3 and PINK proteins, which may further increase mitophagy. Together our results imply core mechanisms of energy deficit in several inherited metabolic disorders.

  13. Optimisation of culture conditions with respect to biotin requirement for the production of recombinant avidin in Pichia pastoris.

    Science.gov (United States)

    Jungo, Carmen; Urfer, Julien; Zocchi, Andrea; Marison, Ian; von Stockar, Urs

    2007-01-20

    Due to its very high affinity to biotin, avidin is one of the most widely exploited proteins in modern biotechnological and biomedical applications. Since biotin is an essential vitamin for the growth of many microorganisms, we examined the effect of biotin deficiency on growth for a recombinant Pichia pastoris strain expressing and secreting a recombinant glycosylated avidin. The results showed that biotin deficiency lowers growth rate and biomass yield for P. pastoris. Substitution of biotin in the medium by the two structurally unrelated compounds, aspartic acid and oleic acid, which do not bind to recombinant avidin was analyzed quantitatively. These two compounds had a growth promoting effect in biotin-deficient medium, but did not replace biotin completely. Indeed, in chemostat culture, wash-out occurred after about six liquid residence times and recombinant avidin productivity was lowered. However, addition of low amounts of biotin (20 microg L(-1) of biotin for a cell density of 8 g L(-1)) resulted in stable chemostat cultures on methanol with the production of recombinant biotin-free avidin. The specific avidin production rate was 22 microg g(-1) h(-1) at a dilution rate of 0.06 h(-1).

  14. Selective inhibition of Biotin Protein Ligase from Staphylococcus aureus*

    Science.gov (United States)

    Soares da Costa, Tatiana P.; Tieu, William; Yap, Min Y.; Pendini, Nicole R.; Polyak, Steven W.; Sejer Pedersen, Daniel; Morona, Renato; Turnidge, John D.; Wallace, John C.; Wilce, Matthew C. J.; Booker, Grant W.; Abell, Andrew D.

    2012-01-01

    There is a well documented need to replenish the antibiotic pipeline with new agents to combat the rise of drug resistant bacteria. One strategy to combat resistance is to discover new chemical classes immune to current resistance mechanisms that inhibit essential metabolic enzymes. Many of the obvious drug targets that have no homologous isozyme in the human host have now been investigated. Bacterial drug targets that have a closely related human homologue represent a new frontier in antibiotic discovery. However, to avoid potential toxicity to the host, these inhibitors must have very high selectivity for the bacterial enzyme over the human homolog. We have demonstrated that the essential enzyme biotin protein ligase (BPL) from the clinically important pathogen Staphylococcus aureus could be selectively inhibited. Linking biotin to adenosine via a 1,2,3 triazole yielded the first BPL inhibitor selective for S. aureus BPL over the human equivalent. The synthesis of new biotin 1,2,3-triazole analogues using click chemistry yielded our most potent structure (Ki 90 nm) with a >1100-fold selectivity for the S. aureus BPL over the human homologue. X-ray crystallography confirmed the mechanism of inhibitor binding. Importantly, the inhibitor showed cytotoxicity against S. aureus but not cultured mammalian cells. The biotin 1,2,3-triazole provides a novel pharmacophore for future medicinal chemistry programs to develop this new antibiotic class. PMID:22437830

  15. Selective inhibition of biotin protein ligase from Staphylococcus aureus.

    Science.gov (United States)

    Soares da Costa, Tatiana P; Tieu, William; Yap, Min Y; Pendini, Nicole R; Polyak, Steven W; Sejer Pedersen, Daniel; Morona, Renato; Turnidge, John D; Wallace, John C; Wilce, Matthew C J; Booker, Grant W; Abell, Andrew D

    2012-05-18

    There is a well documented need to replenish the antibiotic pipeline with new agents to combat the rise of drug resistant bacteria. One strategy to combat resistance is to discover new chemical classes immune to current resistance mechanisms that inhibit essential metabolic enzymes. Many of the obvious drug targets that have no homologous isozyme in the human host have now been investigated. Bacterial drug targets that have a closely related human homologue represent a new frontier in antibiotic discovery. However, to avoid potential toxicity to the host, these inhibitors must have very high selectivity for the bacterial enzyme over the human homolog. We have demonstrated that the essential enzyme biotin protein ligase (BPL) from the clinically important pathogen Staphylococcus aureus could be selectively inhibited. Linking biotin to adenosine via a 1,2,3 triazole yielded the first BPL inhibitor selective for S. aureus BPL over the human equivalent. The synthesis of new biotin 1,2,3-triazole analogues using click chemistry yielded our most potent structure (K(i) 90 nM) with a >1100-fold selectivity for the S. aureus BPL over the human homologue. X-ray crystallography confirmed the mechanism of inhibitor binding. Importantly, the inhibitor showed cytotoxicity against S. aureus but not cultured mammalian cells. The biotin 1,2,3-triazole provides a novel pharmacophore for future medicinal chemistry programs to develop this new antibiotic class.

  16. Engineering of a Bacillus subtilis strain with adjustable levels of intracellular biotin for secretory production of functional streptavidin.

    Science.gov (United States)

    Wu, Sau-Ching; Wong, Sui-Lam

    2002-03-01

    Streptavidin is a biotin-binding protein which has been widely used in many in vitro and in vivo applications. Because of the ease of protein recovery and availability of protease-deficient strains, the Bacillus subtilis expression-secretion system is an attractive system for streptavidin production. However, attempts to produce streptavidin using B. subtilis face the problem that cells overproducing large amounts of streptavidin suffer poor growth, presumably because of biotin deficiency. This problem cannot be solved by supplementing biotin to the culture medium, as this will saturate the biotin binding sites in streptavidin. We addressed this dilemma by engineering a B. subtilis strain (WB800BIO) which overproduces intracellular biotin. The strategy involves replacing the natural regulatory region of the B. subtilis chromosomal biotin biosynthetic operon (bioWAFDBIorf2) with an engineered one consisting of the B. subtilis groE promoter and gluconate operator. Biotin production in WB800BIO is induced by gluconate, and the level of biotin produced can be adjusted by varying the gluconate dosage. A level of gluconate was selected to allow enhanced intracellular production of biotin without getting it released into the culture medium. WB800BIO, when used as a host for streptavidin production, grows healthily in a biotin-limited medium and produces large amounts (35 to 50 mg/liter) of streptavidin, with over 80% of its biotin binding sites available for future applications.

  17. Biotin status affects nickel allergy via regulation of interleukin-1beta production in mice.

    Science.gov (United States)

    Kuroishi, Toshinobu; Kinbara, Masayuki; Sato, Naoki; Tanaka, Yukinori; Nagai, Yasuhiro; Iwakura, Yoichiro; Endo, Yasuo; Sugawara, Shunji

    2009-05-01

    Biotin, a water-soluble B complex vitamin, is possibly involved in chronic inflammatory diseases, although the detailed mechanisms are unclear. In this study, we investigated the effects of biotin status on nickel (Ni) allergy in mice. Mice were fed a basal or biotin-deficient (BD) diet for 8 wk and sensitized with an intraperitoneal injection of NiCl(2) and lipopolysaccharide. Ten days after sensitization, NiCl(2) was intradermally injected into pinnas and ear swelling was measured. For in vitro analysis, we cultured a murine macrophage cell line, J774.1, under a biotin-sufficient (C, meaning control) or BD condition for 4 wk and analyzed interleukin (IL)-1 production. Significantly higher ear swelling was induced in BD mice than C mice. Adaptive transfer of splenocytes from both C and BD mice induced Ni allergy in unsensitized mice. Regardless of donor mice, ear swelling was significantly higher in BD recipient mice than C recipient mice. Ni allergy was not induced in either C or BD IL-1(-/-) mice. Splenocytes from BD mice produced a significantly higher amount of IL-1beta than those from C mice. Production and mRNA expression of IL-1beta were significantly higher in BD J774.1 cells than in C cells. Biotin supplementation inhibited the augmentation of IL-1beta production in vitro. In vivo supplementation of biotin in drinking water dose-dependently decreased ear swelling in C and BD mice. These results indicate that biotin status affects Ni allergy in the elicitation phase via the upregulation of IL-1beta production in mice, suggesting that biotin supplementation may have therapeutic effects on human metal allergy.

  18. Novel biosensor system model based on fluorescence quenching by a fluorescent streptavidin and carbazole-labeled biotin.

    Science.gov (United States)

    Zhu, Xianwei; Shinohara, Hiroaki; Miyatake, Ryuta; Hohsaka, Takahiro

    2016-10-01

    In the present study, a novel molecular biosensor system model was designed by using a couple of the fluorescent unnatural mutant streptavidin and the carbazole-labeled biotin. BODIPY-FL-aminophenylalanine (BFLAF), a fluorescent unnatural amino acid was position-specifically incorporated into Trp120 position of streptavidin by four-base codon method. On the other hand, carbazole-labeled biotin was synthesized as a quencher for the fluorescent Trp120BFLAF mutant streptavidin. The fluorescence of fluorescent Trp120BFLAF mutant streptavidin was decreased as we expected when carbazole-labeled biotin was added into the mutant streptavidin solution. Furthermore, the fluorescence decrease of Trp120BFLAF mutant streptavidin with carbazole-labeled biotin (100 nM) was recovered by the competitive addition of natural biotin. This result demonstrated that by measuring the fluorescence quenching and recovery, a couple of the fluorescent Trp120BFLAF mutant streptavidin and the carbazole-labeled biotin were successfully applicable for quantification of free biotin as a molecular biosensor system. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  19. One-step radiolabelled biotin scintigraphy in patients with suspected vertebral infections

    International Nuclear Information System (INIS)

    Lazzeri, E.; Erba, P.; Chinol, M.; Tascini, C.; Menichetti, F.; Paganelli, G.; Mariani, G.

    2003-01-01

    Full text: Biotin (B), or vitamin H, belonging to the B-complex group is utilized by bacteria for acid synthesis by acetyl-CoA carboxylase. We evaluated the diagnostic potential per se of radiolabeled biotin without avidin pre-targeting, in patients with suspected vertebral bacterial infections. We evaluated 31 patients for suspected spine infection. All patients were selected on clinical ground, blood chemistry findings, back pain and fever. Patients were injected i.v. with 500 μg of DTPA-conjugated Biotin labelled with 111In (110-130 MBq); planar and SPECT scans were recorded starting 90 min post-injection. All patients underwent MR, or CT and some patients were imaged with either 99mTc-HMPAO-WBC and/or 67Ga-citrate. Diagnostic-quality imaging was obtained at 90 min and 4 hr after 111In-DTPA-Biotin injection. We observed only 2 false-negative results, while 24 studies were true-positive (4 performed during follow-up) and 10 true-negative (1 during follow-up) (91% sensitivity, 100% specificity). Either MR, CT or 99mTc-HMPAO-WBC had high proportions of either false-negative, false-positive or equivocal results (sensitivity/specificity around 50%). These preliminary results outline the high diagnostic potential of one-step 111In-DTPA-Biotin scintigraphy without avidin pre-targeting) in patients with suspected vertebral infection. The high true-positive and true-negative rate suggests that this system displays some specificity for bacterial infections. The high diagnostic accuracy of 111In-Biotin scintigraphy seems to be independent from antibiotic therapy, thus making this method very helpful relative to other imaging modalities in the clinical decision on starting proper therapy and for monitoring the efficacy of treatment. (author)

  20. Endogenous biotin expression in renal and testicular tumours and literature review.

    Science.gov (United States)

    Fahmy, Nader; Woo, Mark; Alameldin, Mona; Lee, Joe King; MacDonald, Kyle; Goneau, Lee W; Cadieux, Peter; Burton, Jeremy; Pautler, Stephen

    2014-07-01

    The aim of this study was to examine endogenous biotin levels in tumour specimens collected from patients with renal and testicular tumours and compare them to the surrounding non-neoplastic surgical margin. Frozen samples were obtained from the Ontario Tumour Bank. Renal and testicular tumour tissue were included in this study. Normal tissue from the negative surgical margins of each tumour served as a control. Biotin detection in tissue specimens was determined using immunohistochemistry (IHC). Specimens collected from 56 patients (36 men and 20 women) were included in this study. Histopathology of the 52 renal tumours included 31 (60%) conventional type RCC, 5 (10%) chromophobe RCC, 5 (10%) papillary RCC, 1 (2%) oncocytoma and 10 (19%) upper tract urothelial carcinoma (UC). The 4 testicular tumours included 1 seminomatous (25%) germ cell tumour and 3 (75%) non seminomatous germ cell tumours. No biotin signal was perceived in all tested tumour samples. Endogenous biotin expression was detected in the matching non-neoplastic surgical margin of tested renal tissues. This lack of staining may prove to be a valuable tool in future studies.

  1. Highly stabilized and photoluminescence enhancement of ZnS:Mn2+ nanoparticles in biotin matrix

    International Nuclear Information System (INIS)

    Keshari, Ashish K.; Pandey, Avinash C.

    2009-01-01

    We synthesized the ZnS:Mn 2+ nanoparticles passivated by biocompatible layer, namely, biotin by chemical precipitation route and studied their temporal evolution for size, structure, optical, and photoluminescence stability. To monitor the structural and optoelectronic properties of the nanoparticles with time, we have characterized the grown product by x-ray diffraction, small angle x-ray scattering, UV visible, and photoluminescence spectroscopic techniques at a regular interval for a period of three months. Results showed that the properties of nanophosphors capped with biotin are remaining the same even after 3 months. Energy dispersive x-ray analysis of 3 month aged sample shows long time compatibility between ZnS:Mn 2+ nanoparticles and the biotin. This is also confirmed by electron microscopy that the growth of the nanoparticles is strongly arrested by the biotin. X-ray photoelectron spectra were also recorded to show the chemical state of the elements. Enhanced ratio of Zn 2p to Mn 2p peaks in the x-ray photoelectron spectra of ZnS:Mn 2+ nanoparticles shows that the Mn 2+ ions are incorporated within ZnS host matrix. We found that biotin capping will enhance the luminescence from ZnS:Mn 2+ nanoparticles as compared to without capped particles. Absence of biotin will gradually degrade the luminescence upon aging while drastic degradation in luminescence intensity was observed after annealing. Properties show that biotin also protected the nanoparticles from any environmental attack

  2. Biotin enhances ATP synthesis in pancreatic islets of the rat, resulting in reinforcement of glucose-induced insulin secretion.

    Science.gov (United States)

    Sone, Hideyuki; Sasaki, Yuka; Komai, Michio; Toyomizu, Masaaki; Kagawa, Yasuo; Furukawa, Yuji

    2004-02-13

    Previous studies showed that biotin enhanced glucose-induced insulin secretion. Changes in the cytosolic ATP/ADP ratio in the pancreatic islets participate in the regulation of insulin secretion by glucose. In the present study we investigated whether biotin regulates the cytosolic ATP/ADP ratio in glucose-stimulated islets. When islets were stimulated with glucose plus biotin, the ATP/ADP ratio increased to approximately 160% of the ATP/ADP ratio in islets stimulated with glucose alone. The rate of glucose oxidation, assessed by CO(2) production, was also about 2-fold higher in islets treated with biotin. These increasing effects of biotin were proportional to the effects seen in insulin secretion. There are no previous reports of vitamins, such as biotin, directly affecting ATP synthesis. Our data indicate that biotin enhances ATP synthesis in islets following the increased rate of substrate oxidation in mitochondria and that, as a consequence of these events, glucose-induced insulin release is reinforced by biotin.

  3. Radioimmunoassay for chicken avidin. Comparison with a (/sup 14/C)biotin-binding method

    Energy Technology Data Exchange (ETDEWEB)

    Kulomaa, M S; Elo, H A; Tuohimaa, P J [Tampere Univ. of Tech. (Finland)

    1978-11-01

    A double-antibody solid-phase radioimmunoassay for chicken avidin is reported. Avidin was labelled with /sup 125/I by the chloramine-T method. The bound and free avidin were separated with a second antibody bound to a solid matrix. In the logit-log scale the standard curve was linear from 1-2 to 100-200ng of avidin/ml. Cross-reaction of ovalbumin was less than 0.015%. Saturation of biotin-binding sites of avidin with an excess of biotin decreased radioimmunoassay values by about 15%. Recovery studies indicated that avidin can be assayed from all chicken tissues studied with radioimmunoassay, whereas the (/sup 14/C)biotin/bentonite method gave poor recoveries for avidin in the liver and kidney. Radioimmunoassay and the (/sup 14/C)biotin/bentonite method gave similar concentrations for oviduct avidin.

  4. The enzymes of biotin dependent CO2 metabolism: What structures reveal about their reaction mechanisms

    Science.gov (United States)

    Waldrop, Grover L; Holden, Hazel M; Maurice, Martin St

    2012-01-01

    Biotin is the major cofactor involved in carbon dioxide metabolism. Indeed, biotin-dependent enzymes are ubiquitous in nature and are involved in a myriad of metabolic processes including fatty acid synthesis and gluconeogenesis. The cofactor, itself, is composed of a ureido ring, a tetrahydrothiophene ring, and a valeric acid side chain. It is the ureido ring that functions as the CO2 carrier. A complete understanding of biotin-dependent enzymes is critically important for translational research in light of the fact that some of these enzymes serve as targets for anti-obesity agents, antibiotics, and herbicides. Prior to 1990, however, there was a dearth of information regarding the molecular architectures of biotin-dependent enzymes. In recent years there has been an explosion in the number of three-dimensional structures reported for these proteins. Here we review our current understanding of the structures and functions of biotin-dependent enzymes. In addition, we provide a critical analysis of what these structures have and have not revealed about biotin-dependent catalysis. PMID:22969052

  5. Preparation of 166 Dy/166 Ho DTPA-bis biotin as a system of In vivo generator

    International Nuclear Information System (INIS)

    Jimenez V, M.R.

    2003-01-01

    The objective of this work was to synthesize the complex 166 Dy/ 166 Ho - diethylen triamine pentaacetic-bis Biotin ( 166 Dy/ 166 Ho DTPA-bis Biotin) to evaluate its potential as a new radiopharmaceutical in directed radiotherapy. The Dysprosium-166 was obtained for neutron irradiation of 164 Dy 2 0 3 in the TRIGA Mark III reactor. The labelled was carried out in aqueous solution to p H 8.0 for addition of 166 Dy Cl 3 to the diethylen triamine pentaacetic-α, ω-bis Biotin (DTPA-bis Biotin). The radiochemical purity was determined for HPLC and ITLC. The biological integrity of the marked biotin is evaluated by the biological recognition of the avidin for HPLC - molecular exclusion with and without avidin addition. The studies of stability in vitro were made in dilutions of saline solution to 0.9% and with human serum at 37 C incubated 1 and 24 hours. The complex 166 Dy/ 166 Ho DTPA-bis Biotin was obtained with a radiochemical purity of 99.1 ± 0.6%. The biological recognition of the complex 166 Dy/ 166 Ho DTPA-bis Biotin for the avidin it doesn't affect the labelling procedure. The studies in vitro demonstrated that the 166 Dy/ 166 Ho DTPA-bis Biotin is stable after the dilution in saline solution and in human serum that there is not translocation of the one radionuclide subsequent son to the beta decay of the 166 Dy that could produce the 166 Ho 3+ liberation. The studies of Biodistribution in healthy mice demonstrated that the one complex 166 Dy/ 166 Ho DTPA-bis Biotin have a high renal distribution. In conclusion the radiolabelled biotin in this investigation has the appropriate properties to be used as an In vivo generator system stable for directed radiotherapy. (Author)

  6. Structural characterization of core-bradavidin in complex with biotin

    Science.gov (United States)

    Agrawal, Nitin; Määttä, Juha A. E.; Kulomaa, Markku S.; Hytönen, Vesa P.; Johnson, Mark S.; Airenne, Tomi T.

    2017-01-01

    Bradavidin is a tetrameric biotin-binding protein similar to chicken avidin and bacterial streptavidin, and was originally cloned from the nitrogen-fixing bacteria Bradyrhizobium diazoefficiens. We have previously reported the crystal structure of the full-length, wild-type (wt) bradavidin with 138 amino acids, where the C-terminal residues Gly129-Lys138 (“Brad-tag”) act as an intrinsic ligand (i.e. Gly129-Lys138 bind into the biotin-binding site of an adjacent subunit within the same tetramer) and has potential as an affinity tag for biotechnological purposes. Here, the X-ray structure of core-bradavidin lacking the C-terminal residues Gly114-Lys138, and hence missing the Brad-tag, was crystallized in complex with biotin at 1.60 Å resolution [PDB:4BBO]. We also report a homology model of rhodavidin, an avidin-like protein from Rhodopseudomonas palustris, and of an avidin-like protein from Bradyrhizobium sp. Ai1a-2, both of which have the Brad-tag sequence at their C-terminus. Moreover, core-bradavidin V1, an engineered variant of the original core-bradavidin, was also expressed at high levels in E. coli, as well as a double mutant (Cys39Ala and Cys69Ala) of core-bradavidin (CC mutant). Our data help us to further engineer the core-bradavidin–Brad-tag pair for biotechnological assays and chemical biology applications, and provide deeper insight into the biotin-binding mode of bradavidin. PMID:28426764

  7. Structural characterization of core-bradavidin in complex with biotin.

    Directory of Open Access Journals (Sweden)

    Nitin Agrawal

    Full Text Available Bradavidin is a tetrameric biotin-binding protein similar to chicken avidin and bacterial streptavidin, and was originally cloned from the nitrogen-fixing bacteria Bradyrhizobium diazoefficiens. We have previously reported the crystal structure of the full-length, wild-type (wt bradavidin with 138 amino acids, where the C-terminal residues Gly129-Lys138 ("Brad-tag" act as an intrinsic ligand (i.e. Gly129-Lys138 bind into the biotin-binding site of an adjacent subunit within the same tetramer and has potential as an affinity tag for biotechnological purposes. Here, the X-ray structure of core-bradavidin lacking the C-terminal residues Gly114-Lys138, and hence missing the Brad-tag, was crystallized in complex with biotin at 1.60 Å resolution [PDB:4BBO]. We also report a homology model of rhodavidin, an avidin-like protein from Rhodopseudomonas palustris, and of an avidin-like protein from Bradyrhizobium sp. Ai1a-2, both of which have the Brad-tag sequence at their C-terminus. Moreover, core-bradavidin V1, an engineered variant of the original core-bradavidin, was also expressed at high levels in E. coli, as well as a double mutant (Cys39Ala and Cys69Ala of core-bradavidin (CC mutant. Our data help us to further engineer the core-bradavidin-Brad-tag pair for biotechnological assays and chemical biology applications, and provide deeper insight into the biotin-binding mode of bradavidin.

  8. Biotin protein ligase from Candida albicans: expression, purification and development of a novel assay.

    Science.gov (United States)

    Pendini, Nicole R; Bailey, Lisa M; Booker, Grant W; Wilce, Matthew C J; Wallace, John C; Polyak, Steven W

    2008-11-15

    Biotin protein ligase (BPL) is an essential enzyme responsible for the activation of biotin-dependent enzymes through the covalent attachment of biotin. In yeast, disruption of BPL affects important metabolic pathways such as fatty acid biosynthesis and gluconeogenesis. This makes BPL an attractive drug target for new antifungal agents. Here we report the cloning, recombinant expression and purification of BPL from the fungal pathogen Candida albicans. The biotin domains of acetyl CoA carboxylase and pyruvate carboxylase were also cloned and characterised as substrates for BPL. A novel assay was established thereby allowing examination of the enzyme's properties. These findings will facilitate future structural studies as well as screening efforts to identify potential inhibitors.

  9. Infection imaging with 99mTc-biotin in patients with prosthetic hip replacement

    International Nuclear Information System (INIS)

    Villa, G.; Mariani, G.; Augeri, C.; Pipino, F.; Paganelli, G.; Chinol, M.

    2002-01-01

    Full text: Although the incidence of infection in prosthetic hip joint replacements has decreased from about 10-15 % to about 0.5-2 % over the last 20 years, the total number of infections has actually increased because of the large number of patients undergoing the procedure. The most frequent clinical presentation of this complication is functional impairment and pain, with or without fever and other signs and/or symptoms of infection. The main is differentiating true infection from simple loosening with inflammation of the implanted stem. Scintigraphy with radiolabeled autologous leukocytes (WBC) represents the 'gold standard' nuclear medicine procedure for imaging infection. However, this procedure is time-consuming, expensive, and involves some biological hazard. Preliminary data, obtained during validation of the avidin/111In-biotin approach, have suggested some potential of 111ln-biotin per se to accumulate at sites of infection. In this pilot study we explored the potential of 99mTc-biotin as an infection imaging agent in pts with orthopedic infections. N4-lys-biotin was labeled with 1110 MBq. Sixteen pts bearing a total of 20 prosthetic hip replacements were enrolled in the study (9 women and 7 men, mean age 73.2 yrs). Eight pts had previously undergone removal of their hip prosthesis because of infection, while infection was suspected in the remaining 8 pts. Scintigraphy was recorded 20 min, then 1, 4 and 24 hr after the i.v. injection of 99mTc-biotin. Within 48 hrs of the 99mTc-biotin study, all pts also underwent scintigraphy with 99mTc-HMPAO-WBC. Out of the 20 hips evaluated, 15 turned out to be infected while in the remaining 5 cases pain was only caused by bone-prosthetic loosening and/or conditions other than infection. In 12/15 infected sites scintigraphy was concordantly positive with both procedures, 99mTc-biotin yielding higher target-to-nontarget ratios than 99mTc-HMPAO-WBC in 4 cases and similar values in the other cases. Discordant patterns

  10. Preparation and in vitro evaluation of ''no-carrier-added'' 18F-labeled biotin

    International Nuclear Information System (INIS)

    Najafi, A.; Peterson, A.

    1993-01-01

    This paper will describe the preparation of ''no-carrieradded'' 18 F-labeled biotin where the radiolabel bound to an aromatic moiety is described. This has been accomplished by preparation of [ 18 F]fluoro-benzylbromide (yield 20-30%) and its reaction with biotin-LC-hydrazide. This yielded ''no-carrier-added'' radiolabeled biotin (5-10%) which was then purified by reversed phase HPLC. The pure product was found to bind to avidin, thereby demonstrating retention of its biological integrity. Thus this product is potentially useful for imaging tumor tissue following injection of avidin coupled MoAbs. (author)

  11. Preparation and in vitro evaluation of ''no-carrier-added'' 18F-labeled biotin

    International Nuclear Information System (INIS)

    Najafi, A.; Peterson, A.

    1993-01-01

    This paper will describe the preparation of ''no-carrieradded'' 18 F-labeled biotin where the radiolabel bound to an aromatic moiety is described. This has been accomplished by preparation of [ 18 F]fluorobenzylbromide (yield 20-30%) and its reaction with biotin-LC-hydrazide. This yielded ''nocarrier-added'' radiolabeled biotin(5-10%) which was then purified by reversed phase HPLC. The pure product was found to bind to avidin, thereby demonstrating retention of its biological integrity. Thus this product is potentially useful for imaging tumor tissue following injection of avidin coupled MoAbs. (Author)

  12. A Canonical Biotin Synthesis Enzyme, 8-Amino-7-Oxononanoate Synthase (BioF), Utilizes Different Acyl Chain Donors in Bacillus subtilis and Escherichia coli.

    Science.gov (United States)

    Manandhar, Miglena; Cronan, John E

    2018-01-01

    BioF (8-amino-7-oxononanoate synthase) is a strictly conserved enzyme that catalyzes the first step in assembly of the fused heterocyclic rings of biotin. The BioF acyl chain donor has long been thought to be pimeloyl-CoA. Indeed, in vitro the Escherichia coli and Bacillus sphaericus enzymes have been shown to condense pimeloyl-CoA with l-alanine in a pyridoxal 5'-phosphate-dependent reaction with concomitant CoA release and decarboxylation of l-alanine. However, recent in vivo studies of E. coli and Bacillus subtilis suggested that the BioF proteins of the two bacteria could have different specificities for pimelate thioesters in that E. coli BioF may utilize either pimeloyl coenzyme A (CoA) or the pimelate thioester of the acyl carrier protein (ACP) of fatty acid synthesis. In contrast, B. subtilis BioF seemed likely to be specific for pimeloyl-CoA and unable to utilize pimeloyl-ACP. We now report genetic and in vitro data demonstrating that B. subtilis BioF specifically utilizes pimeloyl-CoA. IMPORTANCE Biotin is an essential vitamin required by mammals and birds because, unlike bacteria, plants, and some fungi, these organisms cannot make biotin. Currently, the biotin included in vitamin tablets and animal feeds is made by chemical synthesis. This is partly because the biosynthetic pathways in bacteria are incompletely understood. This paper defines an enzyme of the Bacillus subtilis pathway and shows that it differs from that of Escherichia coli in the ability to utilize specific precursors. These bacteria have been used in biotin production and these data may aid in making biotin produced by biotechnology commercially competitive with that produced by chemical synthesis. Copyright © 2017 American Society for Microbiology.

  13. Ultrastructural and biochemical detection of biotin and biotinylated polypeptides in Schistosoma mansoni

    Directory of Open Access Journals (Sweden)

    Santos P.R.P.

    1997-01-01

    Full Text Available Biotinylation is proposed for the identification of surface proteins in Schistosoma mansoni using the streptavidin-HRP conjugate for the detection of labeled polypeptides. However, control samples also showed several endogenous biotinylated polypeptides. In an attempt to determine the possibility of nonspecific binding between the streptavidin-HRP conjugate and polypeptides from S. mansoni, the conjugate was blocked with biotinamidecaproate-N-hydroxysuccinimide ester (BcapNHS before biotin-streptavidin blotting. No bands were detected on the nitrocellulose sheet, demonstrating the specific recognition of biotin by the streptavidin present in the conjugate. Whole cercariae and cercarial bodies and tails showed several endogenous biotinylated polypeptides. The biotin concentration was 13 µg/190,000 cercariae. Adult worms presented less endogenous biotinylated polypeptides than cercariae. These results may be due to changes in the environment from aerobic to anaerobic conditions when cercarial bodies (schistosomula are transformed into adult worms and a decrease in CO2 production may occur. Cercariae, cercarial bodies and adult male worms were examined by transmission electron microscopy employing an avidin-colloidal gold conjugate for the detection of endogenous biotin. Gold particles were distributed mainly on the muscle fibers, but dispersed granules were observed in the tegument, mitochondria and cytosol. The discovery of endogenous biotin in S. mansoni should be investigated in order to clarify the function of this vitamin in the parasite

  14. Voltammetric investigation of avidin-biotin complex formation using an electroactive bisbiotinyl compound

    International Nuclear Information System (INIS)

    Sugawara, Kazuharu; Shirotori, Tatsuya; Hirabayashi, George; Kamiya, Naoto; Kuramitz, Hideki; Tanaka, Shunitz

    2004-01-01

    Formation of avidin-biotin complex was investigated using bisbiotinyl thionine (BBT) by means of voltammetric techniques. Thionine is an electroactive compound and has two amino groups that are necessary for the reaction with a biotinylation reagent. The biotinylation of thionine produces a new reagent with two biotin moieties at each end of thionine. Three BBTs of different lengths of the spacer that connects the biotin moiety to the thionine moiety were prepared. The avidin-biotin binding assay was achieved by measuring the electrode response of the thionine moiety in BBT. The binding affinity and the conformation of complex, which depended on the length of spacer, are discussed. BBT in which the spacer is shortest (BBT-S, distance between carbonyl group of the two biotin moieties: 11 A) binds with only one avidin molecule. BBT with medium length of spacer (BBT-M, 28.8 A) forms the complex with two avidin molecules. BBT with the longest spacer (BBT-L, 46.6 A) allows binding with two avidin molecules as well as intramolecular binding within one avidin molecule. The affinity constants of BBT-S, BBT-M and BBT-L for avidin were estimated to be 7.0 x 10 12 M -1 , 3.2 x 10 12 M -1 and 4.0 x 10 12 M -1 , respectively

  15. A replaceable liposomal aptamer for the ultrasensitive and rapid detection of biotin

    Science.gov (United States)

    Sung, Tzu-Cheng; Chen, Wen-Yih; Shah, Pramod; Chen, Chien-Sheng

    2016-02-01

    Biotin is an essential vitamin which plays an important role for maintaining normal physiological function. A rapid, sensitive, and simple method is necessary to monitor the biotin level. Here, we reported a replacement assay for the detection of biotin using a replaceable liposomal aptamer. Replacement assay is a competitive assay where a sample analyte replaces the labeled competitor of analyte out of its biorecognition element on a surface. It is user friendly and time-saving because of washing free. We used aptamer as a competitor, not a biorecognition element as tradition. To label aptamers, we used cholesterol-conjugated aptamers to tag signal-amplifying-liposomes. Without the need of conjugation procedure, aptamers can be easily incorporated into the surface of dye-encapsulating liposomes. Two aptamers as competitors of biotin, ST-21 and ST-21M with different affinities to streptavidin, were studied in parallel for the detection of biotin using replacement assays. ST-21 and ST-21M aptamers reached to limits of detection of 1.32 pg/80 μl and 0.47 pg/80 μl, respectively. The dynamic ranges of our assays using ST-21 and ST-21M aptamers were seven and four orders of magnitude, respectively. This assay can be completed in 20 minutes without washing steps. These results were overall better than previous reported assays.

  16. Dual functionalized graphene oxide serves as a carrier for delivering oligohistidine- and biotin-tagged biomolecules into cells.

    Science.gov (United States)

    Jana, Batakrishna; Mondal, Goutam; Biswas, Atanu; Chakraborty, Indrani; Saha, Abhijit; Kurkute, Prashant; Ghosh, Surajit

    2013-11-01

    A versatile method of dual chemical functionalization of graphene oxide (GO) with Tris-[nitrilotris(acetic acid)] (Tris-NTA) and biotin for cellular delivery of oligohistidine- and biotin-tagged biomolecules is reported. Orthogonally functionalized GO surfaces with Tris-NTA and biotin to obtain a dual-functionalized GO (DFGO) are prepared and characterized by various spectroscopic and microscopic techniques. Fluorescence microscopic images reveal that DFGO surfaces are capable of binding oligohistidine-tagged biomolecules/proteins and avidin/biotin-tagged biomolecules/proteins orthogonally. The DFGO nanoparticles are non-cytotoxic in nature and can deliver oligohistidine- and biotin-tagged biomolecules simultaneously into the cell. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Enterohemorrhagic Escherichia coli senses low biotin status in the large intestine for colonization and infection

    Science.gov (United States)

    Yang, Bin; Feng, Lu; Wang, Fang; Wang, Lei

    2015-01-01

    Enterohemorrhagic Escherichia coli (EHEC) is an important foodborne pathogen that infects humans by colonizing the large intestine. Here we identify a virulence-regulating pathway in which the biotin protein ligase BirA signals to the global regulator Fur, which in turn activates LEE (locus of enterocyte effacement) genes to promote EHEC adherence in the low-biotin large intestine. LEE genes are repressed in the high-biotin small intestine, thus preventing adherence and ensuring selective colonization of the large intestine. The presence of this pathway in all nine EHEC serotypes tested indicates that it is an important evolutionary strategy for EHEC. The pathway is incomplete in closely related small-intestinal enteropathogenic E. coli due to the lack of the Fur response to BirA. Mice fed with a biotin-rich diet show significantly reduced EHEC adherence, indicating that biotin might be useful to prevent EHEC infection in humans. PMID:25791315

  18. Avidin-biotin system: a small library of cysteine biotinylated derivatives designed for the [99mTc(N)(PNP)]2+ metal fragment

    International Nuclear Information System (INIS)

    Bolzati, Cristina; Caporale, Andrea; Agostini, Stefania; Carta, Davide; Cavazza-Ceccato, Mario; Refosco, Fiorenzo; Tisato, Francesco; Schievano, Elisabetta; Bandoli, Giuliano

    2007-01-01

    Using the avidin-biotin system as model, we investigate here the effective application of [Tc(N)L(PNP)] +/0 technology (L=N-functionalized cysteine [O - ,S - ]; PNP=aminodiphosphine) to the preparation of target-specific radiopharmaceuticals. A series of 99m Tc-nitrido complexes containing functionalized biotin ligands was prepared and their biological profile was determined. To minimize the steric and the electronic influences of the Tc-carrying complex on the biotin-avidin receptor interaction, the following N-functionalized cysteine-biotin derivatives were synthesized: (1) Biot-CysOSH; (2) Biot-Abu-CysOSH; (3) Biot-Abz-CysOSH; (4) Biot-L-(Ac)Lys-CysOSH; (5) Biot-D-(Ac)Lys-CysOSH; (6) Biot-Glu-CysOSH. The asymmetrical nitrido-Tc(V) 99g/99m Tc(N)(Biot-X-CysOS)(PNP3) (X=spacer) complexes, where PNP3 was N,N-bis-[(dimethoxypropyl)phosphinoethyl] methoxy-ethylamine, were obtained by simultaneous addition of PNP3 and the relevant biotinylated ligand to a solution containing a 99m Tc-nitrido precursor (yields >95%). In all cases, a mixture of syn- and anti isomers was observed. In vitro challenge experiments with glutathione and cysteine indicated that no transchelation reactions occurred. Assessment of the in vitro binding to avidin of the complexes revealed that only the complexes containing Biot-Abu-CysOS and Biot-Glu-CysOS ligand maintained a good affinity for the concentrator. Stability studies carried out in human and mouse plasma as well as in rat and mouse liver homogenate evidenced a rapid enzymatic degradation for the 99m Tc(N)(Biot-Abu-CysOS)(PNP3) complex, whereas the 99m Tc(N)(Biot-Glu-CysOS)(PNP3) one was stable in all conditions. Tissue biodistribution in normal Balb/C mice of the most stable candidate showed a rapid clearance both from the blood and the other tissues. The activity was eliminated both through the hepatobiliary system and the urinary tract

  19. CONTAIN calculations of direct containment heating in the Surry plant

    International Nuclear Information System (INIS)

    Williams, D.C.; Louie, D.L.Y.

    1988-01-01

    The draft NUREG-1150 risk analysis performed for the Surry plant identified direct containment heating (DCH) as a potentially dominant contributor to the total public risk associated with this plant. At that time, however, detailed mechanistic calculations of DCH loads were unavailable. Subsequently, a series of analyses of DCH scenarios using the CONTAIN-DCH code was performed in order to put the treatment of DCH on a firmer basis in the final draft of NUREG-1150. The present paper describes some of the results obtained for the Surry plant. A developmental model for DCH has been incorporated into CONTAIN code. This model includes mechanistic treatments of reasonably well-understood phenomena (e.g., heat and mass transfer), together with a parametric treatment of poorly understood phenomena for which mechanistic models are unavailable (e.g., debris de-entrainment from the gas stream due to debris-structure interactions). The DCH model was described in an earlier report, but the present version incorporates a number of advances, including treatment of the chemical equilibria involved in the iron-steam reaction

  20. Biotin starvation causes mitochondrial protein hyperacetylation and partial rescue by the SIRT3-like deacetylase Hst4p

    Science.gov (United States)

    Madsen, Christian T.; Sylvestersen, Kathrine B.; Young, Clifford; Larsen, Sara C.; Poulsen, Jon W.; Andersen, Marianne A.; Palmqvist, Eva A.; Hey-Mogensen, Martin; Jensen, Per B.; Treebak, Jonas T.; Lisby, Michael; Nielsen, Michael L.

    2015-01-01

    The essential vitamin biotin is a covalent and tenaciously attached prosthetic group in several carboxylases that play important roles in the regulation of energy metabolism. Here we describe increased acetyl-CoA levels and mitochondrial hyperacetylation as downstream metabolic effects of biotin deficiency. Upregulated mitochondrial acetylation sites correlate with the cellular deficiency of the Hst4p deacetylase, and a biotin-starvation-induced accumulation of Hst4p in mitochondria supports a role for Hst4p in lowering mitochondrial acetylation. We show that biotin starvation and knockout of Hst4p cause alterations in cellular respiration and an increase in reactive oxygen species (ROS). These results suggest that Hst4p plays a pivotal role in biotin metabolism and cellular energy homeostasis, and supports that Hst4p is a functional yeast homologue of the sirtuin deacetylase SIRT3. With biotin deficiency being involved in various metabolic disorders, this study provides valuable insight into the metabolic effects biotin exerts on eukaryotic cells. PMID:26158509

  1. m-[125I]iodoaniline: a useful reagent for radiolabeling biotin

    International Nuclear Information System (INIS)

    Khawli, L.A.; Kassis, A.I.

    1992-01-01

    Biotinyl-m-[ 125 I]iodoanilide (BIA) was synthesized by coupling biotin to m-[ 125 I]iodoaniline via a mixed anhydride reaction. m-[ 125 I]Iodoaniline was produced from the tin precursor, which was prepared using a palladium catalyzed reaction of hexabutylditin with m-bromoaniline. The radioiodinated BIA derivative is characterized by a stable amide and/or intact ureido group on the biotin molecule, it may thus be a useful carrier for targeting radionuclides to avidin-conjugated antibodies previously localized on tumors. (author)

  2. Study on synthesis, kit formulation and chemical kinetics of dissociation of 99mTc labeled PnAO biotin complex

    International Nuclear Information System (INIS)

    Afshan, A.; Jafri, S.R.A.; Maecke, H.

    2004-01-01

    Full text: A bifunctional ligand of PnAO-biotin has recently been synthesized, with a better percentage yield of 63% in the presence of newly developed coupling agent 0-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexaflorophos phate (HATU). Then lyophilized kit with 150μg of PnAObiotin has been developed and labeled with high specific activity of technetium-99m (2500-3000MBq) to get maximum radiochemical purity of 99mTc-PnAO-biotin complex i.e. > 97%. The association of avidin and streptavidin is among the strongest known non-covalent protein ligand interaction Ka 1015 M-1 and 1013 M-1 respectively. We measured the dissociation rate constant of PnAO-biotin from avidin and streptavidin challenged with excess of cold biotin. For the separation of bound and free-labeled biotin we employed ultrafilteration technique. The results of these experiments demonstrated that the non-covalent binding between 99mTc-PnAO-biotin with avidin and 99mTc-PnAO-biotin with streptavidin is more than 99%. Both biotin-binding proteins exhibited a faster initial phase and the rate of dissociation of 99mTc-PnAO-biotin with avidin is found to be 8.2x10-8 at 250C and 2.6x10-7 at 370C while the rate of dissociation 99mTc-PnAO-biotin from streptavidin is found to be 6x10-7 at 250C and 1.06x10-6 at 370C. The in-vitro study of the kinetics of dissociation exhibits the strong interaction of 99mTc-PnAO-biotin complex with both proteins, which suggests that this bifunctional PnAO-biotin ligand can be used for tumor localization with monoclonal antibodies to achieve high tumor to non-tumor ratio. (author)

  3. A Rhizavidin Monomer with Nearly Multimeric Avidin-Like Binding Stability Against Biotin Conjugates.

    Science.gov (United States)

    Lee, Jeong Min; Kim, Jung A; Yen, Tzu-Chi; Lee, In Hwan; Ahn, Byungjun; Lee, Younghoon; Hsieh, Chia-Lung; Kim, Ho Min; Jung, Yongwon

    2016-03-01

    Developing a monomeric form of an avidin-like protein with highly stable biotin binding properties has been a major challenge in biotin-avidin linking technology. Here we report a monomeric avidin-like protein-enhanced monoavidin-with off-rates almost comparable to those of multimeric avidin proteins against various biotin conjugates. Enhanced monoavidin (eMA) was developed from naturally dimeric rhizavidin by optimally maintaining protein rigidity during monomerization and additionally shielding the bound biotin by diverse engineering of the surface residues. eMA allowed the monovalent and nonperturbing labeling of head-group-biotinylated lipids in bilayer membranes. In addition, we fabricated an unprecedented 24-meric avidin probe by fusing eMA to a multimeric cage protein. The 24-meric avidin and eMA were utilized to demonstrate how artificial clustering of cell-surface proteins greatly enhances the internalization rates of assembled proteins on live cells. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Biotin IgM Antibodies in Human Blood: A Previously Unknown Factor Eliciting False Results in Biotinylation-Based Immunoassays

    Science.gov (United States)

    Chen, Tingting; Hedman, Lea; Mattila, Petri S.; Jartti, Laura; Jartti, Tuomas; Ruuskanen, Olli; Söderlund-Venermo, Maria; Hedman, Klaus

    2012-01-01

    Biotin is an essential vitamin that binds streptavidin or avidin with high affinity and specificity. As biotin is a small molecule that can be linked to proteins without affecting their biological activity, biotinylation is applied widely in biochemical assays. In our laboratory, IgM enzyme immuno assays (EIAs) of µ-capture format have been set up against many viruses, using as antigen biotinylated virus like particles (VLPs) detected by horseradish peroxidase-conjugated streptavidin. We recently encountered one serum sample reacting with the biotinylated VLP but not with the unbiotinylated one, suggesting in human sera the occurrence of biotin-reactive antibodies. In the present study, we search the general population (612 serum samples from adults and 678 from children) for IgM antibodies reactive with biotin and develop an indirect EIA for quantification of their levels and assessment of their seroprevalence. These IgM antibodies were present in 3% adults regardless of age, but were rarely found in children. The adverse effects of the biotin IgM on biotinylation-based immunoassays were assessed, including four inhouse and one commercial virus IgM EIAs, showing that biotin IgM do cause false positivities. The biotin can not bind IgM and streptavidin or avidin simultaneously, suggesting that these biotin-interactive compounds compete for the common binding site. In competitive inhibition assays, the affinities of biotin IgM antibodies ranged from 2.1×10−3 to 1.7×10−4 mol/L. This is the first report on biotin antibodies found in humans, providing new information on biotinylation-based immunoassays as well as new insights into the biomedical effects of vitamins. PMID:22879954

  5. Intersections of pathways involving biotin and iron relative to therapeutic mechanisms for progressive multiple sclerosis.

    Science.gov (United States)

    Heidker, Rebecca M; Emerson, Mitchell R; LeVine, Steven M

    2016-12-01

    While there are a variety of therapies for relapsing remitting multiple sclerosis (MS), there is a lack of treatments for progressive MS. An early study indicated that high dose biotin therapy has beneficial effects in approximately 12-15% of patients with progressive MS. The mechanisms behind the putative improvements seen with biotin therapy are not well understood, but have been postulated to include: 1) improving mitochondrial function which is impaired in MS, 2) increasing synthesis of lipids and cholesterol to facilitate remyelination, and 3) affecting gene expression. We suggest one reason that a greater percentage of patients with MS didn't respond to biotin therapy is the inaccessibility or lack of other nutrients, such as iron. In addition to biotin, iron (or heme) is necessary for energy production, biosynthesis of cholesterol and lipids, and for some protective mechanisms. Both biotin and iron are required for myelination during development, and by inference, remyelination. However, iron can also play a role in the pathology of MS. Increased deposition of iron can occur in some CNS structures possibly promoting oxidative damage while low iron levels can occur in other areas. Thus, the potential, detrimental effects of iron need to be considered together with the need for iron to support metabolic demands associated with repair and/or protective processes. We propose the optimal utilization of iron may be necessary to maximize the beneficial effects of biotin. This review will examine the interactions between biotin and iron in pathways that may have therapeutic or pathogenic implications for MS.

  6. Faradaic Impedance Spectroscopy for Detection of Small Molecules Binding using the Avidin-Biotin Model

    International Nuclear Information System (INIS)

    Yoetz-Kopelman, Tal; Ram, Yaron; Freeman, Amihay; Shacham-Diamand, Yosi

    2015-01-01

    The changes in the Faradaic impedance of gold/biomolecules system due to specific binding of small molecule to a significantly larger binding protein molecule were investigated. The biotin (244.31 Da) - avidin (66000 Da) couple was used as a model for small ligand - binding protein biorecognition. The study was carried out under open circuit potential in the presence of [Fe(CN) 6 ] −3/−4 redox couple. An equivalent electrical circuit was proposed and used for the interpretation of the recorded impedance spectra. Adsorption of thiolated avidin increased the electron transfer resistance, R ct , by a factor of about 7.5 while subsequent addition of biotin within the concentration range of 4.1-40.9 nM reduced the value of R ct by amount proportional to the biotin concentration. The addition of biotin did not affect, however, the equivalent double layer capacitance or other equivalent circuit parameters. A simple model based on effective surface coverage by the avidin molecules and the effect of the added biotin on electron transfer through the coated surface is proposed. A model for the minimum detection limit based on the random distribution of the binding protein and its dimensions is proposed

  7. Distributed biotin-streptavidin transcription roadblocks for mapping cotranscriptional RNA folding.

    Science.gov (United States)

    Strobel, Eric J; Watters, Kyle E; Nedialkov, Yuri; Artsimovitch, Irina; Lucks, Julius B

    2017-07-07

    RNA folding during transcription directs an order of folding that can determine RNA structure and function. However, the experimental study of cotranscriptional RNA folding has been limited by the lack of easily approachable methods that can interrogate nascent RNA structure at nucleotide resolution. To address this, we previously developed cotranscriptional selective 2΄-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq) to simultaneously probe all intermediate RNA transcripts during transcription by stalling elongation complexes at catalytically dead EcoRIE111Q roadblocks. While effective, the distribution of elongation complexes using EcoRIE111Q requires laborious PCR using many different oligonucleotides for each sequence analyzed. Here, we improve the broad applicability of cotranscriptional SHAPE-Seq by developing a sequence-independent biotin-streptavidin (SAv) roadblocking strategy that simplifies the preparation of roadblocking DNA templates. We first determine the properties of biotin-SAv roadblocks. We then show that randomly distributed biotin-SAv roadblocks can be used in cotranscriptional SHAPE-Seq experiments to identify the same RNA structural transitions related to a riboswitch decision-making process that we previously identified using EcoRIE111Q. Lastly, we find that EcoRIE111Q maps nascent RNA structure to specific transcript lengths more precisely than biotin-SAv and propose guidelines to leverage the complementary strengths of each transcription roadblock in cotranscriptional SHAPE-Seq. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  8. Clinical validation of the avidin/indium-111 biotin approach for imaging infection/inflammation in orthopaedic patients

    International Nuclear Information System (INIS)

    Lazzeri, E.; Molea, N.; Bodei, L.; Bianchi, R.; Manca, M.; Marchetti, S.; Consoli, V.; Chinol, M.; Paganelli, G.; Mariani, G.

    1999-01-01

    We report here the results of a validation study of the avidin/indium-111 biotin approach in patients with skeletal lesions. This study involved 54 patients with orthopaedic conditions: 20 patients with intermediate suspected osteomyelitis of the trunk, 19 patients with infection/inflammation of prosthetic joint replacements, and 15 patients with suspected osteomyelitis of appendicular bones. Avidin (3 mg) was injected as an i.v. bolus, followed 4 h later by 111 In-biotin; imaging was acquired 30 min and 16-18 h after administration of 111 In-biotin. Technetium-99m hexamethylpropylene amine oxime ( 99m Tc-HMPAO)-labelled leucocyte scintigraphy was performed in 39/54 patients. The overall sensitivity of the avidin/ 111 In-biotin scan was 97.7% (versus 88.9% for 99m Tc-HMPAO leucocyte scintigraphy). While the diagnostic performance of avidin/ 111 In-biotin scintigraphy was similar to that of 99m Tc-HMPAO leucocyte scintigraphy in patients with prosthetic joint replacements or osteomyelitis of appendicular bones, the avidin/ 111 In-biotin approach clearly performed better than 99m Tc-HMPAO leucocyte scintigraphy in patients with suspected osteomyelitis of the trunk (100% sensitivity, specificity and accuracy versus 50% sensitivity, 100% specificity and 66.7% accuracy for 99m Tc-HMPAO-leucocyte scintigraphy). These results demonstrate the feasibility of the avidin/ 111 In-biotin approach for imaging sites of infection/inflammation in the clinical setting. Although no systematic advantages of avidin/ 111 In-biotin scintigraphy were found versus 99m Tc-HMPAO leucocyte scintigraphy, the newer scintigraphic method is more practicable and involves lower biological risk for the operators. (orig.)

  9. Fc-specific biotinylation of antibody using an engineered photoactivatable Z–Biotin and its biosensing application

    International Nuclear Information System (INIS)

    Yang, Hong-Ming; Bao, Ru-Meng; Yu, Chang-Mei; Lv, Yan-Na; Zhang, Wei-Fen; Tang, Jin-Bao

    2017-01-01

    The development of a site-specific and covalent attachment methodology is crucial for antibody–biotin conjugates to preserve the antigen-binding ability of antibodies and yield homogeneous products. In this study, an engineered photoactivatable Z-domain variant [an UV-active amino acid benzoylphenylalanine (Bpa) was genetically incorporated into the Z-domain] carrying one biotin molecule (Z_B_p_a–Biotin) was prepared by employing aminoacyl-tRNA synthetase/suppressor tRNA and Avitag/BirA techniques. The site-specific and covalent attachment of IgG–biotin conjugates, viz. photo-biotinylated IgG, was successfully achieved after UV exposure by combining the inherent Fc-binding capability of the Z-domain with the formation of covalent bond by the photo-crosslinker. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis assay showed that more than 90% of IgGs conjugated with Z_B_p_a–Biotin molecules suffered 3 h UV irradiation. Further pepsin digestion analysis confirmed that the Z_B_p_a–Biotin was conjugated to the Fc fragment of IgG without interference. We took the tumor biomarker carcinoembryoic antigen (CEA) as model to evaluate the detection efficiency of the site-specific photo-biotinylated IgG in biosensing application using surface plasmon resonance (SPR) technology. The photo-biotinylated IgG coated surface gave a limit of detection (LOD) of 2 ng mL"-"1, is 5-fold lower than that of the randomly NHS-biotinylated IgG (10 ng mL"-"1). Given that the (strept)avidin–biotin complex is extensively used in immunoassays, the proposed method for biotinylated IgG provides a powerful approach to further expand related applications. - Highlights: • A photoactivable Z_B_p_a–Biotin was fabricated by aaRS/tRNA and Avitag/BirA techniques. • A approach for Fc-specific photo-biotinylated IgG via Z_B_p_a–Biotin was proposed. • The photo-biotinylated IgG was used to fabricate an immunosensor for detecting CEA. • It gave a LOD of 2 ng mL"-"1 CEA

  10. Fc-specific biotinylation of antibody using an engineered photoactivatable Z–Biotin and its biosensing application

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Hong-Ming; Bao, Ru-Meng; Yu, Chang-Mei; Lv, Yan-Na; Zhang, Wei-Fen; Tang, Jin-Bao, E-mail: tangjb@wfmc.edu.cn

    2017-01-01

    The development of a site-specific and covalent attachment methodology is crucial for antibody–biotin conjugates to preserve the antigen-binding ability of antibodies and yield homogeneous products. In this study, an engineered photoactivatable Z-domain variant [an UV-active amino acid benzoylphenylalanine (Bpa) was genetically incorporated into the Z-domain] carrying one biotin molecule (Z{sub Bpa}–Biotin) was prepared by employing aminoacyl-tRNA synthetase/suppressor tRNA and Avitag/BirA techniques. The site-specific and covalent attachment of IgG–biotin conjugates, viz. photo-biotinylated IgG, was successfully achieved after UV exposure by combining the inherent Fc-binding capability of the Z-domain with the formation of covalent bond by the photo-crosslinker. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis assay showed that more than 90% of IgGs conjugated with Z{sub Bpa}–Biotin molecules suffered 3 h UV irradiation. Further pepsin digestion analysis confirmed that the Z{sub Bpa}–Biotin was conjugated to the Fc fragment of IgG without interference. We took the tumor biomarker carcinoembryoic antigen (CEA) as model to evaluate the detection efficiency of the site-specific photo-biotinylated IgG in biosensing application using surface plasmon resonance (SPR) technology. The photo-biotinylated IgG coated surface gave a limit of detection (LOD) of 2 ng mL{sup -1}, is 5-fold lower than that of the randomly NHS-biotinylated IgG (10 ng mL{sup -1}). Given that the (strept)avidin–biotin complex is extensively used in immunoassays, the proposed method for biotinylated IgG provides a powerful approach to further expand related applications. - Highlights: • A photoactivable Z{sub Bpa}–Biotin was fabricated by aaRS/tRNA and Avitag/BirA techniques. • A approach for Fc-specific photo-biotinylated IgG via Z{sub Bpa}–Biotin was proposed. • The photo-biotinylated IgG was used to fabricate an immunosensor for detecting CEA. • It gave a LOD

  11. An improved synthesis of a fluorophosphonate–polyethylene glycol–biotin probe and its use against competitive substrates

    Science.gov (United States)

    Amidon, Gordon L

    2013-01-01

    Summary The fluorophosphonate (FP) moiety attached to a biotin tag is a prototype chemical probe used to quantitatively analyze and enrich active serine hydrolases in complex proteomes in an approach called activity-based protein profiling (ABPP). In this study we have designed a novel synthetic route to a known FP probe linked by polyethylene glycol to a biotin tag (FP–PEG–biotin). Our route markedly increases the efficiency of the probe synthesis and overcomes several problems of a prior synthesis. As a proof of principle, FP–PEG–biotin was evaluated against isolated protein mixtures and different rat-tissue homogenates, showing its ability to specifically target serine hydrolases. We also assessed the ability of FP–PEG–biotin to compete with substrates that have high enzyme turnover rates. The reduced protein-band intensities resulting in these competition studies demonstrate a new application of FP-based probes seldom explored before. PMID:23400700

  12. An improved synthesis of a fluorophosphonate–polyethylene glycol–biotin probe and its use against competitive substrates

    Directory of Open Access Journals (Sweden)

    Hao Xu

    2013-01-01

    Full Text Available The fluorophosphonate (FP moiety attached to a biotin tag is a prototype chemical probe used to quantitatively analyze and enrich active serine hydrolases in complex proteomes in an approach called activity-based protein profiling (ABPP. In this study we have designed a novel synthetic route to a known FP probe linked by polyethylene glycol to a biotin tag (FP–PEG–biotin. Our route markedly increases the efficiency of the probe synthesis and overcomes several problems of a prior synthesis. As a proof of principle, FP–PEG–biotin was evaluated against isolated protein mixtures and different rat-tissue homogenates, showing its ability to specifically target serine hydrolases. We also assessed the ability of FP–PEG–biotin to compete with substrates that have high enzyme turnover rates. The reduced protein-band intensities resulting in these competition studies demonstrate a new application of FP-based probes seldom explored before.

  13. Effect of biotin on milk performance of dairy cattle: a meta-analysis.

    Science.gov (United States)

    Chen, B; Wang, C; Wang, Y M; Liu, J X

    2011-07-01

    A meta-analysis of the effect of biotin on production outcomes of dairy cattle was conducted following a literature review. A total of 11 studies from 9 papers, with information on the milk production and composition data from a total number of 238 cows were extracted and analyzed using meta-analysis software in Stata. Estimated size of effect of biotin was calculated for dry matter intake (DMI), milk production, and composition. Heterogeneity was not significant for all of the parameters (the highest I(2)=12%). Therefore, fixed effects models were used for analysis. With the addition of biotin to lactating dairy cattle, DMI and milk production increased by 0.87 and 1.66 kg/d. No significant effect on percentage of milk fat and milk protein was observed. Additionally, Begg's test indicated no evidence of substantial publication bias for all variables. The influence analysis shows that the removal of any study did not change the direction or significance of the point estimates. It can be concluded that the use of biotin supplements increases DMI and milk yield in lactating dairy cows. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  14. Fc-specific biotinylation of antibody using an engineered photoactivatable Z-Biotin and its biosensing application.

    Science.gov (United States)

    Yang, Hong-Ming; Bao, Ru-Meng; Yu, Chang-Mei; Lv, Yan-Na; Zhang, Wei-Fen; Tang, Jin-Bao

    2017-01-01

    The development of a site-specific and covalent attachment methodology is crucial for antibody-biotin conjugates to preserve the antigen-binding ability of antibodies and yield homogeneous products. In this study, an engineered photoactivatable Z-domain variant [an UV-active amino acid benzoylphenylalanine (Bpa) was genetically incorporated into the Z-domain] carrying one biotin molecule (Z Bpa -Biotin) was prepared by employing aminoacyl-tRNA synthetase/suppressor tRNA and Avitag/BirA techniques. The site-specific and covalent attachment of IgG-biotin conjugates, viz. photo-biotinylated IgG, was successfully achieved after UV exposure by combining the inherent Fc-binding capability of the Z-domain with the formation of covalent bond by the photo-crosslinker. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis assay showed that more than 90% of IgGs conjugated with Z Bpa -Biotin molecules suffered 3 h UV irradiation. Further pepsin digestion analysis confirmed that the Z Bpa -Biotin was conjugated to the Fc fragment of IgG without interference. We took the tumor biomarker carcinoembryoic antigen (CEA) as model to evaluate the detection efficiency of the site-specific photo-biotinylated IgG in biosensing application using surface plasmon resonance (SPR) technology. The photo-biotinylated IgG coated surface gave a limit of detection (LOD) of 2 ng mL -1 , is 5-fold lower than that of the randomly NHS-biotinylated IgG (10 ng mL -1 ). Given that the (strept)avidin-biotin complex is extensively used in immunoassays, the proposed method for biotinylated IgG provides a powerful approach to further expand related applications. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Biotin uptake by mouse and human pancreatic beta cells/islets: a regulated, lipopolysaccharide-sensitive carrier-mediated process

    Science.gov (United States)

    Ghosal, Abhisek; Sekar, Thillai V.

    2014-01-01

    Biotin is essential for the normal function of pancreatic beta cells. These cells obtain biotin from their surroundings via transport across their cell membrane. Little is known about the uptake mechanism involved, how it is regulated, and how it is affected by internal and external factors. We addressed these issues using the mouse-derived pancreatic beta-TC-6 cells and freshly isolated mouse and human primary pancreatic beta cells as models. The results showed biotin uptake by pancreatic beta-TC-6 cells occurs via a Na+-dependent, carrier-mediated process, that is sensitive to desthiobiotin, as well as to pantothenic acid and lipoate; the process is also saturable as a function of concentration (apparent Km = 22.24 ± 5.5 μM). These cells express the sodium-dependent multivitamin transporter (SMVT), whose knockdown (with doxycycline-inducible shRNA) led to a sever inhibition in biotin uptake. Similarly, uptake of biotin by mouse and human primary pancreatic islets is Na+-dependent and carrier-mediated, and both cell types express SMVT. Biotin uptake by pancreatic beta-TC-6 cells is also adaptively regulated (via transcriptional mechanism) by extracellular substrate level. Chronic treatment of pancreatic beta-TC-6 cells with bacterial lipopolysaccharides (LPS) leads to inhibition in biotin uptake. This inhibition is mediated via a Toll-Like receptor 4-mediated process and involves a decrease in membrane expression of SMVT. These findings show, for the first time, that pancreatic beta cells/islets take up biotin via a specific and regulated carrier-mediated process, and that the process is sensitive to the effect of LPS. PMID:24904078

  16. Clinical feasibility of two-step streptavidin/111In-biotin scintigraphy in patients with suspected vertebral osteomyelitis

    International Nuclear Information System (INIS)

    Lazzeri, Elena; Erba, Paola A.; Volterrani, Duccio; Bottoni, Antonio; Mariani, Giuliano; Pauwels, Ernest K.J.; Manca, Mario; Bodei, Lisa; Trippi, Donatella; Cristofani, Renza; Consoli, Vincenzo; Palestro, Christopher J.

    2004-01-01

    Streptavidin accumulates at sites of inflammation and infection as a result of increased capillary permeability. In addition to being utilised by bacteria for their own growth, biotin forms a stable, high-affinity non-covalent complex with avidin. The objective of this investigation was to determine the diagnostic performance of two-step streptavidin/ 111 In-biotin imaging for evaluating patients with suspected vertebral osteomyelitis. We evaluated 55 consecutive patients with suspected vertebral osteomyelitis (34 women and 21 men aged 27-86 years), within 2 weeks after the onset of clinical symptoms. Thirty-two of the patients underwent magnetic resonance imaging (MRI) and 24, computed tomography (CT). DTPA-conjugated biotin was radiolabelled by incubating 500 μg of DTPA-biotin with 111 MBq of 111 In-chloride. Two-step scintigraphy was performed by first infusing 3 mg streptavidin intravenously, followed 4 h later by 111 In-biotin. Imaging was begun 60 min later. Streptavidin/ 111 In-biotin scintigraphy was positive in 32/34 patients with spinal infection (94.12% sensitivity). The study was negative in 19/21 patients without infection (95.24% specificity). The corresponding results for MRI and CT were 54.17% and 35.29% (sensitivity), and 75% and 57.14% (specificity), respectively. All statistical parameters of diagnostic performance (Youden's J index, kappa measure of agreement with correct classification, accuracy, sensitivity, specificity, positive likelihood and negative likelihood) were clearly better for streptavidin/ 111 In-biotin scintigraphy than for either MRI or CT. Streptavidin/ 111 In-biotin scintigraphy is highly sensitive and specific for detecting vertebral osteomyelitis in the first 2 weeks after the onset of clinical symptoms, and is potentially very useful for guiding clinical decisions on instituting appropriate therapy. (orig.)

  17. Clinical validation of the avidin/indium-111 biotin approach for imaging infection/inflammation in orthopaedic patients

    Energy Technology Data Exchange (ETDEWEB)

    Lazzeri, E.; Molea, N.; Bodei, L.; Bianchi, R. [Regional Centre of Nuclear Medicine, University of Pisa, Pisa (Italy); Manca, M.; Marchetti, S.; Consoli, V. [Institute of Orthopedics, University of Pisa, Pisa (Italy); Chinol, M.; Paganelli, G. [Nuclear Medicine Service, European Institute of Oncology, Milan (Italy); Mariani, G. [Nuclear Medicine Service, DIMI, University of Genoa, Genoa (Italy)

    1999-06-01

    We report here the results of a validation study of the avidin/indium-111 biotin approach in patients with skeletal lesions. This study involved 54 patients with orthopaedic conditions: 20 patients with intermediate suspected osteomyelitis of the trunk, 19 patients with infection/inflammation of prosthetic joint replacements, and 15 patients with suspected osteomyelitis of appendicular bones. Avidin (3 mg) was injected as an i.v. bolus, followed 4 h later by {sup 111}In-biotin; imaging was acquired 30 min and 16-18 h after administration of {sup 111}In-biotin. Technetium-99m hexamethylpropylene amine oxime ({sup 99m}Tc-HMPAO)-labelled leucocyte scintigraphy was performed in 39/54 patients. The overall sensitivity of the avidin/{sup 111}In-biotin scan was 97.7% (versus 88.9% for {sup 99m}Tc-HMPAO leucocyte scintigraphy). While the diagnostic performance of avidin/{sup 111}In-biotin scintigraphy was similar to that of {sup 99m}Tc-HMPAO leucocyte scintigraphy in patients with prosthetic joint replacements or osteomyelitis of appendicular bones, the avidin/{sup 111}In-biotin approach clearly performed better than {sup 99m}Tc-HMPAO leucocyte scintigraphy in patients with suspected osteomyelitis of the trunk (100% sensitivity, specificity and accuracy versus 50% sensitivity, 100% specificity and 66.7% accuracy for {sup 99m}Tc-HMPAO-leucocyte scintigraphy). These results demonstrate the feasibility of the avidin/{sup 111}In-biotin approach for imaging sites of infection/inflammation in the clinical setting. Although no systematic advantages of avidin/{sup 111}In-biotin scintigraphy were found versus {sup 99m}Tc-HMPAO leucocyte scintigraphy, the newer scintigraphic method is more practicable and involves lower biological risk for the operators. (orig.) With 3 figs., 2 tabs., 56 refs.

  18. Magnetically separable polymer (Mag-MIP) for selective analysis of biotin in food samples.

    Science.gov (United States)

    Uzuriaga-Sánchez, Rosario Josefina; Khan, Sabir; Wong, Ademar; Picasso, Gino; Pividori, Maria Isabel; Sotomayor, Maria Del Pilar Taboada

    2016-01-01

    This work presents an efficient method for the preparation of magnetic nanoparticles modified with molecularly imprinted polymers (Mag-MIP) through core-shell method for the determination of biotin in milk food samples. The functional monomer acrylic acid was selected from molecular modeling, EGDMA was used as cross-linking monomer and AIBN as radical initiator. The Mag-MIP and Mag-NIP were characterized by FTIR, magnetic hysteresis, XRD, SEM and N2-sorption measurements. The capacity of Mag-MIP for biotin adsorption, its kinetics and selectivity were studied in detail. The adsorption data was well described by Freundlich isotherm model with adsorption equilibrium constant (KF) of 1.46 mL g(-1). The selectivity experiments revealed that prepared Mag-MIP had higher selectivity toward biotin compared to other molecules with different chemical structure. The material was successfully applied for the determination of biotin in diverse milk samples using HPLC for quantification of the analyte, obtaining the mean value of 87.4% recovery. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Virus immobilization on biomaterial scaffolds through biotin-avidin interaction for improving bone regeneration.

    Science.gov (United States)

    Hu, Wei-Wen; Wang, Zhuo; Krebsbach, Paul H

    2016-02-01

    To spatially control therapeutic gene delivery for potential tissue engineering applications, a biotin-avidin interaction strategy was applied to immobilize viral vectors on biomaterial scaffolds. Both adenoviral vectors and gelatin sponges were biotinylated and avidin was applied to link them in a virus-biotin-avidin-biotin-material (VBABM) arrangement. The tethered viral particles were stably maintained within scaffolds and SEM images illustrated that viral particles were evenly distributed in three-dimensional (3D) gelatin sponges. An in vivo study demonstrated that transgene expression was restricted to the implant sites only and transduction efficiency was improved using this conjugation method. For an orthotopic bone regeneration model, adenovirus encoding BMP-2 (AdBMP2) was immobilized to gelatin sponges before implanting into critical-sized bone defects in rat calvaria. Compared to gelatin sponges with AdBMP2 loaded in a freely suspended form, the VBABM method enhanced gene transfer and bone regeneration was significantly improved. These results suggest that biotin-avidin immobilization of viral vectors to biomaterial scaffolds may be an effective strategy to facilitate tissue regeneration. Copyright © 2013 John Wiley & Sons, Ltd.

  20. Effect of biotin on transcription levels of key enzymes and glutamate efflux in glutamate fermentation by Corynebacterium glutamicum.

    Science.gov (United States)

    Cao, Yan; Duan, Zuoying; Shi, Zhongping

    2014-02-01

    Biotin is an important factor affecting the performance of glutamate fermentation by biotin auxotrophic Corynebacterium glutamicum and glutamate is over-produced only when initial biotin content is controlled at suitable levels or initial biotin is excessive but with Tween 40 addition during fermentation. The transcription levels of key enzymes at pyruvate, isocitrate and α-ketoglutarate metabolic nodes, as well as transport protein (TP) of glutamate were investigated under the conditions of varied biotin contents and Tween 40 supplementation. When biotin was insufficient, the genes encoding key enzymes and TP were down-regulated in the early production phase, in particular, the transcription level of isocitrate dehydrogenase (ICDH) which was only 2% of that of control. Although the cells' morphology transformation and TP level were not affected, low transcription level of ICDH led to lower final glutamate concentration (64 g/L). When biotin was excessive, the transcription levels of key enzymes were at comparable levels as those of control with ICDH as an exception, which was only 3-22% of control level throughout production phase. In this case, little intracellular glutamate accumulation (1.5 mg/g DCW) and impermeable membrane resulted in non glutamate secretion into broth, even though the quantity of TP was more than 10-folds of control level. Addition of Tween 40 when biotin was excessive stimulated the expression of all key enzymes and TP, intracellular glutamate content was much higher (10-12 mg/g DCW), and final glutamate concentration reached control level (75-80 g/L). Hence, the membrane alteration and TP were indispensable in glutamate secretion. Biotin and Tween 40 influenced the expression level of ICDH and glutamate efflux, thereby influencing glutamate production.

  1. Conformational flexibility of avidin: the influence of biotin binding

    International Nuclear Information System (INIS)

    Soledad Celej, M.; Montich, Guillermo G.; Fidelio, Gerardo D.

    2004-01-01

    Ligand binding to proteins is a key process in cell biochemistry. The interaction usually induces modifications in the unfolding thermodynamic parameters of the macromolecule due to the coupling of unfolding and binding equilibria. In addition, these modifications can be attended by changes in protein structure and/or conformational flexibility induced by ligand binding. In this work, we have explored the effect of biotin binding on conformation and dynamic properties of avidin by using infrared spectroscopy including kinetics of hydrogen/deuterium exchange. Our results, along with previously thermodynamic published data, indicate a clear correlation between thermostability and protein compactness. In addition, our results also help to interpret the thermodynamic binding parameters of the exceptionally stable biotin:AVD complex

  2. Functionality screen of streptavidin mutants by non-denaturing SDS-PAGE using biotin-4-fluorescein.

    Science.gov (United States)

    Humbert, Nicolas; Ward, Thomas R

    2008-01-01

    Site-directed mutagenesis or directed evolution of proteins often leads to the production of inactive mutants. For streptavidin and related proteins, mutations may lead to the loss of their biotin-binding properties. With high-throughput screening methodologies in mind, it is imperative to detect, prior to the high-density protein production, the bacteria that produce non-functional streptavidin isoforms. Based on the incorporation of biotin-4-fluorescein in streptavidin mutants present in Escherichia coli bacterial extracts, we detail a functional screen that allows the identification of biotin-binding streptavidin variants. Bacteria are cultivated in a small volume, followed by a rapid treatment of the cells; biotin-4-fluorescein is added to the bacterial extract and loaded on an Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis (SDS-PAGE) under non-denaturing conditions. Revealing is performed using a UV transilluminator. This screen is thus easy to implement, cheap and requires only readily available equipment.

  3. Purification of CD47-streptavidin fusion protein from bacterial lysate using biotin-agarose affinity chromatography.

    Science.gov (United States)

    Salehi, Nasrin; Peng, Ching-An

    2016-07-08

    CD47 is a widely expressed transmembrane glycoprotein that modulates the activity of a plethora of immune cells via its extracellular domain. Therefore, CD47 plays important roles in the regulation of immune responses and may serve as targets for the development of immunotherapeutic agents. To make sure CD47 functionality is intact under the process of protein conjugation, CD47-streptavidin fusion protein was expressed and purified because it can easily bind to biotin-tagged materials via the unique biotin-streptavidin affinity. In this study, gene sequences of CD47 extracellular domain (CD47ECD) and core streptavidin (coreSA) with a total 834 bp were inserted into pET20b plasmid to construct recombinant plasmid encoding CD47-SA fusion gene. After bacteria transformation, the CD47-SA fusion protein was expressed by isopropyl-β-d-thiogalactopyranoside (IPTG) induction. The collected bacteria lysate was loaded on biotinylated agarose to proceed the purification of CD47-SA fusion protein. Due to the unexpected high affinity between biotin and coreSA, standard washing and elution approaches (e.g., varying pH, using biotin, and applying guanidine hydrochloride) reported for biotin-streptavidin affinity chromatography were not able to separate the target fusion protein. Instead, using low concentration of the non-ionic detergent Triton X-100 followed with alkaline buffer could efficiently weaken the binding between biotin and coreSA, thereby eluting out CD47-SA fusion protein from the biotin agarose column. The purified CD47-SA fusion protein was further characterized by molecular biology methods and its antiphagocytic functionality was confirmed by the phagocytosis assay. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:949-958, 2016. © 2016 American Institute of Chemical Engineers.

  4. An efficient synthesis of 1α,25-dihydroxyvitamin D3 LC-biotin.

    Science.gov (United States)

    Kattner, Lars; Bernardi, Dan

    2017-10-01

    In recent years the apparent impact of vitamin D deficiency on human health has gained increased awareness. Consequently, the development of appropriate assays to measure the status of medicinally most relevant vitamin D metabolites in human blood, serum or relevant tissue is continuously being improved. Particularly, assaying of 1α,25-dihydroxyvitamin D 3 , in turn considered as the most active metabolite, is mainly indicated in disorders leading to calcaemia or those resulting from an impaired 1α-hydroxylation of 25-hydroxyvitamin D 3 . Thus, in some competitive protein binding and ELISA assays, biotin-linked 1α,25-dihydroxyvitamin D 3 (1α,25-dihydroxyvitamin D 3 LC-biotin) is employed for measurement of actual calicitriol concentration. A new efficient synthesis of 1α,25-dihydroxyvitamin D 3 LC-biotin is described, starting with readily available vitamin D 2 , and combining a classical approach to access 1α,25-dihydroxyvitamin D 3 , appropriate OH-protective group transformations, and a C-3-O-alkylation, suitable to connect the biotin-linker in a reliable, selective and high yielding strategy. The developed methodology is applicable to the synthesis of a wide variety of C-3-OH-linked vitamin D 3 and D 2 derivatives. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Biotin increases glucokinase expression via soluble guanylate cyclase/protein kinase G, adenosine triphosphate production and autocrine action of insulin in pancreatic rat islets.

    Science.gov (United States)

    Vilches-Flores, Alonso; Tovar, Armando R; Marin-Hernandez, Alvaro; Rojas-Ochoa, Alberto; Fernandez-Mejia, Cristina

    2010-07-01

    Besides its role as a carboxylase prosthetic group, biotin has important effects on gene expression. However, the molecular mechanisms through which biotin exerts these effects are largely unknown. We previously found that biotin increases pancreatic glucokinase expression. We have now explored the mechanisms underlying this effect. Pancreatic islets from Wistar rats were treated with biotin, in the presence or absence of different types of inhibitors. Glucokinase mRNA and 18s rRNA abundance were determined by real-time PCR. Adenosine triphosphate (ATP) content was analyzed by fluorometry. Biotin treatment increased glucokinase mRNA abundance approximately one fold after 2 h; the effect was sustained up to 24 h. Inhibition of soluble guanylate cyclase or protein kinase G (PKG) signalling suppressed biotin-induced glucokinase expression. The cascade of events downstream of PKG in biotin-mediated gene transcription is not known. We found that inhibition of insulin secretion with diazoxide or nifedipine prevented biotin-stimulated glucokinase mRNA increase. Biotin treatment increased islet ATP content (control: 4.68+/-0.28; biotin treated: 6.62+/-0.26 pmol/islet) at 30 min. Inhibition of PKG activity suppressed the effects of biotin on ATP content. Insulin antibodies or inhibitors of phosphoinositol-3-kinase/Akt insulin signalling pathway prevented biotin-induced glucokinase expression. The nucleotide 8-Br-cGMP mimicked the biotin effects. We propose that the induction of pancreatic glucokinase mRNA by biotin involves guanylate cyclase and PKG activation, which leads to an increase in ATP content. This induces insulin secretion via ATP-sensitive potassium channels. Autocrine insulin, in turn, activates phosphoinositol-3-kinase/Akt signalling. Our results offer new insights into the pathways that participate in biotin-mediated gene expression. (c) 2010 Elsevier Inc. All rights reserved.

  6. Effect of feeding biotin on milk production and hoof health in lactating dairy cows: a quantitative assessment.

    Science.gov (United States)

    Lean, I J; Rabiee, A R

    2011-03-01

    Objectives of this study were to critically review randomized controlled trials, evaluate the effectiveness of supplementation with biotin on milk yield and composition and hoof health in lactating dairy cows, explore sources of heterogeneity among studies, and evaluate publication bias. Quantitative assessments can increase the statistical power with which we study the effect of treatments, such as biotin, on outcomes. A total of 9 papers, with 6 production and 3 hoof health studies, met the eligibility criteria for meta-analysis. Eight studies evaluated various hoof lesions in biotin-supplemented cows that did not meet the inclusion criteria. Eleven comparisons were made of milk production responses to biotin treatment. Data extracted included the number of cows in control and treatment groups, measures of variance of responses (standard error or standard deviation) and P-values. Other data obtained included the duration of treatment before and after calving, parity, breed of cow, type and dose of biotin, delivery method of supplementation, and types of diets. Biotin increased milk production by 1.29 kg per head per day (95% confidence interval=0.35 to 2.18 kg) with no evidence of heterogeneity (I(2)=0.0%). Treatment did not affect milk fat or protein percentages, and a trend to increase fat and protein yields was observed. Milk production and composition results were not influenced by duration of treatment before calving, parity, or diet type. Assessment of biotin supplementation on hoof health indicated that more studies had improved rather than negative or neutral outcomes. The effect of biotin treatment on milk production was relatively large and the effects on fat and protein yields, although not significant, were consistent in direction and magnitude with the milk response. The hoof health responses to biotin should encourage further studies to more effectively define the nature of these responses using consistent criteria for examination of hoof conditions

  7. Competitive enzyme immunoassay for human chorionic somatomammotropin using the avidin-biotin system

    International Nuclear Information System (INIS)

    Rappuoli, R.; Leoncini, P.; Tarli, P.; Neri, P.

    1981-01-01

    Human chorionic somatomammotropin (HCS) is determined by an enzyme immunoassay where HCS competes with biotin-labeled HCS for insolubilized anti-HCS antibodies. Enzyme-labeled avidin is then used to reveal the amount of bound HCS. The system proves to be sensitive (1 ng/ml of HCS can be detected) and results agree with radioimmunoassay determinations (correlation coefficient = 0.979). Kinetics of the avidin-biotin reaction and coating of polystyrene wells are also investigated

  8. Purification, crystallization and preliminary crystallographic analysis of biotin protein ligase from Staphylococcus aureus

    International Nuclear Information System (INIS)

    Pendini, Nicole R.; Polyak, Steve W.; Booker, Grant W.; Wallace, John C.; Wilce, Matthew C. J.

    2008-01-01

    The biotin protein ligase from S. aureus has been overexpressed in E. coli, purified, crystallized by the hanging-drop vapour-diffusion method and analysed using X-ray diffraction. Biotin protein ligase from Staphylococcus aureus catalyses the biotinylation of acetyl-CoA carboxylase and pyruvate carboxylase. Recombinant biotin protein ligase from S. aureus has been cloned, expressed and purified. Crystals were grown using the hanging-drop vapour-diffusion method using PEG 8000 as the precipitant at 295 K. X-ray diffraction data were collected to 2.3 Å resolution from crystals using synchrotron X-ray radiation at 100 K. The diffraction was consistent with the tetragonal space group P4 2 2 1 2, with unit-cell parameters a = b = 93.665, c = 131.95

  9. Comparing Planting Tools for Container Longleaf Pine

    Science.gov (United States)

    Daniel J. Leduc; James D. Haywood; Shi-Jean Susana Sung

    2011-01-01

    We examined if compressing the soil to make a planting hole with a custom-built, solid round dibble versus coring the soil with a commercially available tube dibble influenced container-grown longleaf pine seedling development differently. Seven teen months after planting, the planting tool did not significantly affect root collar diameter, shoot or root mass, root-to-...

  10. Growth hormone-releasing peptide-biotin conjugate stimulates myocytes differentiation through insulin-like growth factor-1 and collagen type I.

    Science.gov (United States)

    Lim, Chae Jin; Jeon, Jung Eun; Jeong, Se Kyoo; Yoon, Seok Jeong; Kwon, Seon Deok; Lim, Jina; Park, Keedon; Kim, Dae Yong; Ahn, Jeong Keun; Kim, Bong-Woo

    2015-09-01

    Based on the potential beneficial effects of growth hormone releasing peptide (GHRP)-6 on muscle functions, a newly synthesized GHRP-6-biotin conjugate was tested on cultured myoblast cells. Increased expression of myogenic marker proteins was observed in GHRP-6-biotin conjugate-treated cells. Additionally, increased expression levels of insulin-like growth factor-1 and collagen type I were observed. Furthermore, GHRP-6-biotin conjugate-treated cells showed increased metabolic activity, as indicated by increased concentrations of energy metabolites, such as ATP and lactate, and increased enzymatic activity of lactate dehydrogenase and creatine kinase. Finally, binding protein analysis suggested few candidate proteins, including desmin, actin, and zinc finger protein 691 as potential targets for GHRP6-biotin conjugate action. These results suggest that the newly synthesized GHRP-6-biotin conjugate has myogenic stimulating activity through, at least in part, by stimulating collagen type I synthesis and several key proteins. Practical applications of the GHRP-6-biotin conjugate could include improving muscle condition.

  11. Screening of carnitine and biotin deficiencies on tandem mass spectrometry.

    Science.gov (United States)

    Hagiwara, Shin-Ichiro; Kubota, Mitsuru; Nambu, Ryusuke; Kagimoto, Seiichi

    2017-04-01

    It is important to assess pediatric patients for nutritional deficiency when they are receiving specific interventions, such as enteral feeding. We focused on measurement of C0 and 3-hydroxyisovalerylcarnitine (C5-OH) with tandem mass spectrometry (MS/MS), which is performed as part of the newborn mass screening. The purpose of this study was to investigate the usefulness of MS/MS for screening carnitine and biotin deficiencies. Forty-two children (24 boys, 18 girls) were enrolled between December 2013 and December 2015. Blood tests, including measurement of serum free carnitine via the enzyme cycling method, and acylcarnitine analysis on MS/MS of dried blood spot (DBS), were performed for the evaluation of nutrition status. Median patient age was 2 years (range, 2 months-14 years). Mean serum free carnitine was 41.8 ± 19.2 μmol/L. In six of the 42 patients, serum free carnitine was 1.00 nmol/L. Therapy-resistant eczema was improved by treatment with additional biotin and a non-hydrolyzed formula. C0 and C5-OH, measured on MS/MS of DBS, were useful for screening carnitine and biotin deficiencies. © 2016 Japan Pediatric Society.

  12. Biotin starvation causes mitochondrial protein hyperacetylation and partial rescue by the SIRT3-like deacetylase Hst4p

    DEFF Research Database (Denmark)

    Madsen, Christian Toft; Sylvestersen, Kathrine Beck; Young, Clifford

    2015-01-01

    deficiency. Upregulated mitochondrial acetylation sites correlate with the cellular deficiency of the Hst4p deacetylase, and a biotin-starvation-induced accumulation of Hst4p in mitochondria supports a role for Hst4p in lowering mitochondrial acetylation. We show that biotin starvation and knockout of Hst4p...... cause alterations in cellular respiration and an increase in reactive oxygen species (ROS). These results suggest that Hst4p plays a pivotal role in biotin metabolism and cellular energy homeostasis, and supports that Hst4p is a functional yeast homologue of the sirtuin deacetylase SIRT3. With biotin...

  13. High doses of biotin in chronic progressive multiple sclerosis: a pilot study.

    Science.gov (United States)

    Sedel, Frédéric; Papeix, Caroline; Bellanger, Agnès; Touitou, Valérie; Lebrun-Frenay, Christine; Galanaud, Damien; Gout, Olivier; Lyon-Caen, Olivier; Tourbah, Ayman

    2015-03-01

    No drug has been found to have any impact on progressive multiple sclerosis (MS). Biotin is a vitamin acting as a coenzyme for carboxylases involved in key steps of energy metabolism and fatty acids synthesis. Among others, biotin activates acetylCoA carboxylase, a potentially rate-limiting enzyme in myelin synthesis. The aim of this pilot study is to assess the clinical efficacy and safety of high doses of biotin in patients suffering from progressive MS. Uncontrolled, non-blinded proof of concept study 23 consecutive patients with primary and secondary progressive MS originated from three different French MS reference centers were treated with high doses of biotin (100-300mg/day) from 2 to 36 months (mean=9.2 months). Judgement criteria varied according to clinical presentations and included quantitative and qualitative measures. In four patients with prominent visual impairment related to optic nerve injury, visual acuity improved significantly. Visual evoked potentials in two patients exhibited progressive reappearance of P100 waves, with normalization of latencies in one case. Proton magnetic resonance spectroscopy (H-MRS) in one case showed a progressive normalization of the Choline/Creatine ratio. One patient with left homonymous hemianopia kept on improving from 2 to 16 months following treatment׳s onset. Sixteen patients out of 18 (89%) with prominent spinal cord involvement were considered as improved as confirmed by blinded review of videotaped clinical examination in 9 cases. In all cases improvement was delayed from 2 to 8 months following treatment׳s onset. These preliminary data suggest that high doses of biotin might have an impact on disability and progression in progressive MS. Two double-blind placebo-controlled trials are on going. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  14. Dual production of poly(3-hydroxybutyrate) and glutamate using variable biotin concentrations in Corynebacterium glutamicum.

    Science.gov (United States)

    Jo, Sung-Jin; Leong, Chean Ring; Matsumoto, Ken'ichiro; Taguchi, Seiichi

    2009-04-01

    We previously synthesized poly(3-hydroxybutyrate) [P(3HB)] in recombinant Corynebacterium glutamicum, a prominent producer of amino acids. In this study, a two-step cultivation was established for the dual production of glutamate and P(3HB) due to the differences in the optimal concentration of biotin. Glutamate was extracellularly produced first under the biotin-limited condition of 0.3 microg/L. Production was then shifted to P(3HB) by addition of biotin to a total concentration of 9 microg/L. The final products obtained were 18 g/L glutamate and 36 wt% of P(3HB).

  15. Containment/surveillance concepts for international safeguards in reprocessing plants

    International Nuclear Information System (INIS)

    Bleck, M.E.; Cameron, C.P.; Camp, A.L.

    1980-01-01

    This paper examines the potential role of advanced containment/surveillance instrumentation systems for international safeguards in reprocessing plants. Several conceptual systems for the surveillance of containment boundary penetrations in a reference reprocessing plant are described and evaluated. The results of the evaluation aid in understanding the potential capabilities and limitations of containment/surveillance as an international safeguards concept in this type of facility

  16. On the radioprotective effect of biotin (vitamin H)

    International Nuclear Information System (INIS)

    Perepelkin, S.R.; Egorova, N.D.; Katsitadze, V.A.

    1977-01-01

    It has been shown on rats exposed to a whole-body X-irradiation with 600 R that food rich in vitamin H (biotin) causes a radioprotective effect. Most effective were the vitamin doses of 4.0 and 8.0 μg/rat

  17. Biotin augments acetyl CoA carboxylase 2 gene expression in the hypothalamus, leading to the suppression of food intake in mice.

    Science.gov (United States)

    Sone, Hideyuki; Kamiyama, Shin; Higuchi, Mutsumi; Fujino, Kaho; Kubo, Shizuka; Miyazawa, Masami; Shirato, Saya; Hiroi, Yuka; Shiozawa, Kota

    2016-07-29

    It is known that biotin prevents the development of diabetes by increasing the functions of pancreatic beta-cells and improving insulin sensitivity in the periphery. However, its anti-obesity effects such as anorectic effects remain to be clarified. Acetyl CoA carboxylase (ACC), a biotin-dependent enzyme, has two isoforms (ACC1 and ACC2) and serves to catalyze the reaction of acetyl CoA to malonyl CoA. In the hypothalamus, ACC2 increases the production of malonyl CoA, which acts as a satiety signal. In this study, we investigated whether biotin increases the gene expression of ACC2 in the hypothalamus and suppresses food intake in mice administered excessive biotin. Food intake was significantly decreased by biotin, but plasma regulators of appetite, including glucose, ghrelin, and leptin, were not affected. On the other hand, biotin notably accumulated in the hypothalamus and enhanced ACC2 gene expression there, but it did not change the gene expression of ACC1, malonyl CoA decarboxylase (a malonyl CoA-degrading enzyme), and AMP-activated protein kinase α-2 (an ACC-inhibitory enzyme). These findings strongly suggest that biotin potentiates the suppression of appetite by upregulating ACC2 gene expression in the hypothalamus. This effect of biotin may contribute to the prevention of diabetes by biotin treatment. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Evaluation of the avidin/biotin-liposome system injected in pleural space and peritoneum for drug delivery to mediastinal lymph nodes

    Science.gov (United States)

    Medina-Velazquez, Luis Alberto

    The avidin/biotin-liposome system is a new modality recently developed for targeting lymph nodes through the lymphatic system after local injection in a cavity as the route of delivery. In this dissertation we show that the avidin/biotin-liposome system has potential advantages over the injection of only liposomes for targeting lymph nodes. A goal of this dissertation was to evaluate the potential of pleural space as a route of transport for the targeting of mediastinal nodes. Another objective was to study the role of the injected dose of the avidin/biotin-liposome system for targeting mediastinal nodes. Dose, volume, site and sequence of injection of the agents were studied as factors that play an important role in the lymphatic targeting and in the organ distribution of liposomes after intracavitary injection of the avidin/biotin-liposome system. The hypothesis tested in this dissertation was that intracavitary injection of the avidin/biotin-liposome system in pleural space and/or peritoneum results in high levels of mediastinal node targeting with a significant reduction of unfavorable organ distribution when compared with the injection of only liposomes. The specific aims of this dissertation were: (1) to determine the pharmacokinetics, mediastinal node targeting, and biodistribution of avidin and biotin-liposomes injected individually in pleural and peritoneal space, (2) to determine the effect of injected dose and volume on the targeting of mediastinal nodes after intrapleural injection of the avidin/biotin-liposome system, and (3) to evaluate the dose effect of the avidin/biotin-liposome system on the targeting of mediastinal nodes and the lymphatics that drain the peritoneum and pleural space by injecting one agent in peritoneum and the corresponding agent in pleural space, and vice versa. To perform these studies, scintigraphic images were acquired with a gamma camera to non-invasively follow the pharmacokinetics and organ uptake of the avidin/biotin

  19. Peptide nucleic acid probe for protein affinity purification based on biotin-streptavidin interaction and peptide nucleic acid strand hybridization.

    Science.gov (United States)

    Tse, Jenny; Wang, Yuanyuan; Zengeya, Thomas; Rozners, Eriks; Tan-Wilson, Anna

    2015-02-01

    We describe a new method for protein affinity purification that capitalizes on the high affinity of streptavidin for biotin but does not require dissociation of the biotin-streptavidin complex for protein retrieval. Conventional reagents place both the selectively reacting group (the "warhead") and the biotin on the same molecule. We place the warhead and the biotin on separate molecules, each linked to a short strand of peptide nucleic acid (PNA), synthetic polymers that use the same bases as DNA but attached to a backbone that is resistant to attack by proteases and nucleases. As in DNA, PNA strands with complementary base sequences hybridize. In conditions that favor PNA duplex formation, the warhead strand (carrying the tagged protein) and the biotin strand form a complex that is held onto immobilized streptavidin. As in DNA, the PNA duplex dissociates at moderately elevated temperature; therefore, retrieval of the tagged protein is accomplished by a brief exposure to heat. Using iodoacetate as the warhead, 8-base PNA strands, biotin, and streptavidin-coated magnetic beads, we demonstrate retrieval of the cysteine protease papain. We were also able to use our iodoacetyl-PNA:PNA-biotin probe for retrieval and identification of a thiol reductase and a glutathione transferase from soybean seedling cotyledons. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. Alkaloid-Containing Plants Poisonous to Cattle and Horses in Europe.

    Science.gov (United States)

    Cortinovis, Cristina; Caloni, Francesca

    2015-12-08

    Alkaloids, nitrogen-containing secondary plant metabolites, are of major interest to veterinary toxicology because of their occurrence in plant species commonly involved in animal poisoning. Based on epidemiological data, the poisoning of cattle and horses by alkaloid-containing plants is a relatively common occurrence in Europe. Poisoning may occur when the plants contaminate hay or silage or when forage alternatives are unavailable. Cattle and horses are particularly at risk of poisoning by Colchicum autumnale (meadow saffron), Conium maculatum (poison hemlock), Datura stramonium (jimson weed), Equisetum palustre (marsh horsetail), Senecio spp. (ragwort and groundsel) and Taxus baccata (European yew). This review of poisonous alkaloid-containing plants describes the distribution of these plants, conditions under which poisoning occurs, active toxic principles involved and subsequent clinical signs observed.

  1. Effect of streptavidin-biotin on endothelial vasoregulation and leukocyte adhesion.

    Science.gov (United States)

    Chan, Bernard P; Reichert, William M; Truskey, George A

    2004-08-01

    The current study examines whether the adhesion promoting arginine-glycine-aspartate-streptavidin mutant (RGD-SA) also affects two important endothelial cell (EC) functions in vitro: vasoregulation and leukocyte adhesion. EC adherent to surfaces via fibronectin (Fn) or Fn plus RGD-SA were subjected to laminar shear flow and media samples were collected over a period of 4h to measure the concentration of nitric oxide (NO), prostacyclin (PGI(2)), and endothelin-1 (ET-1). Western blot analysis was used to quantify the levels of endothelial-derived nitric oxide synthase (eNOS) and cyclooxygenase II (COX II). In a separate set of experiments, fluorescent polymorphonuclear leukocyte (PMN) adhesion to EC was quantified for EC with and without exposure to flow preconditioning. When cell adhesion was supplemented with the SA-biotin system, flow-induced production of NO and PGI(2) increased significantly relative to cells adherent on Fn alone. Previous exposure of EC to shear flow also significantly decreased PMN attachment to SA-biotin supplemented EC, but only after 2h of exposure to shear flow. The observed decrease in PMN-EC adhesion was negated by NG-nitro-L-arginine methyl ester (L-NAME), an antagonist of NO synthesis, but not by indomethacin, an inhibitor to PGI(2) synthesis, indicating the induced effect of PMN-EC interaction is primarily NO-dependent. Results from this study suggest that the use of SA-biotin to supplement EC adhesion encourages vasodilation and PMN adhesion in vitro under physiological shear-stress conditions. We postulate that the presence of SA-biotin more efficiently transmits the shear-stress signal and amplifies the downstream events including the NO and PGI(2) release and leukocyte-EC inhibition. These results may have ramifications for reducing thrombus-induced vascular graft failure.

  2. Effect of exogenous fatty acids on biotin deprived death of Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Shimada, Shoji; Kuraishi, Hiroshi; Aida, Ko

    1978-01-01

    The effect of exogeneous fatty acids on cell growth and death of the biotin-requiring yeast Saccharomyces cerevisiae BA-1 was examined with respect to the mechanism of synthetic pathway of fatty acid under biotin starvation. At a growth temperature of 30 0 C, exogeneous unsaturated fatty acids, such as palmitoleic, oleic, linoleic, and linolenic acids which promote the cell growth and suppress death effectively, were incorporated intactly into the cellular fatty acids, whereas the saturated fatty acid, palmitic acid, which supports growth but some what inhibits death, was once incorporated, and about 60% of incorporated palmitic acid was found to be desaturated. However, at an elevated temperature of 36 0 C, even palmitic acid showed similar effects to unsaturated fatty acids in cell growth and death; following by an increased desaturation of palmitic acid. Thus the data indicate that palmitic aicd, as well as unsaturated fatty acids directly compensate for the deficiency of endogenously synthesized fatty acids caused by biotin starvation. (auth.)

  3. Electrochemical biotin detection based on magnetic beads and a new magnetic flow cell for screen printed electrode.

    Science.gov (United States)

    Biscay, Julien; González García, María Begoña; Costa García, Agustín

    2015-01-01

    The use of the first flow-cell for magnetic assays with an integrated magnet is reported here. The flow injection analysis system (FIA) is used for biotin determination. The reaction scheme is based on a one step competitive assay between free biotin and biotin labeled with horseradish peroxidase (B-HRP). The mixture of magnetic beads modified with streptavidin (Strep-MB), biotin and B-HRP is left 15 min under stirring and then a washing step is performed. After that, 100 μL of the mixture is injected and after 30s 100 μL of 3,3',5,5'-Tetramethylbenzidine (TMB) is injected and the FIAgram is recorded applying a potential of -0.2V. The linear range obtained is from 0.01 to 1 nM of biotin and the sensitivity is 758 nA/nM. The modification and cleaning of the electrode are performed in an easy way due to the internal magnet of the flow cell. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Fluorescent, thermo-responsive biotin-P(NIPAAm-co-NDAPM)- b-PCL micelles for cell-tracking and drug delivery

    International Nuclear Information System (INIS)

    Li Yongyong; Zhang Xianzheng; Cheng Han; Zhu Jingling; Li Unnam; Cheng Sixue; Zhuo Renxi

    2007-01-01

    An amphiphilic, biotinylated poly(N-isopropylacrylamide-co-N-(3-dimethylamino propyl)methacrylamide)-block- poly(ε-caprolactone) (biotin-P(NIPAAm-co-NDAPM)- b-PCL) block copolymer was synthesized. The cytotoxicity study showed that the copolymer exhibited no apparent cytotoxicity. In aqueous solution, biotin-P(NIPAAm-co-NDAPM)- b-PCL copolymer was able to self-assemble into micelles of around 60 nm in diameter with a critical micellar concentration (CMC) of 36 mg l -1 . Biotin-P(NIPAAm- co-NDAPM)-b-PCL micelles were thermo-responsive and the cloud point temperature was at 36.5 deg. C. The fluorescent group, fluorescein isothiocyanate (FITC) was further introduced to label the biotin-P(NIPAAm-co-NDAPM)- b-PCL copolymer. A cell internalization experiment was conducted and it was found that the fluorescent micelles could be internalized into the cells. The drug release behavior of drug-loading micelles was also examined and the drug-loaded biotin-P(NIPAAm-co-NDAPM)- b-PCL micelles showed slow drug release at 27 deg. C and fast drug release at 37 deg. C

  5. Protein labelling with avidin-biotin systems; Radiomarcado de proteinas con sistemas avidina-biotina

    Energy Technology Data Exchange (ETDEWEB)

    Hernandez B, B E

    1998-06-01

    The stability of connection in avidin-biotin system is very important due to the quadruple connections with avidin established with the same number of biotin molecules, which can amplify damage on cancer cells and increase specific activity of radio immuno conjugate in white cell. If between the first and second step (Ac Mo-biotin + avidin) enough time is left so that the monoclonal antibody accumulates in a therapeutic concentration required for the tumor or cancerous cells, then upon application of the third step (biotin-DTPA-{sup 153} Sm) it is hoped that in the first 30 minutes after application, only radioactivity remains with tumor. However, so that the amount radioactivity is enough to destroy a tumor, it would be necessary to use {sup 153} Sm with an activity of approximately 370 GBq (10 Ci)/ (mg). Since {sup 99m} Tc has similar chemistry to that of the {sup 188} Re, it is possible to propose their conjugates with biotin-avidin-Ac Mo-{sup 188} Re as a powerful option for therapeutic applications, this is, recommending the use of biotinylated labelled monoclonal antibody and the further injection of avidin to decrease of desirable effects on several other organs and bone marrow and high specific and selective action on tumor. On the other hand, we postulate the hypothesis in the sense that {sup 188} Re complexes tend to be more stable than those of {sup 99m} Tc, probably due to their metabolism, in which radioactivity of {sup 188} Re, not captured by tumor, is cleared easily from blood stream which results in a decrease of total and liver total dose in patient. (Author).

  6. Osseoconductivity of a Specific Streptavidin-Biotin-Fibronectin Surface Coating of Biotinylated Titanium Implants - A Rabbit Animal Study.

    Science.gov (United States)

    Kämmerer, Peer W; Lehnert, Michael; Al-Nawas, Bilal; Kumar, Vinay V; Hagmann, Sebastien; Alshihri, Abdulmonem; Frerich, Bernhard; Veith, Michael

    2015-10-01

    Biofunctionalized implant surfaces may accelerate bony integration and increase long-term stability. The aim of the study was to evaluate the osseous reaction toward biomimetic titanium implants surfaces coated with quasicovalent immobilized fibronectin in an in vivo animal model. A total of 84 implants (uncoated [control 1, n = 36], streptavidin-biotin coated [test 1, n = 24], streptavidin-biotin-fibronectin coated [test 2, n = 24]) were inserted 1 mm supracortically in the proximal tibia of 12 rabbits. The samples were examined after 3 and 6 weeks. Total bone-implant contact (tBIC; %), bone-implant contact in the cortical (cBIC; %) and in the spongious bone (sBIC; %) as well as the percentage of linear bone fill (PLF; %) were evaluated. After 3 weeks, streptavidin-biotin-fibronectin implants had a significant higher sBIC (p = .043) and PLF (p = .007) compared with the uncoated samples. After 6 weeks, this difference was significant for tBIC (p = .016) and cBIC (p biotin-coated implants showed less bone growth at both time points of all examined parameters when compared with their counterparts (all p biotin-fibronectin system on smooth surface titanium shows a beneficial faster osseous healing in vivo. Besides, an antifouling effect of the streptavidin-biotin coating was proven. © 2015 Wiley Periodicals, Inc.

  7. Vitamin B12-Containing Plant Food Sources for Vegetarians

    Science.gov (United States)

    Watanabe, Fumio; Yabuta, Yukinori; Bito, Tomohiro; Teng, Fei

    2014-01-01

    The usual dietary sources of Vitamin B12 are animal-derived foods, although a few plant-based foods contain substantial amounts of Vitamin B12. To prevent Vitamin B12 deficiency in high-risk populations such as vegetarians, it is necessary to identify plant-derived foods that contain high levels of Vitamin B12. A survey of naturally occurring plant-derived food sources with high Vitamin B12 contents suggested that dried purple laver (nori) is the most suitable Vitamin B12 source presently available for vegetarians. Furthermore, dried purple laver also contains high levels of other nutrients that are lacking in vegetarian diets, such as iron and n-3 polyunsaturated fatty acids. Dried purple laver is a natural plant product and it is suitable for most people in various vegetarian groups. PMID:24803097

  8. Vitamin B12-Containing Plant Food Sources for Vegetarians

    Directory of Open Access Journals (Sweden)

    Fumio Watanabe

    2014-05-01

    Full Text Available The usual dietary sources of Vitamin B12 are animal-derived foods, although a few plant-based foods contain substantial amounts of Vitamin B12. To prevent Vitamin B12 deficiency in high-risk populations such as vegetarians, it is necessary to identify plant-derived foods that contain high levels of Vitamin B12. A survey of naturally occurring plant-derived food sources with high Vitamin B12 contents suggested that dried purple laver (nori is the most suitable Vitamin B12 source presently available for vegetarians. Furthermore, dried purple laver also contains high levels of other nutrients that are lacking in vegetarian diets, such as iron and n-3 polyunsaturated fatty acids. Dried purple laver is a natural plant product and it is suitable for most people in various vegetarian groups.

  9. Alkaloid-Containing Plants Poisonous to Cattle and Horses in Europe

    Directory of Open Access Journals (Sweden)

    Cristina Cortinovis

    2015-12-01

    Full Text Available Alkaloids, nitrogen-containing secondary plant metabolites, are of major interest to veterinary toxicology because of their occurrence in plant species commonly involved in animal poisoning. Based on epidemiological data, the poisoning of cattle and horses by alkaloid-containing plants is a relatively common occurrence in Europe. Poisoning may occur when the plants contaminate hay or silage or when forage alternatives are unavailable. Cattle and horses are particularly at risk of poisoning by Colchicum autumnale (meadow saffron, Conium maculatum (poison hemlock, Datura stramonium (jimson weed, Equisetum palustre (marsh horsetail, Senecio spp. (ragwort and groundsel and Taxus baccata (European yew. This review of poisonous alkaloid-containing plants describes the distribution of these plants, conditions under which poisoning occurs, active toxic principles involved and subsequent clinical signs observed.

  10. Nuclear power plant with a containment

    International Nuclear Information System (INIS)

    Barthelmes, C.P.

    1982-01-01

    In nuclear power plants there is usually a containment incorporating components bearing activity. If in the cladding free hydrogen develops, controlled oxidation must be ensured by means of a recombination device, in order to prevent oxyhydrogen explosions. For this purpose, a permanent thoroughmixing of the gases in the containment is required. This can be achieved by vertical shafts reaching to at least half the height of the containment and provided with heating devices to initiate the gas circulation by the stack effect. These heating devices mainly serve as thermal recombinator. (orig.) [de

  11. Preclinical evaluation of a 68Ga-labeled biotin analogue for applications in islet transplantation

    International Nuclear Information System (INIS)

    Eriksson, Olof; Carlsson, Fredrik; Blom, Elisabeth; Sundin, Anders; Långström, Bengt; Korsgren, Olle; Velikyan, Irina

    2012-01-01

    Introduction: Islet transplantation is a promising treatment for type 1 diabetes mellitus, but the fate of the cells after intraportal infusion is unclear. It is therefore imperative to develop novel techniques for noninvasive imaging and quantification of events following islet transplantation. Methods: Small islet-like microbeads, avidin-covered agarose resins (AARs), were used as a model system for islet transplantation. Capability for specific [ 68 Ga]Ga-DOTA-(PEG) 2 -biotin uptake and retention for either AARs or human islets conjugated with avidin by means of a heparin scaffold was studied in vitro. Biodistribution of the novel positron emission tomography (PET) tracer [ 68 Ga]Ga-DOTA-(PEG) 2 -biotin was evaluated in mice treated by intraportal transplantation of AARs by μPET/computed tomography and ex vivo organ distribution and compared with control mice. Results: AARs had high capability to bind [ 68 Ga]Ga-DOTA-(PEG) 2 -biotin, close to 50% of administrated tracer/μl in vitro (>0.25 MBq/μl). Avidin-tagged human islets could bind on average 2.2% of administered tracer/μl. Specificity (>90%) and retention (>90% after 1 h) were high for both AARs and avidin-tagged islets. Hepatic tracer uptake and retention were increased in mice transplanted with AARs [standardized uptake value (SUV)=2.6] compared to the untreated group (SUV=1.4). In vivo uptake of tracer to AARs was blocked by preadministration of unlabeled biotin. Conclusions: Avidin-tagged islet-like objects can be tracked in hepatic volume after intraportal transplantation by using [ 68 Ga]Ga-DOTA-(PEG) 2 -biotin and PET.

  12. Improved tumor localization with (strept)avidin and labeled biotin as a substitute for antibody

    International Nuclear Information System (INIS)

    Hnatowich, D.J.; Fritz, B.; Virzi, F.; Mardirossian, G.; Rusckowski, M.

    1993-01-01

    We have investigated tumor localization with labeled biotin administered subsequent to unlabeled and unconjugated streptavidin. Nude mice bearing anti-CEA tumors (LS174T) received 10 μg of 111 In-labeled anti-CEA antibody (C110) or 111 In-labeled streptavidin with sacrifice 5 h later. In an examination of pretargeting, other animals received 50 μg of unlabeled streptavidin followed 3 h later with 1 μg of 111 In-labeled biotin (EB 1 ) and sacrifice 2 h later. The biodistribution of labeled streptavidin was similar to that of labeled specific antibody except for lower blood and higher kidney levels. Tumor levels were also lower with labeled streptavidin but, because of still lower levels in liver and blood, the tumor/normal tissue ratios were improved. When unlabeled streptavidin was administered and followed by labeled biotin (pretargeting), tumor levels were further reduced modestly; however, normal tissue levels were greatly reduced such that the tumor/blood and tumor/liver ratios were 10.6 and 2.2 vs 1.5 and 0.5 for the specific antibody. Improvements were seen in all tissues sampled with the exception of kidney and muscle. A further control of labeled biotin alone showed minimal accumulation in all tissues with the exception of kidneys. In conclusion, the accumulation of (strept)avidin by passive diffusion in tumor may be comparable, at early times, to the accumulation of specific antibody. By combining the administration of unlabeled (strept)avidin with labeled biotin, tumor targeting may potentially be improved. (author)

  13. Pharmacokinetics and pharmacodynamics of MD1003 (high-dose biotin) in the treatment of progressive multiple sclerosis.

    Science.gov (United States)

    Peyro Saint Paul, Laure; Debruyne, Danièle; Bernard, Delphine; Mock, Donald M; Defer, Gilles L

    2016-01-01

    Multiple sclerosis (MS) is a chronic, potentially highly disabling neurological disorder. No disease-modifying treatments are approved in the progressive and not active forms of the disease. High doses of biotin were tested in an open-label pilot study involving 23 patients with progressive MS and reported positive results. A randomized, double-blind, placebo-controlled trial in 154 progressive MS patients confirmed the beneficial effect of MD1003 (high-dose biotin) on reversing or stabilizing disability progression, with a good safety profile. It is proposed that MD1003 in progressive MS 1) increases energy production in demyelinated axons and/or 2) enhances myelin synthesis in oligodendrocytes. Biotin is highly bioavailable; absorption and excretion are rapid. The major route of elimination is urinary excretion. A high oral dose of biotin seems generally well tolerated but a few important safety concerns were identified: 1) teratogenicity in one species and 2) interference with some biotin-based laboratory immunoassays. The animal toxicity data are limited at such high doses. Further preclinical studies would be useful to address the mechanism of action of MD1003. Assessment of clinical benefit duration in responders will be also very important to set. Results of randomized, placebo-controlled trial are reassuring and provide hope for the treatment of progressive MS.

  14. Biotin-tagged proteins: Reagents for efficient ELISA-based serodiagnosis and phage display-based affinity selection.

    Science.gov (United States)

    Verma, Vaishali; Kaur, Charanpreet; Grover, Payal; Gupta, Amita; Chaudhary, Vijay K

    2018-01-01

    The high-affinity interaction between biotin and streptavidin has opened avenues for using recombinant proteins with site-specific biotinylation to achieve efficient and directional immobilization. The site-specific biotinylation of proteins carrying a 15 amino acid long Biotin Acceptor Peptide tag (BAP; also known as AviTag) is effected on a specific lysine either by co-expressing the E. coli BirA enzyme in vivo or by using purified recombinant E. coli BirA enzyme in the presence of ATP and biotin in vitro. In this paper, we have designed a T7 promoter-lac operator-based expression vector for rapid and efficient cloning, and high-level cytosolic expression of proteins carrying a C-terminal BAP tag in E. coli with TEV protease cleavable N-terminal deca-histidine tag, useful for initial purification. Furthermore, a robust three-step purification pipeline integrated with well-optimized protocols for TEV protease-based H10 tag removal, and recombinant BirA enzyme-based site-specific in vitro biotinylation is described to obtain highly pure biotinylated proteins. Most importantly, the paper demonstrates superior sensitivities in indirect ELISA with directional and efficient immobilization of biotin-tagged proteins on streptavidin-coated surfaces in comparison to passive immobilization. The use of biotin-tagged proteins through specific immobilization also allows more efficient selection of binders from a phage-displayed naïve antibody library. In addition, for both these applications, specific immobilization requires much less amount of protein as compared to passive immobilization and can be easily multiplexed. The simplified strategy described here for the production of highly pure biotin-tagged proteins will find use in numerous applications, including those, which may require immobilization of multiple proteins simultaneously on a solid surface.

  15. Chicken genome analysis reveals novel genes encoding biotin-binding proteins related to avidin family

    Directory of Open Access Journals (Sweden)

    Nordlund Henri R

    2005-03-01

    Full Text Available Abstract Background A chicken egg contains several biotin-binding proteins (BBPs, whose complete DNA and amino acid sequences are not known. In order to identify and characterise these genes and proteins we studied chicken cDNAs and genes available in the NCBI database and chicken genome database using the reported N-terminal amino acid sequences of chicken egg-yolk BBPs as search strings. Results Two separate hits showing significant homology for these N-terminal sequences were discovered. For one of these hits, the chromosomal location in the immediate proximity of the avidin gene family was found. Both of these hits encode proteins having high sequence similarity with avidin suggesting that chicken BBPs are paralogous to avidin family. In particular, almost all residues corresponding to biotin binding in avidin are conserved in these putative BBP proteins. One of the found DNA sequences, however, seems to encode a carboxy-terminal extension not present in avidin. Conclusion We describe here the predicted properties of the putative BBP genes and proteins. Our present observations link BBP genes together with avidin gene family and shed more light on the genetic arrangement and variability of this family. In addition, comparative modelling revealed the potential structural elements important for the functional and structural properties of the putative BBP proteins.

  16. Discovery of a cyclic 6 + 6 hexamer of d-biotin and formaldehyde

    DEFF Research Database (Denmark)

    Lisbjerg, Micke; Jessen, Bo M.; Rasmussen, Brian

    2014-01-01

    The discovery of receptors using templated synthesis enables the selection of strong receptors from complex mixtures. In this contribution we describe a study of the condensation of d-biotin and formaldehyde in acidic water. We have discovered that halide anions template the formation of a single...... isomer of a 6 + 6 macrocycle. The macrocycle (biotin[6]uril) is water-soluble, chiral and binds halide anions (iodide, bromide and chloride) with selectivity for iodide in water, and it can be isolated on a gram scale in a one-pot reaction in 63% yield....

  17. Adding Biotin to Parenteral Nutrition Solutions Without Lipid Accelerates the Growth of Candida albicans.

    Science.gov (United States)

    Kuwahara, Takashi; Kaneda, Shinya; Shimono, Kazuyuki

    2016-01-01

    We have previously demonstrated that Candida albicans requires multivitamins (MVs) or lipid to increase rapidly in parenteral nutrition (PN) solutions. In this study, in detail, the effects of vitamins on the growth of C. albicans in PN solutions without lipid were investigated. In the 1st experiment, a commercial PN solution without lipid was supplemented with water-soluble vitamins (SVs: vitamins B1, B2, B6, B12 and C, folic acid, nicotinamide, biotin and panthenol), water-insoluble vitamins (IVs: vitamins A, D, E and K) or both (MVs). In the 2nd experiment, the test solutions were prepared by supplementing the PN solution with one of each or all of the SVs. In the 3rd experiment, another commercial peripheral PN (PPN) solution without lipid was supplemented with SVs, nicotinic acid, biotin or both nicotinic acid and biotin. In each of the experiments, a specified number of C. albicans organisms was added to each test solution, and all of the test solutions were allowed to stand at room temperature (23-26ºC). The number of C. albicans was counted at 0, 24, 48 and 72 hours after the addition of the organism. In the 1st experiment, the C. albicans increased rapidly in the PN solution supplemented with the SVs, but increased slowly without the SVs, regardless of the addition of the IVs. In the 2nd experiment, the C. albicans increased rapidly in the PN solution supplemented with the SVs or biotin, but increased slowly with each of the other water-soluble vitamins. In the 3rd experiment, the C. albicans increased rapidly in the PPN solution supplemented with the SVs or biotin, but increased slowly with the addition of nicotinic acid. These results suggested that adding MVs or SVs to PN solutions without lipid promotes the growth of C. albicans, and that this effect is mostly attributable to biotin.

  18. Conjugation of biotin-coated luminescent quantum dots with single domain antibody-rhizavidin fusions

    Directory of Open Access Journals (Sweden)

    Jinny L. Liu

    2016-06-01

    Full Text Available Straightforward and effective methods are required for the bioconjugation of proteins to surfaces and particles. Previously we demonstrated that the fusion of a single domain antibody with the biotin binding molecule rhizavidin provided a facile method to coat biotin-modified surfaces with a highly active and oriented antibody. Here, we constructed similar single domain antibody—rhizavidin fusions as well as unfused rhizavidin with a His-tag. The unfused rhizavidin produced efficiently and its utility for assay development was demonstrated in surface plasmon resonance experiments. The single domain antibody-rhizavidin fusions were utilized to coat quantum dots that had been prepared with surface biotins. Preparation of antibody coated quantum dots by this means was found to be both easy and effective. The prepared single domain antibody-quantum dot reagent was characterized by surface plasmon resonance and applied to toxin detection in a fluoroimmunoassay sensing format.

  19. Structural characterization of the Mycobacterium tuberculosis biotin biosynthesis enzymes 7,8-diaminopelargonic acid synthase and dethiobiotin synthetase .

    Science.gov (United States)

    Dey, Sanghamitra; Lane, James M; Lee, Richard E; Rubin, Eric J; Sacchettini, James C

    2010-08-10

    Mycobacterium tuberculosis (Mtb) depends on biotin synthesis for survival during infection. In the absence of biotin, disruption of the biotin biosynthesis pathway results in cell death rather than growth arrest, an unusual phenotype for an Mtb auxotroph. Humans lack the enzymes for biotin production, making the proteins of this essential Mtb pathway promising drug targets. To this end, we have determined the crystal structures of the second and third enzymes of the Mtb biotin biosynthetic pathway, 7,8-diaminopelargonic acid synthase (DAPAS) and dethiobiotin synthetase (DTBS), at respective resolutions of 2.2 and 1.85 A. Superimposition of the DAPAS structures bound either to the SAM analogue sinefungin or to 7-keto-8-aminopelargonic acid (KAPA) allowed us to map the putative binding site for the substrates and to propose a mechanism by which the enzyme accommodates their disparate structures. Comparison of the DTBS structures bound to the substrate 7,8-diaminopelargonic acid (DAPA) or to ADP and the product dethiobiotin (DTB) permitted derivation of an enzyme mechanism. There are significant differences between the Mtb enzymes and those of other organisms; the Bacillus subtilis DAPAS, presented here at a high resolution of 2.2 A, has active site variations and the Escherichia coli and Helicobacter pylori DTBS have alterations in their overall folds. We have begun to exploit the unique characteristics of the Mtb structures to design specific inhibitors against the biotin biosynthesis pathway in Mtb.

  20. The function of single containment and double containment of PWR nuclear power plant

    International Nuclear Information System (INIS)

    Chen Weijing.

    1985-01-01

    The function and structures of single containment and double containment of PWR nuclear power plant were described briefiy. The dissimilarites of diffent type of containments, which effects the impact of environment are discused. The impact of environment, effected by 'source term', containment gas leak rate and diffusion pattern of the released gas, under different operating condition is analysed. Especially, the impact of environment under LOCA accident is fully analysed

  1. 177Lu-DTPA-BIS-BIOTIN Binding of Octreotide-dextran-avidinated PANC-1 Cell Lines in Vitro

    International Nuclear Information System (INIS)

    Deng Xinrong; Zhai Shizhen; Shen Yijia; Luo Zhifu; Du Jin

    2011-01-01

    Tyr3-octreotide, dextran-40 and avidin were used to prepare octreotide-dextran-avidin (TOC-Dx 40 -Av). DTPA-BIS-BIOTIN was labelled with 177 Lu. The in vitro somatostatin receptor binding study was carried out by pretargeted method using TOC-Dx 40 -Av and 177 Lu-DTPA-BIS-BIOTIN. The 24 well cell culture plates were prepared with PANC-1 cell monolayer and then incubated with TOC-Dx 40 -Av. After two washed with PBS, the cells were incubated with different concentration of 177 Lu-DTPA-BIS-BIOTIN (48.8 ∼ 391 pmol). Cells uptake was evaluated with γ counter. The results showed that the chemical purity of TOC-Dx 40 -Av was over 99%. The results also showed that TOC-Dx 40 -Av remained high receptor binding affinity to somatostatin receptor which indicated that TOC- Dx 40 -Av could bind to 177 Lu-DTPA-BIS-BIOTIN with the molar ratio of 1 : 1 on the cell surface. (authors)

  2. A simple elution strategy for biotinylated proteins bound to streptavidin conjugated beads using excess biotin and heat.

    Science.gov (United States)

    Cheah, Joleen S; Yamada, Soichiro

    2017-12-02

    Protein-protein interactions are the molecular basis of cell signaling. Recently, proximity based biotin identification (BioID) has emerged as an alternative approach to traditional co-immunoprecipitation. In this protocol, a mutant biotin ligase promiscuously labels proximal binding partners with biotin, and resulting biotinylated proteins are purified using streptavidin conjugated beads. This approach does not require preservation of protein complexes in vitro, making it an ideal approach to identify transient or weak protein complexes. However, due to the high affinity bond between streptavidin and biotin, elution of biotinylated proteins from streptavidin conjugated beads requires harsh denaturing conditions, which are often incompatible with downstream processing. To effectively release biotinylated proteins bound to streptavidin conjugated beads, we designed a series of experiments to determine optimal binding and elution conditions. Interestingly, the concentrations of SDS and IGEPAL-CA630 during the incubation with streptavidin conjugated beads were the key to effective elution of biotinylated proteins using excess biotin and heating. This protocol provides an alternative method to isolate biotinylated proteins from streptavidin conjugated beads that is suitable for further downstream analysis. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Connecting DNA Origami Structures Using the Biotin-Streptavidin ...

    African Journals Online (AJOL)

    Abstract. This work made use of the strong interaction between biotin and streptavidin to connect designed DNA origami structures. The caDNAno software was used to design a 6 layer 3D origami cross-like structure. Selected DNA strands at the engineered attachment sites on the DNA origami structure were biotinylated.

  4. Biotin carboxylases in mitochondria and the cytosol from skeletal and cardiac muscle as detected by avidin binding

    NARCIS (Netherlands)

    Kirkeby, S.; Moe, D.; Bøg-Hansen, T. C.; van Noorden, C. J.

    1993-01-01

    Biotin carboxylases in mammalian cells are regulatory enzymes in lipogenesis and gluconeogenesis. In this study, endogenous biotin in skeletal and cardiac muscle was detected using avidin conjugated with alkaline phosphatase and applied in high concentrations to muscle sections. The avidin binding

  5. Targeting demyelination and virtual hypoxia with high-dose biotin as a treatment for progressive multiple sclerosis.

    Science.gov (United States)

    Sedel, Frédéric; Bernard, Delphine; Mock, Donald M; Tourbah, Ayman

    2016-11-01

    Progressive multiple sclerosis (MS) is a severely disabling neurological condition, and an effective treatment is urgently needed. Recently, high-dose biotin has emerged as a promising therapy for affected individuals. Initial clinical data have shown that daily doses of biotin of up to 300 mg can improve objective measures of MS-related disability. In this article, we review the biology of biotin and explore the properties of this ubiquitous coenzyme that may explain the encouraging responses seen in patients with progressive MS. The gradual worsening of neurological disability in patients with progressive MS is caused by progressive axonal loss or damage. The triggers for axonal loss in MS likely include both inflammatory demyelination of the myelin sheath and primary neurodegeneration caused by a state of virtual hypoxia within the neuron. Accordingly, targeting both these pathological processes could be effective in the treatment of progressive MS. Biotin is an essential co-factor for five carboxylases involved in fatty acid synthesis and energy production. We hypothesize that high-dose biotin is exerting a therapeutic effect in patients with progressive MS through two different and complementary mechanisms: by promoting axonal remyelination by enhancing myelin production and by reducing axonal hypoxia through enhanced energy production. This article is part of the Special Issue entitled 'Oligodendrocytes in Health and Disease'. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  6. Structural characterization of Staphylococcus aureus biotin protein ligase and interaction partners: An antibiotic target

    OpenAIRE

    Pendini, Nicole R; Yap, Min Y; Polyak, Steven W; Cowieson, Nathan P; Abell, Andrew; Booker, Grant W; Wallace, John C; Wilce, Jacqueline A; Wilce, Matthew C J

    2013-01-01

    The essential metabolic enzyme biotin protein ligase (BPL) is a potential target for the development of new antibiotics required to combat drug-resistant pathogens. Staphylococcus aureus BPL (SaBPL) is a bifunctional protein, possessing both biotin ligase and transcription repressor activities. This positions BPL as a key regulator of several important metabolic pathways. Here, we report the structural analysis of both holo- and apo-forms of SaBPL using X-ray crystallography. We also present ...

  7. Structural and functional studies of the biotin protein ligase from Aquifex aeolicus reveal a critical role for a conserved residue in target specificity.

    Science.gov (United States)

    Tron, Cecile M; McNae, Iain W; Nutley, Margaret; Clarke, David J; Cooper, Alan; Walkinshaw, Malcolm D; Baxter, Robert L; Campopiano, Dominic J

    2009-03-20

    Biotin protein ligase (BPL; EC 6.3.4.15) catalyses the formation of biotinyl-5'-AMP from biotin and ATP, and the succeeding biotinylation of the biotin carboxyl carrier protein. We describe the crystal structures, at 2.4 A resolution, of the class I BPL from the hyperthermophilic bacteria Aquifex aeolicus (AaBPL) in its ligand-free form and in complex with biotin and ATP. The solvent-exposed beta- and gamma-phosphates of ATP are located in the inter-subunit cavity formed by the N- and C-terminal domains. The Arg40 residue from the conserved GXGRXG motif is shown to interact with the carboxyl group of biotin and to stabilise the alpha- and beta-phosphates of the nucleotide. The structure of the mutant AaBPL R40G in both the ligand-free and biotin-bound forms reveals that the mutated loop has collapsed, thus hindering ATP binding. Isothermal titration calorimetry indicated that the presence of biotin is not required for ATP binding to wild-type AaBPL in the absence of Mg(2+), and the binding of biotin and ATP has been determined to occur via a random but cooperative process. The affinity for biotin is relatively unaffected by the R40G mutation. In contrast, the thermodynamic data indicate that binding of ATP to AaBPL R40G is very weak in the absence or in the presence of biotin. The AaBPL R40G mutant remains catalytically active but shows poor substrate specificity; mass spectrometry and Western blot studies revealed that the mutant biotinylates both the target A. aeolicus BCCPDelta67 fragment and BSA, and is subject to self-biotinylation.

  8. Electrochemical evaluation of avidin-biotin interaction on self-assembled gold electrodes

    International Nuclear Information System (INIS)

    Ding, S.-J.; Chang, B.-W.; Wu, C.-C.; Lai, M.-F.; Chang, H.-C.

    2005-01-01

    The avidin-biotin interaction on 11-mercaptoundecanoic acid self-assembled gold electrodes was investigated by means of cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The interfacial properties of the modified electrodes were evaluated in the presence of the Fe(China) 6 3-/4- couple redox as a probe. A simple equivalent circuit model with a constant phase element was used to interpret the obtained impedance spectra. The results of cyclic voltammetry showed that the voltammetric behavior of the redox probe was influenced by the electrode surface modification. It is evident that the accumulation of treated substances and the binding of biotin to avidin on the electrode surface resulted in the increasing electron-transfer resistance and the decreasing capacitance. The changes in the electron-transfer resistance on the avidin-modified electrodes were more sensitive than that in the capacitance while detecting biotin over the 2-10 μg/mL concentration. The detection amount can be as low as 20 ng/mL based on the electron-transfer resistance that presented the change of 4.3 kΩ without the use of labels. The development of a rapid, facile, and sensitive method for the quantitation of nanogram quantities of biomolecules utilizing EIS may be achieved

  9. Concrete containment vessels (CCV) for nuclear power plants, (1)

    International Nuclear Information System (INIS)

    Ibe, Yukimi; Kitajima, Masatake

    1977-01-01

    Containment vessels (CV) and the construction of concrete containment vessels (CCV) for nuclear power plants are described generally, and their use and techniques in foreign countries are illustrated, in connection with the introduction of CCV to Japanese nuclear power plants. The introduction deals with the construction plan of Japanese nuclear power plants, and with the difficulties in the steel CV for large scale construction. The investigations, tests and researches are not yet sufficient. The prompt establishment of safety supported by technical criteria, analytical methods and experiments is desired. The second part deals with the consideration for aseismatic design, construction, function and characteristics of CCV. The classification and currently employed CCV, which is mainly reinforced concrete containment vessels (RCCV), are described, and the typical CCV employed for BWR is illustrated. Further, the typical arrangement of reinforcing steels at the cylindrical portion and the dome portion of RCCV is illustrated. The third part deals with the present state of CCV abroad. A prestressed concrete containment vessel (PCCV) of Turkey Point power plant is illustrated as a typical example of CCV. The tests reported in the international meeting for the design, construction and operation of concrete pressure vessels and concrete containment vessels at York University in England in 1975 are reviewed. Typical examples of the design conditions, the size and form, and the construction procedure for PCCV and RCCV abroad are reviewed. (Iwakiri, K.)

  10. NeutrAvidin Functionalization of CdSe/CdS Quantum Nanorods and Quantification of Biotin Binding Sites using Biotin-4-Fluorescein Fluorescence Quenching.

    Science.gov (United States)

    Lippert, Lisa G; Hallock, Jeffrey T; Dadosh, Tali; Diroll, Benjamin T; Murray, Christopher B; Goldman, Yale E

    2016-03-16

    We developed methods to solubilize, coat, and functionalize with NeutrAvidin elongated semiconductor nanocrystals (quantum nanorods, QRs) for use in single molecule polarized fluorescence microscopy. Three different ligands were compared with regard to efficacy for attaching NeutrAvidin using the "zero-length cross-linker" 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). Biotin-4-fluorescene (B4F), a fluorophore that is quenched when bound to avidin proteins, was used to quantify biotin binding activity of the NeutrAvidin coated QRs and biotin binding activity of commercially available streptavidin coated quantum dots (QDs). All three coating methods produced QRs with NeutrAvidin coating density comparable to the streptavidin coating density of the commercially available quantum dots (QDs) in the B4F assay. One type of QD available from the supplier (ITK QDs) exhibited ∼5-fold higher streptavidin surface density compared to our QRs, whereas the other type of QD (PEG QDs) had 5-fold lower density. The number of streptavidins per QD increased from ∼7 streptavidin tetramers for the smallest QDs emitting fluorescence at 525 nm (QD525) to ∼20 tetramers for larger, longer wavelength QDs (QD655, QD705, and QD800). QRs coated with NeutrAvidin using mercaptoundecanoicacid (MUA) and QDs coated with streptavidin bound to biotinylated cytoplasmic dynein in single molecule TIRF microscopy assays, whereas Poly(maleic anhydride-alt-1-ocatdecene) (PMAOD) or glutathione (GSH) QRs did not bind cytoplasmic dynein. The coating methods require optimization of conditions and concentrations to balance between substantial NeutrAvidin binding vs tendency of QRs to aggregate and degrade over time.

  11. Computational redesign of bacterial biotin carboxylase inhibitors using structure-based virtual screening of combinatorial libraries.

    Science.gov (United States)

    Brylinski, Michal; Waldrop, Grover L

    2014-04-02

    As the spread of antibiotic resistant bacteria steadily increases, there is an urgent need for new antibacterial agents. Because fatty acid synthesis is only used for membrane biogenesis in bacteria, the enzymes in this pathway are attractive targets for antibacterial agent development. Acetyl-CoA carboxylase catalyzes the committed and regulated step in fatty acid synthesis. In bacteria, the enzyme is composed of three distinct protein components: biotin carboxylase, biotin carboxyl carrier protein, and carboxyltransferase. Fragment-based screening revealed that amino-oxazole inhibits biotin carboxylase activity and also exhibits antibacterial activity against Gram-negative organisms. In this report, we redesigned previously identified lead inhibitors to expand the spectrum of bacteria sensitive to the amino-oxazole derivatives by including Gram-positive species. Using 9,411 small organic building blocks, we constructed a diverse combinatorial library of 1.2×10⁸ amino-oxazole derivatives. A subset of 9×10⁶ of these compounds were subjected to structure-based virtual screening against seven biotin carboxylase isoforms using similarity-based docking by eSimDock. Potentially broad-spectrum antibiotic candidates were selected based on the consensus ranking by several scoring functions including non-linear statistical models implemented in eSimDock and traditional molecular mechanics force fields. The analysis of binding poses of the top-ranked compounds docked to biotin carboxylase isoforms suggests that: (1) binding of the amino-oxazole anchor is stabilized by a network of hydrogen bonds to residues 201, 202 and 204; (2) halogenated aromatic moieties attached to the amino-oxazole scaffold enhance interactions with a hydrophobic pocket formed by residues 157, 169, 171 and 203; and (3) larger substituents reach deeper into the binding pocket to form additional hydrogen bonds with the side chains of residues 209 and 233. These structural insights into drug-biotin

  12. Artificial Metalloenzymes Based on the Biotin-Streptavidin Technology: Challenges and Opportunities.

    Science.gov (United States)

    Heinisch, Tillmann; Ward, Thomas R

    2016-09-20

    The biotin-streptavidin technology offers an attractive means to engineer artificial metalloenzymes (ArMs). Initiated over 50 years ago by Bayer and Wilchek, the biotin-(strept)avidin techonology relies on the exquisite supramolecular affinity of either avidin or streptavidin for biotin. This versatile tool, commonly referred to as "molecular velcro", allows nearly irreversible anchoring of biotinylated probes within a (strept)avidin host protein. Building upon a visionary publication by Whitesides from 1978, several groups have been exploiting this technology to create artificial metalloenzymes. For this purpose, a biotinylated organometallic catalyst is introduced within (strept)avidin to afford a hybrid catalyst that combines features reminiscent of both enzymes and organometallic catalysts. Importantly, ArMs can be optimized by chemogenetic means. Combining a small collection of biotinylated organometallic catalysts with streptavidin mutants allows generation of significant diversity, thus allowing optimization of the catalytic performance of ArMs. Pursuing this strategy, the following reactions have been implemented: hydrogenation, alcohol oxidation, sulfoxidation, dihydroxylation, allylic alkylation, transfer hydrogenation, Suzuki cross-coupling, C-H activation, and metathesis. In this Account, we summarize our efforts in the latter four reactions. X-ray analysis of various ArMs based on the biotin-streptavidin technology reveals the versatility and commensurability of the biotin-binding vestibule to accommodate and interact with transition states of the scrutinized organometallic transformations. In particular, streptavidin residues at positions 112 and 121 recurrently lie in close proximity to the biotinylated metal cofactor. This observation led us to develop a streamlined 24-well plate streptavidin production and screening platform to optimize the performance of ArMs. To date, most of the efforts in the field of ArMs have focused on the use of purified

  13. Pimelic acid, the first precursor of the Bacillus subtilis biotin synthesis pathway, exists as the free acid and is assembled by fatty acid synthesis.

    Science.gov (United States)

    Manandhar, Miglena; Cronan, John E

    2017-05-01

    Biotin synthetic pathways are readily separated into two stages, synthesis of the seven carbon α, ω-dicarboxylic acid pimelate moiety and assembly of the fused heterocyclic rings. The biotin pathway genes responsible for pimelate moiety synthesis vary widely among bacteria whereas the ring synthesis genes are highly conserved. Bacillus subtilis seems to have redundant genes, bioI and bioW, for generation of the pimelate intermediate. Largely consistent with previous genetic studies it was found that deletion of bioW caused a biotin auxotrophic phenotype whereas deletion of bioI did not. BioW is a pimeloyl-CoA synthetase that converts pimelic acid to pimeloyl-CoA. The essentiality of BioW for biotin synthesis indicates that the free form of pimelic acid is an intermediate in biotin synthesis although this is not the case in E. coli. Since the origin of pimelic acid in Bacillus subtilis is unknown, 13 C-NMR studies were carried out to decipher the pathway for its generation. The data provided evidence for the role of free pimelate in biotin synthesis and the involvement of fatty acid synthesis in pimelate production. Cerulenin, an inhibitor of the key fatty acid elongation enzyme, FabF, markedly decreased biotin production by B. subtilis resting cells whereas a strain having a cerulenin-resistant FabF mutant produced more biotin. In addition, supplementation with pimelic acid fully restored biotin production in cerulenin-treated cells. These results indicate that pimelic acid originating from fatty acid synthesis pathway is a bona fide precursor of biotin in B. subtilis. © 2017 John Wiley & Sons Ltd.

  14. Pi overlapping ring systems contained in a homogeneous assay: a novel homogeneous assay for antigens

    Science.gov (United States)

    Kidwell, David A.

    1993-05-01

    A novel immunoassay, Pi overlapping ring systems contained in a homogeneous assay (PORSCHA), is described. This assay relies upon the change in fluorescent spectral properties that pyrene and its derivatives show with varying concentration. Because antibodies and other biomolecules can bind two molecules simultaneously, they can change the local concentration of the molecules that they bind. This concentration change may be detected spectrally as a change in the fluorescence emission wavelength of an appropriately labeled biomolecule. Several tests of PORSCHA have been performed which demonstrate this principle. For example: with streptavidin as the binding biomolecule and a biotin labeled pyrene derivative, the production of the excimer emitting at 470 nm is observed. Without the streptavidin present, only the monomer emitting at 378 and 390 nm is observed. The ratio of monomer to excimer provides the concentration of unlabeled biotin in the sample. Approximately 1 ng/mL of biotin may be detected with this system using a 50 (mu) l sample (2 X 10-16 moles biotin). The principles behind PORSCHA, the results with the streptavidin/biotin system are discussed and extensions of the PORSCHA concept to antibodies as the binding partner and DNA in homogeneous assays are suggested.

  15. Development of a formulation for the preparation of 99m Tc-Ida-bis-Biotin complex

    International Nuclear Information System (INIS)

    Gutierrez B, L.C.

    2000-01-01

    The radiopharmaceuticals of diagnostic use incorporate the radioisotope to an organic or inorganic molecule which goes selectively to the interest organ, to an a physiologic or metabolic process of the body with a simple and quantitatively interpretable kinetics. The 99m Tc occupies 80% from total of the studies realized in the world by the optimum combination of physical half-life (6 h), radionuclide quantity (ng) and high energy emission which allows to obtain results with the greatest information. Actually, in Nuclear Medicine, the research strategies are directed to the use of 'premarkers systems' based in the antibody administration, separated from radionuclide through the use of the avidin/biotin system. According to these considerations it was developed the 99m Tc-IDA-bis-Biotine complex as a new radiopharmaceutical which improves the diagnostic image of infectious core and tumorals. The IDA-biotin compound was synthesised and characterized by its melting point, IR spectroscopy, NMR, MS, UV and High-resolution liquid chromatography (HRLC). With base in an experimental factorial design those variables were established which influence in the radiochemical purity of the radiopharmaceutical which allowed to determine the reaction conditions, pH 9 at environmental temperature (22 Celsius degrees) and the optimum concentrations of the formulation components. IDA-biotine 1.0 mg, stannous chloride 0.1 mg and gluconate 15 mg as weak binding linking were realized to the lyophilized product quality control tests like: stability and radiochemical purity. The analytical techniques used UV spectrophotometry and HRLC were validated. The studies of biodistribution of the 99m Tc-Ida-bis-biotin complex were realized in healthy laboratory animals, showing stability 'In vivo' with renal purification. (Author)

  16. Structural characterization of Staphylococcus aureus biotin protein ligase and interaction partners: an antibiotic target.

    Science.gov (United States)

    Pendini, Nicole R; Yap, Min Y; Traore, D A K; Polyak, Steven W; Cowieson, Nathan P; Abell, Andrew; Booker, Grant W; Wallace, John C; Wilce, Jacqueline A; Wilce, Matthew C J

    2013-06-01

    The essential metabolic enzyme biotin protein ligase (BPL) is a potential target for the development of new antibiotics required to combat drug-resistant pathogens. Staphylococcus aureus BPL (SaBPL) is a bifunctional protein, possessing both biotin ligase and transcription repressor activities. This positions BPL as a key regulator of several important metabolic pathways. Here, we report the structural analysis of both holo- and apo-forms of SaBPL using X-ray crystallography. We also present small-angle X-ray scattering data of SaBPL in complex with its biotin-carboxyl carrier protein substrate as well as the SaBPL:DNA complex that underlies repression. This has revealed the molecular basis of ligand (biotinyl-5'-AMP) binding and conformational changes associated with catalysis and repressor function. These data provide new information to better understand the bifunctional activities of SaBPL and to inform future strategies for antibiotic discovery. © 2013 The Protein Society.

  17. Lipopolysaccharide inhibits colonic biotin uptake via interference with membrane expression of its transporter: a role for a casein kinase 2-mediated pathway.

    Science.gov (United States)

    Lakhan, Ram; Said, Hamid M

    2017-04-01

    Biotin (vitamin B7), an essential micronutrient for normal cellular functions, is obtained from both dietary sources as well as gut microbiota. Absorption of biotin in both the small and large intestine is via a carrier-mediated process that involves the sodium-dependent multivitamin transporter (SMVT). Although different physiological and molecular aspects of intestinal biotin uptake have been delineated, nothing is known about the effect of LPS on the process. We addressed this issue using in vitro (human colonic epithelial NCM460 cells) and in vivo (mice) models of LPS exposure. Treating NCM460 cells with LPS was found to lead to a significant inhibition in carrier-mediated biotin uptake. Similarly, administration of LPS to mice led to a significant inhibition in biotin uptake by native colonic tissue. Although no changes in total cellular SMVT protein and mRNA levels were observed, LPS caused a decrease in the fraction of SMVT expressed at the cell surface. A role for casein kinase 2 (CK2) (whose activity was also inhibited by LPS) in mediating the endotoxin effects on biotin uptake and on membrane expression of SMVT was suggested by findings that specific inhibitors of CK2, as well as mutating the putative CK2 phosphorylation site (Thr 78 Ala) in the SMVT protein, led to inhibition in biotin uptake and membrane expression of SMVT. This study shows for the first time that LPS inhibits colonic biotin uptake via decreasing membrane expression of its transporter and that these effects likely involve a CK2-mediated pathway.

  18. Purification, crystallization and preliminary crystallographic analysis of biotin protein ligase from Staphylococcus aureus.

    Science.gov (United States)

    Pendini, Nicole R; Polyak, Steve W; Booker, Grant W; Wallace, John C; Wilce, Matthew C J

    2008-06-01

    Biotin protein ligase from Staphylococcus aureus catalyses the biotinylation of acetyl-CoA carboxylase and pyruvate carboxylase. Recombinant biotin protein ligase from S. aureus has been cloned, expressed and purified. Crystals were grown using the hanging-drop vapour-diffusion method using PEG 8000 as the precipitant at 295 K. X-ray diffraction data were collected to 2.3 A resolution from crystals using synchrotron X-ray radiation at 100 K. The diffraction was consistent with the tetragonal space group P4(2)2(1)2, with unit-cell parameters a = b = 93.665, c = 131.95.

  19. Pseudo-immunolabelling with the avidin-biotin-peroxidase complex (ABC) due to the presence of endogenous biotin in retinal Müller cells of goldfish and salamander

    NARCIS (Netherlands)

    Bhattacharjee, J.; Nunes Cardozo, B.; Kamphuis, W.; Kamermans, M.; Vrensen, G. F.

    1997-01-01

    Immunodetection techniques are dependent on enzyme-protein conjugates for the visualisation of antigen-antibody complexes. One of the most widely used is the avidin-biotin-peroxidase complex (ABC) method. The present study demonstrates that direct treatment of goldfish and salamander retinal

  20. Nuclear power plant containment construction

    International Nuclear Information System (INIS)

    Schabert, H.P.; Danisch, R.; Strickroth, E.

    1975-01-01

    The Nuclear Power Plant Containment Construction includes the spherical steel safety enclosure for the reactor and the equipment associated with the reactor and requiring this type of enclosure. This steel enclosure is externally structurally protected against accident by a concrete construction providing a foundation for the steel enclosure and having a cylindrical wall and a hemispherical dome, these parts being dimensioned to form an annular space surrounding the spherical steel enclosure, the latter and the concrete construction heretofore being concentrically arranged with respect to each other. In the disclosed construction the two parts are arranged with their vertical axis horizontally offset from each other so that opposite to the offsetting direction of the concrete construction a relatively large space is formed in the now asymmetrical annular space in which reactor auxiliary equipment not requiring enclosure by the steel containment vessel or safety enclosure, may be located outside of the steel containment vessel and inside of the concrete construction where it is structurally protected by the latter

  1. A simple and rapid ultra-high-performance liquid chromatography-tandem mass spectrometry method to determine plasma biotin in hemodialysis patients.

    Science.gov (United States)

    Yagi, Shigeaki; Nishizawa, Manabu; Ando, Itiro; Oguma, Shiro; Sato, Emiko; Imai, Yutaka; Fujiwara, Masako

    2016-08-01

    A simple, rapid, and selective method for determination of plasma biotin was developed using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). After single-step protein precipitation with methanol, biotin and stable isotope-labeled biotin as an internal standard (IS) were chromatographed on a pentafluorophenyl stationary-phase column (2.1 × 100 mm, 2.7 μm) under isocratic conditions using 10 mm ammonium formate-acetonitrile (93:7, v/v) at a flow rate of 0.6 mL/min. The total chromatographic runtime was 5 min for each injection. Detection was performed in a positive electrospray ionization mode by monitoring selected ion transitions at m/z 245.1/227.0 and 249.1/231.0 for biotin and the IS, respectively. The calibration curve was linear in the range of 0.05-2 ng/mL using 300 μL of plasma. The intra- and inter-day precisions were all biotin concentrations in hemodialysis patients. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  2. Periodic inspection of CANDU nuclear power plant containment components

    International Nuclear Information System (INIS)

    1989-09-01

    This Standard is one in a series intended to provide uniform requirements for CANDU nuclear power plants. It provides requirements for the periodic inspection of containment components including the containment pressure suppression systems

  3. Reproductive performance and oviductal expression of avidin and avidin-related protein-2 in young and old broiler breeder hens orally exposed to supplementary biotin.

    Science.gov (United States)

    Daryabari, H; Akhlaghi, A; Zamiri, M J; Mianji, G Rahimi; Pirsaraei, Z Ansari; Deldar, H; Eghbalian, A N

    2014-09-01

    Published data on the probable involvement of avidin and avidin-related protein-2 (AVR2) in sustaining sperm viability in sperm storage tubules in 38-wk-old turkeys, and the high affinity of avidin or its analogs to biotin suggest that supplementary biotin may increase oviductal avidin and AVR2 expression, thereby attenuating the adverse effect of aging on hen reproductive performance. Broiler breeder hens (n = 120) were randomly assigned to receive 0 (T0), 0.30 (T1), or 0.45 (T2) mg of biotin/L of drinking water from 30 to 33 (young) and 53 to 56 (old) wk of age, and artificially inseminated to determine their reproductive performance. At the end of each period of biotin administration, 8 hens from each treatment group were killed for RNA extraction from the uterovaginal junction. Egg production was lower in the old hens (44%) compared with the young ones (82%), and biotin supplementation increased egg production only in the latter. Administering supplementary biotin to young hens increased their oviductal expression of AVR2, which was much higher in the old hens (1.0 and 4.6 for young and old groups, respectively). Fertility rate was not different between young and old hens, and was increased (4.4%) at the higher level of biotin supplementation. Hatchability and hatchling quality were not affected by biotin supplementation. Embryonic mortality between 17 to 21 d of incubation was higher in young (5.2%) compared with old (1.4%) birds. Egg fertility rate showed a moderate correlation (P biotin supplementation on AVR2 expression, and the relationship between biotin administration and oviductal expression of avidin and AVR2 was dependent on the hen's age, being higher in the young hens. © 2014 Poultry Science Association Inc.

  4. Design of reactor containment systems for nuclear power plants. Safety guide

    International Nuclear Information System (INIS)

    2008-01-01

    This Safety Guide was prepared under the IAEA programme for safety standards for nuclear power plants. It is a revision of the Safety Guide on Design of the Reactor Containment Systems in Nuclear Power Plants (Safety Series No. 50-Sg-D1) issued in 1985 and supplements the Safety Requirements publication on Safety of Nuclear Power Plants: Design. The present Safety Guide was prepared on the basis of a systematic review of the relevant publications, including the Safety of Nuclear Power Plants: Design, the Safety fundamentals publication on The Safety of Nuclear Installations, Safety Guides, INSAG Reports, a Technical Report and other publications covering the safety of nuclear power plants. 1.2. The confinement of radioactive material in a nuclear plant, including the control of discharges and the minimization of releases, is a fundamental safety function to be ensured in normal operational modes, for anticipated operational occurrences, in design basis accidents and, to the extent practicable, in selected beyond design basis accidents. In accordance with the concept of defence in depth, this fundamental safety function is achieved by means of several barriers and levels of defence. In most designs, the third and fourth levels of defence are achieved mainly by means of a strong structure enveloping the nuclear reactor. This structure is called the 'containment structure' or simply the 'containment'. This definition also applies to double wall containments. 1.3. The containment structure also protects the reactor against external events and provides radiation shielding in operational states and accident conditions. The containment structure and its associated systems with the functions of isolation, energy management, and control of radionuclides and combustible gases are referred to as the containment systems

  5. Design of reactor containment systems for nuclear power plants. Safety guide

    International Nuclear Information System (INIS)

    2004-01-01

    This Safety Guide was prepared under the IAEA programme for safety standards for nuclear power plants. It is a revision of the Safety Guide on Design of the Reactor Containment Systems in Nuclear Power Plants (Safety Series No. 50-Sg-D1) issued in 1985 and supplements the Safety Requirements publication on Safety of Nuclear Power Plants: Design. The present Safety Guide was prepared on the basis of a systematic review of the relevant publications, including the Safety of Nuclear Power Plants: Design, the Safety fundamentals publication on The Safety of Nuclear Installations, Safety Guides, INSAG Reports, a Technical Report and other publications covering the safety of nuclear power plants. 1.2. The confinement of radioactive material in a nuclear plant, including the control of discharges and the minimization of releases, is a fundamental safety function to be ensured in normal operational modes, for anticipated operational occurrences, in design basis accidents and, to the extent practicable, in selected beyond design basis accidents. In accordance with the concept of defence in depth, this fundamental safety function is achieved by means of several barriers and levels of defence. In most designs, the third and fourth levels of defence are achieved mainly by means of a strong structure enveloping the nuclear reactor. This structure is called the 'containment structure' or simply the 'containment'. This definition also applies to double wall containments. 1.3. The containment structure also protects the reactor against external events and provides radiation shielding in operational states and accident conditions. The containment structure and its associated systems with the functions of isolation, energy management, and control of radionuclides and combustible gases are referred to as the containment systems

  6. Improved tumor localization with (strept)avidin and labeled biotin as a substitute for antibody

    International Nuclear Information System (INIS)

    Hnatowich, D.J.; Fritz, B.; Virzi, F.; Mardirossian, G.; Rusckowski, M.

    1993-01-01

    We have investigated tumor localization with labeled biotin administered subsequent to unlabeled and unconjugated streptavidin. Nude mice bearing anti-CEA tumors (LS174T) received 10 μg of 111 In-labeled anti-CEA antibody (C110) or 111 In-labeled streptavidin with sacrifice 5 h later. In an examination of pretargeting, other animals received 50 μg of unlabeled streptavidin followed 3 h later with 1 μg of 111 In-labeled biotin (EB 1 ) and sacrifice 2 h later. The biodistribution of labeled streptavidin was similar to that of labeled specific antibody except for lower blood and higher kidney levels. Tumor levels were also lower with labeled streptavidin but, because of still lower levels in liver and blood, the tumor/normal tissue ratios were improved. When unlabeled streptavidin was administered and followed by labeled biotin (pretargeting), tumor levels were further reduced modestly; however, normal tissue levels were greatly reduced such that the tumor/blood and tumor/liver ratios were 10.6 and 2.2 vs 1.5 and 0.5 for the specific antibody. Improvements were seen in all tissues sampled with the exception of kidney and muscle. A further control of labeled biotin alone (without the preinjection of streptavidin) showed minimal accumulations in all tissues with the exception of kidneys. In conclusion, the accumulation of (strept)avidin by passive diffusion in tumor may be comparable, at early times, to the accumulation of specific antibody. (author)

  7. Steam explosions-induced containment failure studies for Swiss nuclear power plants

    Energy Technology Data Exchange (ETDEWEB)

    Zuchuat, O.; Schmocker, U. [Swiss Federal Nuclear Safety Inspectorate, Villigen (Switzerland); Esmaili, H.; Khatib-Rahbar, M.

    1998-01-01

    The assessment of the consequences of both in-vessel and ex-vessel energetic fuel-coolant interaction for Beznau (a Westinghouse pressurized water reactor with a large, dry containment), Goesgen (a Siemens/KWU pressurized water reactor with a large, dry containment) and Leibstadt (a General Electric boiling water reactor-6 with a free standing steel, MARK-III containment) nuclear power plants is presented in this paper. The Conditional Containment Failure Probability of the steel containment of these Swiss nuclear power plants is determined based on different probabilistic approaches. (author)

  8. High dietary biotin levels affect the footpad and hock health of broiler chickens reared at different stocking densities and litter conditions.

    Science.gov (United States)

    Sun, Z W; Fan, Q H; Wang, X X; Guo, Y M; Wang, H J; Dong, X

    2017-06-01

    Responses to stocking density (SD), dietary biotin concentration and litter condition were evaluated on 2016 Ross 308 male broilers in the fattening period (day 22-day 42). The birds were placed in 48 pens with either dry or wet litter to simulate the final stocking density of 30 kg (12 broilers/m 2 ; normal stocking density, NSD) and 40 kg (16 broilers/m 2 ; high stocking density, HSD) of body weight (BW)/m 2 floor space. A corn-soybean meal-based diet was supplemented with biotin to provide a normal (NB; 155 μg/kg) or high (HB, 1521 μg/kg) level of dietary biotin. There were six repetitions per treatment. The inappropriate moisture content of litter associated with HSD was avoided (p  0.05) for growth and processing yield. In HSD, the biotin effect (gains, FCR) was significantly higher (p chickens, and a significant interaction (p density, biotin supplementation and litter condition existed from 35 to 42 days of age. Taken together, increasing dietary biotin improves the performance and well-being of broiler chickens stocked at high densities in litter-independent and litter-dependent manners respectively. Journal of Animal Physiology and Animal Nutrition © 2016 Blackwell Verlag GmbH.

  9. Aging management of containment structures in nuclear power plants

    International Nuclear Information System (INIS)

    Naus, D.J.; Oland, C.B.; Ellingwood, B.R.; Graves, H.L. III; Norris, W.E.

    1996-01-01

    Research is being conducted by Oak Ridge National Laboratory under US nuclear regulatory commission (USNRC) sponsorship to address aging management of nuclear power plant containment and other safety-related structures. Documentation is being prepared to provide the USNRC with potential structural safety issues and acceptance criteria for use in continued service evaluations of nuclear power plants. Accomplishments include development of a structural materials information center containing data and information on the time variation of 144 material properties under the influence of pertinent environmental stressors or aging factors, evaluation of models for potential concrete containment degradation factors, development of a procedure to identify critical structures and degradation factors important to aging management, evaluations of non-destructive evaluation techniques, assessments of European and North American repair practices for concrete, review of parameters affecting corrosion of metals embedded in concrete, and development of methodologies for making current condition assessments and service life predictions of new or existing reinforced concrete structures in nuclear power plants. (orig.)

  10. Aging management of containment structures in nuclear power plants

    International Nuclear Information System (INIS)

    Naus, D.J.; Oland, C.B.; Ellingwood, B.R.

    1994-01-01

    Research is being conducted by Oak Ridge National Laboratory under U.S. Nuclear Regulatory Commission sponsorship to address aging management of nuclear power plant containment and other safety-related structures. Documentation is being prepared to provide the US-NRC with potential structural safety issues and acceptance criteria for use in continued service evaluations of nuclear power plants. Accomplishments include development of a Structural Materials Information Center containing data and information on the time variation of 144 material properties under the influence of pertinent environmental stressors or aging factors, evaluation of models for potential concrete containment degradation factors, development of a procedure to identify critical structures and degradation factors important to aging management, evaluations of nondestructive evaluation techniques, assessments of European and North American repair practices for concrete, review of parameters affecting corrosion of metals embedded in concrete, and development of methodologies for making current condition assessments and service life predictions of new or existing reinforced concrete structures in nuclear power plants. (author). 29 refs., 2 figs

  11. Aging management of containment structures in nuclear power plants

    International Nuclear Information System (INIS)

    Naus, D.J.; Oland, C.B.; Ellingwood, B.R.; Graves, H.L. III; Norris, W.E.

    1994-01-01

    Research is being conducted by ORNL under US Nuclear Regulatory Commission (USNRC) sponsorship to address aging management of nuclear power plant containment and other safety-related structures. Documentation is being prepared to provide the USNRC with potential structural safety issues and acceptance criteria for use in continued service evaluations of nuclear power plants. Accomplishments include development of a Structural Materials Information Center containing data and information on the time variation of 144 material properties under the influence of pertinent environmental stressors or aging factors, evaluation of models for potential concrete containment degradation factors, development of a procedure to identify critical structures and degradation factors important to aging management, evaluations of nondestructive evaluation techniques. assessments of European and North American repair practices for concrete, review of parameters affecting corrosion of metals embedded in concrete, and development of methodologies for making current condition assessments and service life predictions of new or existing reinforced concrete structures in nuclear power plants

  12. An analysis of avidin, biotin and their interaction at attomole levels by voltammetric and chromatographic techniques

    Czech Academy of Sciences Publication Activity Database

    Kizek, René; Masařík, Michal; Kramer, Karl J.; Potěšil, David; Bailey, M.; Howard, John A.; Klejdus, Bořivoj; Mikelová, Radka; Adam, V.; Trnková, Libuše; Jelen, František

    2005-01-01

    Roč. 381, č. 6 (2005), s. 1167-1178 ISSN 1618-2642 R&D Projects: GA ČR(CZ) GP525/04/P132; GA ČR(CZ) GA203/02/0422; GA MŠk(CZ) LN00A081; GA AV ČR(CZ) IAA1163201 Institutional research plan: CEZ:AV0Z50040507 Keywords : avidin * biotin * avidin- biotin technology Subject RIV: BO - Biophysics Impact factor: 2.695, year: 2005

  13. In vitro cytotoxicity of the ternary PAMAM G3–pyridoxal–biotin bioconjugate

    Directory of Open Access Journals (Sweden)

    Uram Ł

    2013-12-01

    Full Text Available Łukasz Uram, Magdalena Szuster, Krzysztof Gargasz, Aleksandra Filipowicz, Elżbieta Wałajtys-Rode, Stanisław Wołowiec Cosmetology Department, University of Information Technology and Management in Rzeszów, Rzeszów, Poland Abstract: A third-generation polyamidoamine dendrimer (PAMAM G3 was used as a macromolecular carrier for pyridoxal and biotin. The binary covalent bioconjugate of G3, with nine molecules of biotin per one molecule of G3 (G39B, and the ternary covalent bioconjugate of G3, with nine biotin and ten pyridoxal molecules (G39B10P, were synthesized. The biotin and pyridoxal residues of the bioconjugate were available for carboxylase and transaminase enzymes, as demonstrated in the conversion of pyruvate to oxaloacetate and alanine to pyruvate, respectively, by in vitro monitoring of the reactions, using 1H nuclear magnetic resonance spectroscopy. The toxicity of the ternary bioconjugate (BC-PAMAM was studied in vitro on BJ human normal skin fibroblasts and human squamous cell carcinoma (SCC-15 cell cultures in comparison with PAMAM G3, using three cytotoxicity assays (XTT, neutral red, and crystal violet and an estimation of apoptosis by confocal microscopy detection. The tests have shown that BC-PAMAM has significantly lower cytotoxicity compared with PAMAM. Nonconjugated PAMAM was not cytotoxic at concentrations up to 5 µM (NR and 10 µM (XTT, and BC-PAMAM was not cytotoxic up to 50 µM (both assays for both cell lines. It has been also found that normal fibroblasts were more sensitive than SCC to both PAMAM and BC-PAMAM. The effect of PAMAM and BC-PAMAM on the initiation of apoptosis (PAMAM in fibroblasts at 5 µM and BC-PAMAM at 10 µM in both cell lines corresponded with cytotoxicity assays for both cell lines. We concluded that normal fibroblasts are more sensitive to the cytotoxic effects of the PAMAM G3 dendrimer and that modification of its surface cationic groups by substitution with biologically active molecules

  14. Effects of Biotin Supplementation in the Diet on Adipose Tissue cGMP Concentrations, AMPK Activation, Lipolysis, and Serum-Free Fatty Acid Levels.

    Science.gov (United States)

    Boone-Villa, Daniel; Aguilera-Méndez, Asdrubal; Miranda-Cervantes, Adriana; Fernandez-Mejia, Cristina

    2015-10-01

    Several studies have shown that pharmacological concentrations of biotin decrease hyperlipidemia. The molecular mechanisms by which pharmacological concentrations of biotin modify lipid metabolism are largely unknown. Adipose tissue plays a central role in lipid homeostasis. In the present study, we analyzed the effects of biotin supplementation in adipose tissue on signaling pathways and critical proteins that regulate lipid metabolism, as well as on lipolysis. In addition, we assessed serum fatty acid concentrations. Male BALB/cAnN Hsd mice were fed a control or a biotin-supplemented diet (control: 1.76 mg biotin/kg; supplemented: 97.7 mg biotin/kg diet) over 8 weeks postweaning. Compared with the control group, biotin-supplemented mice showed an increase in the levels of adipose guanosine 3',5'-cyclic monophosphate (cGMP) (control: 30.3±3.27 pmol/g wet tissue; supplemented: 49.5±3.44 pmol/g wet tissue) and of phosphorylated forms of adenosine 5'-monophosphate-activated protein kinase (AMPK; 65.2%±1.06%), acetyl-coenzyme A (CoA), carboxylase-1 (196%±68%), and acetyl-CoA carboxylase-2 (78.1%±18%). Serum fatty acid concentrations were decreased (control: 1.12±0.04 mM; supplemented: 0.91±0.03 mM), and no change in lipolysis was found (control: 0.29±0.05 μmol/mL; supplemented: 0.33±0.08 μmol/mL). In conclusion, 8 weeks of dietary biotin supplementation increased adipose tissue cGMP content and protein expression of the active form of AMPK and of the inactive forms of acetyl-CoA carboxylase-1 and acetyl-CoA carboxylase-2. Serum fatty acid levels fell, and no change in lipolysis was observed. These findings provide insight into the effects of biotin supplementation on adipose tissue and support its use in the treatment of dyslipidemia.

  15. Fluorescent nanoscale detection of biotin-streptavidin interaction using near-field scanning optical microscopy

    International Nuclear Information System (INIS)

    Park, Hyun Kyu; Chung, Bong Hyun; Gokarna, Anisha; Hulme, John P; Park, Hyun Gyu

    2008-01-01

    We describe a nanoscale strategy for detecting biotin-streptavidin binding using near-field scanning optical microscopy (NSOM) that exploits the fluorescence properties of single polydiacetylene (PDA) liposomes. NSOM is more useful to observe nanomaterials having optical properties with the help of topological information. We synthesized amine-terminated 10,12-pentacosadiynoic acid (PCDA) monomer (PCDA-NH 2 ) and used this derivatized monomer to prepare PCDA liposomes. PCDA-NH 2 liposomes were immobilized on an aldehyde-functionalized glass surface followed by photopolymerization by using a 254 nm light source. To measure the biotin-streptavidin binding, we conjugated photoactivatable biotin to immobilized PCDA-NH 2 liposomes by UV irradiation (365 nm) and subsequently allowed them to interact with streptavidin. We analyzed the fluorescence using a fluorescence scanner and observed single liposomes using NSOM. The average height and NSOM signal observed in a single liposome after binding were ∼31.3 to 8.5 ± 0.5 nm and 0.37 to 0.16 ± 0.6 kHz, respectively. This approach, which has the advantage of not requiring a fluorescent label, could prove highly beneficial for single molecule detection technology

  16. Biotinidase Resistant 68Gallium-Radioligand Based on Biotin/Avidin Interaction for Pretargeting: Synthesis and Preclinical Evaluation.

    Science.gov (United States)

    Prakash, Surbhi; Hazari, Puja Panwar; Meena, Virendra Kumar; Jaswal, Ambika; Khurana, Harleen; Kukreti, Shrikant; Mishra, Anil Kumar

    2016-11-16

    A new macrocyclic system 2,2'-(12-amino-11,13-dioxo-1,4,7,10-tetraazacyclotridecane-4,7-diyl)diacetic acid (ATRIDAT) was designed for coordinating metals in +2 and +3 oxidation states particularly 68 Ga(III), for PET imaging. ATRIDAT was conjugated to d-biotin for pretargeting via biotin-avidin interaction. This model provides high tumor targeting efficiency and stability to biotinidase activity leading to modest signal amplification at the tumor site. Cyclization of triethylenetetramine with protected diethylamino malonate resulted in the formation of 13 membered diamide ring. d-Biotin was then anchored on the pendant amine rendering α-methyne carbon to the biotinamide bond which blocks the biotinidase enzyme activity. Biotinidase stability assay showed remarkable stability toward the action of biotinidase with ∼95% remaining intact after treatment following 4 h. Binding affinity experiments such as HABA assay, competitive displacement studies with d-biotin and CD showed high binding affinity of the molecule with avidin in nanomolar range. Biotin conjugate was successfully radiolabeled with 68 Ga(III) with radiolabeling efficiency of ∼70% and then purified to get 99.9% radiochemical yield. IC 50 of the compound was found to be 2.36 mM in HEK cell line and 0.82 mM in A549 as assessed in MTT assay. In biodistribution studies, the major route of excretion was found to be renal. Significant uptake of 4.15 ± 0.35% was observed in tumor in the avidin pretreated mouse at 1 h. μPET images also showed a high tumor to muscle ratio of 26.8 and tumor to kidney ratio of 1.74 at 1 h post-injection after avidin treatment.

  17. Use of chromium picolinate and biotin in the management of type 2 diabetes: an economic analysis.

    Science.gov (United States)

    Fuhr, Joseph P; He, Hope; Goldfarb, Neil; Nash, David B

    2005-08-01

    This paper addresses the potential economic benefits of chromium picolinate plus biotin (Diachrome) use in people with Type 2 diabetes (T2DM). The economic model was developed to estimate the impact on health care systems' costs by improved HbA1C levels with chromium picolinate plus biotin (Diachrome). Lifetimes cost savings were estimated by adjusting a benchmark from the literature, using a price index to adjust for inflation. The cost of diabetes is highly dependent on the HbA1C level with higher initial levels and higher annual increments increasing the cost. Improvement in glycemic control has proven to be cost-effective in delaying the onset and progression of T2DM, reducing the risk for diabetes-associated complications and lowering utilization and cost of care. Chromium picolinate plus biotin (Diachrome) showed greater improvement of glycemic control in poorly controlled T2DM patients (HbA(1C) > or = 10%) compared to their better controlled counterparts (HbA(1C) diabetes without heart diseases or hypertension, to 5,435 dollars for a poorly controlled patient with diabetes, heart disease, and hypertension. Average 3-year cost savings was estimated to be between 3.9 billion dollars and 52.9 billion dollars for the 16.3 million existing patients with diabetes. Chromium picolinate plus biotin (Diachrome) use among the 1.17 million newly diagnosed patients with T2DM each year could deliver lifetime cost savings of 42 billion dollars, or 36,000 dollars per T2DM patient. Affordable, safe, and convenient, chromium picolinate plus biotin (Diachrome) could prove to be a cost-effective complement to existing pharmacological therapies for controlling T2DM.

  18. Protein detection on biotin-derivatized polyallylamine by optical microring resonators

    NARCIS (Netherlands)

    Ullien, D.; Harmsma, P.J.; Chakkalakkal Abdulla, S.M.C.; Boer, B.M. de; Bosma, D.; Sudhölter, E.J.R.; Smet, L.C.P.M. de; Jager, W.F.

    2014-01-01

    Silicon optical microring resonators (MRRs) are sensitive devices that can be used for biosensing. We present a novel biosensing platform based on the application of polyelectrolyte (PE) layers on such MRRs. The top PE layer was covalently labeled with biotin to ensure binding sites for antibodies

  19. Two-monoclonal-antibody sandwich-type assay for thyrotropin, with use of an avidin-biotin separation technique

    International Nuclear Information System (INIS)

    Odell, W.D.; Griffin, J.; Zahradnik, R.

    1986-01-01

    We have developed a sensitive, specific, noncompetitive, sandwich-type radioimmunoassay for human thyrotropin (hTSH), which can be performed in 30 min. The assay involves two monoclonal antibodies, selected for high affinity and specificity and also for reaction against antigenic sites on hTSH that are distal from each other. One of these antibodies is labeled with 125 I; the other is conjugated covalently to biotin. Polystyrene beads were also conjugated covalently to biotin. After conjugation, the beads were incubated with avidin. These beads represent a rapid, simple method for separating hTSH-bound antibody from free antibody. The biotin-antibody-hTSH- 125 I-labeled antibody complexes bind to the beads and hTSH concentration is directly related to counts per minute. This assay can detect hTSH at a concentration of 0.06 milli-unit/L in serum

  20. The Infatuation With Biotin Supplementation: Is There Truth Behind Its Rising Popularity? A Comparative Analysis of Clinical Efficacy versus Social Popularity.

    Science.gov (United States)

    Soleymani, Teo; Lo Sicco, Kristen; Shapiro, Jerry

    2017-05-01

    Biotin, also known as Vitamin B7 or vitamin H, is a water-soluble B vitamin that acts as an essential cofactor for several carboxylases involved in the cellular metabolism of fatty acids, amino acids, and gluconeogenesis. Although there exists an incredible amount of social media hype and market advertising touting its efficacy for the improvement of hair quantity and quality, biotin's efficacy for hair remains largely unsubstantiated in scientific literature. We reviewed all pertinent scientific literature regarding the efficacy of biotin supplementation for hair growth and quality improvement, and we also investigated its popularity in society defined as a function of market analytics. To date, there have been no clinical trials conducted to investigate the efficacy of biotin supplementation for the treatment of alopecia of any kind, nor has there been any randomized controlled trial to study its effect on hair quality and quantity in human subjects. Because of the lack of clinical evidence, its use to improve hair quantity or quality is not routinely recommended. However, societal infatuation with biotin supplementation is not only propagated by its glamorization in popular media, its popularity is vastly disproportionate to the insufficient clinical evidence supporting it's efficacy in hair improvement. In other words, biotin supplements are quite "in vogue", without there being any real reason to be so. J Drugs Dermatol. 2017;16(5):496-500..

  1. Chlorine-containing natural compounds in higher plants

    DEFF Research Database (Denmark)

    Engvild, Kjeld Christensen

    1986-01-01

    More than 130 chlorine-containing compounds have been isolated from higher plants and ferns; about half are polyacetylenes, thiophenes and sesquiterpene lactones from the Asteraceae. A chlorinated chlorophyll may be an important part of photosystem 1. High biological activity is found in 4...

  2. Monodisperse measurement of the biotin-streptavidin interaction strength in a well-defined pulling geometry.

    Directory of Open Access Journals (Sweden)

    Steffen M Sedlak

    Full Text Available The widely used interaction of the homotetramer streptavidin with the small molecule biotin has been intensively studied by force spectroscopy and has become a model system for receptor ligand interaction. However, streptavidin's tetravalency results in diverse force propagation pathways through the different binding interfaces. This multiplicity gives rise to polydisperse force spectroscopy data. Here, we present an engineered monovalent streptavidin tetramer with a single cysteine in its functional subunit that allows for site-specific immobilization of the molecule, orthogonal to biotin binding. Functionality of streptavidin and its binding properties for biotin remain unaffected. We thus created a stable and reliable molecular anchor with a unique high-affinity binding site for biotinylated molecules or nanoparticles, which we expect to be useful for many single-molecule applications. To characterize the mechanical properties of the bond between biotin and our monovalent streptavidin, we performed force spectroscopy experiments using an atomic force microscope. We were able to conduct measurements at the single-molecule level with 1:1-stoichiometry and a well-defined geometry, in which force exclusively propagates through a single subunit of the streptavidin tetramer. For different force loading rates, we obtained narrow force distributions of the bond rupture forces ranging from 200 pN at 1,500 pN/s to 230 pN at 110,000 pN/s. The data are in very good agreement with the standard Bell-Evans model with a single potential barrier at Δx0 = 0.38 nm and a zero-force off-rate koff,0 in the 10-6 s-1 range.

  3. Monodisperse measurement of the biotin-streptavidin interaction strength in a well-defined pulling geometry.

    Science.gov (United States)

    Sedlak, Steffen M; Bauer, Magnus S; Kluger, Carleen; Schendel, Leonard C; Milles, Lukas F; Pippig, Diana A; Gaub, Hermann E

    2017-01-01

    The widely used interaction of the homotetramer streptavidin with the small molecule biotin has been intensively studied by force spectroscopy and has become a model system for receptor ligand interaction. However, streptavidin's tetravalency results in diverse force propagation pathways through the different binding interfaces. This multiplicity gives rise to polydisperse force spectroscopy data. Here, we present an engineered monovalent streptavidin tetramer with a single cysteine in its functional subunit that allows for site-specific immobilization of the molecule, orthogonal to biotin binding. Functionality of streptavidin and its binding properties for biotin remain unaffected. We thus created a stable and reliable molecular anchor with a unique high-affinity binding site for biotinylated molecules or nanoparticles, which we expect to be useful for many single-molecule applications. To characterize the mechanical properties of the bond between biotin and our monovalent streptavidin, we performed force spectroscopy experiments using an atomic force microscope. We were able to conduct measurements at the single-molecule level with 1:1-stoichiometry and a well-defined geometry, in which force exclusively propagates through a single subunit of the streptavidin tetramer. For different force loading rates, we obtained narrow force distributions of the bond rupture forces ranging from 200 pN at 1,500 pN/s to 230 pN at 110,000 pN/s. The data are in very good agreement with the standard Bell-Evans model with a single potential barrier at Δx0 = 0.38 nm and a zero-force off-rate koff,0 in the 10-6 s-1 range.

  4. Containment pressure monitoring method after severe accident in nuclear power plant

    International Nuclear Information System (INIS)

    Luo Chuanjie; Zhang Shishui

    2011-01-01

    The containment atmosphere monitoring system in nuclear power plant was designed on the basis of design accident. But containment pressure will increase greatly in a severe accident, and pressure instrument in the containment can't satisfy the monitoring requirement. A new method to monitor the pressure change in the containment after a severe accident was considered, through which accident soften methods can be adopted. Under present technical condition, adding a pressure monitoring channel out of containment for post-severe accident is a considerable method. Daya Bay Nuclear Power Plant implemented this modification, by which the containment release time can be delayed during severe accident, and nuclear safety can be increased. After analysis, this method is safe and feasible. (authors)

  5. Liver biopsy as diagnostic method for poisoning by swainsonine-containing plants

    Science.gov (United States)

    With the aim to investigate the use of hepatic biopsies for the diagnosis of poisoning by swainsonine-containing plants, dry leaves of Ipomoea marcellia containing 0.02% of swainsonine were administered to goats. Group I, with six goats, ingested 4g/kg of dry plant (0.8mg of swainsonina/kg) until th...

  6. Plant-based Paste Fermented by Lactic Acid Bacteria and Yeast: Functional Analysis and Possibility of Application to Functional Foods

    Directory of Open Access Journals (Sweden)

    Shinsuke Kuwaki

    2012-01-01

    Full Text Available A plant-based paste fermented by lactic acid bacteria and yeast (fermented paste was made from various plant materials. The paste was made of fermented food by applying traditional food-preservation techniques, that is, fermentation and sugaring. The fermented paste contained major nutrients (carbohydrates, proteins, and lipids, 18 kinds of amino acids, and vitamins (vitamin A, B 1 B 2 , B 6 , B 12 , E, K, niacin, biotin, pantothenic acid, and folic acid. It contained five kinds of organic acids, and a large amount of dietary fiber and plant phytochemicals. Sucrose from brown sugar, used as a material, was completely resolved into glucose and fructose. Some physiological functions of the fermented paste were examined in vitro. It was demonstrated that the paste possessed antioxidant, antihypertensive, antibacterial, anti-inflammatory, anti-allergy and anti-tyrosinase activities in vitro. It was thought that the fermented paste would be a helpful functional food with various nutrients to help prevent lifestyle diseases.

  7. Containment and release management applied to a PWR ice condenser plant

    International Nuclear Information System (INIS)

    Lehner, J.; Neogy, P.

    1991-01-01

    As part of a US Nuclear Regulatory Commission program focusing on the accident management capabilities of operating nuclear plants, Brookhaven National Laboratory (BNL) is carrying out a series of studies of containment and release management (CRM) for each of the five containment types used in US commercial power plants. The purpose of CRM is to maintain the integrity of the containment as long as possible following a severe accident and minimize the release of radioactivity to the environment

  8. Antibody-guided three-step therapy for high grade glioma with yttrium-90 biotin

    International Nuclear Information System (INIS)

    Paganelli, G.; Grana, C.; Chinol, M.; Cremonesi, M.; De Cicco, C.; Zoboli, S.; De Braud, F.; Robertson, C.; Zurrida, S.; Veronesi, U.; Casadio, C.; Siccardi, A.G.

    1999-01-01

    While the incidence of brain tumours seems to be increasing, median survival in patients with glioblastoma remains less than 1 year, despite improved diagnostic imaging and neurosurgical techniques, and innovations in treatment. We have developed an avidin-biotin pre-targeting approach for delivering therapeutic radionuclides to gliomas, using anti-tenascin monoclonal antibodies, which seems potentially effective for treating these tumours. We treated 48 eligible patients with histologically confirmed grade III or IV glioma and documented residual disease or recurrence after conventional treatment. Three-step radionuclide therapy was performed by intravenous administration of 35 mg/m 2 of biotinylated anti-tenascin monoclonal antibody (1st step), followed 36 h later by 30 mg of avidin and 50 mg of streptavidin (2nd step), and 18-24 h later by 1-2 mg of yttrium-90-labelled biotin (3rd step). 90 Y doses of 2.22-2.96 GBq/m 2 were administered; maximum tolerated dose (MTD) was determined at 2.96 GBq/m 2 . Tumour mass reduction (>25%-100%), documented by computed tomography or magnetic resonance imaging, occurred in 12/48 patients (25%), with 8/48 having a duration of response of at least 12 months. At present, 12 patients are still in remission, comprising four with a complete response, two with a parital response, two with a minor response and four with stable disease. Median survival from 90 Y treatment is 11 months for grade IV glioblastoma and 19 months for grade III anaplastic gliomas. Avidin-biotin based three-step radionuclide therapy is well tolerated at the dose of 2.2 GBq/m 2 , allowing the injection of 90 Y-biotin without bone marrow transplantation. This new approach interferes with the progression of high-grade glioma and may produce tumour regression in patients no longer responsive to other therapies. (orig.)

  9. In vivo evaluation of an anti-PSMA antibody conjugated with varying numbers of biotin molecules in a pretargeting protocol

    International Nuclear Information System (INIS)

    Wilbur, D.S.; Hamlin, D.K.; Quinn, J.; Vessella, R.L.

    2003-01-01

    An investigation has been conducted to determine the effect of varying the number of biotin molecules conjugated with an anti-PSMA antibody (mAb) as part of our studies to optimize biotinylated antibodies and radiolabeled streptavidin in pretargeting protocols for Targeted Radionuclide Therapy of prostate cancer. In the investigation, the anti-PSMA antibody 107-1A4 was biotinylated with varying amounts of biotinamidocaproate N-hydroxysuccinimide ester. This procedure resulted in obtaining 107-1A4 with 2.3, 4.5, and 6.8 biotin conjugated as measured by the standard HABA assay. The biotinylated 107-1A4 was radioiodinated and was evaluated in a pretargeting protocol in athymic mice bearing LNCaP human tumor xenografts. In the protocol, 50 μg biotinylated [ 125 I]107-1A4 was injected, followed 48h later by 25 μg of avidin for blood clearance, and 1h after that 20 μg of radiolabeled succinylated recombinant streptavidin ([ 13 1I]sSAv) was administered. The tumor localization and tissue distribution was evaluated at 24, 48, and 72h post [ 131 I]sSAv injection. With 2.3 biotin/mAb, an approximate 1:1 molar ratio (4-5 pmol/g) of sSAv/mAb was obtained at all three time points. With 4.5 biotin/mAb, a 1:1 ratio was observed at 24h, but approx. 2: 1 was observed at 48 and 72h pi. With 6.8 biotin/mAb, sSAv/mAb ratios of approximately 1.5:1; 2:1; and 3:1 were obtained at 24, 48, and 72h pi respectively. The amount of sSAv localized in the tumor was nearly the same (4-5 pmol/g) when 107-1A4 had 2.3 or 4.5 biotin conjugated, but decreased to 3-4.5 pmol/g with 6.8 biotin conjugated. Because the highest levels of co-localized sSAv was found with the lowest number of biotin conjugates, the observed differences in ratios of sSAv/mAb may be best explained as differences in internalization, and degradation of mAb and protease resistant sSAv. In duplicate experiments, similar results were obtained with biotinylated 107-1A4 F(ab') 2 , but not with an mAb to a non-internalizing antigen

  10. Country report: Italy (Chinol). Pre-clinical evaluation of a new biotin-DOTA conjugate labeled with 90Y for application in pretargeting clinical protocols

    International Nuclear Information System (INIS)

    Chinol, Marco

    2010-01-01

    In the attempt to improve the therapeutic efficacy of radiolabeled mAbs in cancer radioimmunotherapy, various studies have examined the concept of tumor pretargeting. The so called three-step pretargeting technique, employing the avidin–biotin system, was applied in phase I-II clinical trials showing low toxicity and therapeutic efficacy. The final step of the pretargeting protocols consists in the systemic injection of radiolabeled biotin. The worldwide recognized “successful association” is between 90 Y and the tetraazamacrocycle DOTA chelator chemically bound to biotin. Improvements in the structure of the biotin-DOTA conjugate have been reported by our group following a novel approach which simplified the synthetic pattern by reducing the amide group to a methylene group, thus transforming the amide into a secondary amine without affecting the length of the biotin side arm. Preliminary in-vitro experiments, previously published by our group, indicated the potential of the new conjugate. Based on our previous experience with avidin-based pre-targeting followed 90 Y-DOTA-biotin in the locoregional treatment of peritoneal carcinomatosis and malignant glioma suggested that similar radionuclide therapy might be worth investigating as a partial replacement of external beam radiotherapy in breast cancer. We have developed IART® the Intra-operative Avidination for Radionuclide Therapy that relies on the avidin-biotin binding system. In fact, the “avidination” of the anatomical area of the tumor with native avidin, directly injected by the surgeon into and around the tumor bed, provides a target for the radiolabeled biotin intravenously (iv) injected one day later. In order to optimize the overall strategy, further efforts were needed to optimize the use of the labeled new biotin conjugate and to elucidate its chemical and biological properties. In the first 18 months of this CRP, the pre-clinical evaluation of this new reduced biotinamidohexylamine

  11. Y2O3: Eu,Zn nanocrystals as a fluorescent probe for the detection of biotin

    International Nuclear Information System (INIS)

    Ganjkhanlou, Y.; Hosseinnia, A.; Kazemzad, M.; Khanlarkhani, A.; Moghaddam, A.B.

    2012-01-01

    We report on the application of nanocrystals (NCs) of the type Y 2 O 3 : Eu,Zn as a probe for the fluorescent detection of biotin in aqueous solution. The NCs were dispersed in water in the presence of various surface modifiers including mercaptoethanol (ME), monoethanolamine and ethylene glycol. Both the absorbance of surfactant and the stability of the suspensions were investigated in order to optimize the experimental conditions. ME is found to be the most suitable surfactant for stabilization of the suspended NCs. Their photoluminescence intensity is found to be quenched by biotin. The Stern-Volmer constant for the quenching process is 7.6 x 10 3 M -1 . This NC probe can be applied to the detection of biotin in the 1-60 μM concentration range with detection limit of 1.89 μM. The possible mechanisms of quenching also are discussed. (author)

  12. Characterization of in-containment cables for nuclear plant life extension

    International Nuclear Information System (INIS)

    DuCharme, A.R.; Bustard, L.D.

    1988-01-01

    Electrical cable is made by a large number of manufacturers and used for a variety of applications in nuclear plants. cables have been identified in the Monticello and Surry Pilot Plant life extension studies and the NRC Nuclear Plant Aging Research Program as components important to the economic and safety aspects of life extension. Currently, fitness for service is largely determined by preoperational testing. The US Department of Energy is supporting work at Sandia National Laboratories to assess the technical basis for the life extension of cables found inside containment at US nuclear plants. The work is being performed in coordination with the Nuclear Management and Resource Council's (NUMARC) NUPLEX Working Group. The initial task of this effort is to characterize the design attributes of in-containment cables. This has been completed via development of a data base depicting the manufacturer, type, material composition, use, qualification, and relative popularity of cables installed in containment. Other ongoing work is focused on a review of cable operational experience and assessment of the issues affecting cable life extension. In the long term, the work aims to identify the technical criteria and life extension strategies needed to support continued cable qualification by nuclear plant owner/operators. 7 refs., 4 tabs

  13. EFSA NDA Panel (EFSA Panel on Dietetic Products, Nutrition and Allergies), 2014. Scientific Opinion on Dietary Reference Values for biotin

    DEFF Research Database (Denmark)

    Tetens, Inge

    2014-01-01

    Following a request from the European Commission, the Panel on Dietetic Products, Nutrition and Allergies (NDA) derived Dietary Reference Values (DRVs) for biotin. Biotin is a water-soluble vitamin which serves as a co-factor for several carboxylases that play critical roles in the synthesis...

  14. Intravenous siRNA of brain cancer with receptor targeting and avidin-biotin technology.

    Science.gov (United States)

    Xia, Chun-Fang; Zhang, Yufeng; Zhang, Yun; Boado, Ruben J; Pardridge, William M

    2007-12-01

    The effective delivery of short interfering RNA (siRNA) to brain following intravenous administration requires the development of a delivery system for transport of the siRNA across the brain capillary endothelial wall, which forms the blood-brain barrier in vivo. siRNA was delivered to brain in vivo with the combined use of a receptor-specific monoclonal antibody delivery system, and avidin-biotin technology. The siRNA was mono-biotinylated on either terminus of the sense strand, in parallel with the production of a conjugate of the targeting MAb and streptavidin. Rat glial cells (C6 or RG-2) were permanently transfected with the luciferase gene, and implanted in the brain of adult rats. Following the formation of intra-cranial tumors, the rats were treated with a single intravenous injection of 270 microg/kg of biotinylated siRNA attached to a transferrin receptor antibody via a biotin-streptavidin linker. The intravenous administration of the siRNA caused a 69-81% decrease in luciferase gene expression in the intracranial brain cancer in vivo. Brain delivery of siRNA following intravenous administration is possible with siRNAs that are targeted to brain with the combined use of receptor specific antibody delivery systems and avidin-biotin technology.

  15. The synthesis and characterization of biotin-silver-dendrimer nanocomposites as novel bioselective labels

    Energy Technology Data Exchange (ETDEWEB)

    Maly, J; Lampova, H; Semeradtova, A; Stofik, M [Faculty of Science, University of J E Purkynje, 40096 Usti nad Labem (Czech Republic); Kovacik, L, E-mail: malyjalga@seznam.c [Institute of Cellular Biology and Pathology, First Faculty of Medicine, Charles University in Prague (Czech Republic)

    2009-09-23

    This paper presents a synthesis of a novel nanoparticle label with selective biorecognition properties based on a biotinylated silver-dendrimer nanocomposite (AgDNC). Two types of labels, a biotin-AgDNC (bio-AgDNC) and a biotinylated AgDNC with a poly(ethylene)glycol spacer (bio-PEG-AgDNC), were synthesized from a generation 7 (G7) hydroxyl-terminated ethylenediamine-core-type (2-carbon core) PAMAM dendrimer (DDM) by an N,N'-dicyclohexylcarbodiimide (DDC) biotin coupling and a NaBH{sub 4} silver reduction method. Synthesized conjugates were characterized by several analytical methods, such as UV-vis, FTIR, AFM, TEM, ELISA, HABA assay and SPR. The results show that stable biotinylated nanocomposites can be formed either with internalized silver nanoparticles (AgNPs) in a DMM polymer backbone ('type I') or as externally protected ('type E'), depending on the molar ratio of the silver/DMM conjugate and type of conjugate. Furthermore, the selective biorecognition function of the biotin is not affected by the AgNPs' synthesis step, which allows a potential application of silver nanocomposite conjugates as biospecific labels in various bioanalytical assays, or potentially as fluorescence cell biomarkers. An exploitation of the presented label in the development of electrochemical immunosensors is anticipated.

  16. Photoacoustic imaging of tumor targeting with biotin conjugated nanostructured phthalocyanine assemblies

    Science.gov (United States)

    Lee, Seunghyun; Li, Xingshu; Lee, Dayoung; Yoon, Juyoung; Kim, Chulhong

    2018-02-01

    Visualizing biological markers and delivering bioactive agents to living organisms are important to biological research. In recent decades, photoacoustic imaging (PAI) has been significantly improved in the area of molecular imaging, which provides high-resolution volume imaging with high optical absorption contrast. To demonstrate the ability of nanoprobes to target tumors using PAI, we synthesize convertible nanostructured agents with strong photothermal and photoacoustic properties and linked the nanoprobe with biotin to target tumors in small animal model. Interestingly, these nanoprobes allow partial to disassemble in the presence of targeted proteins that switchable photoactivity, thus the nanoprobes provides a fluorescent-cancer imaging with high signal-to-background ratios. The proposed nanoprobe produce a much stronger PA signal compared to the same concentration of methylene blue (MB), which is widely used in clinical study and contrast agent for PAI. The biotin conjugated nanoprobe has high selectivity for biotin receptor positive cancer cells such as A549 (human lung cancer). Then we subsequently examined the PA properties of the nanoprobe that are inherently suitable for in vivo PAI. After injecting of the nanoprobe via intravenous method, we observed the mice's whole body by PA imaging and acquired the PA signal near the cancer. The PA signal increased linearly with time after injection and the fluorescence signal near the cancer was confirmed by fluorescence imaging. The ability to target a specific cancer of the nanoprobe was well verified by PA imaging. This study provides valuable perspective on the advancement of clinical translations and in the design of tumor-targeting phototheranostic agents that could act as new nanomedicines.

  17. Magnetic molecularly imprinted polymer for the isolation and detection of biotin and biotinylated biomolecules.

    Science.gov (United States)

    Ben Aissa, A; Herrera-Chacon, A; Pupin, R R; Sotomayor, M D P T; Pividori, M I

    2017-02-15

    Magnetic separation based on biologically-modified magnetic particles is a preconcentration procedure commonly integrated in magneto actuated platforms for the detection of a huge range of targets. However, the main drawback of this material is the low stability and high cost. In this work, a novel hybrid molecularly-imprinted polymer with magnetic properties is presented with affinity towards biotin and biotinylated biomolecules. During the synthesis of the magneto core-shell particles, biotin was used as a template. The characterization of this material by microscopy techniques including SEM, TEM and confocal microscopy is presented. The application of the magnetic-MIPs for the detection of biotin and biotinylated DNA in magneto-actuated platforms is also described for the first time. The magnetic-MIP showed a significant immobilization capacity of biotinylated molecules, giving rise to a cheaper and a robust method (it is not required to be stored at 4°C) with high binding capacity for the separation and purification under magnetic actuation of a wide range of biotinylated molecules, and their downstream application including determination of their specific targets. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Effect of biotin supplementation on meat quality of F1 Wagyu/Black Angus feedlot steers of known genotype.

    Science.gov (United States)

    Lawrence, R J; Doyle, J C; Elliott, R; Norton, B W; Loxton, I

    2007-10-01

    Biotin (D-biotin) was supplemented to F1 Wagyu/Black Angus steers fed a wheat-based ration to evaluate the effect on meat quality. One hundred and eight steers of known Wagyu sire lines were assigned to three biotin treatments (0, 10 and 20mg/head/day) with each treatment replicated four times using an unfasted liveweight of 410.5kg (±24.42 SD). Biotin supplementation had no effect (P>0.05) on beef marbling standard at either the 5/6th or 10/11th rib quartering site, 10/11th rib intra-muscular fat percentage, intra-muscular fat fatty acid composition or adipose melting points. Wagyu genotype had an effect (P<0.05) on beef marbling standard and intra-muscular fat percentage at the 10/11th rib, inter-muscular and intra-muscular melting point and fatty acid composition of intra-muscular fat. A significant (P<0.001) but poor correlation existed between beef marbling standard and intra-muscular fat percentage (R(2)=0.198). Total conjugated linoleic acid had a highly significantly (P<0.0001) positive correlation to intra-muscular fat percentage (R(2)=0.446).

  19. Periodic protein adsorption at the gold/biotin aqueous solution interface: evidence of kinetics with time delay

    Science.gov (United States)

    Neff, H.; Laborde, H. M.; Lima, A. M. N.

    2016-11-01

    An oscillatory molecular adsorption pattern of the protein neutravidin from aqueous solution onto gold, in presence of a pre-deposited self assembled mono-molecular biotin film, is reported. Real time surface Plasmon resonance sensing was utilized for evaluation of the adsorption kinetics. Two different fractions were identified: in the initial phase, protein molecules attach irreversibly onto the Biotin ligands beneath towards the jamming limit, forming a neutravidin-biotin fraction. Afterwards, the growth rate exhibits distinct, albeit damped adsorption-desorption oscillations over an extended time span, assigned to a quasi reversibly bound fraction. These findings agree with, and firstly confirm a previously published model, proposing macro-molecular adsorption with time delay. The non-linear dynamic model is applicable to and also resembles non-damped oscillatory binding features of the hetero-catalytic oxidation of carbon monoxide molecules on platinum in the gas phase. An associated surface residence time can be linked to the dynamics and time scale required for self-organization.

  20. Quantitation of biotinylated compounds by a solid-phase assay using a /sup 125/I-labelled biotin derivative

    Energy Technology Data Exchange (ETDEWEB)

    Smith, P J; Warren, R M; Holt, C von

    1987-05-11

    The biotin analogue biotinylglycyltyrosine has been synthesized and labelled to a specific activity of 2000 Ci/mmol with /sup 125/I. This analogue has been used in conjunction with immobilized streptavidin in an assay which detects as little as 1 fmol biotin or biotinylated molecules in solution. The determination of biotinylated insulin in a tissue extract and the quantitation of a transcription assay are given as examples. (Auth.). 12 refs.; 4 figs.; 3 tabs.

  1. Flavin-containing monooxygenases in plants: looking beyond detox.

    Science.gov (United States)

    Schlaich, Nikolaus L

    2007-09-01

    Flavin-containing monooxygenases (FMOs) are known in bacteria, yeast and mammals where they catalyze the transfer of one atom of molecular O(2) to low molecular weight substrates. The predominant physiological function of animal FMOs appears to be detoxification of a vast spectrum of xenobiotics but until recently very little was known about the function of FMOs in plants. In the last two to three years, genetic and biochemical characterization has shown that plant FMOs can catalyze specific steps in the biosynthesis of auxin or in the metabolism of glucosinolates, and, furthermore, have a role in pathogen defence. Thus, plant FMOs hint that further FMO functions might be identified also in non-plant organisms and could stimulate novel research in this area.

  2. Construction of pegylated multilayer architectures via (strept)avidin/biotin interactions

    International Nuclear Information System (INIS)

    Dai Zhifei; Wilson, John T.; Chaikof, Elliot L.

    2007-01-01

    Pegylated multilayer architectures were fabricated as films on planar substrates, as shells on colloidal particles, or as free-standing hollow capsules using layer-by-layer (LbL) self-assembly of biotinylated poly-L-lysine (PLL) and (strept)avidin. Poly(ethylene glycol) (PEG) was incorporated into the multilayer architectures by assembly with biotin-derivatized poly(L-lysine)-g-poly(ethylene glycol)(PPB). Stepwise growth of multilayers was followed by UV-vis spectroscopy and the formation of core-shells and hollow capsules characterized by means of confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM). Both absorbance and TEM data suggest that approximately two layers of FITC-avidin were adsorbed with each surface deposition. In contrast, use of unmodified PLL did not lead to formation of multilayer coatings, confirming that (strept)avidin-biotin interactions were responsible for film growth even in the presence of electrostatic repulsive forces between PLL and avidin and the steric hindrance of associated PEG chains. This technique provides new opportunities for the generation of robust films with tailored interfacial binding and transport properties

  3. Country report: Italy (Chinol). Pre-clinical evaluation of a new biotin-DOTA conjugate labeled with {sup 90}Y for application in pretargeting clinical protocols

    Energy Technology Data Exchange (ETDEWEB)

    Chinol, Marco

    2010-07-01

    In the attempt to improve the therapeutic efficacy of radiolabeled mAbs in cancer radioimmunotherapy, various studies have examined the concept of tumor pretargeting. The so called three-step pretargeting technique, employing the avidin–biotin system, was applied in phase I-II clinical trials showing low toxicity and therapeutic efficacy. The final step of the pretargeting protocols consists in the systemic injection of radiolabeled biotin. The worldwide recognized “successful association” is between {sup 90}Y and the tetraazamacrocycle DOTA chelator chemically bound to biotin. Improvements in the structure of the biotin-DOTA conjugate have been reported by our group following a novel approach which simplified the synthetic pattern by reducing the amide group to a methylene group, thus transforming the amide into a secondary amine without affecting the length of the biotin side arm. Preliminary in-vitro experiments, previously published by our group, indicated the potential of the new conjugate. Based on our previous experience with avidin-based pre-targeting followed {sup 90}Y-DOTA-biotin in the locoregional treatment of peritoneal carcinomatosis and malignant glioma suggested that similar radionuclide therapy might be worth investigating as a partial replacement of external beam radiotherapy in breast cancer. We have developed IART® the Intra-operative Avidination for Radionuclide Therapy that relies on the avidin-biotin binding system. In fact, the “avidination” of the anatomical area of the tumor with native avidin, directly injected by the surgeon into and around the tumor bed, provides a target for the radiolabeled biotin intravenously (iv) injected one day later. In order to optimize the overall strategy, further efforts were needed to optimize the use of the labeled new biotin conjugate and to elucidate its chemical and biological properties. In the first 18 months of this CRP, the pre-clinical evaluation of this new reduced biotinamidohexylamine

  4. Plant life management of the ACR-1000 Concrete containment structure

    International Nuclear Information System (INIS)

    Abrishami, H.H.; Ricciuti, R.; Elgohary, M.

    2009-01-01

    The Ageing of reinforced concrete structures due to service conditions, aggressive environments, or accidents may cause their strength, serviceability and durability to decrease over time. For a new plant, a Plant Life Management (PLiM) program should start in the design process and then continues through the plant operation and decommissioning. Hence, PLiM must provide not only Ageing Management program (AMP) but also provide requirements on material characteristic and design criteria as well. The purpose of this paper is to present the Plant Life Management (PLiM) strategy for the concrete containment structure of the ACR-10001 (Advanced CANDU Reactor) designed by AECL. The ACR-1000 is designed for a 100-year plant life including 60-year operating life and an additional 40-year decommissioning period. The approach adopted for the PLiM strategy of the concrete containment structure is a preventive one, key areas being: 1) design methodology, 2) material performance and 3) ageing management program. During the design phase, in addition to strength and serviceability, durability, throughout the service life and decommissioning phase of the ACR-1000 structure, is a major consideration. Factors affecting durability design include: a) concrete performance, b) structural application, and c) consideration of environmental conditions. In addition to addressing the design methodology and material performance requirements, a systematic approach for the ageing management program for the concrete containment structure is presented. (authors)

  5. Double loading and unloading plant for EHB containers

    Energy Technology Data Exchange (ETDEWEB)

    Arntz, H.; Kalle, N.; Miebach, K.

    1986-04-01

    An integrated haulage system was installed for the shafts of the 'Eschweiler Bergwerksverein', Emil Mayrisch colliery and Anna colliery for the purpose of cost reduction. Transport problems resulting from different floor levels and track gauges were solved by means of a double loading and unloading plant for EHB containers at the 860 m level of Anna mine. The mode of operation, the hydraulic system, and the control system of the loading and unloading plant are described.

  6. NeutrAvidin Functionalization of CdSe/CdS Quantum Nanorods and Quantification of Biotin Binding Sites using Biotin-4-Fluorescein Fluorescence Quenching

    OpenAIRE

    Lippert, Lisa G.; Hallock, Jeffrey T.; Dadosh, Tali; Diroll, Benjamin T.; Murray, Christopher B.; Goldman, Yale E.

    2016-01-01

    We developed methods to solubilize, coat, and functionalize with NeutrAvidin elongated semiconductor nanocrystals (quantum nanorods, QRs) for use in single molecule polarized fluorescence microscopy. Three different ligands were compared with regard to efficacy for attaching NeutrAvidin using the “zero-length cross-linker” 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide (EDC). Biotin-4-fluorescene (B4F), a fluorophore that is quenched when bound to avidin proteins, was used to quantify bioti...

  7. Modeling of hydrogen behaviour in a PWR nuclear power plant containment with the CONTAIN code

    International Nuclear Information System (INIS)

    Bobovnik, G.; Kljenak, I.

    2001-01-01

    Hydrogen behavior in the containment during a severe accident in a two-loop Westinghouse-type PWR nuclear power plant was simulated with the CONTAIN code. The accident was initiated with a cold-leg break of the reactor coolant system in a steam generator compartment. In the input model, the containment is represented with 34 cells. Beside hydrogen concentration, the containment atmosphere temperature and pressure and the carbon monoxide concentration were observed as well. Simulations were carried out for two different scenarios: with and without successful actuation of the containment spray system. The highest hydrogen concentration occurs in the containment dome and near the hydrogen release location in the early stages of the accident. Containment sprays do not have a significant effect on hydrogen stratification.(author)

  8. Genes encoding biotin carboxylase subunit of acetyl-CoA carboxylase from Brassica napus and parental species: cloning, expression patterns, and evolution

    Science.gov (United States)

    Comparative genomics is a useful tool to investigate gene and genome evolution. Biotin carboxylase (BC), an important subunit of heteromeric ACCase that is a rate-limiting enzyme in fatty acid biosynthesis in dicots, catalyzes ATP, biotin-carboxyl-carrier protein and CO2 to form carboxybiotin-carbo...

  9. Uptake of biotin by Chlamydia Spp. through the use of a bacterial transporter (BioY and a host-cell transporter (SMVT.

    Directory of Open Access Journals (Sweden)

    Derek J Fisher

    Full Text Available Chlamydia spp. are obligate intracellular Gram-negative bacterial pathogens that cause disease in humans and animals. Minor variations in metabolic capacity between species have been causally linked to host and tissue tropisms. Analysis of the highly conserved genomes of Chlamydia spp. reveals divergence in the metabolism of the essential vitamin biotin with genes for either synthesis (bioF_2ADB and/or transport (bioY. Streptavidin blotting confirmed the presence of a single biotinylated protein in Chlamydia. As a first step in unraveling the need for divergent biotin acquisition strategies, we examined BioY (CTL0613 from C. trachomatis 434/Bu which is annotated as an S component of the type II energy coupling-factor transporters (ECF. Type II ECFs are typically composed of a transport specific component (S and a chromosomally unlinked energy module (AT. Intriguingly, Chlamydia lack recognizable AT modules. Using (3H-biotin and recombinant E. coli expressing CTL0613, we demonstrated that biotin was transported with high affinity (a property of Type II ECFs previously shown to require an AT module and capacity (apparent K(m of 3.35 nM and V(max of 55.1 pmol×min(-1×mg(-1. Since Chlamydia reside in a host derived membrane vacuole, termed an inclusion, we also sought a mechanism for transport of biotin from the cell cytoplasm into the inclusion vacuole. Immunofluorescence microscopy revealed that the mammalian sodium multivitamin transporter (SMVT, which transports lipoic acid, biotin, and pantothenic acid into cells, localizes to the inclusion. Since Chlamydia also are auxotrophic for lipoic and pantothenic acids, SMVT may be subverted by Chlamydia to move multiple essential compounds into the inclusion where BioY and another transporter(s would be present to facilitate transport into the bacterium. Collectively, our data validates the first BioY from a pathogenic organism and describes a two-step mechanism by which Chlamydia transport biotin

  10. Uptake of Biotin by Chlamydia Spp. through the Use of a Bacterial Transporter (BioY) and a Host-Cell Transporter (SMVT)

    Science.gov (United States)

    Fisher, Derek J.; Fernández, Reinaldo E.; Adams, Nancy E.; Maurelli, Anthony T.

    2012-01-01

    Chlamydia spp. are obligate intracellular Gram-negative bacterial pathogens that cause disease in humans and animals. Minor variations in metabolic capacity between species have been causally linked to host and tissue tropisms. Analysis of the highly conserved genomes of Chlamydia spp. reveals divergence in the metabolism of the essential vitamin biotin with genes for either synthesis (bioF_2ADB) and/or transport (bioY). Streptavidin blotting confirmed the presence of a single biotinylated protein in Chlamydia. As a first step in unraveling the need for divergent biotin acquisition strategies, we examined BioY (CTL0613) from C. trachomatis 434/Bu which is annotated as an S component of the type II energy coupling-factor transporters (ECF). Type II ECFs are typically composed of a transport specific component (S) and a chromosomally unlinked energy module (AT). Intriguingly, Chlamydia lack recognizable AT modules. Using 3H-biotin and recombinant E. coli expressing CTL0613, we demonstrated that biotin was transported with high affinity (a property of Type II ECFs previously shown to require an AT module) and capacity (apparent K(m) of 3.35 nM and V(max) of 55.1 pmol×min−1×mg−1). Since Chlamydia reside in a host derived membrane vacuole, termed an inclusion, we also sought a mechanism for transport of biotin from the cell cytoplasm into the inclusion vacuole. Immunofluorescence microscopy revealed that the mammalian sodium multivitamin transporter (SMVT), which transports lipoic acid, biotin, and pantothenic acid into cells, localizes to the inclusion. Since Chlamydia also are auxotrophic for lipoic and pantothenic acids, SMVT may be subverted by Chlamydia to move multiple essential compounds into the inclusion where BioY and another transporter(s) would be present to facilitate transport into the bacterium. Collectively, our data validates the first BioY from a pathogenic organism and describes a two-step mechanism by which Chlamydia transport biotin from the

  11. Rapid and specific biotin labelling of the erythrocyte surface antigens of both cultured and ex-vivo Plasmodium parasites

    Directory of Open Access Journals (Sweden)

    Thompson Joanne

    2007-05-01

    Full Text Available Abstract Background Sensitive detection of parasite surface antigens expressed on erythrocyte membranes is necessary to further analyse the molecular pathology of malaria. This study describes a modified biotin labelling/osmotic lysis method which rapidly produces membrane extracts enriched for labelled surface antigens and also improves the efficiency of antigen recovery compared with traditional detergent extraction and surface radio-iodination. The method can also be used with ex-vivo parasites. Methods After surface labelling with biotin in the presence of the inhibitor furosemide, detergent extraction and osmotic lysis methods of enriching for the membrane fractions were compared to determine the efficiency of purification and recovery. Biotin-labelled proteins were identified on silver-stained SDS-polyacrylamide gels. Results Detergent extraction and osmotic lysis were compared for their capacity to purify biotin-labelled Plasmodium falciparum and Plasmodium chabaudi erythrocyte surface antigens. The pellet fraction formed after osmotic lysis of P. falciparum-infected erythrocytes is notably enriched in suface antigens, including PfEMP1, when compared to detergent extraction. There is also reduced co-extraction of host proteins such as spectrin and Band 3. Conclusion Biotinylation and osmotic lysis provides an improved method to label and purify parasitised erythrocyte surface antigen extracts from both in vitro and ex vivo Plasmodium parasite preparations.

  12. Experimental study of biotin-avidin pretargeting technique for anti-CEA McAb radioimmunoimaging

    International Nuclear Information System (INIS)

    Sun Jianzhong; Zhu Chengmo; Guan Liang; Li Biao; Zhang Jixian; Shi Ailan; Zhang Suyin

    1996-01-01

    Biotin-avidin pretargeting technique was used in promoting the diagnostic efficacy of anti-CEA McAb radioimmunoimaging. CEA McAb was conjugated with biotin McAb (B-McAb), streptavidin (SA) was labeled with 131 I ( 131 I-SA) and DTPA-biotin with 111 In( 111 In-DTPA-B). Experimental human colonic tumor bearing nude mice were used. Two step method: B-McAb was preinjected, followed by 131 I SA 48h later, 24, 48, 96 and 120 h postinjection, γ-imaging and biodistribution were studied. Three step method: B-McAb was preinjected, followed by cold SA 24h later and 111 In-DTPA-B another 24h later. 2,6,24 and 48h postinjection, γ-imaging and biodistribution were also studied. Two step method: T/NT of all organs in experimental group was significantly increased compared with controls. The blood T/NT in experimental group and control group at 24 and 120h was 1.11:0.42 and 8.58:3.51, respectively. Tumor % ID/g in all organs slightly decreased compared with direct group. In γ-imaging radioactivity has been accumulated in tumor site as early as 24h, while only slightly visualized or non-visualized in controls. Three step method: in experimental group the blood T/NT reached 4.19 at 2 h, whereas all was < 1.37 at each phase of controls, the T/NT of all organs was also higher in experimental grouped than in controls. The tumor % ID/g in experimental group was 9.72% at 2h and 3.65% at 48h whereas % ID/g in controls in all phases was <3.07. The tumor clearly visualized at 2h and clearer at 48h in γ-imaging. In controls, the tumor was slightly visualized also to early stage, but faded away later on. Biotin-avidin pretargeting technique can elevate the T/NT ratio and decrease the blood background. Early imaging was obtained with better imaging quality

  13. Transfer of the high-GC cyclohexane carboxylate degradation pathway from Rhodopseudomonas palustris to Escherichia coli for production of biotin.

    Science.gov (United States)

    Bernstein, Jeffrey R; Bulter, Thomas; Liao, James C

    2008-01-01

    This work demonstrates the transfer of the five-gene cyclohexane carboxylate (CHC) degradation pathway from the high-GC alphaproteobacterium Rhodopseudomonas palustris to Escherichia coli, a gammaproteobacterium. The degradation product of this pathway is pimeloyl-CoA, a key metabolite in E. coli's biotin biosynthetic pathway. This pathway is useful for biotin overproduction in E. coli; however, the expression of GC-rich genes is troublesome in this host. When the native R. palustris CHC degradation pathway is transferred to a DeltabioH pimeloyl-CoA auxotroph of E. coli, it is unable to complement growth in the presence of CHC. To overcome this expression problem we redesigned the operon with decreased GC content and removed stretches of high-GC intergenic DNA which comprise the 5' untranslated region of each gene, replacing these features with shorter low-GC sequences. We show this synthetic construct enables growth of the DeltabioH strain in the presence of CHC. When the synthetic degradation pathway is overexpressed in conjunction with the downstream genes for biotin biosynthesis, we measured significant accumulation of biotin in the growth medium, showing that the pathway transfer is successfully integrated with the host metabolism.

  14. Study on alternatives of inertisation of nuclear power plant containment

    International Nuclear Information System (INIS)

    Baron, J.H.; Zarate, S.M.

    1998-01-01

    In the course of a severe accident in a nuclear power plant, the hydrogen generation and other flammable gases, during the core degradation phase and the interaction corium-concrete, could produce the failure of the containment by overpressure of by combustion. According to the analysis of the potential effects of hydrogen evolution, following accidents inside the containment trough a Defense-in depth principle, which attempts to assure that the containment must not fail catastrophically, two techniques have been evaluated: a: Inertisation pre-accident and b: Inertisation post-accident. The technique of inertisation pre-accident consists in replacing the air of the containment with inert-gas like nitrogen (N 2 ) or carbon dioxide (CO 2 ) during the normal operation. The inertisation post-accident in combination with early venting system consists in replacing the air of the containment with inert-gas like nitrogen (N 2 ) or carbon dioxide (CO 2 ), immediately after the beginning of the accident, while the radioactivity is still negligible inside the containment. A system of inertisation pre-accident with nitrogen is used on BWR Mark I and Mark II. Investigations on the inertisation post-accident of the containment atmosphere during severe accidents have been carried out with different objectives from principles of the decade of 1980. Studies concerning hydrogen problem for the nuclear power plants Atucha I and CAREM-25 have permitted to know that the hydrogen generation during an accidental sequence with core degradation, would result important, being able to arrive to form explosive mixtures. In the present work, the applicability of the techniques of inertisation is analyzed for the containment of the Atucha I and CAREM-25, considering the particular design characteristics of these plants. (author) [es

  15. Development of Robust and Standardized Cantilever Sensors Based on Biotin/Neutravidin Coupling for Antibody Detection

    Directory of Open Access Journals (Sweden)

    Christoph Gerber

    2013-04-01

    Full Text Available A cantilever-based protein biosensor has been developed providing a customizable multilayer platform for the detection of antibodies. It consists of a biotin-terminated PEG layer pre-functionalized on the gold-coated cantilever surface, onto which NeutrAvidin is adsorbed through biotin/NeutrAvidin specific binding. NeutrAvidin is used as a bridge layer between the biotin-coated surface and the biotinylated biomolecules, such as biotinylated bovine serum albumin (biotinylated BSA, forming a multilayer sensor for direct antibody capture. The cantilever biosensor has been successfully applied to the detection of mouse anti-BSA (m-IgG and sheep anti-BSA(s-IgG antibodies. As expected, the average differential surface stress signals of about 5.7 ± 0.8 ´ 10−3 N/m are very similar for BSA/m-IgG and BSA/s-IgG binding, i.e., they are independent of the origin of the antibody. A statistic evaluation of 112 response curves confirms that the multilayer protein cantilever biosensor shows high reproducibility. As a control test, a biotinylated maltose binding protein was used for detecting specificity of IgG, the result shows a signal of bBSA layer in response to antibody is 5.8 ´ 10−3 N/m compared to bMBP. The pre-functionalized biotin/PEG cantilever surface is found to show a long shelf-life of at least 40 days and retains its responsivity of above 70% of the signal when stored in PBS buffer at 4 °C. The protein cantilever biosensor represents a rapid, label-free, sensitive and reliable detection technique for a real-time protein assay.

  16. The origin of the cooperativity in the streptavidin-biotin system: A computational investigation through molecular dynamics simulations

    Science.gov (United States)

    Liu, Fengjiao; Zhang, John Z. H.; Mei, Ye

    2016-06-01

    Previous experimental study measuring the binding affinities of biotin to the wild type streptavidin (WT) and three mutants (S45A, D128A and S45A/D128A double mutant) has shown that the loss of binding affinity from the double mutation is larger than the direct sum of those from two single mutations. The origin of this cooperativity has been investigated in this work through molecular dynamics simulations and the end-state free energy method using the polarized protein-specific charge. The results show that this cooperativity comes from both the enthalpy and entropy contributions. The former contribution mainly comes from the alternations of solvation free energy. Decomposition analysis shows that the mutated residues nearly have no contributions to the cooperativity. Instead, N49 and S88, which are located at the entry of the binding pocket and interact with the carboxyl group of biotin, make the dominant contribution among all the residues in the first binding shell around biotin.

  17. Design, synthesis, and evaluation of VEGFR-targeted macromolecular MRI contrast agent based on biotin-avidin-specific binding.

    Science.gov (United States)

    Liu, Yongjun; Wu, Xiaoyun; Sun, Xiaohe; Wang, Dan; Zhong, Ying; Jiang, Dandan; Wang, Tianqi; Yu, Dexin; Zhang, Na

    2017-01-01

    Developing magnetic resonance imaging (MRI) contrast agents with high relaxivity and specificity was essential to increase MRI diagnostic sensitivity and accuracy. In this study, the MRI contrast agent, vascular endothelial growth factor receptor (VEGFR)-targeted poly (l-lysine) (PLL)-diethylene triamine pentacetate acid (DTPA)-gadolinium (Gd) (VEGFR-targeted PLL-DTPA-Gd, VPDG), was designed and prepared to enhance the MRI diagnosis capacity of tumor. Biotin-PLL-DTPA-Gd was synthesized first, then, VEGFR antibody was linked to biotin-PLL-DTPA-Gd using biotin-avidin reaction. In vitro cytotoxicity study results showed that VPDG had low toxicity to MCF-7 cells and HepG2 cells at experimental concentrations. In cell uptake experiments, VPDG could significantly increase the internalization rates (61.75%±5.22%) in VEGFR-positive HepG2 cells compared to PLL-DTPA-Gd (PDG) (25.16%±4.71%, P contrast agent and held great potential for molecular diagnosis of tumor.

  18. Molecular dynamics investigations of BioH protein substrate specificity for biotin synthesis.

    Science.gov (United States)

    Xue, Qiao; Cui, Ying-Lu; Zheng, Qing-Chuan; Zhang, Hong-Xing

    2016-05-01

    BioH, an enzyme of biotin synthesis, plays an important role in fatty acid synthesis which assembles the pimelate moiety. Pimeloyl-acyl carrier protein (ACP) methyl ester, which is long known to be a biotin precursor, is the physiological substrate of BioH. Azelayl methyl ester, which has a longer chain than pimeloyl methyl ester, conjugated to ACP is also indeed accepted by BioH with very low rate of hydrolysis. To date, the substrate specificity for BioH and the molecular origin for the experimentally observed rate changes of hydrolysis by the chain elongation have remained elusive. To this end, we have investigated chain elongation effects on the structures by using the fully atomistic molecular dynamics simulations combined with binding free energy calculations. The results indicate that the substrate specificity is determined by BioH together with ACP. The added two methylenes would increase the structural flexibility by protein motions at the interface of ACP and BioH, instead of making steric clashes with the side chains of the BioH hydrophobic cavity. On the other hand, the slower hydrolysis of azelayl substrate is suggested to be associated with the loose of contacts between BioH and ACP, and with the lost electrostatic interactions of two ionic/hydrogen bonding networks at the interface of the two proteins. The present study provides important insights into the structure-function relationships of the complex of BioH with pimeloyl-ACP methyl ester, which could contribute to further understanding about the mechanism of the biotin synthetic pathway, including the catalytic role of BioH.

  19. Pressure test behaviour of embalse nuclear power plant containment structure

    International Nuclear Information System (INIS)

    Bruschi, S.; Marinelli, C.

    1984-01-01

    It's described the structural behaviour of the containment structure during the pressure test of the Embalse plant (CANDU type, 600MW), made of prestressed concrete with an epoxi liner. Displacement, strain, temperature, and pressure measurements of the containment structure of the Embalse Nuclear Power Plant are presented. The instrumentation set up and measurement specifications are described for all variables of interest before, during and after the pressure test. The analytical models to simulate the heat transfer due to sun heating and air convenction and to predict the associated thermal strains and displacements are presented. (E.G.) [pt

  20. Efficient production of α-ketoglutarate in the gdh deleted Corynebacterium glutamicum by novel double-phase pH and biotin control strategy.

    Science.gov (United States)

    Li, Yanjun; Sun, Lanchao; Feng, Jia; Wu, Ruifang; Xu, Qingyang; Zhang, Chenglin; Chen, Ning; Xie, Xixian

    2016-06-01

    Production of L-glutamate using a biotin-deficient strain of Corynebacterium glutamicum has a long history. The process is achieved by controlling biotin at suboptimal dose in the initial fermentation medium, meanwhile feeding NH4OH to adjust pH so that α-ketoglutarate (α-KG) can be converted to L-glutamate. In this study, we deleted glutamate dehydrogenase (gdh1 and gdh2) of C. glutamicum GKG-047, an L-glutamate overproducing strain, to produce α-KG that is the direct precursor of L-glutamate. Based on the method of L-glutamate fermentation, we developed a novel double-phase pH and biotin control strategy for α-KG production. Specifically, NH4OH was added to adjust the pH at the bacterial growth stage and NaOH was used when the cells began to produce acid; besides adding an appropriate amount of biotin in the initial medium, certain amount of additional biotin was supplemented at the middle stage of fermentation to maintain a high cell viability and promote the carbon fixation to the flux of α-KG production. Under this control strategy, 45.6 g/L α-KG accumulated after 30-h fermentation in a 7.5-L fermentor and the productivity and yield achieved were 1.52 g/L/h and 0.42 g/g, respectively.

  1. Preparation, biodistribution, and dosimetry of 188Re-Labeled MoAb ior cea1 and its f(ab')2 fragments by avidin-biotin strategy

    International Nuclear Information System (INIS)

    Ferro-Flores, Guillermina; Pimentel-Gonzalez, Gilmara; Gonzalez-Zavala, Maria Antonia; Murphy, Consuelo Arteaga de; Melendez-Alafort, Laura; Tendilla, Jose I.; Croft, Barbara Y.

    1999-01-01

    The biotinylated monoclonal antibody (MoAb) ior cea1 and its F(ab') 2 fragments were labeled with Re-188 by combination of avidin-biotin strategy. 188 Re-MoAb, 188 Re-MoAb-biotin, 188 Re-F(ab') 2 , and 188 Re-F(ab') 2 -biotin preparations were produced for these studies with specific activities of 1.30±0.18 GBq/mg and from instant freeze-dried kit formulations using ethane-1-hydroxy-1,1-diphosphonic acid (EHDP) as a weak competing ligand. There were no significant differences (p>0.05) between the biodistribution in mice of biotinylated and unbiotinylated 188 Re-labeled immunoconjugates. When avidin was injected as a chase after injection of 188 Re-MoAb-biotin or 188 Re-F(ab') 2 -biotin, the blood radioactivity level decreased approximately 75% (cumulated activity) and the effective dose decreased almost 25% with respect to that of the radioimmunoconjugates in which the chase effect was not used. Our results suggest that 188 Re-labeled biotinylated MoAb ior cea1 and its F(ab') 2 fragments prepared by this method are stable complexes in vivo

  2. Double containment shell for nuclear power plants

    International Nuclear Information System (INIS)

    Sykora, D.

    1977-01-01

    A double containment shell is proposed for nuclear power plants, especially those equipped with pressurized water reactors. The shell offers increased environmental protection from primary circuit accidents. The inner shell is built of steel or concrete while the outer shell is always built of concrete. The space between the two shells is filled with water and is provided with several manholes and with stiffeners designed for compensation for load due to the water hydrostatic pressure. Water serves the airtight separation of the containment shell inside from the environment and the absorption of heat released in a primary circuit accident. In case the inner shell is made of concrete, it is provided with heat-removal tubes in-built in its walls ensuring rapid heat transfer from the inside of the containment to the water in the interwall space. (Z.M.)

  3. MR immuno-imaging study using avidin-biotin pre-targeting system on nude mice grafted with human colorectal carcinoma

    International Nuclear Information System (INIS)

    Chai Qingfen; Huang Qiliu; Xu Yikai; Liu Xian; Wu Yuankui

    2001-01-01

    Objective: To further improve the amount of gadolinium located on tumor, a gadolinium chelate enhanced magnetic resonance imaging pre-targeting with avidin-biotin system technique was adopted and the enhancing characteristics of difference of signal intensity at various scan timing were investigated in author's experiment. Methods: (1) Anti-CEA antibody CL -3 was biotinylated in a mixture with antibody to NHS-LS-biotin with a molar ratio of 1/30-50. (2) After the reaction of GdCl 3 and DTPA-B, the unconjugated gadolinium was removed by chromatography on G-10 column. (3) Steps for pre-targeting tumor: First step, McAb-B was injected intravenously into nude mice on the first day. Second step, avidin (Av) and streptavidin (SA) were injected intraperitoneally 24 hours later. Third step, Gd-DTPA-Bt was injected intravenously 48 hours after the first injection. MRI was performed with plain scans, enhanced scans at 20 minutes, 2 hours, 8 hours, and 24 hours after the third step. Signal intensities of tumor and muscles were measured. The pre-targeting effect was compared with those of Gd-DTPA-McAb and Gd-DTPA. Results: (1) Each monoclonal antibody conjugated with 11-23 biotin and the immuno-activity of biotinylated antibody with 12 biotin/antibody was 94.9%. (2) The enhancing effect of pre-targeting approach was tumor specific. Contrarily that of Gd-DTPA was not. (3) The enhancing rate of signal intensity specificity of pre-targeting approach was 43%, while that of McAb-Gd-DTPA was 17.9% only, so the enhancing ratio was 2.4. Conclusion: Pre-targeting approach using avidin-biotin system improves the amounts of gadolinium locating on tumors and yields a specific enhancing effect. It is a promising modality which promotes the ability of Gd labelled magnetic resonance immuno-imaging in the detection of colon cancer and its recurrence

  4. TRANSGENIC PLANT CONTAINMENT

    Science.gov (United States)

    The new technology using plant genetics to produce chemicals, pharmaceuticals, and therapeuitics in a wide array of new plant forms requires sufficient testing to ensure that these new plant introductions are benign in the environment. A recent effort to provide necessary guidan...

  5. ATP-dependent Conformational Changes Trigger Substrate Capture and Release by an ECF-type Biotin Transporter.

    Science.gov (United States)

    Finkenwirth, Friedrich; Sippach, Michael; Landmesser, Heidi; Kirsch, Franziska; Ogienko, Anastasia; Grunzel, Miriam; Kiesler, Cornelia; Steinhoff, Heinz-Jürgen; Schneider, Erwin; Eitinger, Thomas

    2015-07-03

    Energy-coupling factor (ECF) transporters for vitamins and metal ions in prokaryotes consist of two ATP-binding cassette-type ATPases, a substrate-specific transmembrane protein (S component) and a transmembrane protein (T component) that physically interacts with the ATPases and the S component. The mechanism of ECF transporters was analyzed upon reconstitution of a bacterial biotin transporter into phospholipid bilayer nanodiscs. ATPase activity was not stimulated by biotin and was only moderately reduced by vanadate. A non-hydrolyzable ATP analog was a competitive inhibitor. As evidenced by cross-linking of monocysteine variants and by site-specific spin labeling of the Q-helix followed by EPR-based interspin distance analyses, closure and reopening of the ATPase dimer (BioM2) was a consequence of ATP binding and hydrolysis, respectively. A previously suggested role of a stretch of small hydrophobic amino acid residues within the first transmembrane segment of the S units for S unit/T unit interactions was structurally and functionally confirmed for the biotin transporter. Cross-linking of this segment in BioY (S) using homobifunctional thiol-reactive reagents to a coupling helix of BioN (T) indicated a reorientation rather than a disruption of the BioY/BioN interface during catalysis. Fluorescence emission of BioY labeled with an environmentally sensitive fluorophore was compatible with an ATP-induced reorientation and consistent with a hypothesized toppling mechanism. As demonstrated by [(3)H]biotin capture assays, ATP binding stimulated substrate capture by the transporter, and subsequent ATP hydrolysis led to substrate release. Our study represents the first experimental insight into the individual steps during the catalytic cycle of an ECF transporter in a lipid environment. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Vulnerability analysis in a pwr nuclear power plant containment building

    OpenAIRE

    Musolas Otaño, Antoni Maria

    2013-01-01

    When supervising a nuclear power plant, the containment building is crucial. Its functions are guaranteeing structural integrity and avoiding leaks in case of accident. Both events are considered of high risk. Once a given overpressure is registered inside the containment building, three possible outputs are considered: serviceability, breakdown, and collapse. The aim is the study of vulnerability. The vulnerability of the containment building under an overpressure is described by the conditi...

  7. Pyrrolizidine alkaloid-containing toxic plants (Senecio, Crotalaria, Cynoglossum, Amsinckia, Heliotropium, and Echium spp.).

    Science.gov (United States)

    Stegelmeier, Bryan L

    2011-07-01

    Pyrrolizidine alkaloid (PA)-containing plants are found throughout the world and are probably the most common plant cause of poisoning of livestock, wildlife, and humans. PAs are potent liver toxins that under some conditions can be carcinogenic. This article briefly introduces high-risk North American PA-containing plants, summarizing their toxicity and subsequent pathology. Current diagnostic techniques, treatments, and strategies to avoid losses to PA poisoning are also reviewed. Published by Elsevier Inc.

  8. Rapid Identification and Verification of Indirubin-Containing Medicinal Plants

    OpenAIRE

    Hu, Zhigang; Tu, Yuan; Xia, Ye; Cheng, Peipei; Sun, Wei; Shi, Yuhua; Guo, Licheng; He, Haibo; Xiong, Chao; Chen, Shilin; Zhang, Xiuqiao

    2015-01-01

    Indirubin, one of the key components of medicinal plants including Isatis tinctoria, Polygonum tinctorium, and Strobilanthes cusia, possesses great medicinal efficacy in the treatment of chronic myelocytic leukemia (CML). Due to misidentification and similar name, materials containing indirubin and their close relatives frequently fall prey to adulteration. In this study, we selected an internal transcribed spacer 2 (ITS2) for distinguishing these indirubin-containing species from five of the...

  9. Plant Life Management of the EC6 Concrete Containment Structure

    Energy Technology Data Exchange (ETDEWEB)

    Abrishami, Homayoun; Ricciuti, Rick; Khan, Azhar [CANDU Energy Inc., Mississauga (Canada)

    2012-03-15

    Aging of reinforced concrete structures due to service conditions, aggressive environments, or accidents may cause their strength, serviceability and durability to decrease over time. Due to the complex nature of safety-related structures in nuclear power plants in comparison to other structures, they possess a number of characteristics that make them comparison to other structures, they possess a number of characteristics that make them unique. These characteristics are: thick concrete cross-sections, heavy reinforcement, often one-side access only, subjected to such ageing stresses as irradiation and elevated temperature, in addition to other typical ageing mechanisms (i. e., exposure to freeze/thaw cycles, aggressive chemicals, etc.) that typically affects other types of non-nuclear structures. For a new plant, the Plant Life Management Program (PLiM) should start in the design process and then continues through construction, plant operation and decommissioning. Hence PLiM must provide not only Ageing Management program (AMP) but also provide requirements on material characteristic and the design criteria as well. The purpose of this paper is to present the Plant Life Management (PLiM) strategy for the concrete containment structure of EC6 (Enhanced CANDU 6) Nuclear Power Plant designed by CANDU Energy Inc. The EC6 is designed for 100-year plant life including a 60-year operating life and an additional 40-year decommissioning period of time. The approach adopted for the PLiM strategy of the concrete containment structure is a preventive one, key areas being: 1) design methodology, 2) material performance and 3) life cycle management and ageing management program. In addition to strength and serviceability, durability is a major consideration during the design phase, service life and up to the completion of decommissioning. Factors affecting durability design include: a) concrete performance, b) structural application, and c) consideration of environmental

  10. Plant Life Management of the EC6 Concrete Containment Structure

    International Nuclear Information System (INIS)

    Abrishami, Homayoun; Ricciuti, Rick; Khan, Azhar

    2012-01-01

    Aging of reinforced concrete structures due to service conditions, aggressive environments, or accidents may cause their strength, serviceability and durability to decrease over time. Due to the complex nature of safety-related structures in nuclear power plants in comparison to other structures, they possess a number of characteristics that make them comparison to other structures, they possess a number of characteristics that make them unique. These characteristics are: thick concrete cross-sections, heavy reinforcement, often one-side access only, subjected to such ageing stresses as irradiation and elevated temperature, in addition to other typical ageing mechanisms (i. e., exposure to freeze/thaw cycles, aggressive chemicals, etc.) that typically affects other types of non-nuclear structures. For a new plant, the Plant Life Management Program (PLiM) should start in the design process and then continues through construction, plant operation and decommissioning. Hence PLiM must provide not only Ageing Management program (AMP) but also provide requirements on material characteristic and the design criteria as well. The purpose of this paper is to present the Plant Life Management (PLiM) strategy for the concrete containment structure of EC6 (Enhanced CANDU 6) Nuclear Power Plant designed by CANDU Energy Inc. The EC6 is designed for 100-year plant life including a 60-year operating life and an additional 40-year decommissioning period of time. The approach adopted for the PLiM strategy of the concrete containment structure is a preventive one, key areas being: 1) design methodology, 2) material performance and 3) life cycle management and ageing management program. In addition to strength and serviceability, durability is a major consideration during the design phase, service life and up to the completion of decommissioning. Factors affecting durability design include: a) concrete performance, b) structural application, and c) consideration of environmental

  11. Containment hydrogen removal system for a nuclear power plant

    International Nuclear Information System (INIS)

    Callaghan, V.M.; Flynn, E.P.; Pokora, B.M.

    1984-01-01

    A hydrogen removal system (10) separates hydrogen from the containment atmosphere of a nuclear power plant using a hydrogen permeable membrane separator (30). Water vapor is removed by condenser (14) from a gas stream withdrawn from the containment atmosphere. The gas stream is then compressed by compressor (24) and cooled (28,34) to the operating temperature of the hydrogen permeable membrane separator (30). The separator (30) separates the gas stream into a first stream, rich in hydrogen permeate, and a second stream that is hydrogen depleted. The separated hydrogen is passed through a charcoal adsorber (48) to adsorb radioactive particles that have passed through the hydrogen permeable membrane (44). The hydrogen is then flared in gas burner (52) with atmospheric air and the combustion products vented to the plant vent. The hydrogen depleted stream is returned to containment through a regenerative heat exchanger (28) and expander (60). Energy is extracted from the expander (60) to drive the compressor (24) thereby reducing the energy input necessary to drive the compressor (24) and thus reducing the hydrogen removal system (10) power requirements

  12. Molecular Cloning and Characterization of Two Genes for the Biotin Carboxylase and Carboxyltransferase Subunits of Acetyl Coenzyme A Carboxylase in Myxococcus xanthus

    OpenAIRE

    Kimura, Yoshio; Miyake, Rina; Tokumasu, Yushi; Sato, Masayuki

    2000-01-01

    We have cloned a DNA fragment from a genomic library of Myxococcus xanthus using an oligonucleotide probe representing conserved regions of biotin carboxylase subunits of acetyl coenzyme A (acetyl-CoA) carboxylases. The fragment contained two open reading frames (ORF1 and ORF2), designated the accB and accA genes, capable of encoding a 538-amino-acid protein of 58.1 kDa and a 573-amino-acid protein of 61.5 kDa, respectively. The protein (AccA) encoded by the accA gene was strikingly similar t...

  13. Rapid Identification and Verification of Indirubin-Containing Medicinal Plants.

    Science.gov (United States)

    Hu, Zhigang; Tu, Yuan; Xia, Ye; Cheng, Peipei; Sun, Wei; Shi, Yuhua; Guo, Licheng; He, Haibo; Xiong, Chao; Chen, Shilin; Zhang, Xiuqiao

    2015-01-01

    Indirubin, one of the key components of medicinal plants including Isatis tinctoria, Polygonum tinctorium, and Strobilanthes cusia, possesses great medicinal efficacy in the treatment of chronic myelocytic leukemia (CML). Due to misidentification and similar name, materials containing indirubin and their close relatives frequently fall prey to adulteration. In this study, we selected an internal transcribed spacer 2 (ITS2) for distinguishing these indirubin-containing species from five of their usual adulterants, after assessing identification efficiency of matK, rbcL, psbA-trnH, and ITS2 among these species. The results of genetic distances and neighbor-joining (NJ) phylogenetic tree indicated that ITS2 region is a powerful DNA barcode to accurately identify these indirubin-containing species and discriminate them from their adulterants. Additionally, high performance liquid chromatography (HPLC) was used to verify indirubin in different organs of the above species. The results showed that indirubin had been detected in the leaves of Is. tinctoria, P. tinctorium, S. cusia, and Indigo Naturalis (made from their mixture), but not in their roots, or in the leaves of their adulterants. Therefore, this study provides a novel and rapid method to identify and verify indirubin-containing medicinal plants for effective natural treatment of CML.

  14. Rapid Identification and Verification of Indirubin-Containing Medicinal Plants

    Directory of Open Access Journals (Sweden)

    Zhigang Hu

    2015-01-01

    Full Text Available Indirubin, one of the key components of medicinal plants including Isatis tinctoria, Polygonum tinctorium, and Strobilanthes cusia, possesses great medicinal efficacy in the treatment of chronic myelocytic leukemia (CML. Due to misidentification and similar name, materials containing indirubin and their close relatives frequently fall prey to adulteration. In this study, we selected an internal transcribed spacer 2 (ITS2 for distinguishing these indirubin-containing species from five of their usual adulterants, after assessing identification efficiency of matK, rbcL, psbA-trnH, and ITS2 among these species. The results of genetic distances and neighbor-joining (NJ phylogenetic tree indicated that ITS2 region is a powerful DNA barcode to accurately identify these indirubin-containing species and discriminate them from their adulterants. Additionally, high performance liquid chromatography (HPLC was used to verify indirubin in different organs of the above species. The results showed that indirubin had been detected in the leaves of Is. tinctoria, P. tinctorium, S. cusia, and Indigo Naturalis (made from their mixture, but not in their roots, or in the leaves of their adulterants. Therefore, this study provides a novel and rapid method to identify and verify indirubin-containing medicinal plants for effective natural treatment of CML.

  15. The effects of age on nuclear power plant containment cooling systems

    Energy Technology Data Exchange (ETDEWEB)

    Lofaro, R.; Subudhi, M.; Travis, R.; DiBiasio, A.; Azarm, A. [Brookhaven National Lab., Upton, NY (United States); Davis, J. [Science Applications International Corp., New York, NY (United States)

    1994-04-01

    A study was performed to assess the effects of aging on the performance and availability of containment cooling systems in US commercial nuclear power plants. This study is part of the Nuclear Plant Aging Research (NPAR) program sponsored by the US Nuclear Regulatory Commission. The objectives of this program are to provide an understanding of the aging process and how it affects plant safety so that it can be properly managed. This is one of a number of studies performed under the NPAR program which provide a technical basis for the identification and evaluation of degradation caused by age. The effects of age were characterized for the containment cooling system by reviewing and analyzing failure data from national databases, as well as plant-specific data. The predominant failure causes and aging mechanisms were identified, along with the components that failed most frequently. Current inspection, surveillance, and monitoring practices were also examined. A containment cooling system unavailability analysis was performed to examine the potential effects of aging by increasing failure rates for selected components. A commonly found containment spray system design and a commonly found fan cooler system design were modeled. Parametric failure rates for those components in each system that could be subject to aging were accounted for in the model to simulate the time-dependent effects of aging degradation, assuming no provisions are made to properly manage it. System unavailability as a function of increasing component failure rates was then calculated.

  16. The effects of age on nuclear power plant containment cooling systems

    International Nuclear Information System (INIS)

    Lofaro, R.; Subudhi, M.; Travis, R.; DiBiasio, A.; Azarm, A.; Davis, J.

    1994-04-01

    A study was performed to assess the effects of aging on the performance and availability of containment cooling systems in US commercial nuclear power plants. This study is part of the Nuclear Plant Aging Research (NPAR) program sponsored by the US Nuclear Regulatory Commission. The objectives of this program are to provide an understanding of the aging process and how it affects plant safety so that it can be properly managed. This is one of a number of studies performed under the NPAR program which provide a technical basis for the identification and evaluation of degradation caused by age. The effects of age were characterized for the containment cooling system by reviewing and analyzing failure data from national databases, as well as plant-specific data. The predominant failure causes and aging mechanisms were identified, along with the components that failed most frequently. Current inspection, surveillance, and monitoring practices were also examined. A containment cooling system unavailability analysis was performed to examine the potential effects of aging by increasing failure rates for selected components. A commonly found containment spray system design and a commonly found fan cooler system design were modeled. Parametric failure rates for those components in each system that could be subject to aging were accounted for in the model to simulate the time-dependent effects of aging degradation, assuming no provisions are made to properly manage it. System unavailability as a function of increasing component failure rates was then calculated

  17. Application of containment and release management strategies to PWR dry-containment plants

    International Nuclear Information System (INIS)

    Yang, J.W.; Lehner, J.R.

    1992-06-01

    This report identifies and evaluates accident management strategies that are potentially of value in maintaining containment integrity and controlling the release of radioactivity following a severe accident as a pressurized water reactor with large-dry containment. The strategies are identified using a logic tree structure leading from the safety objectives and safety functions, through the mechanisms that challenge these safety functions, to the strategies. The strategies are applied to severe accident sequences which have one or more of the following characteristics: significant probability of core damage, high consequences, give rise to a number of potential challenges, and include the failure of important safety systems. Zion and Surry are selected as the representative plants for the atmospheric and sub-atmospheric designs, respectively

  18. Containment integrity of SEP plants under combined loads

    International Nuclear Information System (INIS)

    Lo, T.; Nelson, T.A.; Chen, P.Y.; Persinko, D.; Grimes, C.

    1984-06-01

    Because the containment structure is the last barrier against the release of radioactivity, an assessment was undertaken to identify the design weaknesses and estimate the margins of safety for the SEP containments under the postulated, combined loading conditions of a safe shutdown earthquake (SSE) and a design basis accident (DBA). The design basis accident is either a loss-of-coolant accident (LOCA) or a main steam line break (MSLB). The containment designs analyzed consisted of three inverted light-bulb shaped drywells used in boiling water reactor (BWR) systems, and three steel-lined concrete containments and a spherical steel shell used in pressurized water reactor (PWR) systems. These designs cover a majority of the containment types used in domestic operating plants. The results indicate that five of the seven designs are adequate even under current design standards. For the remaining two designs, the possible design weaknesses identified were buckling of the spherical steel shell and over-stress in both the radial and tangential directions in one of the concrete containments near its base

  19. The IPE Database: providing information on plant design, core damage frequency and containment performance

    International Nuclear Information System (INIS)

    Lehner, J.R.; Lin, C.C.; Pratt, W.T.; Su, T.; Danziger, L.

    1996-01-01

    A database, called the IPE Database has been developed that stores data obtained from the Individual Plant Examinations (IPEs) which licensees of nuclear power plants have conducted in response to the Nuclear Regulatory Commission's (NRC) Generic Letter GL88-20. The IPE Database is a collection of linked files which store information about plant design, core damage frequency (CDF), and containment performance in a uniform, structured way. The information contained in the various files is based on data contained in the IPE submittals. The information extracted from the submittals and entered into the IPE Database can be manipulated so that queries regarding individual or groups of plants can be answered using the IPE Database

  20. Aircraft crash upon outer containment of nuclear power plant

    International Nuclear Information System (INIS)

    Abbas, H.; Paul, D.K.; Godbole, P.N.; Nayak, G.C.

    1996-01-01

    In this paper, analysis of an aircraft crash upon an outer containment of a nuclear power plant is presented. The effect of target yielding is considered simultaneously by calculating the reaction time in a time marching scheme. The concrete model employed is capable of predicting the cracking and yielding. The response for different cracking strains and different locations of aircraft strike for different aircraft has been studied. Critical location of aircraft strike for the containment has been investigated. The analytical procedure and the material model used are found to be capable of representing the aircraft impact response of the containment structure. (orig.)

  1. Effect of administration route and dose of streptavidin or biotin on the tumor uptake of radioactivity in intraperitoneal tumor with multistep targeting

    International Nuclear Information System (INIS)

    Zhang Meili; Yao Zhengsheng; Sakahara, Harumi; Saga, Tsuneo; Nakamoto, Yuji; Sato, Noriko; Zhao Songji; Nakada, Hiroshi; Yamashina, Ikuo; Konishi, Junji

    1998-01-01

    The effect of the administration route and dose of streptavidin or biotin on the biodistribution of radioactivity in multistep targeting was studied in nude mice bearing intraperitoneal (IP) colon cancer xenograft. The multistep targeting included a two-step method using biotinylated antibody and radiolabeled streptavidin and a three-step method with radiolabeled biotin based on the two-step method. A monoclonal antibody, MLS128, which recognizes Tn antigen on mucin, was biotinylated and injected intravenously (IV) or IP in nude mice bearing human colon cancer LS180 IP xenografts for pretargeting. In the two-step method, IP-injected streptavidin showed a higher tumor uptake and tumor-to-nontumor ratios than IV-injected streptavidin regardless of administration route of pretargeting. The tumor uptake of radiolabeled streptavidin was increased with a high dose of biotinylated antibody pretargeting, but decreased with an increasing dose of streptavidin. In the three-step targeting, IP injection also gave a higher tumor uptake of radiolabeled biotin than IV injection. In conclusion, IP administration of radiolabeled streptavidin or biotin resulted in more efficient IP tumor targeting with the multistep methods

  2. Optimal use of tandem biotin and V5 tags in ChIP assays

    Directory of Open Access Journals (Sweden)

    Krpic Sanja

    2009-02-01

    Full Text Available Abstract Background Chromatin immunoprecipitation (ChIP assays coupled to genome arrays (Chip-on-chip or massive parallel sequencing (ChIP-seq lead to the genome wide identification of binding sites of chromatin associated proteins. However, the highly variable quality of antibodies and the availability of epitopes in crosslinked chromatin can compromise genomic ChIP outcomes. Epitope tags have often been used as more reliable alternatives. In addition, we have employed protein in vivo biotinylation tagging as a very high affinity alternative to antibodies. In this paper we describe the optimization of biotinylation tagging for ChIP and its coupling to a known epitope tag in providing a reliable and efficient alternative to antibodies. Results Using the biotin tagged erythroid transcription factor GATA-1 as example, we describe several optimization steps for the application of the high affinity biotin streptavidin system in ChIP. We find that the omission of SDS during sonication, the use of fish skin gelatin as blocking agent and choice of streptavidin beads can lead to significantly improved ChIP enrichments and lower background compared to antibodies. We also show that the V5 epitope tag performs equally well under the conditions worked out for streptavidin ChIP and that it may suffer less from the effects of formaldehyde crosslinking. Conclusion The combined use of the very high affinity biotin tag with the less sensitive to crosslinking V5 tag provides for a flexible ChIP platform with potential implications in ChIP sequencing outcomes.

  3. Optimal use of tandem biotin and V5 tags in ChIP assays

    Science.gov (United States)

    Kolodziej, Katarzyna E; Pourfarzad, Farzin; de Boer, Ernie; Krpic, Sanja; Grosveld, Frank; Strouboulis, John

    2009-01-01

    Background Chromatin immunoprecipitation (ChIP) assays coupled to genome arrays (Chip-on-chip) or massive parallel sequencing (ChIP-seq) lead to the genome wide identification of binding sites of chromatin associated proteins. However, the highly variable quality of antibodies and the availability of epitopes in crosslinked chromatin can compromise genomic ChIP outcomes. Epitope tags have often been used as more reliable alternatives. In addition, we have employed protein in vivo biotinylation tagging as a very high affinity alternative to antibodies. In this paper we describe the optimization of biotinylation tagging for ChIP and its coupling to a known epitope tag in providing a reliable and efficient alternative to antibodies. Results Using the biotin tagged erythroid transcription factor GATA-1 as example, we describe several optimization steps for the application of the high affinity biotin streptavidin system in ChIP. We find that the omission of SDS during sonication, the use of fish skin gelatin as blocking agent and choice of streptavidin beads can lead to significantly improved ChIP enrichments and lower background compared to antibodies. We also show that the V5 epitope tag performs equally well under the conditions worked out for streptavidin ChIP and that it may suffer less from the effects of formaldehyde crosslinking. Conclusion The combined use of the very high affinity biotin tag with the less sensitive to crosslinking V5 tag provides for a flexible ChIP platform with potential implications in ChIP sequencing outcomes. PMID:19196479

  4. Use of avidin-biotin-peroxidase complex for measurement of UV lesions in human DNA by microELISA

    Energy Technology Data Exchange (ETDEWEB)

    Leipold, B [Technischen Universitaet Muenchen (Germany, F.R.). Dermatologische Klinik; Remy, W [Max-Planck-Institut fuer Biochemie, Muenchen (Germany, F.R.)

    1984-02-10

    The avidin/biotin system was introduced into the standard enzyme-linked immunosorbent assay (ELISA) to increase its sensitivity for detecting UV lesions in human DNA. Goat anti-rabbit IgG-peroxidase used in the standard ELISA as second antibody was replaced by biotinylated goat anti-rabbit IgG plus the avidin-biotin-peroxidase complex (ABC) reagent. Sensitivity of detection of plate-fixed UV-DNA-antibody complexes was increased about 8-fold and photolesions in human DNA samples irradiated with as low a dose as 1 J/m/sup 2/ UVC or a suberythermal dose of UVB light could be detected.

  5. [Cloning, expression and transcriptional analysis of biotin carboxyl carrier protein gene (accA) from Amycolatopsis mediterranei U32 ].

    Science.gov (United States)

    Lu, Jie; Yao, Yufeng; Jiang, Weihong; Jiao, Ruishen

    2003-02-01

    Acetyl CoA carboxylase (EC 6.4.1.2, ACC) catalyzes the ATP-dependent carboxylation of acetyl CoA to yield malonyl CoA, which is the first committed step in fatty acid synthesis. A pair of degenerate PCR primers were designed according to the conserved amino acid sequence of AccA from M. tuberculosis and S. coelicolor. The product of the PCR amplification, a DNA fragment of 250bp was used as a probe for screening the U32 genomic cosmid library and its gene, accA, coding the biotinylated protein subunit of acetyl CoA carboxylase, was successfully cloned from U32. The accA ORF encodes a 598-amino-acid protein with the calculated molecular mass of 63.7kD, with 70.1% of G + C content. A typical Streptomyces RBS sequence, AGGAGG, was found at the - 6 position upstream of the start codon GTG. Analysis of the deduced amino acid sequence showed the presence of biotin-binding site and putative ATP-bicarbonate interaction region, which suggested the U32 AccA may act as a biotin carboxylase as well as a biotin carrier protein. Gene accA was then cloned into the pET28 (b) vector and expressed solubly in E. coli BL21 (DE3) by 0.1 mmol/L IPTG induction. Western blot confirmed the covalent binding of biotin with AccA. Northern blot analyzed transcriptional regulation of accA by 5 different nitrogen sources.

  6. Thermal-hydraulic calculation and analysis for QNPP (Qinshan Nuclear Power Plant) containment

    International Nuclear Information System (INIS)

    Xie Hui; Zhou Jie; He Yingchao

    1993-01-01

    Three containment thermal-hydraulic codes CONTEMPT-LT/028, CONTEMPT-4/MOD3 and COMPARE are used to compute and analyse the Qinshan Nuclear Power Plant (QNPP) containment response under LOCA or MSLB conditions. An evaluation of the capability of containment of QNPP is given

  7. Biotin-tagged platinum(iv) complexes as targeted cytostatic agents against breast cancer cells.

    Science.gov (United States)

    Muhammad, Nafees; Sadia, Nasreen; Zhu, Chengcheng; Luo, Cheng; Guo, Zijian; Wang, Xiaoyong

    2017-09-05

    A biotin-guided platinum IV complex is highly cytotoxic against breast cancer cells but hypotoxic against mammary epithelial cells. The mono-biotinylated Pt IV complex is superior to the di-biotinylated one and hence a promising drug candidate for the targeted therapy of breast cancer.

  8. General requirements for concrete containment structures for CANDU nuclear power plants

    International Nuclear Information System (INIS)

    1993-07-01

    This standard provides the general requirements used in the design, construction, testing, and commissioning of concrete containment structures for CANDU nuclear power plants designated as class containment and is directed to the owners, designers, manufacturers, fabricators, and constructors of the concrete components and parts

  9. International safeguards for a light-water reactor fuels reprocessing plant: containment and surveillance concepts

    International Nuclear Information System (INIS)

    Cameron, C.P.; Bleck, M.E.

    1980-12-01

    Concepts for containment/surveillance for reprocessing plants are described, conceptual designs are developed, and their effectiveness is evaluated. A technical approach to design of containment/surveillance systems is presented, and design considerations are discussed. This is the second in a series of reports. The first described the basis for the study of international safeguards for reprocessing plants. In this second report, only containment/surveillance is discussed. The third report will discuss the integration of concepts for containment/surveillance and material accountancy

  10. The use of plants containing genotoxic carcinogens as foods and medicine.

    Science.gov (United States)

    Prinsloo, Gerhard; Nogemane, Noluyolo; Street, Renee

    2018-04-05

    In many developing countries, populations rely on traditional medicine for primary health care, which have infiltrated commercial markets globally as natural remedies are generally regarded as safe. Traditional and natural remedies are adapted and expanded in commercial products and product ranges to provide alternatives for various diseases and illnesses. These products resemble very little of the traditional use and application and adverse effects are observed in several cases. Some of the herbs and botanical formulations therefore, are not as safe as are commonly contemplated. This paper discusses some plants that are used as food or medicine. These plants are known to contain chemical components that have been identified as genotoxic carcinogens. Often contradictory results are obtained with beneficial and adverse effects reported. The concentration, biotransformation and metabolism of these compounds, as well as the matrix effect, affect the outcome of these results, therefore not providing a clear picture of the risk associated with the use and consumption of these plants. This paper focuses on plants that are accepted as healthy, however contain compounds that are genotoxic and carcinogenic. We further highlight the risks in use of these plants where thorough studies have been conducted in various food and plant products. Copyright © 2018 Elsevier Ltd. All rights reserved.

  11. Repeat-containing protein effectors of plant-associated organisms

    Directory of Open Access Journals (Sweden)

    Carl H. Mesarich

    2015-10-01

    Full Text Available Many plant-associated organisms, including microbes, nematodes, and insects, deliver effector proteins into the apoplast, vascular tissue, or cell cytoplasm of their prospective hosts. These effectors function to promote colonization, typically by altering host physiology or by modulating host immune responses. The same effectors however, can also trigger host immunity in the presence of cognate host immune receptor proteins, and thus prevent colonization. To circumvent effector-triggered immunity, or to further enhance host colonization, plant-associated organisms often rely on adaptive effector evolution. In recent years, it has become increasingly apparent that several effectors of plant-associated organisms are repeat-containing proteins (RCPs that carry tandem or non-tandem arrays of an amino acid sequence or structural motif. In this review, we highlight the diverse roles that these repeat domains play in RCP effector function. We also draw attention to the potential role of these repeat domains in adaptive evolution with regards to RCP effector function and the evasion of effector-triggered immunity. The aim of this review is to increase the profile of RCP effectors from plant-associated organisms.

  12. Printed biotin-functionalised polythiophene films as biorecognition layers in the development of paper-based biosensors

    Energy Technology Data Exchange (ETDEWEB)

    Ihalainen, Petri, E-mail: petri.ihalainen@abo.fi [Laboratory of Physical Chemistry, Center for Functional Materials, Åbo Akademi University, Turku (Finland); Pesonen, Markus [Physics, Center for Functional Materials, Åbo Akademi University, Turku (Finland); Sund, Pernilla [Laboratory of Polymer Technology, Center for Functional Materials, Åbo Akademi University, Turku (Finland); Viitala, Tapani [Centre for Drug Research, Division of Pharmaceutical Biosciences, University of Helsinki, Helsinki (Finland); Määttänen, Anni; Sarfraz, Jawad [Laboratory of Physical Chemistry, Center for Functional Materials, Åbo Akademi University, Turku (Finland); Wilén, Carl-Erik [Laboratory of Polymer Technology, Center for Functional Materials, Åbo Akademi University, Turku (Finland); Österbacka, Ronald [Physics, Center for Functional Materials, Åbo Akademi University, Turku (Finland); Peltonen, Jouko [Laboratory of Physical Chemistry, Center for Functional Materials, Åbo Akademi University, Turku (Finland)

    2016-02-28

    Highlights: • Inkjet-printed polythiophene films show good adhesion on ultrathin gold films. • Biotin-functionalisation of polythiophene enables specificity towards streptavidin. • Supramolecular biorecognition architectures can be prepared by printing. • The addition of each printed layer can be followed by a change in capacitance. - Abstract: The integration of flexible electronic sensors in clinical diagnostics is visioned to significantly reduce the cost of many diagnostic tests and ultimately make healthcare more accessible. This study concentrates on the characterisation of inkjet-printed bio-functionalised polythiophene films on paper-based ultrathin gold film (UTGF) electrodes and their possible application as biorecognition layers. Physicochemical surface properties (topography, chemistry, and wetting) and electrochemical characteristics of water-soluble regioirregular tetraethylene-glycol polythiophene (TEGPT) and biotin-functionalised TEGPT (b-TEGPT) films were examined and compared. In addition, their specificity towards streptavidin protein was tested. The results show that stable supramolecular biorecognition layers of insulating b-TEGPT and streptavidin were successfully fabricated on a paper-based UTGF by inkjet-printing. Good adhesion of thiophene to UTGF can be attributed to covalent linkage between sulphur and gold, whereas the stability of the streptavidin layer is due to the high affinity between biotin and streptavidin. The device introduced can be utilised in the development of biosensors for clinically relevant analytes e.g. for detecting complementary DNA oligomers or antibody–antigen complexes.

  13. Purification of ammonia-containing trap waters from atomic power plant by ozone treatment

    International Nuclear Information System (INIS)

    Grachok, M.A.; Prokudina, S.A.; Shulyat'ev, M.I.

    1990-01-01

    The aim of research was to study the process of ozonation of ammonia-containing trap waters from the Kursk Atomic Power Plant both on the model solutions and on real ones. Different factors (pH of the medium, temperature, concentration of the initial substances) have been studied for their effect on ozonation of aqueous ammonia solutions, model solutions of trap waters from the Kursk Atomic Power Plant as well as ammonia-containing trap waters and liquid radioactive wastes delivered to special water treatment at the Kursk Atomic Power Plant. It is shown that in all the cases the highest rate of ammonia oxidation by ozone is observed in the alkaline medium (pH 1.4-11.0) and at 55 deg C. The obtained results have shown that a method of ozonation followed by evaporation of water to be purified can be used to treat ammonia-containing waters from atomic power plant

  14. Direct containment heating integral effects tests in geometries of European nuclear power plants

    International Nuclear Information System (INIS)

    Meyer, Leonhard; Albrecht, Giancarlo; Caroli, Cataldo; Ivanov, Ivan

    2009-01-01

    The DISCO test facility at Forschungszentrum Karlsruhe (FZK) has been used to perform experiments to investigate direct containment heating (DCH) effects during a severe accident in European nuclear power plants, comprising the EPR, the French 1300 MWe plant P'4, the VVER-1000 and the German Konvoi plant. A high-temperature iron-alumina melt is ejected by steam into scaled models of the respective reactor cavities and the containment vessel. Both heat transfer from dispersed melt and combustion of hydrogen lead to containment pressurization. The main experimental findings are presented and critical parameters are identified. The consequences of DCH are limited in reactors with no direct pathway between the cavity and the containment dome (closed pit). The situation is more severe for reactors which do have a direct pathway between the cavity and the containment (open pit). The experiments showed that substantial fractions of corium may be dispersed into the containment in such cases, if the pressure in the reactor coolant system is elevated at the time of RPV failure. Primary system pressures of 1 or 2 MPa are sufficient to lead to full scale DCH effects. Combustion of the hydrogen produced by oxidation as well as the hydrogen initially present appears to be the crucial phenomenon for containment pressurization.

  15. Direct containment heating integral effects tests in geometries of European nuclear power plants

    Energy Technology Data Exchange (ETDEWEB)

    Meyer, Leonhard [Forschungszentrum Karlsruhe (FZK), Postfach 3640, 76021 Karlsruhe (Germany)], E-mail: meyer@iket.fzk.de; Albrecht, Giancarlo [Forschungszentrum Karlsruhe (FZK), Postfach 3640, 76021 Karlsruhe (Germany); Caroli, Cataldo [Institut de Radioprotection et de Surete Nucleaire, BP 17, 92262 Fontenay-aux-Roses Cedex (France); Ivanov, Ivan [Technical University of Sofia, BG-1797 Sofia (Bulgaria)

    2009-10-15

    The DISCO test facility at Forschungszentrum Karlsruhe (FZK) has been used to perform experiments to investigate direct containment heating (DCH) effects during a severe accident in European nuclear power plants, comprising the EPR, the French 1300 MWe plant P'4, the VVER-1000 and the German Konvoi plant. A high-temperature iron-alumina melt is ejected by steam into scaled models of the respective reactor cavities and the containment vessel. Both heat transfer from dispersed melt and combustion of hydrogen lead to containment pressurization. The main experimental findings are presented and critical parameters are identified. The consequences of DCH are limited in reactors with no direct pathway between the cavity and the containment dome (closed pit). The situation is more severe for reactors which do have a direct pathway between the cavity and the containment (open pit). The experiments showed that substantial fractions of corium may be dispersed into the containment in such cases, if the pressure in the reactor coolant system is elevated at the time of RPV failure. Primary system pressures of 1 or 2 MPa are sufficient to lead to full scale DCH effects. Combustion of the hydrogen produced by oxidation as well as the hydrogen initially present appears to be the crucial phenomenon for containment pressurization.

  16. In Vivo Roles of Fatty Acid Biosynthesis Enzymes in Biosynthesis of Biotin and α-Lipoic Acid in Corynebacterium glutamicum.

    Science.gov (United States)

    Ikeda, Masato; Nagashima, Takashi; Nakamura, Eri; Kato, Ryosuke; Ohshita, Masakazu; Hayashi, Mikiro; Takeno, Seiki

    2017-10-01

    For fatty acid biosynthesis, Corynebacterium glutamicum uses two type I fatty acid synthases (FAS-I), FasA and FasB, in addition to acetyl-coenzyme A (CoA) carboxylase (ACC) consisting of AccBC, AccD1, and AccE. The in vivo roles of the enzymes in supplying precursors for biotin and α-lipoic acid remain unclear. Here, we report genetic evidence demonstrating that the biosynthesis of these cofactors is linked to fatty acid biosynthesis through the FAS-I pathway. For this study, we used wild-type C. glutamicum and its derived biotin vitamer producer BFI-5, which was engineered to express Escherichia coli bioBF and Bacillus subtilis bioI Disruption of either fasA or fasB in strain BFI-5 led to decreased production of biotin vitamers, whereas its amplification contributed to increased production, with a larger impact of fasA in both cases. Double disruptions of fasA and fasB resulted in no biotin vitamer production. The acc genes showed a positive effect on production when amplified simultaneously. Augmented fatty acid biosynthesis was also reflected in pimelic acid production when carbon flow was blocked at the BioF reaction. These results indicate that carbon flow down the FAS-I pathway is destined for channeling into the biotin biosynthesis pathway, and that FasA in particular has a significant impact on precursor supply. In contrast, fasB disruption resulted in auxotrophy for lipoic acid or its precursor octanoic acid in both wild-type and BFI-5 strains. The phenotypes were fully complemented by plasmid-mediated expression of fasB but not fasA These results reveal that FasB plays a specific physiological role in lipoic acid biosynthesis in C. glutamicum IMPORTANCE For the de novo biosynthesis of fatty acids, C. glutamicum exceptionally uses a eukaryotic multifunctional type I fatty acid synthase (FAS-I) system comprising FasA and FasB, in contrast to most bacteria, such as E. coli and B. subtilis , which use an individual nonaggregating type II fatty acid synthase

  17. Nuclear containment systems and in-service inspection status of Korea nuclear power plants

    Energy Technology Data Exchange (ETDEWEB)

    Jihong, Park; Jaekeun, Hong; Banuk, Park [Korea Institute of Machinery and Materials, Dept. of Authorized Test and Evaluation, Kyungnam (Korea, Republic of)

    2007-07-01

    20 unit nuclear power plants in Korea have been operated and maintained since the first unit started in commercial service in 1978. Most recently 4 units were under construction and several units were planned to be constructed. by industries. 4 types of nuclear containment systems have been constructed until now: first, metal containments, then pre-stressed concrete containments with grouted tendon systems, followed by pre-stressed concrete containments with un-grouted tendon systems, and Korea standard nuclear containments. All the nuclear containments should be inspected periodically. Therefore for periodic in-service inspection, several appropriate technical requirements should be applied differently depending on the specific nuclear containment types. With the changes of times, nuclear containment systems have undergone a remarkable change, and finally nuclear containment system of Korea standard nuclear power plant was settled down, and as a matter of course it dominates the trend of present and future nuclear containment systems. Overall in-service inspection results of most Korea nuclear containments have not showed any serious evidence of degradation.

  18. Wool-waste as organic nutrient source for container-grown plants

    Energy Technology Data Exchange (ETDEWEB)

    Zheljazkov, Valtcho D. [Mississippi State University, North Mississippi Research and Extension Center, Verona, MS 38879 (United States)], E-mail: vj40@pss.msstate.edu; Stratton, Glenn W [Department of Plant and Animal Sciences and Department of Environmental Sciences, Nova Scotia Agricultural College, Truro, NS, B2N 5E3 (Canada); Pincock, James [Department of Chemistry, Dalhousie University, Halifax, NS, B3H 4J3 (Canada); Butler, Stephanie [Department of Plant and Animal Sciences and Department of Environmental Sciences, Nova Scotia Agricultural College, Truro, NS, B2N 5E3 (Canada); Jeliazkova, Ekaterina A [Mississippi State University, Department of Plant and Soil Sciences, Mississippi State, MS 39762 (United States); Nedkov, Nedko K [Research Institute for Roses and Aromatic Crops, 49 Osvobojdenie Blv., Kazanluk (Bulgaria); Gerard, Patrick D [Department of Applied Economics and Statistics, Clemson University, Clemson, SC 29634 (United States)

    2009-07-15

    A container experiment was conducted to test the hypothesis that uncomposted wool wastes could be used as nutrient source and growth medium constituent for container-grown plants. The treatments were: (1) rate of wool-waste application (0 or unamended control, 20, 40, 80, and 120 g of wool per 8-in. pot), (2) growth medium constituents [(2.1) wool plus perlite, (2.2) wool plus peat, and (2.3) wool plus peat plus perlite], and (3) plant species (basil and Swiss chard). A single addition of 20, 40, 80, or 120 g of wool-waste to Swiss chard (Beta vulgaris L.) and basil (Ocimum basilicum L.) in pots with growth medium provided four harvests of Swiss chard and five harvests of basil. Total basil yield from the five harvests was 1.6-5 times greater than the total yield from the unamended control, while total Swiss chard yield from the four harvests was 2-5 times greater relative to the respective unamended control. The addition of wool-waste to the growth medium increased Swiss chard and basil tissue N, and NO{sub 3}-N and NH{sub 4}-N in growth medium relative to the unamended control. Scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) microanalysis of wool fibers sampled at the end of the experiments indicated various levels of decomposition, with some fibers retaining their original surface structure. Furthermore, most of the wool fibers' surfaces contained significant concentrations of S and much less N, P, or K. SEM/EDX revealed that some plant roots grow directly on wool-waste fibers suggesting either (1) root directional growth towards sites with greater nutrient concentration and/or (2) a possible role for roots or root exudates in wool decomposition. Results from this study suggest that uncomposted wool wastes can be used as soil amendment, growth medium constituent, and nutrient source for container-grown plants.

  19. Wool-waste as organic nutrient source for container-grown plants

    International Nuclear Information System (INIS)

    Zheljazkov, Valtcho D.; Stratton, Glenn W.; Pincock, James; Butler, Stephanie; Jeliazkova, Ekaterina A.; Nedkov, Nedko K.; Gerard, Patrick D.

    2009-01-01

    A container experiment was conducted to test the hypothesis that uncomposted wool wastes could be used as nutrient source and growth medium constituent for container-grown plants. The treatments were: (1) rate of wool-waste application (0 or unamended control, 20, 40, 80, and 120 g of wool per 8-in. pot), (2) growth medium constituents [(2.1) wool plus perlite, (2.2) wool plus peat, and (2.3) wool plus peat plus perlite], and (3) plant species (basil and Swiss chard). A single addition of 20, 40, 80, or 120 g of wool-waste to Swiss chard (Beta vulgaris L.) and basil (Ocimum basilicum L.) in pots with growth medium provided four harvests of Swiss chard and five harvests of basil. Total basil yield from the five harvests was 1.6-5 times greater than the total yield from the unamended control, while total Swiss chard yield from the four harvests was 2-5 times greater relative to the respective unamended control. The addition of wool-waste to the growth medium increased Swiss chard and basil tissue N, and NO 3 -N and NH 4 -N in growth medium relative to the unamended control. Scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) microanalysis of wool fibers sampled at the end of the experiments indicated various levels of decomposition, with some fibers retaining their original surface structure. Furthermore, most of the wool fibers' surfaces contained significant concentrations of S and much less N, P, or K. SEM/EDX revealed that some plant roots grow directly on wool-waste fibers suggesting either (1) root directional growth towards sites with greater nutrient concentration and/or (2) a possible role for roots or root exudates in wool decomposition. Results from this study suggest that uncomposted wool wastes can be used as soil amendment, growth medium constituent, and nutrient source for container-grown plants.

  20. Study of Containment Vent Strategies During Severe Accident Progression for the CANDU6 Plant

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Youngho; Ahn, K. I. [KAERI, Daejeon (Korea, Republic of)

    2015-05-15

    In March, 2011, Fukushima daichi nuclear power plants experienced a long term station blackout. Severe core damage occurred and a large amount of radioactive materials are released outside of the plants. After this terrible accident Nuclear Safety and Security Commission (NSSC) enforced to increase nuclear safety for all operating plants in Korea. To increase plant safety, both hardware reinforcement and software improvement are encouraged. Hardware reinforcement includes the preparation of the external water injection paths to the RCS and the spent fuel pool, a filtered containment venting system (CFVS), and AC power generating truck. Software improvement includes the increase of the effectiveness of the severe accident management guidance (SAMG) and plant staff training. To comply with NSSC's request, Wolsong Unit 1 has fulfilled the hardware reinforcement including the installation of a CFVS and started the extension of a SAMG to the low power and shutdown operation mode. Current SAMG deals accident occurred during full power operation only. The CFVS is designed to open and to close isolation valves manually. It does not require AC power. The operation of the CFVS prevents the reactor containment building failure due to the over-pressurization but it may release radioactive materials out of the reactor containment building. This paper discusses the radiological source terms for the containment vent strategy during severe accident progression which occurred during shutdown operation mode. This work is a part of the development of shutdown SAMG.. The CFVS is an effective means to control the containment pressure when the local air coolers are unavailable. Radioactive materials may release through the CFVS, but their amounts are reduced significantly. The alternative means, i.e., containment vent through the ventilation system which does not have an effective filter, is not a good choice to control the containment condition. It can maintain the containment

  1. Float level switch for a nuclear power plant containment vessel

    International Nuclear Information System (INIS)

    Powell, J.G.

    1993-01-01

    This invention is a float level switch used to sense rise or drop in water level in a containment vessel of a nuclear power plant during a loss of coolant accident. The essential components of the device are a guide tube, a reed switch inside the guide tube, a float containing a magnetic portion that activates a reed switch, and metal-sheathed, ceramic-insulated conductors connecting the reed switch to a monitoring system outside the containment vessel. Special materials and special sealing techniques prevent failure of components and allow the float level switch to be connected to a monitoring system outside the containment vessel. 1 figures

  2. Float level switch for a nuclear power plant containment vessel

    Science.gov (United States)

    Powell, James G.

    1993-01-01

    This invention is a float level switch used to sense rise or drop in water level in a containment vessel of a nuclear power plant during a loss of coolant accident. The essential components of the device are a guide tube, a reed switch inside the guide tube, a float containing a magnetic portion that activates a reed switch, and metal-sheathed, ceramic-insulated conductors connecting the reed switch to a monitoring system outside the containment vessel. Special materials and special sealing techniques prevent failure of components and allow the float level switch to be connected to a monitoring system outside the containment vessel.

  3. Electrochemical DNA biosensor based on avidin-biotin conjugation for influenza virus (type A) detection

    Science.gov (United States)

    Chung, Da-Jung; Kim, Ki-Chul; Choi, Seong-Ho

    2011-09-01

    An electrochemical DNA biosensor (E-DNA biosensor) was fabricated by avidin-biotin conjugation of a biotinylated probe DNA, 5'-biotin-ATG AGT CTT CTA ACC GAG GTC GAA-3', and an avidin-modified glassy carbon electrode (GCE) to detect the influenza virus (type A). An avidin-modified GCE was prepared by the reaction of avidin and a carboxylic acid-modified GCE, which was synthesized by the electrochemical reduction of 4-carboxyphenyl diazonium salt. The current value of the E-DNA biosensor was evaluated after hybridization of the probe DNA and target DNA using cyclic voltammetry (CV). The current value decreased after the hybridization of the probe DNA and target DNA. The DNA that was used follows: complementary target DNA, 5'-TTC GAC CTC GGT TAG AAG ACT CAT-3' and two-base mismatched DNA, 5'-TTC GAC AGC GGT TAT AAG ACT CAT-3'.

  4. In type 1 diabetics, high-dose biotin may compensate for low hepatic insulin exposure, promoting a more normal expression of glycolytic and gluconeogenic enyzymes and thereby aiding glycemic control.

    Science.gov (United States)

    McCarty, Mark F

    2016-10-01

    In type 1 diabetics, hepatic exposure to insulin is chronically subnormal even in the context of insulin therapy; as a result, expression of glycolytic enzymes is decreased, and that of gluconeogenic enzymes is enhanced, resulting in a physiologically inappropriate elevation of hepatic glucose output. Subnormal expression of glucokinase (GK) is of particular importance in this regard. Possible strategies for correcting this perturbation of hepatic enzyme expression include administration of small molecule allosteric activators of GK, as well as a procedure known as chronic intermittent intravenous insulin therapy (CIIIT); however, side effects accompany the use of GK activators, and CIIIT is time and labor intensive. Alternatively, administration of high-dose biotin has potential for modulating hepatic enzyme expression in a favorable way. Studies in rodents and in cultured hepatocytes demonstrate that, in the context of low insulin exposure, supra-physiological levels of biotin induce increased expression of GK while suppressing that of the key gluconeogenic enzyme phosphoenolpyruvate carboxykinase. These effects may be a downstream consequence of the fact that biotin down-regulates mRNA expression of FOXO1; insulin's antagonism of the activity of this transcription factor is largely responsible for its modulatory impact on hepatic glycolysis and gluconeogenesis. Hence, high-dose biotin may compensate for subnormal insulin exposure by suppressing FOXO1 levels. High-dose biotin also has the potential to oppose hepatic steatosis by down-regulating SREBP-1 expression. Two pilot trials of high-dose biotin (16 or 2mg per day) in type 1 diabetics have yielded promising results. There is also some reason to suspect that high-dose biotin could aid control of diabetic neuropathy and nephropathy via its stimulatory effect on cGMP production. Owing to the safety, good tolerance, moderate expense, and current availability of high-dose biotin, this strategy merits more

  5. Importance of water quality in container plant production

    Science.gov (United States)

    John M. Ruter

    2013-01-01

    High substrate pH is a major problem for producers of container-grown plants and seedlings. The primary cause of high substrate pH is irrigation water with high alkalinity. Alkalinity is defined as the capacity of water to neutralize acids. Some alkalinity in irrigation water is beneficial as it serves as a buffer to large swings in pH levels, but high alkalinity in...

  6. Plasmon-induced photoelectrochemical biosensor for in situ real-time measurement of biotin-streptavidin binding kinetics under visible light irradiation

    International Nuclear Information System (INIS)

    Guo, Jingchun; Oshikiri, Tomoya; Ueno, Kosei; Shi, Xu; Misawa, Hiroaki

    2017-01-01

    We developed a localized surface plasmon-induced visible light-responsive photoelectrochemical (PEC) biosensor using a titanium dioxide (TiO_2) photoelectrode loaded with gold nanoislands (AuNIs) for in situ real-time measurement of biotin-streptavidin association. As a proof of concept, self-assembled thiol-terminated biotin molecules bound on a AuNIs/TiO_2 photoelectrode were successfully utilized to explore the photocurrent response to streptavidin-modified gold nanoparticle (STA-AuNP) solutions. This plasmon-induced PEC biosensor is simple and easy to miniaturize. Additionally, the PEC biosensor achieves highly sensitive measurements under only visible light irradiation and prevents the UV-induced damage of samples. Furthermore, a novel approach has been proposed to realize the real-time monitoring of biotin-STA binding affinities and kinetics by analyzing the PEC sensing characteristics. This PEC biosensor and novel analysis method could provide a new approach for the specific electrical detection and real-time kinetic measurements for clinical diagnostics and drug development. - Highlights: • A plasmon-induced visible light-responsive photoelectrochemical biosensor is developed and the system can be miniaturized.

  7. Plasmon-induced photoelectrochemical biosensor for in situ real-time measurement of biotin-streptavidin binding kinetics under visible light irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Jingchun; Oshikiri, Tomoya; Ueno, Kosei; Shi, Xu [Research Institute for Electronic Science, Hokkaido University, Sapporo 001-0021 (Japan); Misawa, Hiroaki, E-mail: misawa@es.hokudai.ac.jp [Research Institute for Electronic Science, Hokkaido University, Sapporo 001-0021 (Japan); Department of Applied Chemistry & Institute of Molecular Science, National Chiao Tung University, Hsinchu 30010, Taiwan (China)

    2017-03-08

    We developed a localized surface plasmon-induced visible light-responsive photoelectrochemical (PEC) biosensor using a titanium dioxide (TiO{sub 2}) photoelectrode loaded with gold nanoislands (AuNIs) for in situ real-time measurement of biotin-streptavidin association. As a proof of concept, self-assembled thiol-terminated biotin molecules bound on a AuNIs/TiO{sub 2} photoelectrode were successfully utilized to explore the photocurrent response to streptavidin-modified gold nanoparticle (STA-AuNP) solutions. This plasmon-induced PEC biosensor is simple and easy to miniaturize. Additionally, the PEC biosensor achieves highly sensitive measurements under only visible light irradiation and prevents the UV-induced damage of samples. Furthermore, a novel approach has been proposed to realize the real-time monitoring of biotin-STA binding affinities and kinetics by analyzing the PEC sensing characteristics. This PEC biosensor and novel analysis method could provide a new approach for the specific electrical detection and real-time kinetic measurements for clinical diagnostics and drug development. - Highlights: • A plasmon-induced visible light-responsive photoelectrochemical biosensor is developed and the system can be miniaturized.

  8. Phase II trial of yttrium-90-DOTA-biotin pretargeted by NR-LU-10 antibody/streptavidin in patients with metastatic colon cancer

    International Nuclear Information System (INIS)

    Knox, S J.; Goris, M L.; Tempero, M.; Weiden, P L.; Gentner, L.; Breitz, H.; Adams, G. P.; Axworthy, D.; Gaffigan, S.; Bryan, K.; Fisher, Darrell R.; Colcher, D; Horak, I D.; Weiner, L M.

    1999-01-01

    A Phase II study of yttrium-90-tetra-azacyclododecanetetra-acetic acid-biotin (Y-90-DOTA-biotin) pretargeted by NR-LU-10 antibody/streptavidin (SA) was performed. The primary objectives of the study were to evaluate the efficacy and safety of this therapy in patients with metastatic colon cancer. Twenty-five patients were treated with a single dose of 110 mCi/m 2 (mean administered dose, 106.5-10.3 mCi/m 2 ) of Y-90-DOTA-biotin. There were three components of the therapy. Patients first received NR-LU-10/SA on day 1. A clearing agent (biotin-galactose-human serum albumin) was administered 48 h after the NR-LU-10/SA to remove residual circulating unbound NR-LU-10/SA. Lastly, 24 h after administration of clearing agent, patients received biotin-DOTA-labeled with 110 mCi/m 2 Y-90. All three components of the therapy were administered i.v. Both hematological and nonhematological toxicities were observed. Diarrhea was the most frequent grade 4 nonhematological toxicity (16%; with 16% grade 3 diarrhea). Hematological toxicity was less severe with 8% grade 3 and 8% grade 4 neutropenia and 8% grade 3 and 16% grade 4 thrombocytopenia. The overall response rate was 8%. Two partial responders had freedom from progression of 16 weeks. Four patients (16%) had stable disease with freedom from progression of 10-20 weeks. Despite the relatively disappointing results of this study in terms of therapeutic efficacy and toxicity, proof of principle was obtained for the pretargeting approach. In addition, valuable new information was obtained about normal tissue tolerance to low-dose-rate irradiation that will help to provide useful guidelines for future study designs

  9. Advancements in Root Growth Measurement Technologies and Observation Capabilities for Container-Grown Plants

    Directory of Open Access Journals (Sweden)

    Lesley A. Judd

    2015-07-01

    Full Text Available The study, characterization, observation, and quantification of plant root growth and root systems (Rhizometrics has been and remains an important area of research in all disciplines of plant science. In the horticultural industry, a large portion of the crops grown annually are grown in pot culture. Root growth is a critical component in overall plant performance during production in containers, and therefore it is important to understand the factors that influence and/or possible enhance it. Quantifying root growth has varied over the last several decades with each method of quantification changing in its reliability of measurement and variation among the results. Methods such as root drawings, pin boards, rhizotrons, and minirhizotrons initiated the aptitude to measure roots with field crops, and have been expanded to container-grown plants. However, many of the published research methods are monotonous and time-consuming. More recently, computer programs have increased in use as technology advances and measuring characteristics of root growth becomes easier. These programs are instrumental in analyzing various root growth characteristics, from root diameter and length of individual roots to branching angle and topological depth of the root architecture. This review delves into the expanding technologies involved with expertly measuring root growth of plants in containers, and the advantages and disadvantages that remain.

  10. Advancements in Root Growth Measurement Technologies and Observation Capabilities for Container-Grown Plants.

    Science.gov (United States)

    Judd, Lesley A; Jackson, Brian E; Fonteno, William C

    2015-07-03

    The study, characterization, observation, and quantification of plant root growth and root systems (Rhizometrics) has been and remains an important area of research in all disciplines of plant science. In the horticultural industry, a large portion of the crops grown annually are grown in pot culture. Root growth is a critical component in overall plant performance during production in containers, and therefore it is important to understand the factors that influence and/or possible enhance it. Quantifying root growth has varied over the last several decades with each method of quantification changing in its reliability of measurement and variation among the results. Methods such as root drawings, pin boards, rhizotrons, and minirhizotrons initiated the aptitude to measure roots with field crops, and have been expanded to container-grown plants. However, many of the published research methods are monotonous and time-consuming. More recently, computer programs have increased in use as technology advances and measuring characteristics of root growth becomes easier. These programs are instrumental in analyzing various root growth characteristics, from root diameter and length of individual roots to branching angle and topological depth of the root architecture. This review delves into the expanding technologies involved with expertly measuring root growth of plants in containers, and the advantages and disadvantages that remain.

  11. Requirements for the coatings of a nuclear power plant containment building

    International Nuclear Information System (INIS)

    Orantie, K.; Kuosa, H.; Haekkae-Roennholm, E.

    2001-06-01

    The report presents the criteria for the inside coatings of nuclear power plant containment buildings including: radiation resistance, decontamination, chemical resistance in accident situations and fire resistance

  12. Synthesis of Biotin Linkers with the Activated Triple Bond Donor [p-(N-propynoylaminotoluic Acid] (PATA for Efficient Biotinylation of Peptides and Oligonucleotides

    Directory of Open Access Journals (Sweden)

    Martina Jezowska

    2012-11-01

    Full Text Available Biotin is an important molecule for modern biological studies including, e.g., cellular transport. Its exclusive affinity to fluorescent streptavidin/avidin proteins allows ready and specific detection. As a consequence methods for the attachment of biotin to various biological targets are of high importance, especially when they are very selective and can also proceed in water. One useful method is Hüisgen dipolar [3+2]-cycloaddition, commonly referred to as “click chemistry”. As we reported recently, the activated triple bond donor p-(N-propynoylaminotoluic acid (PATA gives excellent results when used for conjugations at submicromolar concentrations. Thus, we have designed and synthesized two biotin linkers, with different lengths equipped with this activated triple bond donor and we proceeded with biotinylation of oligonucleotides and C-myc peptide both in solution and on solid support with excellent yields of conversion.

  13. Aedes albopictus (Diptera: Culicidae) Oviposition Preference as Influenced by Container Size and Buddleja davidii Plants.

    Science.gov (United States)

    Davis, Timothy J; Kline, Daniel L; Kaufman, Phillip E

    2016-03-01

    Aedes albopictus (Skuse) is a container-breeding mosquito commonly found in residential areas of its range in the United States. Mosquitoes are known to utilize flowering plants for sugar acquisition. Limited information is known about the influences on oviposition site selection, outside of container size. Residential areas are often landscaped with a variety of flowering plants and are known to provide numerous sizes of potential larval developmental sites for container-breeding mosqutioes. Through screened enclosure and field studies, the oviposition preference of Ae. albopictus for containers of three selected sizes (473, 946 and 1,892 ml) and the influence of flowering butterfly bush (Buddleja davidii Franchett cultivar 'Guinevere') plants were examined. Our results document that significantly more eggs were oviposited in the largest containers. Additionally, significantly more eggs were oviposited in containers adjacent to flowering butterfly bushes than in those without a flowering butterfly bush. Finally, our results document that flowering butterfly bushes exerted greater influence over Ae. albopictus oviposition decisions than did container size. Our findings can be applied to several aspects of Ae. albopictus surveillance and control.

  14. Phenomenological uncertainty analysis of early containment failure at severe accident of nuclear power plant

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Su Won

    2011-02-15

    The severe accident has inherently significant uncertainty due to wide range of conditions and performing experiments, validation and practical application are extremely difficult because of its high temperature and pressure. Although internal and external researches were put into practice, the reference used in Korean nuclear plants were foreign data of 1980s and safety analysis as the probabilistic safety assessment has not applied the newest methodology. Also, it is applied to containment pressure formed into point value as results of thermal hydraulic analysis to identify the probability of containment failure in level 2 PSA. In this paper, the uncertainty analysis methods for phenomena of severe accident influencing early containment failure were developed, the uncertainty analysis that apply Korean nuclear plants using the MELCOR code was performed and it is a point of view to present the distribution of containment pressure as a result of uncertainty analysis. Because early containment failure is important factor of Large Early Release Frequency(LERF) that is used as representative criteria of decision-making in nuclear power plants, it was selected in this paper among various modes of containment failure. Important phenomena of early containment failure at severe accident based on previous researches were comprehended and methodology of 7th steps to evaluate uncertainty was developed. The MELCOR input for analysis of the severe accident reflected natural circulation flow was developed and the accident scenario for station black out that was representative initial event of early containment failure was determined. By reviewing the internal model and correlation for MELCOR model relevant important phenomena of early containment failure, the uncertainty factors which could affect on the uncertainty were founded and the major factors were finally identified through the sensitivity analysis. In order to determine total number of MELCOR calculations which can

  15. Elimination of the containment spray additive for Vogtle electric generating plant

    International Nuclear Information System (INIS)

    Lowery, K.G.

    1995-01-01

    This paper discusses the details for elimination of the spray additive portion of the containment spray system (CSS) in a pressurized water reactor (PWR) power plant. A particular emphasis is placed on nuclear power plant design associated with operation and maintenance (O and M), cost control strategies, and reliability initiatives. The CSS is an engineered safeguard system that functions to reduce reactor containment building pressure and temperature and the quantity of airborne fission products in the containment atmosphere subsequent to a loss-of-coolant accident (LOCA). Pressure and temperature reduction is accomplished by spraying water into the containment building atmosphere. Sodium hydroxide (NaOH) is added to the containment spray water to increase its pH. Results of recent studies on the behavior of fission products in the post-LOCA containment environment have demonstrated that the iodine removal can be effectively performed by boric acid sprays without the NaOH additive and by deposition on the internal surfaces of the containment building. Thus, the NaOH, the SAT, the chemical injection system (eductor) which delivers the additive to the spray system, and the related testing and maintenance required by the Technical Specifications can be eliminated. The NaOH will be replaced by TSP in baskets in the containment sump area. The TSP is needed for pH control during the recirculation phase following a LOCA. The deletion of the requirement for the SAT will result in a reduction of regulatory requirements in that the level of surveillance will be reduced. The safety analysis acceptance limits will still be met

  16. Advanced conceptual design report: T Plant secondary containment and leak detection upgrades. Project W-259

    International Nuclear Information System (INIS)

    Hookfin, J.D.

    1995-01-01

    The T Plant facilities in the 200-West Area of the Hanford site were constructed in the early 1940s to produce nuclear materials in support of national defense activities. T Plant includes the 271-T facility, the 221-T facility, and several support facilities (eg, 2706-T), utilities, and tanks/piping systems. T Plant has been recommended as the primary interim decontamination facility for the Hanford site. Project W-259 will provide capital upgrades to the T Plant facilities to comply with Federal and State of Washington environmental regulations for secondary containment and leak detection. This document provides an advanced conceptual design concept that complies with functional requirements for the T Plant Secondary Containment and Leak Detection upgrades

  17. A solid-phase technique for preparation of no-carrier-added technetium-99m radiopharmaceuticals: application to the streptavidin/biotin system

    International Nuclear Information System (INIS)

    Dunn-Dufault, Robert; Pollak, Alfred; Fitzgerald, Jane; Thornback, John R.; Ballinger, James R.

    2000-01-01

    A high effective specific activity (HESA) formulation of a biotin-containing 99m Tc ligand [RP488: dimethyl-Gly-Ser-Cys(Acm)-Lys(Biotin)-Gly] conveniently prepared from solid phase was compared to a typical low effective specific activity (LESA) solution formulation to demonstrate improved targeting to streptavidin in an in vitro assay and in an in vivo rat model. RP488 was coupled to a maleimide-functionalized polyethylene glycol resin via a thiol ether linkage and labeled with 99m Tc-gluconate at room temperature, followed by elution of the HESA 99m Tc-RP488 in saline (minimum specific activity ∼ 1000 TBq/mmol by amino acid analysis). Both HESA and LESA 99m Tc-RP488 labeled at > 90% purity. In vitro, HESA 99m Tc-RP488 incubated with streptavidin-agarose was bound quantitatively, but there was competition from addition of increasing amounts of cold RP488. In rats, radiotracer uptake was evident at the site of implantation of streptavidin-agarose beads for the HESA dose, less uptake of low effective specific activity (LESA) material, and no appreciable uptake in the control rats of the LESA or HESA dose. The target-to-background ratio for HESA 99m Tc-RP488 was 5.4 times that of the control. The solid-phase technology offers a convenient way to prepare high specific activity receptor-targeting 99m Tc radiopharmaceuticals

  18. 7 CFR 340.2 - Groups of organisms which are or contain plant pests and exemptions.

    Science.gov (United States)

    2010-01-01

    ... (Continued) ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE INTRODUCTION OF ORGANISMS... plant pest in § 340.1. GROUP Viroids Superkingdom Prokaryotae Kingdom Virus All members of groups containing plant viruses, and all other plant and insect viruses Kingdom Monera Division Bacteria Family...

  19. Aging of steel containments and liners in nuclear power plants

    International Nuclear Information System (INIS)

    Naus, D.J.; Oland, C.B.; Ellingwood, B.; Norris, W.E.

    1998-02-01

    Aging of the containment pressure boundary in light water reactor plants is being addressed to understand the significant factors relating occurrence of corrosion efficacy of inspection and structural capacity reduction of steel containments and liners of concrete containments. and to make recommendations on use of risk models in regulatory decisions. Current regulatory in-service inspection requirements are reviewed and a summary of containment related degradation experience is presented. Current and emerging nondestructive examination techniques and a degradation assessment methodology for characterizing and quantifying the amount of damage present are described. Quantitative tools for condition assessment of aging structures using time dependent structural reliability analysis methods are summarized. Such methods provide a framework for addressing the uncertainties attendant to aging in the decision process. Results of this research provide a means for establishing current and estimating future structural capacity margins of containments, and to address the significance of incidences of reported containment degradation

  20. Relationship between molecular structure and supramolecular morphology of DODA-EO2-biotin and related lipids

    NARCIS (Netherlands)

    Huetz, P.; van Neuren, S.; Ringler, P.; Kremer, F.; van Breemen, J.F.L.; Wagenaar, A.; Engberts, J.B.F.N.; Fraaije, J.G E M; Brisson, A.

    1997-01-01

    We have recently reported that a biotinylated lipid molecule, called DODA-EO2-biotin, forms tubular lipid structures upon hydration, which act as a matrix for the formation of ordered helical arrays of streptavidin as well as for secondary macromolecular recognition reactions involving biotinylated

  1. Oral administration of supplementary biotin differentially influences the fertility rate and oviductal expression of avidin and avidin-related protein-2 in low- and high-fertility broiler line hens.

    Science.gov (United States)

    Daryabari, H; Akhlaghi, A; Zamiri, M J; Pirsaraei, Z Ansari; Mianji, G Rahimi; Deldar, H; Eghbalian, A N

    2015-02-01

    Probable involvement of avidin and avidin-related protein-2 (AVR2) in sperm viability in the sperm storage tubules of turkeys has been suggested. The high affinity of biotin to avidin and its analogs is also well documented. The present study aimed to determine the effect of oral biotin on reproductive performance and oviductal mRNA expression of avidin and AVR2 in 2 broiler hen lines with different fertility rates. Low-fertility (line B) and high-fertility (line D) hens (n=144) were randomly allotted to receive 0 (T0), 0.30 (T1), or 0.45 (T2) mg/L biotin in drinking water from 30 through 33 wk of age. The reproductive performance of the hens was evaluated using artificial insemination. At the end of the treatment period, 24 hens per line were killed to assay the expression of avidin and AVR2 in the uterovaginal junction. Supplementary biotin increased egg production from 73.5% for T0 to 87.8% for T2. Hens administered with biotin in line B, but not in line D, showed an increase (8.4%) in fertility rate. Hatchability, chick quality, and overall embryonic mortality were not different among the experimental groups. Real-time PCR data showed that both avidin (P=0.0013) and AVR2 (Pbiotin×line interaction effect, where low-fertility line B hens receiving the high biotin level recorded respectively a 3.9 and 15.3% increase in avidin and AVR2 mRNA expression, although biotin did not affect these traits in line D hens. Control hens in line D had a dramatically higher AVR2 expression record (7.4-fold) compared with the control hens in line B. The correlation coefficients of fertility rate and avidin expression were 0.73 and 0.66 in lines B and D, respectively. However, the correlation of fertility and AVR2 (r=0.65) was significant for line D hens only. Overall, fertility rate and oviductal expression of avidin and AVR2 were dichotomously affected by oral biotin in low- and high-fertility line hens, where only low-fertility birds showed improvements in these attributes.

  2. A role of the transcriptional regulator LldR (NCgl2814) in glutamate metabolism under biotin-limited conditions in Corynebacterium glutamicum.

    Science.gov (United States)

    Supkulsutra, Tanyanut; Maeda, Tomoya; Kumagai, Kosuke; Wachi, Masaaki

    2013-01-01

    Corynebacterium glutamicum is a Gram-positive, rod-shaped, aerobic bacterium used for the fermentative production of L-glutamate. LldR (NCgl2814) is known as a repressor for ldhA and lldD encoding lactate dehydrogenases. LdhA is responsible for production of L-lactate, while LldD is for its assimilation. Since L-lactate production was observed as a by-product of glutamate production under biotin-limited conditions, LldR might play a regulatory role in the glutamate metabolism. Here for the first time, we investigated effects of overproduction or deletion of LldR on the glutamate metabolism under biotin-limited conditions in C. glutamicum. It was found that glutamate production under biotin-limited conditions was decreased by overproduction of LldR. In the wild-type cells, L-lactate was produced in the first 24 h and it was re-consumed thereafter. On the other hand, in the overproduced cells, L-lactate was produced like the wild type, but it was not re-consumed. This means that L-lactate assimilation, which is catalyzed by LldD, was suppressed by the overproduction of LldR, but L-lactate production, which is catalyzed by LdhA, was not affected, indicating that LldR mainly controls the expression of lldD but not of ldhA under biotin-limited conditions. This was confirmed by quantitative real-time RT-PCR. From these results, it is suggested that L-lactate metabolism, which is controlled by LldR, has a buffering function of the pyruvate pool for glutamate production.

  3. Biotin-targeted Pluronic(®) P123/F127 mixed micelles delivering niclosamide: A repositioning strategy to treat drug-resistant lung cancer cells.

    Science.gov (United States)

    Russo, Annapina; Pellosi, Diogo Silva; Pagliara, Valentina; Milone, Maria Rita; Pucci, Biagio; Caetano, Wilker; Hioka, Noboru; Budillon, Alfredo; Ungaro, Francesca; Russo, Giulia; Quaglia, Fabiana

    2016-09-10

    With the aim to develop alternative therapeutic tools for the treatment of resistant cancers, here we propose targeted Pluronic(®) P123/F127 mixed micelles (PMM) delivering niclosamide (NCL) as a repositioning strategy to treat multidrug resistant non-small lung cancer cell lines. To build multifunctional PMM for targeting and imaging, Pluronic(®) F127 was conjugated with biotin, while Pluronic(®) P123 was fluorescently tagged with rhodamine B, in both cases at one of the two hydroxyl end groups. This design intended to avoid any interference of rhodamine B on biotin exposition on PMM surface, which is a key fundamental for cell trafficking studies. Biotin-decorated PMM were internalized more efficiently than non-targeted PMM in A549 lung cancer cells, while very low internalization was found in NHI3T3 normal fibroblasts. Biotin-decorated PMM entrapped NCL with good efficiency, displayed sustained drug release in protein-rich media and improved cytotoxicity in A549 cells as compared to free NCL (Pbiotin-decorated PMM carrying NCL at low doses demonstrated a significantly higher cytotoxicity than free NCL in CPr-A549. These results point at NCL-based regimen with targeted PMM as a possible second-line chemotherapy for lung cancer showing cisplatin or multidrug resistance. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Avidin-biotin-based approach to forming heterotypic cell clusters and cell sheets on a gas-permeable membrane

    Energy Technology Data Exchange (ETDEWEB)

    Hamon, M; Ozawa, T; Montagne, K; Kojima, N; Ishii, R; Sakai, Y [Institute of Industrial Science (IIS), University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505 (Japan); Yamaguchi, S; Nagamune, T [Department of Bioengineering, Graduate School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan); Ushida, T, E-mail: mzh0026@auburn.edu [Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan)

    2011-09-15

    Implantation of sheet-like liver tissues is a promising method in hepatocyte-based therapies, because angiogenesis is expected to occur upon implantation from the surrounding tissues. In this context, we introduce here a new methodology for the formation of a functional thick hepatic tissue usable for cell sheet technology. First, we report the formation of composite tissue elements in suspension culture. Composite elements were composed of human hepatoma Hep G2 cells and mouse NIH/3T3 fibroblasts which are important modulators for thick-tissue formation. To overcome the very low attachment and organization capability between different cells in suspension, we synthesized a new cell-to-cell binding molecule based on the avidin-biotin binding system that we previously applied to attach hepatocytes on artificial substrata. This newly synthesized biotin-conjugated biocompatible anchoring molecule was inserted in the plasma membrane of both cell types. NIH/3T3 cells were further conjugated with avidin and incubated with biotin-presenting Hep G2 cells to form highly composite tissue elements. Then, we seeded those elements on highly gas-permeable membranes at their closest packing density to induce the formation of a thick, composite, functional hepatic tissue without any perfusion. This methodology could open a new way to engineer implantable thick liver tissue sheets where different cell types are spatially organized and well supplied with oxygen.

  5. Novel SLC19A3 Promoter Deletion and Allelic Silencing in Biotin-Thiamine-Responsive Basal Ganglia Encephalopathy.

    Directory of Open Access Journals (Sweden)

    Irene Flønes

    Full Text Available Biotin-thiamine responsive basal ganglia disease is a severe, but potentially treatable disorder caused by mutations in the SLC19A3 gene. Although the disease is inherited in an autosomal recessive manner, patients with typical phenotypes carrying single heterozygous mutations have been reported. This makes the diagnosis uncertain and may delay treatment.In two siblings with early-onset encephalopathy dystonia and epilepsy, whole-exome sequencing revealed a novel single heterozygous SLC19A3 mutation (c.337T>C. Although Sanger-sequencing and copy-number analysis revealed no other aberrations, RNA-sequencing in brain tissue suggested the second allele was silenced. Whole-genome sequencing resolved the genetic defect by revealing a novel 45,049 bp deletion in the 5'-UTR region of the gene abolishing the promoter. High dose thiamine and biotin therapy was started in the surviving sibling who remains stable. In another patient two novel compound heterozygous SLC19A3 mutations were found. He improved substantially on thiamine and biotin therapy.We show that large genomic deletions occur in the regulatory region of SLC19A3 and should be considered in genetic testing. Moreover, our study highlights the power of whole-genome sequencing as a diagnostic tool for rare genetic disorders across a wide spectrum of mutations including non-coding large genomic rearrangements.

  6. Criticality safety evaluation of Rocky Flats Plant one-gallon shipping containers

    International Nuclear Information System (INIS)

    Shaw, M.E.

    1991-12-01

    Criticality safety calculations have been performed to provide an analytical basis for handling, storage and transport of Rocky Flats Plant (RFP) one-gallon shipping containers. A mass limit was establish for metal (solid uranium or plutonium) and slurries (undissolved U or Pu solids in a ''mud,'' ''sludge,'' or ''slurry''). A separate volume limit was developed for plutonium solutions (liquids, either aqueous or organic, containing no visible undissolved solids)

  7. Criticality safety evaluation of Rocky Flats Plant one-gallon shipping containers

    Energy Technology Data Exchange (ETDEWEB)

    Shaw, M.E.

    1991-12-01

    Criticality safety calculations have been performed to provide an analytical basis for handling, storage and transport of Rocky Flats Plant (RFP) one-gallon shipping containers. A mass limit was establish for metal (solid uranium or plutonium) and slurries (undissolved U or Pu solids in a ``mud,`` ``sludge,`` or ``slurry``). A separate volume limit was developed for plutonium solutions (liquids, either aqueous or organic, containing no visible undissolved solids).

  8. Generic Containment: Detailed comparison of containment simulations performed on plant scale

    International Nuclear Information System (INIS)

    Kelm, St.; Klauck, M.; Beck, S.; Allelein, H.-J.; Preusser, G.; Sangiorgi, M.; Klein-Hessling, W.; Bakalov, I.; Bleyer, A.; Bentaib, A.; Kljenak, I.; Stempniewicz, M.; Kostka, P.; Morandi, S.; Ada del Corno, B.; Bratfisch, C.; Risken, T.; Denk, L.; Parduba, Z.; Paci, S.

    2014-01-01

    Highlights: • Consequent implementation of the recommendations derived from the OECD/NEA ISP-47. • Phenomenological code-to-code comparison performed on plant scale. • Systematic identification and elimination of the user effect. • Identification of fundamental differences in the model basis. • Application to PAR system analysis. - Abstract: One outcome of the OECD/NEA ISP-47 activity was the recommendation to elaborate a ‘Generic Containment’ in order to allow comparing and rating the results obtained by different lumped-parameter models on plant scale. Within the European SARNET2 project ( (http://www.sar-net.eu)), such a Generic Containment nodalisation, based on a German PWR (1300 MW el ), was defined. This agreement on the nodalisation allows investigating the remaining differences among the results, especially the ‘user-effect’, related to the modelling choices, as well as fundamental differences in the underlying model basis in detail. The methodology applied in order to compare the different code predictions consisted of a series of three benchmark steps with increasing complexity as well as a systematic comparison of characteristic variables and observations. This paper summarises the benchmark series, the lessons learned during specifying the steps, comparing and discussing the results and finally gives an outlook on future steps

  9. One-pot production of 18F-biotin by conjugation with 18F-FDG for pre-targeted imaging: Synthesis and radio-labelling of a PEGylated precursor

    International Nuclear Information System (INIS)

    Simpson, Michael; Trembleau, Laurent; Cheyne, Richard W.; Smith, Tim A.D.

    2011-01-01

    The biotin-avidin affinity system is exploited in pre-targeted imaging using avidin-conjugated antibodies. 18 F-FDG is available at all PET centres. 18 F-FDG forms oximes by reaction with oxyamine. Herein we describe the synthesis of oxyamine-funtionalised biotin, its 18 F-labelling by conjugation with 18 F-FDG and confirm its ability to interact with avidin.

  10. Comparative evaluation of Bis(thiosemicarbazone)- Biotin and Met-ac-TE3A for tumor imaging

    Science.gov (United States)

    Singh, Sweta; Tiwari, Anjani K.; Varshney, Raunak; Mathur, R.; Shukla, Gauri; Bag, N.; Singh, B.; Mishra, Anil K.

    2016-01-01

    2,2‧,2″-(11-(2-((4-mercapto-1-methoxy-1-oxobutan-2-yl)amino)-2-oxoethyl)-1,4,8,11-tetraaza cyclotetradecane-1,4,8-triyl)triacetic acid, Met-ac-TE3A and (E)-N-methyl-2-((E)-3-(2-(2-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoyl)hydrazinecarbono-thioyl)hydrazonobutan-2-ylidene)hydrazinecarbothioamide, Bis(thiosemicarbazone)- Biotin were synthesized and evaluated for imaging application. The pharmacokinetics of these ligands were determined by tracer methods. In vitro human serum stability of 99mTc Met-ac-TE3A/99mTc Bis(thiosemicarbazone)-Biotin after 24 h was found to be 96.5% and 97.0% respectively. Blood kinetics of both ligands in normal rabbits showed biphasic clearance pattern. Ex vivo biodistribution study revealed significant initial tumor uptake and high tumor/muscles ratio which is a pre-requisite condition for a ligand to work as SPECT-radiopharmaceutical for tumor imaging.

  11. Mitigating Capability Analysis during LOCA for Korean Standard Nuclear Power Plants in Containment Integrity

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Young; Park, Soo Yong; Kim, D. H.; Song, Y. M. [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

    2007-07-01

    The objective of this paper is to establish Containment spray operational technical bases for the typical Korean Standard Nuclear Power plants (Ulchin units 3 and 4) by modeling the plant, and analyzing a loss of coolant accident (LOCA) using the MAAP code. The severe accident phenomena at nuclear power plants have large uncertainties. For the integrity of the reactor vessel and containment safety against severe accidents, it is essential to understand severe accident sequences and to assess the accident progression accurately by computer codes. Furthermore, it is important to attain the capability to analyze a advanced nuclear reactor design for a severe accident prevention and mitigation.

  12. Pyrrolizidine alkaloid-containing toxic plants (Scenecio, Crotalaria, Cynoglossum, Amsinckia, Heliotropium, and Echium spp.)

    Science.gov (United States)

    Pyrrolizidine alkaloid (PA) containing plants are found throughout the world and are probably the most common plant cause of poisoning of livestock, wildlife and humans. PAs are potent liver toxins that under some conditions can be carcinogenic. The objective of this paper is to briefly introduce hi...

  13. CE/Bechtel design containment response to severe accident phenomenology: A comparison among several combustion engineering plants

    International Nuclear Information System (INIS)

    Khalil, Y.F.; Schneider, R.E.

    1995-01-01

    The objectives of this paper are to: (1) discuss the types of severe accident phenomena that drive containment failure modes in CE plants and (2) contribute to the current state of knowledge of CE/Bechtel-design containment response to severe accident phenomenology. The second objective is addressed by providing a comparative study of containment response to severe accidents among several CE plants including Millstone Unit 2 (MP2), Palisades (Consumers Power), Calvert Cliffs (Baltimore Gas and Electric Company), Palo Verde (Arizona Public Service), and SONGS Units 2 and 3 (Southern California Edison). The motivation for addressing the second objective is based on the current lack of comprehensive literature on CE/Bechtel design containment failure modes and mechanisms for accidents that progress beyond the design basis limits. The first part of this paper addresses severe accident phenomena-related failure mechanisms in CE/Bechtel-designed containments. The second part of this work provides a comparative study of containment response among several CE plants

  14. A survey of repair practices for nuclear power plant containment metallic pressure boundaries

    Energy Technology Data Exchange (ETDEWEB)

    Oland, C.B.; Naus, D.J. [Oak Ridge National Lab., TN (United States)

    1998-05-01

    The Nuclear Regulatory Commission has initiated a program at the Oak Ridge National Laboratory to provide assistance in their assessment of the effects of potential degradation on the structural integrity and leaktightness of metal containment vessels and steel liners of concrete containments in nuclear power plants. One of the program objectives is to identify repair practices for restoring metallic containment pressure boundary components that have been damaged or degraded in service. This report presents issues associated with inservice condition assessments and continued service evaluations and identifies the rules and requirements for the repair and replacement of nonconforming containment pressure boundary components by welding or metal removal. Discussion topics include base and welding materials, welding procedure and performance qualifications, inspection techniques, testing methods, acceptance criteria, and documentation requirements necessary for making acceptable repairs and replacements so that the plant can be returned to a safe operating condition.

  15. A survey of repair practices for nuclear power plant containment metallic pressure boundaries

    International Nuclear Information System (INIS)

    Oland, C.B.; Naus, D.J.

    1998-05-01

    The Nuclear Regulatory Commission has initiated a program at the Oak Ridge National Laboratory to provide assistance in their assessment of the effects of potential degradation on the structural integrity and leaktightness of metal containment vessels and steel liners of concrete containments in nuclear power plants. One of the program objectives is to identify repair practices for restoring metallic containment pressure boundary components that have been damaged or degraded in service. This report presents issues associated with inservice condition assessments and continued service evaluations and identifies the rules and requirements for the repair and replacement of nonconforming containment pressure boundary components by welding or metal removal. Discussion topics include base and welding materials, welding procedure and performance qualifications, inspection techniques, testing methods, acceptance criteria, and documentation requirements necessary for making acceptable repairs and replacements so that the plant can be returned to a safe operating condition

  16. STP-ECRTS - THERMAL AND GAS ANALYSES FOR SLUDGE TRANSPORT AND STORAGE CONTAINER (STSC) STORAGE AT T PLANT

    Energy Technology Data Exchange (ETDEWEB)

    CROWE RD; APTHORPE R; LEE SJ; PLYS MG

    2010-04-29

    The Sludge Treatment Project (STP) is responsible for the disposition of sludge contained in the six engineered containers and Settler tank within the 105-K West (KW) Basin. The STP is retrieving and transferring sludge from the Settler tank into engineered container SCS-CON-230. Then, the STP will retrieve and transfer sludge from the six engineered containers in the KW Basin directly into a Sludge Transport and Storage Containers (STSC) contained in a Sludge Transport System (STS) cask. The STSC/STS cask will be transported to T Plant for interim storage of the STSC. The STS cask will be loaded with an empty STSC and returned to the KW Basin for loading of additional sludge for transportation and interim storage at T Plant. CH2MHILL Plateau Remediation Company (CHPRC) contracted with Fauske & Associates, LLC (FAI) to perform thermal and gas generation analyses for interim storage of STP sludge in the Sludge Transport and Storage Container (STSCs) at T Plant. The sludge types considered are settler sludge and sludge originating from the floor of the KW Basin and stored in containers 210 and 220, which are bounding compositions. The conditions specified by CHPRC for analysis are provided in Section 5. The FAI report (FAI/10-83, Thermal and Gas Analyses for a Sludge Transport and Storage Container (STSC) at T Plant) (refer to Attachment 1) documents the analyses. The process considered was passive, interim storage of sludge in various cells at T Plant. The FATE{trademark} code is used for the calculation. The results are shown in terms of the peak sludge temperature and hydrogen concentrations in the STSC and the T Plant cell. In particular, the concerns addressed were the thermal stability of the sludge and the potential for flammable gas mixtures. This work was performed with preliminary design information and a preliminary software configuration.

  17. Criticality safety evaluation of Rocky Flats Plant one-gallon shipping containers

    Energy Technology Data Exchange (ETDEWEB)

    Shaw, M.E.

    1991-12-01

    Criticality safety calculations have been performed to provide an analytical basis for handling, storage and transport of Rocky Flats Plant (RFP) one-gallon shipping containers. A mass limit was establish for metal (solid uranium or plutonium) and slurries (undissolved U or Pu solids in a mud,'' sludge,'' or slurry''). A separate volume limit was developed for plutonium solutions (liquids, either aqueous or organic, containing no visible undissolved solids).

  18. Ternary WD40 repeat-containing protein complexes: evolution, composition and roles in plant immunity

    Directory of Open Access Journals (Sweden)

    Jimi C. Miller

    2016-01-01

    Full Text Available Plants, like mammals, rely on their innate immune system to perceive and discriminate among the majority of their microbial pathogens. Unlike mammals, plants respond to this molecular dialogue by unleashing a complex chemical arsenal of defense metabolites to resist or evade pathogen infection. In basal or non-host resistance, plants utilize signal transduction pathways to detect non-self, damaged-self and altered-self-associated molecular patterns and translate these danger signals into largely inducible chemical defenses. The WD40 repeat (WDR-containing proteins Gβ and TTG1 are constituents of two independent ternary protein complexes functioning at opposite ends of a plant immune signaling pathway. Gβ and TTG1 are also encoded by single-copy genes that are ubiquitous in higher plants, implying the limited diversity and functional conservation of their respective complexes. In this review, we summarize what is currently known about the evolutionary history of these WDR-containing ternary complexes, their repertoire and combinatorial interactions, and their downstream effectors and pathways in plant defense.

  19. IPE Data Base: Plant design, core damage frequency and containment performance information

    International Nuclear Information System (INIS)

    Lehner, J.; Lin, C.C.; Pratt, W.T.; Su, T.; Danziger, L.

    1995-01-01

    This data base stores data obtained from the Individual Plant Examinations (IPEs) which licensees of nuclear power plants have conducted in response to NRC's Generic Letter GL88-20. The IPE Data Base is a collection of linked files which store information about plant design, core damage frequency, and containment performance in a uniform, structured way. The information contined in the various files is based on data contained in the IPE submittals. The information extracted from the submittals and entered into the IPE Data Base can be maniulated so that queries regarding individual or groups of plants can be answered using the IPE Data Base. The IPE Data Base supports detailed inquiries into the characteristics of individual plants or classes of plants. Progress has been made on the IPE Data Base and it is largely complete. Recent focus has been the development of a user friendly version which is menu driven and allows the user to ask queries of varying complexity easily, without the need to become familiar with particular data base formats or conventions such as those of DBase IV or Microsoft Access. The user can obtain the information he desired by quickly moving through a series of on-screen menus and ''clicking'' on appropriate choices. In this way even a first time user can benefit from the large amount of information stored in the IPE Data Base without the need of a learning period

  20. Preparation of {sup 166} Dy/{sup 166} Ho DTPA-bis biotin as a system of In vivo generator; Preparacion de {sup 166} Dy/{sup 166} Ho DTPA-bis biotina como un sistema de generador In vivo

    Energy Technology Data Exchange (ETDEWEB)

    Jimenez V, M.R

    2003-07-01

    The objective of this work was to synthesize the complex {sup 166} Dy/{sup 166} Ho - diethylen triamine pentaacetic-bis Biotin ({sup 166} Dy/{sup 166} Ho DTPA-bis Biotin) to evaluate its potential as a new radiopharmaceutical in directed radiotherapy. The Dysprosium-166 was obtained for neutron irradiation of {sup 164} Dy{sub 2}0{sub 3} in the TRIGA Mark III reactor. The labelled was carried out in aqueous solution to p H 8.0 for addition of {sup 166} Dy Cl{sub 3} to the diethylen triamine pentaacetic-{alpha}, {omega}-bis Biotin (DTPA-bis Biotin). The radiochemical purity was determined for HPLC and ITLC. The biological integrity of the marked biotin is evaluated by the biological recognition of the avidin for HPLC - molecular exclusion with and without avidin addition. The studies of stability in vitro were made in dilutions of saline solution to 0.9% and with human serum at 37 C incubated 1 and 24 hours. The complex {sup 166} Dy/{sup 166} Ho DTPA-bis Biotin was obtained with a radiochemical purity of 99.1 {+-} 0.6%. The biological recognition of the complex {sup 166} Dy/{sup 166} Ho DTPA-bis Biotin for the avidin it doesn't affect the labelling procedure. The studies in vitro demonstrated that the {sup 166} Dy/{sup 166} Ho DTPA-bis Biotin is stable after the dilution in saline solution and in human serum that there is not translocation of the one radionuclide subsequent son to the beta decay of the {sup 166} Dy that could produce the {sup 166} Ho{sup 3+} liberation. The studies of Biodistribution in healthy mice demonstrated that the one complex {sup 166} Dy/{sup 166} Ho DTPA-bis Biotin have a high renal distribution. In conclusion the radiolabelled biotin in this investigation has the appropriate properties to be used as an In vivo generator system stable for directed radiotherapy. (Author)

  1. Preparation of {sup 166} Dy/{sup 166} Ho DTPA-bis biotin as a system of In vivo generator; Preparacion de {sup 166} Dy/{sup 166} Ho DTPA-bis biotina como un sistema de generador In vivo

    Energy Technology Data Exchange (ETDEWEB)

    Jimenez V, M R

    2003-07-01

    The objective of this work was to synthesize the complex {sup 166} Dy/{sup 166} Ho - diethylen triamine pentaacetic-bis Biotin ({sup 166} Dy/{sup 166} Ho DTPA-bis Biotin) to evaluate its potential as a new radiopharmaceutical in directed radiotherapy. The Dysprosium-166 was obtained for neutron irradiation of {sup 164} Dy{sub 2}0{sub 3} in the TRIGA Mark III reactor. The labelled was carried out in aqueous solution to p H 8.0 for addition of {sup 166} Dy Cl{sub 3} to the diethylen triamine pentaacetic-{alpha}, {omega}-bis Biotin (DTPA-bis Biotin). The radiochemical purity was determined for HPLC and ITLC. The biological integrity of the marked biotin is evaluated by the biological recognition of the avidin for HPLC - molecular exclusion with and without avidin addition. The studies of stability in vitro were made in dilutions of saline solution to 0.9% and with human serum at 37 C incubated 1 and 24 hours. The complex {sup 166} Dy/{sup 166} Ho DTPA-bis Biotin was obtained with a radiochemical purity of 99.1 {+-} 0.6%. The biological recognition of the complex {sup 166} Dy/{sup 166} Ho DTPA-bis Biotin for the avidin it doesn't affect the labelling procedure. The studies in vitro demonstrated that the {sup 166} Dy/{sup 166} Ho DTPA-bis Biotin is stable after the dilution in saline solution and in human serum that there is not translocation of the one radionuclide subsequent son to the beta decay of the {sup 166} Dy that could produce the {sup 166} Ho{sup 3+} liberation. The studies of Biodistribution in healthy mice demonstrated that the one complex {sup 166} Dy/{sup 166} Ho DTPA-bis Biotin have a high renal distribution. In conclusion the radiolabelled biotin in this investigation has the appropriate properties to be used as an In vivo generator system stable for directed radiotherapy. (Author)

  2. DESIGNAND EVALUATION OF TETRA-PAK CONTAINERS' RECYCLING PLANT ON A SMALLSCALE

    OpenAIRE

    Inche Mitma, Jorge; Vergiú Canto, Jorge|; Mavila Hinojoza, Daniel; Godoy Martínez, Manuel; Chung Pinzás, Alfonso

    2014-01-01

    This study deals about the design and evaluation of Tetra Pak containers' recycling plant on a small scale. The basic Plant Engineering was found from the information gathered. Some aspects included were: product design, process design, equipment design and costs evaluation, with the aim of determining its technical, economical and environmental capability for its implementation. El estudio trata sobre el diseño y evaluación de una planta de reciclaje de envases tetra pak a pequeña escala....

  3. Experiments to investigate direct containment heating phenomena with scaled models of the Calvert Cliffs Nuclear Power Plant

    International Nuclear Information System (INIS)

    Blanchat, T.K.; Pilch, M.M.; Allen, M.D.

    1997-02-01

    The Surtsey Test Facility is used to perform scaled experiments simulating High Pressure Melt Ejection accidents in a nuclear power plant (NPP). The experiments investigate the effects of direct containment heating (DCH) on the containment load. The results from Zion and Surry experiments can be extrapolated to other Westinghouse plants, but predicted containment loads cannot be generalized to all Combustion Engineering (CE) plants. Five CE plants have melt dispersal flow paths which circumvent the main mitigation of containment compartmentalization in most Westinghouse PWRs. Calvert Cliff-like plant geometries and the impact of codispersed water were addressed as part of the DCH issue resolution. Integral effects tests were performed with a scale model of the Calvert Cliffs NPP inside the Surtsey test vessel. The experiments investigated the effects of codispersal of water, steam, and molten core stimulant materials on DCH loads under prototypic accident conditions and plant configurations. The results indicated that large amounts of coejected water reduced the DCH load by a small amount. Large amounts of debris were dispersed from the cavity to the upper dome (via the annular gap). 22 refs., 84 figs., 30 tabs

  4. Combined biotin-terpyridine systems : a new versatile bridge between biology, polymer science and metallo-supramolecular chemistry

    NARCIS (Netherlands)

    Hofmeier, H.; Pahnke, J.; Weidl, C.H.; Schubert, U.S.

    2004-01-01

    Biotin, a well-known binding unit for the proteins avidin and streptavidin, was combined with the chelating ligand terpyridine via polymeric and nonpolymeric spacers. An ¿-amino-functionalized terpyridyl-poly(ethylene glycol) was prepared and utilized for complex formation with iron(II), nickel(II),

  5. One-pot production of 18F-biotin by conjugation with 18F-FDG for pre-targeted imaging: synthesis and radio-labelling of a PEGylated precursor.

    Science.gov (United States)

    Simpson, Michael; Trembleau, Laurent; Cheyne, Richard W; Smith, Tim A D

    2011-02-01

    The biotin-avidin affinity system is exploited in pre-targeted imaging using avidin-conjugated antibodies. (18)F-FDG is available at all PET centres. (18)F-FDG forms oximes by reaction with oxyamine. Herein we describe the synthesis of oxyamine-funtionalised biotin, its (18)F-labelling by conjugation with (18)F-FDG and confirm its ability to interact with avidin. Copyright © 2010 Elsevier Ltd. All rights reserved.

  6. Aging of concrete containment structures in nuclear power plants

    International Nuclear Information System (INIS)

    Naus, D.J.; Oland, C.B.; Ellingwood, B.; Mori, Yasuhiro; Arndt, E.G.

    1992-01-01

    Concrete structures play a vital role in the safe operation of all light-water reactor plants in the US Pertinent concrete structures are described in terms of their importance design, considerations, and materials of construction. Degradation factors which can potentially impact the ability of these structures to meet their functional and performance requirements are identified. Current inservice inspection requirements for concrete containments are summarized. A review of the performance history of the concrete components in nuclear power plants is provided. A summary is presented. A summary is presented of the Structural Aging (SAG) Program being conducted at the Oak Ridge National Laboratory for the US Nuclear Regulatory Commission. The SAG Program is addressing the aging management of safety-related concrete structures in nuclear power plants for the purpose of providing improved bases for their continued service. The program consists of a management task and three technical tasks: materials property data base, structural component assessment/repair technologies, and quantitiative methodology for continued service conditions. Objectives and a summary of accomplishments under each of these tasks are presented

  7. Labelling of a biotin-derivative by 99mTc, its purification, and binding-capacity to avidin

    International Nuclear Information System (INIS)

    Pollok, J.M.; Schultes, B.; Oehr, P.

    1990-01-01

    The paper summarizes the procedure of 99m Tc-labelling of NHS-LC-Biotin. It describes the conditions for the cemical reaction, the purification, and the determination of the degree of purity. The binding of the purified product to avidin is at least 94%. (orig.) [de

  8. Immobilized sialyltransferase fused to a fungal biotin-binding protein: Production, properties, and applications.

    Science.gov (United States)

    Kajiwara, Hitomi; Tsunashima, Masako; Mine, Toshiki; Takakura, Yoshimitsu; Yamamoto, Takeshi

    2016-04-01

    A β-galactoside α2,6-sialyltransferase (ST) from the marine bacterium Photobacterium sp. JT-ISH-224 with a broad acceptor substrate specificity was fused to a fungal biotin-binding protein tamavidin 2 (TM2) to produce immobilized enzyme. Specifically, a gene for the fusion protein, in which ST from Photobacterium sp. JT-ISH-224 and TM2 were connected via a peptide linker (ST-L-TM2) was constructed and expressed in Escherichia coli. The ST-L-TM2 was produced in the soluble form with a yield of approximately 15,000 unit/300 ml of the E. coli culture. The ST-L-TM2 was partially purified and part of it was immobilized onto biotin-bearing magnetic microbeads. The immobilized ST-L-TM2 onto microbeads could be used at least seven consecutive reaction cycles with no observed decrease in enzymatic activity. In addition, the optimum pH and temperature of the immobilized enzyme were changed compared to those of a free form of the ST. Considering these results, it was strongly expected that the immobilized ST-L-TM2 was a promising tool for the production of various kind of sialoligosaccharides. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  9. Description of the CONTAIN input model for the Dodewaard nuclear power plant

    International Nuclear Information System (INIS)

    Velema, E.J.

    1992-02-01

    This report describes the ECN standard CONTAIN input model for the Dodewaard Nuclear Power Plant (NPP) that has been developed by ECN. This standard input model will serve as a basis for analyses of the phenomena which may occur inside the Dodewaard containment in the event of a postulated severe accident. Boundary conditions for specific containment analyses can easily be implemented in the input model. as a result ECN will be able to respond quickly on requests for analyses from the utilities of the authorities. The report also includes brief descriptions of the Dodewaard NPP and the CONTAIN computer program. (author). 7 refs.; 5 figs.; 3 tabs

  10. Non-destructive evaluation of containment walls in nuclear power plants

    Science.gov (United States)

    Garnier, V.; Payan, C.; Lott, M.; Ranaivomanana, N.; Balayssac, J. P.; Verdier, J.; Larose, E.; Zhang, Y.; Saliba, J.; Boniface, A.; Sbartai, Z. M.; Piwakowski, B.; Ciccarone, C.; Hafid, H.; Henault, J. M.; Buffet, F. Ouvrier

    2017-02-01

    Two functions are regularly tested on containment walls in order to anticipate a possible accident. The first is mechanical to resist a possible internal over-pressure and the second is to prevent leakage. The AAPR reference accident is the rupture of a pipe in the primary circuit of a nuclear plant. In this case, the pressure and temperature can reach 5 bar and 180°C in 20 seconds. The national project `Non-destructive testing of the containment structures of nuclear plants' aims at studying the non-destructive techniques capable to evaluate the concrete properties and its damaging and cracks. This 4-year-project is segmented into two parts. The first consists in developing and selecting the most relevant NDEs in the laboratory to reach these goals. These evaluations are developed in conditions representing the real conditions of the stresses generated during ten-yearly visits of the plants or those related to an accident. The second part consists in applying the selected techniques to two containment structures under pressure. The first structure is proposed by ONERA and the second is a mockup of a containment wall on a 1/3 scale made by EDF within the VeRCoRs project. Communication is focused on the part of the project that concerns the damage and crack process characterization by means of NDT. The tests are done in 3 or 4 points bending in order to study the cracks' generation, their propagation, as well as their opening and closing. The main ultrasonic techniques developed concern linear or non-linear acoustic: acoustic emission [1], Locadiff [2], energy diffusion, surface wave's velocity and attenuation, DAET [3]. The recorded data contribute to providing the mapping of the investigated parameters, either in volume, in surface or globally. Digital image correlation is an important additional asset to validate the coherence of the data. The spatial normalization of the data in the specimen space allows proposing algorithms on the combination of the

  11. Printed biotin-functionalised polythiophene films as biorecognition layers in the development of paper-based biosensors

    Science.gov (United States)

    Ihalainen, Petri; Pesonen, Markus; Sund, Pernilla; Viitala, Tapani; Määttänen, Anni; Sarfraz, Jawad; Wilén, Carl-Erik; Österbacka, Ronald; Peltonen, Jouko

    2016-02-01

    The integration of flexible electronic sensors in clinical diagnostics is visioned to significantly reduce the cost of many diagnostic tests and ultimately make healthcare more accessible. This study concentrates on the characterisation of inkjet-printed bio-functionalised polythiophene films on paper-based ultrathin gold film (UTGF) electrodes and their possible application as biorecognition layers. Physicochemical surface properties (topography, chemistry, and wetting) and electrochemical characteristics of water-soluble regioirregular tetraethylene-glycol polythiophene (TEGPT) and biotin-functionalised TEGPT (b-TEGPT) films were examined and compared. In addition, their specificity towards streptavidin protein was tested. The results show that stable supramolecular biorecognition layers of insulating b-TEGPT and streptavidin were successfully fabricated on a paper-based UTGF by inkjet-printing. Good adhesion of thiophene to UTGF can be attributed to covalent linkage between sulphur and gold, whereas the stability of the streptavidin layer is due to the high affinity between biotin and streptavidin. The device introduced can be utilised in the development of biosensors for clinically relevant analytes e.g. for detecting complementary DNA oligomers or antibody-antigen complexes.

  12. The scheme optimization and management innovation for the first containment integrated in-service test of nuclear power plant

    International Nuclear Information System (INIS)

    Wang Haiwei; Yang Gang

    2014-01-01

    The containment integrated test is a large-scale, high risk and very difficult test in pressurized water reactor nuclear power plants. By simulating peak pressure inside the containment in DESIGN-BASIS accident conditions, measuring the total leakage rate of the containment with the peak pressure, and implementing the structure inspection test on several pressure levels, the containment's performance can be verified. Containment integrated test is an important witness point supervised by NNSA. The test results crucially decide the reactor to be started or not. The containment integrated test in 301 overhaul is the first in-service test of Unit 3. By the experience of the same 6 former tests in Qinshan Second Nuclear Power Plant and the feedback from other plants, the test scheme get more scientific and the organization management more standardized. This article discusses the containment integrated test in 301 overhaul and summarizes the experience to provide some references for the following containment integrated tests in the future. (authors)

  13. The Use of Biotin to Demonstrate Immunohistochemistry, Western Blotting, and Dot Blots in University Practical Classes

    Science.gov (United States)

    Millar, Thomas James; Knighton, Ronald; Chuck, Jo-Anne

    2012-01-01

    Immunological detection of proteins is an essential method to demonstrate to undergraduate biology students, however, is often difficult in resource and time poor student laboratory sessions. This method describes a failsafe method to rapidly and economically demonstrate this technique using biotinylated proteins or biotin itself as targets for…

  14. Screening for epitope specificity directly on culture supernatants in the early phase of monoclonal antibody production by an ELISA with biotin-labeled antigen

    DEFF Research Database (Denmark)

    Andersen, Ditte C; Jensen, Charlotte H; Gregersen, Annemette

    2004-01-01

    This report describes an assay for comparison of epitope specificity in groups of monoclonal antibodies against a given antigen. The only prerequisite is the biotin-labeled antigen. One of the monoclonal antibodies is captured onto a plastic surface via a rabbit anti-mouse Ig, and the other...... preincubated with biotinylated antigen. When the two antibodies react with the same epitope subsequent binding of the biotin-labeled antigen is abolished (inhibition). In the cases where no inhibition was observed, the two antibodies were considered to react with distinct, independent epitopes. The obvious...

  15. Major involvement of Na(+) -dependent multivitamin transporter (SLC5A6/SMVT) in uptake of biotin and pantothenic acid by human brain capillary endothelial cells.

    Science.gov (United States)

    Uchida, Yasuo; Ito, Katsuaki; Ohtsuki, Sumio; Kubo, Yoshiyuki; Suzuki, Takashi; Terasaki, Tetsuya

    2015-07-01

    The purpose of this study was to clarify the expression of Na(+) -dependent multivitamin transporter (SLC5A6/SMVT) and its contribution to the supply of biotin and pantothenic acid to the human brain via the blood-brain barrier. DNA microarray and immunohistochemical analyses confirmed that SLC5A6 is expressed in microvessels of human brain. The absolute expression levels of SLC5A6 protein in isolated human and monkey brain microvessels were 1.19 and 0.597 fmol/μg protein, respectively, as determined by a quantitative targeted absolute proteomics technique. Using an antibody-free method established by Kubo et al. (2015), we found that SLC5A6 was preferentially localized at the luminal membrane of brain capillary endothelium. Knock-down analysis using SLC5A6 siRNA showed that SLC5A6 accounts for 88.7% and 98.6% of total [(3) H]biotin and [(3) H]pantothenic acid uptakes, respectively, by human cerebral microvascular endothelial cell line hCMEC/D3. SLC5A6-mediated transport in hCMEC/D3 was markedly inhibited not only by biotin and pantothenic acid, but also by prostaglandin E2, lipoic acid, docosahexaenoic acid, indomethacin, ketoprofen, diclofenac, ibuprofen, phenylbutazone, and flurbiprofen. This study is the first to confirm expression of SLC5A6 in human brain microvessels and to provide evidence that SLC5A6 is a major contributor to luminal uptake of biotin and pantothenic acid at the human blood-brain barrier. In humans, it was unclear (not concluded) about what transport system at the blood-brain barrier (BBB) is responsible for the brain uptakes of two vitamins, biotin and pantothenic acid, which are necessary for brain proper function. This study clarified for the first time that the solute carrier 5A6/Na(+) -dependent multivitamin transporter SLC5A6/SMVT is responsible for the supplies of biotin and pantothenic acid into brain across the BBB in humans. DHA, docosahexaenoic acid; NSAID, non-steroidal anti-inflammatory drug; PGE2, prostaglandin E2. © 2015

  16. Containment nuclear plant structures evaluation by non destructive testing: strategy and results

    OpenAIRE

    GARNIER, Vincent; HENAULT, Jean-Marie; HAFID, Hamid; VERDIER, Jérôme; CHAIX, Jean François; ABRAHAM, Odile; SBARTAÏ, Zoubir Medhi; BALAYSSAC, Jean Pierre; PIWAKOWSKI, Bogdan; VILLAIN, Géraldine; DEROBERT, Xavier; PAYAN, Cédric; RAKOTONARIVO, Sandrine; LAROSE, Eric; SOGBOSSI, Hognon

    2016-01-01

    Containment nuclear plants structures are an ultimate barrier in the event of an accident. Mechanical resistance and tightness are the two functions that they are expected to provide. To evaluate their capacity to perform them, destructive testing cannot be used to characterize the material. Non-Destructive Tests then represent a relevant solution to test concrete and the struc- ture. The article positions NDT within the context of containment structures supervision and maintenance, and prese...

  17. Use of an integrated containment and ultimate heat sink (UHS) response approach to evaluate nuclear power plant modifications

    International Nuclear Information System (INIS)

    Wetzel, M.C.; Vieira, A.T.; Patton, D.C.

    1994-01-01

    Detailed containment and Ultimate Heat Sink (UHS) performance evaluations often are required to support major plant modifications, such as power up-rates and steam generator replacements. These UHS and containment pressure and temperature response evaluations are interrelated. Not only is the containment heat load to the UHS a factor in these evaluations, but other heat loads, such as those from the spent fuel pool, may change as a result of the plant modification and impact containment or UHS response. Our experience is that if an integrated containment/UHS response model is developed prior to the feasibility evaluations for such plant modifications, significant savings in engineering hours can be achieved. This paper presents an overview of such a front-end engineering tool that has been developed and used to support engineering evaluations. 3 refs., 2 figs

  18. 21 CFR 1308.35 - Exemption of certain cannabis plant material, and products made therefrom, that contain...

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 9 2010-04-01 2010-04-01 false Exemption of certain cannabis plant material, and... ENFORCEMENT ADMINISTRATION, DEPARTMENT OF JUSTICE SCHEDULES OF CONTROLLED SUBSTANCES Exempt Cannabis Plant... cannabis plant material, and products made therefrom, that contain tetrahydrocannabinols. (a) Any processed...

  19. Development of an Ion Chamber for Monitoring the Containment of a Nuclear Power Plant

    Energy Technology Data Exchange (ETDEWEB)

    Song, Tae-Yung; Kim, Han-Soo; Park, Se-Hwan; Ha, Jang-Ho [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

    2008-05-15

    Nuclear power plants need many different types of radiation detectors for different purposes. Neutron detectors are installed inside and outside of the core to check the neutron flux. Scintillation detectors are used to check the fission products included in the liquids and gases of plant system. Geiger-Mueller counters are used for the area radiation monitoring. In addition to the above-mentioned detectors, ion chambers are installed to monitor radiation level of the containment. A few ion chambers are located within the reactor containment to monitor radiation level of an accident case. Therefore, the ion chamber should be capable of monitoring high level radiation dose up to 10{sup 7} R/h. Korea Atomic Energy Research Institute (KAERI) developed an ion chamber for monitoring the radiation dose inside the containment.

  20. Containment failure modes preliminary analysis for Atucha-I nuclear power plant during severe accidents

    International Nuclear Information System (INIS)

    Baron, J.; Caballero, C.; Zarate, S.M.

    1997-01-01

    The present work has the objective to analyze the containment behavior of the Atucha-I nuclear power plant during a severe accident, as part of a probabilistic safety assessment (PSA). Initially, a generic description of the containment failure modes considered in other PSAs is performed. Then, the possible containment failure modes for Atucha I are qualitatively analyzed, according to it design peculiarities. These failure modes involve some substantial differences from other PSAs, due to the particular design of Atucha I. Among others, it is studied the influence of: moderator/coolant separation, existence of cooling Zircaloy channels, existence of filling bodies inside the pressure vessel, reactor cavity geometry, on-line refueling mode, and existence of a double shell containment (steel and concrete) with an annular separation room. As a functions of the before mentioning analysis, a series of parameters to be taken into account is defined, on a preliminary basis, for definition of the plant damage states. (author) [es

  1. Synthesis of dendrimer-based biotin radiopharmaceuticals to enhance whole-body clearance

    International Nuclear Information System (INIS)

    Sato, Noriko; Park, Chang W.; Kim, Hyung-Sik; Han, Eui-Sik; Wong, Karen J.; Paik, Ronald S.; Park, Luke S.; Yao Zhengsheng; Carrasquillo, Jorge A.; Paik, Chang H.

    2003-01-01

    To synthesize a biotin radiopharmaceutical that clears rapidly, dendrimer was used as a carrier and conjugated with succinimidyl 3-[ 125 I]iodobenzoate and tetrafluorophenyl norbiotinamidosuccinate. Then, succinic anhydride was used to reduce its pI. In mice, the non-succinylated product showed high liver (67% ID/g) and kidney (44% ID/g) uptakes and whole-body retention (94% ID) at 20 min that persisted for 12 hr. The corresponding organ uptakes (22% and 11% ID/g) and the whole-body retention (47% ID) were drastically reduced by succinylation (p<0.0001). Lysine co-injection further lowered renal uptake

  2. Synthetic assembly of novel avidin-biotin-GlcNAc (ABG) complex as an attractive bio-probe and its interaction with wheat germ agglutinin (WGA).

    Science.gov (United States)

    Kumari, Amrita; Koyama, Tetsuo; Hatano, Ken; Matsuoka, Koji

    2016-10-01

    A tetravalent GlcNAc pendant glycocluster was constructed with terminal biotin through C6 linker. To acquire the multivalent carbohydrate-protein interactions, we synthesized a glycopolymer of tetrameric structure using N-acetyl-d-glucosamine (GlcNAc) as the target carbohydrate by the use of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) as coupling reagent, followed by biotin-avidin complexation leading to the formation of glycocluster of avidin-biotin-GlcNAc conjugate (ABG complex). The dynamic light scattering (DLS) system was implied for size detection and to check the binding affinity of GlcNAc conjugate with a WGA lectin we use fluorometric assay by means of specific excitation of tryptophan at λex 295nm and it was found to be very high Ka∼1.39×10(7) M(-1) in case of ABG complex as compared to GlcNAc only Ka∼1.01×10(4) M(-1) with the phenomenon proven to be due to glycocluster effect. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Matrix-derived combination effect and risk assessment for estragole from basil-containing plant food supplements (PFS)

    NARCIS (Netherlands)

    Berg, van den S.J.P.L.; Klaus, V.; Alhusainy, W.; Rietjens, I.

    2013-01-01

    Basil-containing plant food supplements (PFS) can contain estragole which can be metabolised into a genotoxic and carcinogenic 1'-sulfoxymetabolite. This study describes the inhibition of sulfotransferase (SULT)-mediated bioactivation of estragole by compounds present in basil-containing PFS.

  4. Evaluation of the side effects of poly(epsilon-caprolactone) nanocapsules containing atrazine towards maize plants

    Science.gov (United States)

    Oliveira, Halley; Stolf-Moreira, Renata; Martinez, Cláudia; Sousa, Gustavo; Grillo, Renato; de Jesus, Marcelo; Fraceto, Leonardo

    2015-10-01

    Poly(epsilon-caprolactone) (PCL) nanocapsules have been used as a carrier system for the herbicide atrazine, which is commonly applied to maize. We demonstrated previously that these atrazine containing polymeric nanocapsules were ten-fold more effective in the control of mustard plants (a target species), as compared to a commercial atrazine formulation. Since atrazine can have adverse effects on non-target crops, here we analyzed the effect of encapsulated atrazine on growth, physiological and oxidative stress parameters of soil-grown maize plants (Zea mays L.). One day after the post-emergence treatment with PCL nanocapsules containing atrazine (1 mg mL-1), maize plants presented 15 and 21 % decreases in maximum quantum yield of photosystem II and in net CO2 assimilation rate, respectively, as compared to water-sprayed plants. The same treatment led to a 1.8-fold increase in leaf lipid peroxidation in comparison with control plants. However, all of these parameters were unaffected four and eight days after the application of encapsulated atrazine. These results suggested that the negative effects of atrazine were transient, probably due to the ability of maize plants to detoxify the herbicide. When encapsulated atrazine was applied at a ten-fold lower concentration (0.1 mg mL-1), a dosage that is still effective for weed control, no effects were detected even shortly after application. Regardless of the herbicide concentration, neither pre- nor post-emergence treatment with the PCL nanocapsules carrying atrazine resulted in the development of any macroscopic symptoms in maize leaves, and there were no impacts on shoot growth. Additionally, no effects were observed when plants were sprayed with PCL nanocapsules without atrazine. Overall, these results suggested that the use of PCL nanocapsules containing atrazine did not lead to persistent side effects in maize plants, and that the technique could offer a safe tool for weed control without affecting crop growth.

  5. Evolutionary genomics revealed interkingdom distribution of Tcn1-like chromodomain-containing Gypsy LTR retrotransposons among fungi and plants

    Directory of Open Access Journals (Sweden)

    Blinov Alexander

    2010-04-01

    Full Text Available Abstract Background Chromodomain-containing Gypsy LTR retrotransposons or chromoviruses are widely distributed among eukaryotes and have been found in plants, fungi and vertebrates. The previous comprehensive survey of chromoviruses from mosses (Bryophyta suggested that genomes of non-seed plants contain the clade which is closely related to the retrotransposons from fungi. The origin, distribution and evolutionary history of this clade remained unclear mainly due to the absence of information concerning the diversity and distribution of LTR retrotransposons in other groups of non-seed plants as well as in fungal genomes. Results In present study we preformed in silico analysis of chromodomain-containing LTR retrotransposons in 25 diverse fungi and a number of plant species including spikemoss Selaginella moellendorffii (Lycopodiophyta coupled with an experimental survey of chromodomain-containing Gypsy LTR retrotransposons from diverse non-seed vascular plants (lycophytes, ferns, and horsetails. Our mining of Gypsy LTR retrotransposons in genomic sequences allowed identification of numerous families which have not been described previously in fungi. Two new well-supported clades, Galahad and Mordred, as well as several other previously unknown lineages of chromodomain-containing Gypsy LTR retrotransposons were described based on the results of PCR-mediated survey of LTR retrotransposon fragments from ferns, horsetails and lycophytes. It appeared that one of the clades, namely Tcn1 clade, was present in basidiomycetes and non-seed plants including mosses (Bryophyta and lycophytes (genus Selaginella. Conclusions The interkingdom distribution is not typical for chromodomain-containing LTR retrotransposons clades which are usually very specific for a particular taxonomic group. Tcn1-like LTR retrotransposons from fungi and non-seed plants demonstrated high similarity to each other which can be explained by strong selective constraints and the

  6. The plant-specific impact of different pressurization rates in the probabilistic estimation of containment failure modes

    International Nuclear Information System (INIS)

    Ahn, Kwang Il; Yang, Joon Eon; Ha, Jae Joo

    2003-01-01

    The explicit consideration of different pressurization rates in estimating the probabilities of containment failure modes has a profound effect on the confidence of containment performance evaluation that is so critical for risk assessment of nuclear power plants. Except for the sophisticated NUREG-1150 study, many of the recent containment performance analyses (through level 2 PSAs or IPE back-end analyses) did not take into account an explicit distinction between slow and fast pressurization in their analyses. A careful investigation of both approaches shows that many of the approaches adopted in the recent containment performance analyses exactly correspond to the NUREG-1150 approach for the prediction of containment failure mode probabilities in the presence of fast pressurization. As a result, it was expected that the existing containment performance analysis results would be subjected to greater or less conservatism in light of the ultimate failure mode of the containment. The main purpose of this paper is to assess potential conservatism of a plant-specific containment performance analysis result in light of containment failure mode probabilities

  7. Quercetin and doxorubicin co-encapsulated biotin receptor-targeting nanoparticles for minimizing drug resistance in breast cancer.

    Science.gov (United States)

    Lv, Li; Liu, Chunxia; Chen, Chuxiong; Yu, Xiaoxia; Chen, Guanghui; Shi, Yonghui; Qin, Fengchao; Ou, Jiebin; Qiu, Kaifeng; Li, Guocheng

    2016-05-31

    The combination of a chemotherapeutic drug with a chemosensitizer has emerged as a promising strategy for cancers showing multidrug resistance (MDR). Herein we describe the simultaneous targeted delivery of two drugs to tumor cells by using biotin-decorated poly(ethylene glycol)-b-poly(ε-caprolactone) nanoparticles encapsulating the chemotherapeutic drug doxorubicin and the chemosensitizer quercetin (BNDQ). Next, the potential ability of BNDQ to reverse MDR in vitro and in vivo was investigated. Studies demonstrated that BNDQ was more effectively taken up with less efflux by doxorubicin-resistant MCF-7 breast cancer cells (MCF-7/ADR cells) than by the cells treated with the free drugs, single-drug-loaded nanoparticles, or non-biotin-decorated nanoparticles. BNDQ exhibited clear inhibition of both the activity and expression of P-glycoprotein in MCF-7/ADR cells. More importantly, it caused a significant reduction in doxorubicin resistance in MCF-7/ADR breast cancer cells both in vitro and in vivo, among all the groups. Overall, this study suggests that BNDQ has a potential role in the treatment of drug-resistant breast cancer.

  8. Synthesis and biological investigation of PIM mimics carrying biotin or a fluorescent label for cellular imaging.

    Science.gov (United States)

    Front, Sophie; Bourigault, Marie-Laure; Rose, Stéphanie; Noria, Ségueni; Quesniaux, Valérie F J; Martin, Olivier R

    2013-01-16

    Phosphatidyl inositol mannosides (PIMs) are constituents of the mycobacterial cell wall; these glycolipids are known to exhibit potent inhibitory activity toward the LPS-induced production of cytokines by macrophages, and therefore have potential as anti-inflammatory agents. Recently, heterocyclic analogues of PIMs in which the inositol is replaced by a piperidine (aza-PIM mimics) or a tetrahydropyran moiety (oxa-PIM mimics) have been prepared by short synthetic sequences and shown to retain the biological activity of the parent PIM structures. In this investigation, the aza-PIM analogue was used as a convenient scaffold to link biotin or a fluorescent label (tetramethyl-rhodamine) by way of an aminocaproyl spacer, with the goal of using these conjugates for intracellular localization and for the study of the mechanism of their antiinflammatory action. The synthesis of these compounds is reported, as well as the evaluation of their activities as inhibitors of LPS-induced cytokine production by macrophages (TNFα, IL12p40); preliminary investigations by FACS and confocal microscopy indicated that PIM-biotin conjugate binds to macrophage membranes with rapid kinetics.

  9. Livestock Poisoning with Pyrrolizidine Alkaloid Containing Plants (Senecio, Crotalaria, Cynoglossum, Amsinckia, Heliotropium and Echium spp.)

    Science.gov (United States)

    Pyrrolizidine alkaloids (PAs) are potent liver toxins that have been identified in over 6,000 plants throughout the world. Alkaloids are nitrogen-based compounds with potent biological activity. About half of the identified PAs are toxic and several cause cancer (carcinogenic). PA-containing plants...

  10. Studying Protein-Protein Interactions by Biotin AP-Tagged Pulldown and LTQ-Orbitrap Mass Spectrometry.

    Science.gov (United States)

    Xie, Zhongqiu; Jia, Yuemeng; Li, Hui

    2017-01-01

    The study of protein-protein interactions represents a key aspect of biological research. Identifying unknown protein binding partners using mass spectrometry (MS)-based proteomics has evolved into an indispensable strategy in drug discovery. The classic approach of immunoprecipitation with specific antibodies against the proteins of interest has limitations, such as the need for immunoprecipitation-qualified antibody. The biotin AP-tag pull-down system has the advantage of high specificity, ease of use, and no requirement for antibody. It is based on the high specificity, high affinity interaction between biotin and streptavidin. After pulldown, in-gel tryptic digestion and tandem mass spectrometry (MS/MS) analysis of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) protein bands can be performed. In this work, we provide protocols that can be used for the identification of proteins that interact with FOXM1, a protein that has recently emerged as a potential biomarker and drug target in oncotherapy, as an example. We focus on the pull-down procedure and assess the efficacy of the pulldown with known FOXM1 interactors such as β-catenin. We use a high performance LTQ Orbitrap MSn system that combines rapid LTQ ion trap data acquisition with high mass accuracy Orbitrap analysis to identify the interacting proteins.

  11. Containment response analysis for the PSA (Probabilistic Safety Assessment) of the CAREM-25 nuclear power plant

    International Nuclear Information System (INIS)

    Baron, J.H.

    1997-01-01

    This work is part of the probabilistic safety assessment actually under development for the CAREM-25 nuclear power station, and departs from the accident sequences already obtained and quantified by the Event Trees/Fault Trees techniques. At first, the potential containment failure modes for nuclear stations are listed, based on the experience. Then, the CAREM-25 design peculiarities are analyzed, on their possible influence on the containment behavior during severe accidents. Then plan damage states are defined. Furthermore, containment damage states are also defined, and containment event trees are built for each plant damage state. Those sequences considered representative from the annual probability (those which exceed or probability of IE-09 per year, are used to quantify the combinations of plant damage states/containment damage states, based on the estimation of a vulnerability matrix. (author) [es

  12. Collaborative Student Laboratory Exercise Using FT-IR Spectroscopy for the Kinetics Study of a Biotin Analogue

    Science.gov (United States)

    Leong, Jhaque; Ackroyd, Nathan C.; Ho, Karen

    2014-01-01

    The synthesis of N-methoxycarbonyl-2-imidazolidone, an analogue of biotin, was conducted by organic chemistry students and confirmed using FT-IR and H NMR. Spectroscopy students used FT-IR to measure the rate of hydrolysis of the product and determined the rate constant for the reaction using the integrated rate law. From the magnitude of the rate…

  13. The integrity of NSSS and containment during extended station blackout for Kuosheng BWR plant

    Energy Technology Data Exchange (ETDEWEB)

    Hsu, Keng-Hsien; Yuann, Yng-Ruey; Lin, Ansheng [Atomic Energy Council, Taoyuan City, Taiwan (China). Inst. of Nuclear Energy Research

    2017-11-15

    The Fukushima Daiichi accident occurring on March 11, 2011, reveals that Station Blackout (SBO) may last longer than 8 h. However, the original design may not have sufficient capacity to cope with a SBO for more than 8 h. In view of this, Taiwan Power Company has initiated several enhancements to mitigate the severity of the extended SBO. Based on the improved plant configuration, a SBO coping analysis is performed in this study to assess whether the Kuosheng BWR plant has sufficient capability to cope with SBO for 24 h with respect to maintaining the integrity of the reactor core and containment. The analyses in the Nuclear Steam Supply System (NSSS) and the containment are based on the RETRAN-3D and GOTHIC models, respectively. The flow conditions calculated by RETRAN-3D during the event are retrieved and input to the GOTHIC containment model to determine the containment pressure and temperature response. These boundary conditions include SRV flow rate, SRV flow enthalpy, and total reactor coolant system leakage flow rate.

  14. The integrity of NSSS and containment during extended station blackout for Kuosheng BWR plant

    International Nuclear Information System (INIS)

    Hsu, Keng-Hsien; Yuann, Yng-Ruey; Lin, Ansheng

    2017-01-01

    The Fukushima Daiichi accident occurring on March 11, 2011, reveals that Station Blackout (SBO) may last longer than 8 h. However, the original design may not have sufficient capacity to cope with a SBO for more than 8 h. In view of this, Taiwan Power Company has initiated several enhancements to mitigate the severity of the extended SBO. Based on the improved plant configuration, a SBO coping analysis is performed in this study to assess whether the Kuosheng BWR plant has sufficient capability to cope with SBO for 24 h with respect to maintaining the integrity of the reactor core and containment. The analyses in the Nuclear Steam Supply System (NSSS) and the containment are based on the RETRAN-3D and GOTHIC models, respectively. The flow conditions calculated by RETRAN-3D during the event are retrieved and input to the GOTHIC containment model to determine the containment pressure and temperature response. These boundary conditions include SRV flow rate, SRV flow enthalpy, and total reactor coolant system leakage flow rate.

  15. Design and analysis of new prestressed concrete containment and its passive cooling system for nuclear power plants

    International Nuclear Information System (INIS)

    Tan Xiaoshi; Li Xiaowei; Li Xiaotian; He Shuyan

    2014-01-01

    A new nuclear power plant prestressed concrete containment and its passive cooling system design were proposed for CAP1700 nuclear power plant as an example. The thermal-hydraulic calculation method for the new passive containment cooling system of CAP1700 was introduced and the operating parameters in accident condition were obtained. The result shows that the design of passive containment cooling system for CAP1700 is feasible and can meet the cooling demand in accident condition. Reservoir capacity of tank has a big margin and can be further optimized by calculation. (authors)

  16. Antibodies to biotinylated red blood cells in adults and infants: improved detection, partial characterization, and dependence on red blood cell-biotin dose.

    Science.gov (United States)

    Schmidt, Robert L; Mock, Donald M; Franco, Robert S; Cohen, Robert M; North, Anne K; Cancelas, José A; Geisen, Christof; Strauss, Ronald G; Vlaar, Alexander P; Nalbant, Demet; Widness, John A

    2017-06-01

    Biotin-labeled red blood cells (BioRBCs) are used for in vivo kinetic studies. Because BioRBC dosing occasionally induces antibodies, a sensitive and specific anti-BioRBC detection assay is needed. Aims were to 1) develop a gel card assay to evaluate existing, naturally occurring and BioRBC-induced plasma antibodies, 2) compare gel card and tube agglutination detection results, and 3) test for a relationship of antibody induction and BioRBC dose. Reagent BioRBCs were prepared using sulfo-NHS biotin ranging from densities 18 (BioRBC-18) to 1458 (BioRBC-1458) µg/mL RBCs. Among BioRBC-exposed subjects, gel card and tube agglutination results were concordant in 21 of 22 adults and all 19 infant plasma samples. Gel card antibody detection sensitivity was more than 10-fold greater than tube agglutination. Twelve to 16 weeks after BioRBC exposure, induced anti-antibodies were detected by gel card in three of 26 adults (12%) at reagent densities BioRBC-256 or less, but in none of 41 infants. Importantly, induced anti-BioRBC antibodies were associated with higher BioRBC dose (p = 0.008); no antibodies were detected in 18 subjects who received BioRBC doses less than or equal to BioRBC-18. For noninduced BioRBC antibodies, six of 1125 naïve adults (0.3%) and none of 46 naïve infants demonstrated existing anti-BioRBC antibodies using reagent BioRBC-140 or -162. Existing anti-BioRBCs were all neutralized by biotin compounds, while induced antibodies were not. The gel card assay is more sensitive than the tube agglutination assay. We recommend reagent BioRBC-256 for identifying anti-BioRBCs. Use of a low total RBC biotin label dose (≤ BioRBC-18) may minimize antibody induction. © 2017 AABB.

  17. Development of a immunochromatographic test with avidin-biotin for the detection of antibodies against antigen e of hepatitis B in human plasma

    International Nuclear Information System (INIS)

    Mainet Gonzalez, Damian; Palenzuela Gardon, Daniel O; Diaz Argudin, Tamara

    2007-01-01

    The disappearance of antigen e of hepatitis B in the presence of the plasmatic antibodies against antigen e may indicate a satisfactory therapeutic response in patients with chronic hepatitis B. The immuno-chromatographic test carried out in the diagnosis of diseases use different antibody combinations and may employ the avidin or streptavidin-biotin technology to develop a rapid immuno-chromatographic test for the detection of antibodies anti-antigen e in the plasma. They were detected in the laboratory by means of two fast immuno-chromatographic tests when using in one of them the avidin-biotin technology. These tests are carried out with a one-step competitive inhibition format and amplified or not with avidin-biotin. Monoclonal antibodies against antigen e obtained by cellular hybridization were used. Forty-six plasmatic samples classified as positive and negative to the anti-antigen antibodies were evaluated with a reference immunochromatographic test Advanced QualityTM. The possible expiry time of the biological reagents forming part of these tests were studied with accelerated thermal-stability experiments. The possible interference in the plasma of some of the biochemical compounds used in these trials was analyzed. Four murine monoclonal antibodies anti-antigen e were obtained and only one of them was used in these immunochromatographic tests with an anti-antigen polyclonal antibody conjugated with gold. Both tests and their stable biological reagents discriminated the positive and negative samples to the antibodies anti-antigen e, as well as the commercial test. There was no interference in the biochemical compounds studied in these tests. Both immuno-chromatographic tests made in the laboratory are useful to detect antibodies anti-antigen e in the plasma. The avidin-biotin increased the analytical sensitivity of this type of fast immuno-chromatographic test without altering its performance features. (Author)

  18. Intraoperative avidination for radionuclide treatment as a radiotherapy boost in breast cancer: results of a phase II study with 90Y-labeled biotin

    International Nuclear Information System (INIS)

    Paganelli, Giovanni; De Cicco, Concetta; Carbone, Giuseppe; Pacifici, Monica; Ferrari, Mahila E.; Cremonesi, Marta; Di Dia, Amalia; Pagani, Gianmatteo; Galimberti, Viviana; Luini, Alberto; Leonardi, Maria Cristina; Ferrari, Annamaria; Orecchia, Roberto; De Santis, Rita; Zurrida, Stefano; Veronesi, Umberto